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2020-12-10T09:04:17.050Z

1970-01-01T00:00:00.000Z

237234171

s2

Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds. A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds. Several methods have been described for rapid detection and quantitation of griseofulvin in body fluids and in fermentation media. Most of them are based on spectrophotometry (1, 3, 5-7, 10, 18, 22), spectrofluorometry (2,4,8,13,16), and colorimetry (19,23). Some spectrophotometric methods minimized error due to irrelevant materials by measuring absorbancies of the extracts at several equally spaced wavelengths and calculating the concentration of griseofulvin mathematically (1, 3, 5, 6). Holbrook et al. (11) converted griseofulvin to isogriseofulvin with methanesulfonic acid in methanol and determined its amount by measuring the resulting shift in ultraviolet (UV) absorption. Fischer and Riegelman (9) quantitated griseofulvin and griseofulvin-4'alcohol by measurement of fluorescence directly on thin-layer chromatograms. MacMillan (17) described a sensitive color test to detect dechlorogriseofulvin in the presence of griseofulvin. He reported that dechlorogriseofulvin gave an intense blue-violet color with nitric acid, whereas griseofulvin gave a pale yellow color. A method for griseofulvin determination in fermented broths of Penicillium griseofulvum and P. nigricans has been described in which the antibiotic was extracted from the fermentation broth with chloroform, and iodine was added in stochiometric ratios (24). Compounds which are structurally related to griseofulvin, such as dechlorogriseofulvin, can interfere with the analysis. Rezabek (20) and Kleine-Natrop et al. (14) assayed for griseofulvin on the basis of colony growth of Trichophyton persicolor and Trichophyton rubrum, respectively. Other biological methods for griseofulvin determination were re-ported by Knoll et al. (15) and Stepanisshcheva and Ziserman (21). The present study describes a rapid and accurate method for determination of griseofulvin and dechlorogriseofulvin by thin-layer chromatography (TLC) and gas-liquid chromatography (GLC). MATERIALS AND METHODS A griseofulvin-producing isolate of Penicilliwn urticae was used in this study. The fungus was cultured in 500-ml Erlenmeyer flasks containing 25 g of shredded wheat that was moistened with 50 ml of Mycological Broth (Difco) supplemented with 0.5% each of yeast and malt extract. After 10 to 14 days of growth at 28 C, fungal cultures from each flask were transferred into a Waring Blendor and extracted with 100 ml of chloroform. The chloroform extracts were filtered through anhydrous sodium sulfate and a 10 1uliter sample of the extract was spotted onto thinlayer chromatographic plates (0.25 mm) (MN-Kieselgel G-HR, Brinkman Instruments, Westbury, N.Y.), along with authentic griseofulvin. The plates were developed in chloroform-acetone (93:7, v/v) to a height of 10 cm. They were examined for the presence of griseofulvin or dechlorogriseofulvin, or both, first under long-wave UV light and then in normal light after being sprayed with 50% sulfuric acid and heated at 110 C for 30 min. GLC analyses were made with a Barber Colman series 5000 gas chromatograph equipped with a hydrogen-flame ionization detector and disc integrator. The liquid phases used were 1% QF-1 and 1 to 2% SE-30 coated onto Anakrom ABS 80 mesh (Analab Corp., Hamden, Conn.) by the method of Horning et al. (12). The GLC supports were packed into silanized glass columns. Precautions similar to those taken for steroids (12) were rigorously observed when preparing the GLC columns, column supports, and associated equipment to prevent "active sites" which would have caused decomposition of the antibiotics. Griseofulvin and dechlorogriseofulvin were isolated and purified from chloroform extracts of P. urticae by precipitation from chloroform solution with nhexane, followed by silica gel column chromatography (0.05to 0.20-mm mesh) (Brinkman Instruments, Westbury, New York) of the precipitate with chloroform as the eluting solvent. Fractions (25 ml) were collected automatically and subsequently monitored by GLC for the presence of griseofulvin and dechlorogriseofulvin. The fractions containing griseofulvin and dechlorogriseofulvin were combined, evaporated to dryness, and recrystallized from n-hexane-chloroform solution. Analytical confirmation of the structures of the purified griseofulvin and dechlorogriseofulvin from P. urticae was based on melting points, TLC, GLC, and UV, infrared (IR), nuclear molecular resonance (NMR), and mass spectral analyses. Melting points were taken on a Fischer-Johns melting point apparatus; UV spectra were determined in methanol solution with a model DB-G spectrometer (Beckman Instruments, Inc., Fullerton, Calif.). Infrared spectra were measured with a Perkin-Elmer model 257 spectrometer as a thin film coated onto a KBr window. NMR spectra were performed with a Varian A-60A spectrometer in deuterated chloroform. Samples for mass spectra were introduced into the mass spectrometer by the direct-probe method. RESULTS AND DISCUSSION Griseofulvin and dechlorogriseofulvin were determined accurately in crude extracts from P. urticae by TLC and GLC analysis. These two compounds appeared together as a bright blue fluorescent spot at RF 0.65 on our TLC system. The difference in polarity between these two compounds was insufficient for their complete separation. The limit of detection of griseofulvin by TLC was 0.05 ,ug. Thus, this chromatographic method provided a rapid and sensitive technique for tentative identification of both griseofulvin and dechlorogriseofulvin. Two GLC systems were then used to separate these compounds (Fig. 1, Table 1). This permitted accurate quantitation of the individual compounds without extensive purification. Spectrophotometric and spectrofluorometric methods would not make this distinction on a sample containing a mixture of these compounds. The colorimetric method described by MacMillan (17) would detect both substances in a purified sample mixture, but interfering substances in a crude extract might affect accuracy. No metabolites in the extracts from P. urticae interfered with either the GLC or TLC analyses. The use of TLC is valuable for preliminary screening, since several extracts can be evaluated on one chromatographic plate in minimal time. Extracts that appear to contain griseofulvin or dechlorogriseofulvin, or both, can then be simultaneously analyzed qualitatively and quantitatively by GLC. In addition to being an excellent qualitative and quantitative method for analysis of these compounds, GLC also served as a monitor during purification. Use of GLC showed that griseofulvin and dechlorogriseofulvin were not resolved from each other by silica gel column chromatography; however, they were separated by fractional recrystallization. This was consistent with previously reported data (17). The melting point and UV, IR, NMR, and mass spectra of the metabolite identified as griseofulvin from P. urticae and an authentic griseofulvin standard were compared to confirm that the compound observed on TLC and GLC was griseo-fulvin. No authentic standard for dechlorogriseofulvin was available; however, purified dechlorogriseofulvin from P. urticae was identified by comparison of its UV, IR, NMR, and mass spectra with those of griseofulvin. The melting point (220 C), UV spectra [wavelength (X) maxima in methyl alcohol at 291, 236, and 325 nm], and IR spectra of griseofulvin from P. urticae and authentic griseofulvin were identical. The molecular weights of these compounds, as determined by mass spectroscopy [molecular extinction coefficient (m/e) 352], and of the major fragments (m/e 214 and m/e 138) were also identical. In addition, the mass spectra were identical in their overall fragmentation patterns. The mass spectrum of dechlorogriseofulvin showed parent peak at m/e 318 with major fragmentation at m/e 180 and m/e 138. This is consistent with the spectrum of griseofulvin less chlorine. The UV spectrum of dechlorogriseofulvin was identical with griseofulvin and the IR spectrum had only minor differences. The NMR spectra for griseofulvin from P. urticae and authentic griseofulvin were identical. The NMR spectrum of dechlorogriseofulvin showed the presence of an additional proton at 6.08 parts per million (ppm) which was coupled with a proton at 6.26 ppm (J = 2 Hz). This is consistent with the expected coupling of protons that are meta to each other such as the protons at carbon 5 coupling with the proton replacing the chlorine group at carbon 7 in dechlorogriseofulvin. The absorptions of the two aromatic methoxy groups in griseofulvin (4.00 ppm and 4.05 ppm) were shifted in dechlorogriseofulvin so as to be superimposed at 3.92 ppm. The above analytical data prove that the two compounds, from crude extracts of P. urticae which were analyzed via TLC and GLC, were griseofulvin and dechlorogriseofulvin.

v3-fos

2020-12-10T09:04:12.317Z

1970-05-01T00:00:00.000Z

237231559

s2

Effect of Chloramphenicol on Host-Bacteriophage Relationships in the Lactic Streptococci Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml3. Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 μg/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml3 phage in strain ML3-CMrI, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one. ogous attacking phages but not to phage mls. Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 ,g/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml3 phage in strain ML3-CMrI, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phageresistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one. Studies of bacteriophage resistance in the lactic streptococci are of considerable interest since phage can destroy these bacteria when they are used as starter cultures in the manufacture of cheese (2,6). The use of phage-resistant mutants to circumvent phage attack has been examined in the past, with attention directed towards the employment of mutants that either do not allow phage penetration, or are lysogenic, or exist as phage carriers (5,8,10,11,13,14). Indirect means of obtaining phage-resistant mutants have been described for other bacterial genera, including the method of screening drug-resistant mutants for phage resistance. A recent report (9) indicated that choramphenicol (CM)-resistant mutants of group A streptococci were also resistant to lysis by virulent phage. The biochemical events which result from exposure of growing bacteria to CM, which acts primarily to inhibit protein synthesis, have been the subject of intensive investigation (3). This communication describes the effect of CM treatment on phage-host relationships in lactic streptococci. I (16). Bacteriophages. In phage infection experiments with S. lactis ML3 and its homologous phage, a whey suspension of phage ml s containing 2.4 X 1010 plaqueforming units per ml was used. In testing mutants of ML3 for their spectrum of resistance to heterologous phage types, the collection of S. lactis and S. cremoris phages held by the New Zealand Dairy Research Institute was used. Phage assays were carried out by the agar overlay method (1) on LYP agar (16) plates. To test the reaction of a bacterial strain to a particular phage, a sample of the bacteria was layered in semisolid agar on an LYP agar plate, and the phage suspension was spotted on. Isolation of phage-resistant mutants. Mutant colonies of S. lactis ML3 resistant to phage ml3 were isolated from plates layered with semisolid agar containing about 5 X 108 cells and phage at a multiplicity of 5. These were streaked on plates to purify, 707 they were grown in broth, and the cultures were checked for phage resistance. Isolation of CM-resistant mutants. Mutants of S. lactis ML3 resistant to CM were obtained during the process of continuous transfer of ML3 in LYP broth containing increasing amounts of CM. Other CMresistant mutants of ML3 were isolated from LYP agar plates containing CM which had been spread with about 108 colony-forming units from an ML3 culture previously subjected to mutagenic treatment with either NG (15) or ultraviolet (UV) irradiation. Treatment with NG was carried out as follows. A logphase LYP broth culture received 1,000 ,ug of NG/ml before incubation at 30 C for 20 min. The cells were centrifuged, resuspended in LYP broth, and incubated at 30 C for 3 hr before they were spread on CMcontaining plates. For UV treatment, the cells of a log-phase culture were centrifuged, washed in Ringer solution, and finally resuspended in this medium. A sample (3 ml) of the suspension was placed at a distance of 29 cm from a Hanovia UV-lamp and irradiated for 30 sec. Samples were then withdrawn, incubated at 30 C for 3 hr, and spread on CM-containing plates. Bacterial growth curves. The growth or lysis of logphase LYP broth cultures of S. lactis ML3 and derivative CM-and phage-resistant mutants, in the presence or absence of CM and phage, was determined by viable cell counts and turbidity measurements in a Spectronic 20 colorimeter/spectrophotometer set at 560 nm. One-step growth experiments were carried out as described by Adams (1). Premature lysis of cells was achieved by diluting infected cells into 1.0-ml samples of Ringer solution, held at 4 C, containing two drops of chloroform and 25% (v/v) Chance no. 12 Ballotini beads, followed by shaking on a Mickel disintegrator for 2 hr at 4 C. To determine whether phage penetration of bacterial cells occurred after phage adsorption in the presence of CM, cultures of CM-treated and untreated cells were infected with phage and, after allowing time for adsorption, the bacteria were separated from unadsorbed phage by low-speed centrifugation (4,000 X g for 5 min). The cells were then suspended in a solution containing (per liter): 1 mM MgSO4, 0.1 mM CaCl2, and 0.1 g of gelatin (12). This suspension was agitated in a Waring Blendor for 2 min at 10,000 rev/min before cooling in ice water. Samples were removed for assay of infective centers or for further centrifugation to collect released phage. RESULTS Mutagenesis studies with S. lactis ML3. The killing effects of NG and UV irradiation were examined initially to determine suitable mutagenic doses for treatment of S. lactis ML3. The effect of NG treatment on ML3 is presented as a function of dose ( Fig. 1) and as a function of time (Fig. 2). ML3 was found to be very resistant in comparison with gram-negative bacteria (15). The cells of an NG-treated culture, examined The results of a UV survival experiment ( Fig. 3), carried out with a suspension of ML3 cells in Ringer solution, indicated that irradiation at a distance of 29 cm for 30 sec gave about 90% killing of cells, and this was subsequently chosen as the mutagenic dose. CM resistance and phage resistance in S. lactis ML3. Continuous transfer of ML3 in LYP broth containing increasing concentrations of CM produced, after 73 transfers, a culture able to grow slowly in the presence of 280 jig of CM/ml. At levels of CM above this value, no bacterial growth could be obtained. The natural level of resistance is 2 ,g/ml. A culture of ML3_CMrI, isolated after 66 transfers, grew normally in the presence of 210 ,ug of CM/ml and was chosen for examination in subsequent growth curve studies in the presence or absence of CM and phage ml 3. ML3-CMrI did not revert to CM susceptibility when subcultured in the absence of the antibiotic. During the period of training, after addition of each CM increment, the growing culture was tested for its susceptibility to 25 stock S. lactis and S. cremoris phages, seven of which, under normal conditions, are able to form plaques when spotted on an ML3 culture plated in semi-solid agar on LYP agar. The phage relationships of ML3 and the culture of ML3 enriched for CMr mutants are shown in Table 1. In the course of transfer in CM-containing broth, the ML3 culture lost its resistance to hp phage for a period of 29 transfers; it acquired resistance to z8 phage after 14 transfers and to sk2 phage after 72 transfers; and it became susceptible to pl phage after 50 transfers. Apart from phage-relationship changes, other significant observations made during the course of training were as follows. At a level of 16 jAg of CM/ml, there was a distinct change from slow growth to normal growth in the presence of increasing CM concentrations. At 42 ,ug of CM/ ml and thereafter, there was a marked increase in the number of phage-resistant colonies appearing in the phage ml,3 lytic areas. At levels between 175 and 190 ,g of CM/ml, cultures required 48 hr of incubation at 30 C to yield cell numbers previously obtained in 24 hr. Above 190 MAg of CM/ml, it became necessary to alternate subculture in LYP broth plus the appropriate CM increment, with subculture in normal LYP broth to achieve satisfactory growth in the presence of the increased CM concentration. Single CM-resistant colonies were isolated from plates, containing a range of CM concentrations, that had been spread with NG-treated and UV-irradiated S. lactis ML3 cultures. Each colony was suspended in LYP broth, grown to log phase, and then tested for resistance to both the homologous phage and to CM. In this way, nine mutants resistant to CM concentrations ranging up to 200 ,ug/ml were isolated from the NG-treated culture. All of the mutants tested were susceptible to phage ml3. Sixteen mutants, resistant to similar CM concentrations and sensitive to ml,3, were isolated from the UV-irradiated culture and, in addition, four mutants were isolated at a level of 500 ,g of CM/ml after examination of 200 plates. These four mutants were resistant to phage ml,3. In the course of daily subculture in LYP broth in the absence of CM, they lost their resistance to CM after about 4 days, but retained their resistance to phage ml,3 for about 6 weeks before reversion to phage sensitivity was observed in two of the cultures. An identical number of plates spread with the NGtreated culture and containing the same high concentration of CM failed to yield any CM-resistant mutants. A total of 100 phage-resistant forms of S. lactis ML3 were examined for resistance to CM by subculturing to tubes of LYP broth containing a range of CM concentrations; none of the strains tested had acquired CM resistance in addition to their phage resistance. Effect of CM on the multiplication of S. lactis phage m13. The mutant S. lactis strain, ML3-CMrI, trained to grow in the presence of 210 ,ug of CM/ml, was compared with normal ML3 in a number of growth curve experiments to determine the effect of CM on phage multiplication. Each strain was grown to early log phase, and its subsequent growth was examined both when untreated and after receiving additions of (i) an m13 phage suspension, (ii) CM solution at two different concentration levels, and (iii) ml 3 phage suspension plus CM solution at the two different concentration levels ( Fig. 4 and 5). At a concentration which did not inhibit bacterial growth, CM clearly had no effect on phage multiplication in ML3, and the same concentration did not inhibit phage lysis in ML3_CMrI. On the other hand, at a concentration of 50 ,ug of CM/ml, growth of ML3 was halted, whereas ML3_CMrI continued to grow normally. As would be expected under these conditions, ML3 was not lysed by the phage. However, ML3_CMrI was also not lysed by phage ml 3 in the presence of 50 ,ug of CM/ml, despite the fact that normal cell growth occurred at this concentration. As a control experiment, the effect of CM on phage per se was tested, and no decrease in viability was observed. Adsorption experiments. To determine whether phage was adsorbed to ML3_CMrI in the presence of CM, the titer of free phage in an adsorption mixture of ML3_CMrI plus 50 jug of CM/ml was compared with that in ML3-CMrI during the course of an incubation period of 19 min. There was a rapid adsorption by both strains of approximately 45 % of the plaque-forming units within 2 min. Adsorption continued at a decreased rate until about 90% of the particles were adsorbed by 19 min. Phage penetration. An experiment was carried out to determine whether addition of phage ml3 to ML3-CMrI in the presence of CM resulted in phage adsorption but no penetration. Adsorption mixtures of ML3_CMrI plus phage and ML3-CMrI plus 50 ,ug of CM/ml plus phage were examined, after a 10-min adsorption period, for phage penetration. A phage multiplicity of 0.1 was used. The mixtures were assayed for infective centers at the end of the 10-min adsorption period FIG. 6. One-step growth and premature lysis curves for S. lactis ML3 and ML3-CMrl. The experiments were performed in L YP broth at 30 C, with an input ratio of phage to bacteria of 1:10. The number of infected bacteria was determined by neutralization of unadsorbed phage with antiphage serum, followed by plating at dilutions suitable for plaque counts. and then subjected to blendor treatment before determining survival of infective centers by plaque assay. At the same time, the infected bacteria in both mixtures were centrifuged at 5,000 rev/min, and the supernatants were assayed for any free phage that may have been separated from the bacteria by the blendor treatment. Approximately 94%O of the infective centers in the two adsorption mixtures survived the blendor treatment. The free phage titers in the two supernatants, after the final low-speed centrifugation, were: ML3_CMrI plus phage, 1.9 X 102/ml; ML3_CMrI plus CM plus phage, 1.4 X 102/nl. That is, similar small amounts of phage, probably representing unadsorbed phage, were found in the two mixtures, indicating that adsorption of phage leads to penetration in both cases. Premature lysis and one-step growth curves. The results of one of four such experiments for each of the strains ML3 and ML3-CMrI are depicted in Fig. 6. Strain ML3_CMrI was tested both with and without 50 ,ug of CM/ml added to the culture before addition of phage. In the onestep growth curves for ML3, and ML3_CMrI without CM added, phage m13 showed a latent period of about 25 min, followed by a rapid rise in titer which reached its maximum in 6 min. The average burst size in each case was about 43 particles per cell. The other three experiments revealed similar latent and burst periods, although the actual increase in phage titer was variable. In the presence of CM, however, no increase in phage titer was observed for ML3_CMrI during the course of the one-step growth curve. The premature lysis curves for ML3 and ML3-CMrI revealed similar eclipse periods followed by the usual phage proliferation. No mature phage particles were detected in the ML3_CMrI culture containing added CM during the entire incubation period. As a further check on the fate ofphagepenetrating the host cells in an adsorption mixture of ML3_CMrI plus 50 ,ug of CM/ml plus phage ml3, a test was made for the appearance of lysogenic forms. The mixture was incubated for 4 hr after infection, and samples were then withdrawn and spread on LYP agar plates. Single colonies were selected and tested for resistance to 3 phage. One hundred colonies were examined in this way, and none was phage resistant, indicating that no lysogenic association resulted from infection of ML3_CMrI with m13 phage in the presence of CM. DISCUSSION At least three types of mutation have been shown to affect resistance to CM in S. lactis ML3. The stepwise mutation associated with training host cells to grow in the presence of increasing increments of CM produced forms resistant to CM up to a maximum concentration level of 280 jig/ml. In association with this stepwise mutation, there was a change in the response of the strain to infection with heterologous phage types, generally in the direction of increased resistance to these phages. However, it remained susceptible to the homologous phage. After treatment with NG or UV irradiation, both potent mutagenic agents for many bacterial genera, at levels sufficient to give 90% inactivation of the cells, mutants showing resistance to concentrations of CM up to 200 ,ug/ml could be selected. These one-step mutants remained susceptible to attack by the homologous phage. The third type of mutant was selected at low frequency, and again involved a one-step mutation event. In this case, ML3 yielded, after UV irradiation, mutants resistant to 500 Ag of CM/ml; these mutants were also resistant to phage ml 3. During the course of subsequent daily subculture, some of these mutants reverted to CM sensitivity after four transfers and to phage sensitivity after 43 transfers. Mutants selected for phage resistance were not CM resistant. VOL. 19, 1970 711 Ios It seems likely that there are at least two different loci for CM resistance on the ML3 chromosome: the one for high-level resistance (500 Mg! ml), although not identical with the phage-resistance locus, is associated with it, whereas the one or more concerned in the stepwise mutation are independent of it. The locus affected by the singlestep mutations conferring resistance to CM levels up to 200 ,g/ml may not be the same as that involved in the stepwise mutational events. It has been shown that resistance to CM in Escherichia coli may be due to one of three possible mechanisms (17): (i) resistance of the protein-synthesizing machinery; (ii) decreased permeation to the site of antibiotic action; (iii) inactivation of the drug. The demonstration that CM intereferes with the multiplication of phage in S. lactis ML3_CMrI indicates that the second of these mechanisms does not operate in this case. If ability to inactivate the drug resulted from the mutation to CM-resistance, the mutation would be one in which the necessary enzyme was formed in greater amount or had a higher affinity for the CM substrate. In either case, after addition of CM to the growing culture, the growth rate would be decreased until inactivation was completed. No reduction in growth rate was observed when normally bacteriostatic concentrations of CM were added to strain ML3CMrI. It seemslikely, therefore, that in this strain the mutation to CM resistance confers resistance on the proteinsynthesizing ability of the host cells. At or below a concentration of 2 ug/ml, CM had no effect on the growth of ML3 in LYP broth, and lysis occurred after the usual period of incubation in the presence of phage ml 3. The same was true for strain ML3_CMrI which had been trained to grow in the presence of 280 MAg of CM/ml. When the concentration of CM in each adsorption mixture was raised to 50 Mg/ml, growth of ML3 was inhibited and no phage lysis occurred; phage lysis of ML3_CMrI was also inhibited by this concentration of CM, but cell growth remained unaffected. In analogous experiments (R. J. Richards, Ph.D. thesis, Ohio State University, 1960), it was shown that subinhibitory concentrations of penicillin decreased the time required for lysis initiation by a S. lactis host-phage system in broth, whereas oxytetracycline caused a delay in the onset of lysis, and streptomycin had no effect. The magnitude of the penicillin and oxytetracycline effects increased as the antibiotic concentration increased. Since it could be demonstrated that CM had no effect on phage ml 3 per se, the possibility existed that the CM affected the phage-host interaction in one or more of the following ways. (i) Phage adsorption was inhibited; (ii) phage penetration was inhibited; (iii) penetrating phage established a lysogenic relationship with the host. When cells are infected with temperate phages, either a lytic response or a lysogenic response can occur. In some bacteria, CM brings about an increase in the lysogenic response, apparently by inhibiting the protein synthesis that is required for the lytic response (4). This effect is not specific but is brought about also by other conditions that retard biosynthetic operations. The adsorption and penetration experiments carried out with ML3_CMrI provided evidence that CM at a concentration of 50 ,g/ml did not affect adsorption to and penetration of the host cells by phage ml 3. In addition, it was shown that lysogenic forms did not arise as a result of the phage infection, since surviving cells were not resistant to the homologous phage. The one-step growth and premature lysis experiments confirmed that no intracellular multiplication of phage took place even though phage adsorption and penetration occurred. Thus, any infection that occurred may be termed abortive. It may be concluded that, in an adsorption mixture of ML3_CMrI plus phage m13 plus 50 ,ug of CM/ml, the CM interferes with the synthesis of mature phage particles without affecting the synthesis of host cell material, or that phage is synthesized but is nonviable (defective) when synthesized in the presence of CM. When abortive infection occurs, phage development is halted before the completion of mature phage particles. With a variety of chemical manipulations, it is possible to allow phage development to proceed to completion with the formation of structurally mature phage which, however, are non-infective. This results from the incorporation of an unnatural amino acid or pyrimidine into the protein or nucleic acid of the phage. No defective phage of phage components were detected with an electron microscope after artificial disruption of the phage-infected ML3_CMrI. Phage production is dependent on the host cell metabolism for energy and for the synthesis of raw materials. Therefore, any interference with the energy metabolism or the synthetic enzyme system of the host cell may be expected to have an effect on phage production. However, it is possible to interfere with bacterial multiplication without affecting phage growth, and vice versa, showing that the nutritional requirements for the two processes are not identical. CM inhibition of deoxyribonucleic acid (DNA) synthesis has been observed in phage-infected bacteria (7). Several new enzymes, which are necessary for synthesis of phage DNA, are produced in the first few minutes after phage infection. It is possible that CM inhibits phage DNA synthesis indirectly by blocking the formation of these enzymes. It is concluded that the block in phage development in ML3-CMrI probably involves inhibition of phage component synthesis so that, although phage penetrates the cell, either the genome is not replicated or phage coat proteins are not synthesized. Thus, no mature phage particles are formed. ACKNOWLEDGMENT It is a pleasure to acknowledge the capable technical assistance of Diana Mellor.

v3-fos

2022-07-07T15:02:43.334Z

1970-01-01T00:00:00.000Z

250318563

s2

Characteristics and Productivity of Some Pigeon Breeds in Bangladesh (Aves: Columbidae) This is our hurried responsibility to introduce those pigeon breeds that have economic value. In this case, very common and productive breeds but moderately unknown in history or confusing on their scientific identity need to be focused elaborately. A survey in the country, as well as experiences on these pigeons, inspired to write this valuable fundamental composition. Pet shops, markets, and lofts have sulli (moos-sulli), chuva chandan (chua chandan), bombai (sotobanca/Italian crested mondain), kokah (laugher), and parvin (Australian red) pigeons are carrying a remarkable productivity in the country. Prices of adult and squabs are reasonable to all sorts of people. Introduction Bangladesh is very rich with many varieties of pigeons from the past. Many people kept pigeons with maintaining proper care and breeding. In past, people reared pigeons just for meat purposes (local pigeon) and few were tumblers and highflyer pigeons for flying amusement. (Levi, 1941) described moos-sulli/Syrian coop tumbler with their characteristics in his book 'The Pigeon'. In Bangladesh, this breed is called 'ghia sulli' for its only self-yellow colour. (Kabir, 2014), (Kabir, 2014a), (Kabir, 2015) just compiled some tumbler/roller/highflyer pigeons of Bangladesh where this moos-sulli (so-called ghia sulli) was noticeable. In most of the areas of Bangladesh, this breed is available with its perfect markings. It has no leg feathers and cannot tumble when fly and is considered a beautiful cage bird (Kabir, 2021). Italian sotobanca or crested mondain had been noted by Levi (1941), in his book too. In Bangladesh, one type of pigeon is available which is called bombai pigeon. According to this name, this is an Indian breed (Bombay of India) but it has no concrete history of origin in any book of pigeons. A keen observation about sotobanca, this bombai pigeon shows many similarities with them. So this bombai can be a cross product of sotobanca. (Kabir, 2018) mentioned bombai pigeon in his paper as a successful pigeon breed for starting a firsttime pigeon farm. In 1985, Moragacha village of Kumarkhali upazila of Bangladesh, there was a male black bombai (Italian crested mondain), its pair flew from the launch from getting back from Sirajganj of Bangladesh. This Moragacha village was renowned for tumblers and lotan pigeons at a time, and this village carries a nostalgic event with the author of this paper (Kabir, 2020). Kokah pigeon is a special type of vocalized pigeon, available throughout Bangladesh but in the Rangpur division, this is many (Rahman, 1999). Emperor Akbar had around 20,000 pigeons and among them, 500 were specially sorted out. Kabir (2014), published an article on the Mughal pigeons with the availability of kokah pigeons. In other books, there is no trace about this pigeon but their voice is similar to Thai laugher but phenotypically this breed is different than others. There is no single evidence about "chuva chandan" and "Australian red" breeds in books except only one pdf on the internet (Self, 2022). Rahman (1999), only mentioned just the names of bombai, parvin, and chua chandan pigeons in his book without description. The objective of this study is to enhance for keeping these pigeon breeds for sufficient profit with the fulfilling their hobby. Materials and Methods Sulli, chuva chandan, parvin, bombai, and kokah pigeons were available in pet shops of Kushtia, Saidpur, Dinajpur, and Dhaka Katabon Animal Market Kabir (2013), and pigeon markets of Kushtia, Saidpur, and Mirpur Dhaka, Bangladesh (Kabir, 2014c). Direct observation and survey method provided immense thought about their breeding characteristics, comparison with other breeds, and their acceptance value. Weekly visit in these shops and markets enriched the parameters of this article. Pigeon keepers helped to contribute their year-wise data on the above parameters. This study was completed in the year 2021. Results and Discussion Moos-sulli. Wendell Mitchell Levi (1941), described moos-sulli breed in his book 'The Pigeon' (page 217) in tumbler varieties. In Bangladesh, this pigeon is called 'ghia sulli'. Its colour is very nice like ghee and a popular breed (Table 1; Figure 1). This is not a flying bird; rearers keep in the cage as a show bird. In most of the pet shops, this pigeon is available and the price is reasonable at all. Moreover, its breeding performance, as well as care to the young, is mentionable. In Bangladesh, this is considered a non-tumbling tumbler (Kabir, Hawkeswood, & Makhan, 2020). Its body shape, eye colour, beak, and head size are somewhat a true tumbler. Levi (1941), said this breed into coop-tumbler because it tumbles in a very short space. Among the tumbler group, in Bangladesh, this sulli pigeon is very common, especially in Dhaka city. Till now, there is no mixing record of this breed but sometimes white feathers can be expressed with its yellow plumages (Table 1; Figure 1). Levi (1941), described this breed with muff but in Bangladesh, this bird is totally clean-legged. Based on the shape of the head mostly this breed is plainheaded but crested variety also common. In fact, this is a breed (domestic) (Columba livia domestica) not a species. Figure 1. Ghia sulli (Moos-sulli) (Columba livia domestica) Chuva chandan. This is an Indian breed, same with the moos-sulli. Pigeon belongs to the tumbler family but this is a show breed. Remarkable coloured with yellowish head-neck and the body is shades of grey (Table 1; Figure 2). Commonly found in Bangladesh at a reasonable price and has an enchanting face at a glance. Its eye is always black and clean-legged. Mostly plain-headed but crested variety is also available throughout Bangladesh. Moreover, it cannot tumble. The beak is short and is clean-legged. Breeders do not mix this pigeon with others so that this is a purebreed till now; a very docile and calm pigeon. Its crest is always displayed with peak crest. The breeding biology of this pigeon is mentionable and possible to sell its baby anytime. Based on the nice and exceptional plumage colour, this pigeon could snatch anybody's eyesight. Plain-headed variety is more abundant than the crested ones (Table 1; Figure 2). Acceptance, availability, price, overall reproductive performances are distinguished on their commercial value. Figure 2. Chua chandan (chuva chandan) (Columba livia domestica) Bombai pigeon. When I read in class four, saw a big large-sized black male pigeon (so-called bombai) in my village Moragacha. Its pair flew from the launch when my cousin is returning from Sirajganj of Bangladesh. Then in our village, this male got a pair with other local (indigenous) black female and this pair produced some black and intermediate-sized offspring. In my childhood place, Moragacha village under Kumarkhali upazila of Bangladesh had many nostalgic events with pigeons (Kabir, 2020). My father kept tumblers and loton pigeons in this village. Finally, all of those black bombai pigeons were caught by domestic cats. In many books, I did not find the actual international name of this bombai pigeon. From the name bombai, it is easily understandable that this is an Indian breed. On the internet, after keen searching Italian crested mondain is similar to this bombai pigeon (Beaumont, 1962). It has four self colours-black, red, yellow, and white but black is rare. All are very nice with their broad crest (ear to ear); robust body and very powerful birds at all. Carrying a sharp beak and completely clean-legged (Table 1; Figure 3). Breeding is mentionable but sometimes its huge bodyweight makes hazards in incubation. Alternative use of the pair could ensure proper hatching. It is commonly found in any pet shops and markets of Bangladesh. Legs are comparatively short. Pigeon keeper collects this pigeon without any doubt and does not cross with others so that this is purebred as a whole. Due to heavyweight, they cannot fly more. Figure 3. Red bombai (sotobanca) (Columba livia domestica) Kokah Pigeon. The history of the laugher pigeon is known but on kokah pigeon, this is completely unknown in any books. Rahman (1999), mentioned this pigeon as a kokah breed in his book 'Kingdom of Pigeons' (in Bangla). Its voice is completely similar to Thai laugher pigeon but its physical appearance is the same as with the local pigeon. The plumage colour is brick-red. The beak is very sharp; loose plumages; peak crested and always clean legged (Table 1; Figure 4). Till now, this is purebred at all and found abundant. Price is moderately high (depending on its voice quality) and available in Bangladesh especially Rajshahi, Natore, and Rangpur. After hatching, this pair shows more aggressiveness to each other so alternative use of birds ensures the stability of the young (Rahman, 1999). Its continuous narrow cooing is a long-lasting phenomenon. Early in the morning, it makes a huge sound. Normally small pigeons and body stamina are not very strong. Till now it has no alternative colours except brick-red. In addition, it has no plain-headed and muffed variety. With the brick-red plumages, sometimes black feathers can be seen. Slim bodied with lightweight. The eye colour is orange or red (Table 1; Figure 4). Figure 4. Kokah (laugher) (Columba livia domestica) Parvin. In Bangladesh, this breed is called 'parvin' but on the internet supplement, this is somewhat Australian red/Darpan/Barpan (Self, 2022). Always red or yellow and very few are whites. A normal looked pigeon having a broad crest and muff. Plain-headed variety is not available (Table 1; Figure 5). Medium elongated body and medium to large-sized. It has an affectionate face with reasonable market value; not very common in the pet shop or pigeon markets. Breeding performance is acceptable like other pigeons. Cool tempered pigeon and possible to rear with other pigeons. Yellow coloured eye with small and flattened head. It is show bird and cannot fly well due to muff (Table 1; Figure 5). More or less broad crest and muffed pigeon; it has no other extra-ordinary qualities to distinguish from other breeds. All are self-coloured; this is purebred in Bangladesh. Pigeon breeders normally do not mix this exceptional breed with others for earning a good profit. Conclusion The mentionable five breeds are more pronounced perspective Bangladesh with their economic advantages. If any pigeon keepers rear these pigeons, be economically benefited after fulfilling their hobby. In this regard, this article is carrying remarkable merit. For a long time staying in Bangladesh and for some/many differences we can recognize them in the separate breed and could deliver their name internationally. These pigeons are available in the country, outstanding breeding performance, and the market value of adults and squabs are desirable.

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Inactivation of Vesicular Stomatitis Virus by Disinfectants Twenty-four chemical disinfectants considered to be viricidal were tested. Ten disinfectants were not viricidal for vesicular stomatitis virus within 10 min at 20 C when an LD50 titer of 108.5 virus units per 0.1 ml were to be inactivated. Quantitative inactivation experiments were done with acid, alkaline, and a substituted phenolic disinfectant to determine the kinetics of the virus inactivation. Substituted phenolic disinfectants, halogens, and cresylic and hydrochloric acids were viricidal. Basic compounds such as lye and sodium metasilicate were not viricidal. MATERIALS AND METHODS The techniques used to obtain these data were the same as described previously (8) with the following exceptions. Disinfectant tests. Virus-disinfectant contact times were 5 and 10 min. The 30-min interval was eliminated since 10 min has been established as a maximal practical exposure time for effective viricides in animal disease control work. Speed of inactivation. Experiments were completed with seven time intervals. These were 0.5, 1, 2, 3, 4, 7.5, and 10 min. Additional time intervals of 30 and 120 min and 24 hr were used for NaOH. The disinfectant was diluted at the end of each time interval by the addition of 9 ml of phosphate-buffered saline (PBS) to the disinfectant-virus mixture, and, where acid or base was used, sterile 0.1 N NaOH or 0.1 N HCI was added to neutralize the pH of the PBS. Quantitative determinations of virus titer were measured by using serial 10-fold dilutions of virusdisinfectant mixture in embryonating chicken eggs for each time interval. The titer of virus was estimated by the LD5o method of Reed and Muench (7). Disinfectants. The 24 disinfectants are listed in Table 1. RESULTS Of 24 disinfectants, 10 were not viricidal with the previously described procedure. Alkaline chemicals tested were not viricidal for VSV, e.g., VSV survived for 24 hr in 10% NaOH (pH 12.2). Virulent virus was demonstrated after treatment with 10% KOH (pH 13.3), 10% Na2CO3 (pH 11.1), and 5% Na2SiO3 (pH 12.1) for 10 min. The quatemary ammonium compound, disinfectant G, was not viricidal at concentrations of 0.1 to 5.0%. In addition, sulfuric acid, acetic acid, isopropanol, ethyl alcohol, and Formalin were not viricidal at concentrations tested. The results are listed in Table 2 with the range of concentrations. Identical results were obtained for at least four replications of each disinfectant. The 14 other disinfectants were viricidal, but in some cases at concentrations greater than suggested by the manufacturer. The minimum viricidal concentration and the range of concentrations tested are listed in Table 3. Consistent results were not obtained with disinfectant E. The manufacturer's lot 1 was viricidal, whereas a second lot was not viricidal at the same concentration. The speed of inactivation was determined with NaOH, acetic acid, and disinfectant L. The death curve with NaOH and acetic acid was not linear, being initially rapid, followed by a decrease in rate. Virulent virus was not detectable after 30 min with 5% acetic acid, whereas, with 10% NaOH, it was still virulent after 24 hr. Disinfectant L at 2% concentration was rapidly viricidal and virulent virus was not detectable after 2 min. The virus survived 0.5% Table 1. Table 1. b Results variable depending on lot tested. propanol were not viricidal at a rate rapid enough to be useful against VSV. Alkaline chemicals have been employed as viricides in vesicular disease outbreaks in the United States because of their application for the inactivation of foot-and-mouth disease (FMD) virus. FMD virus was much more susceptible to change in pH as a means of virus destruction than VSV (4). VSV was resistant to destruction by alkaline chemicals in the present study. None of the alkaline chemicals, NaOH, KOH, Na2CO3, or Na2SiO3, was effective against VSV. However, in the United States, when a vesicular condition is found, it must be assumed to be FMD and chemicals used must be recognized as effective against FMD virus until the agent is proved to be VSV. Whereas acids have also been used to control VSV, it was determined that acetic acid was of only marginal value, since variable results were obtained with a 5 % concentration and lower concentrations were not viricidal. The results obtained with mineral acids depended on the particular acid, possibly owing to the different degrees of ionization (pK). The pK values of the mineral acids were hydrochloric acid, 0.784; sulfuric acid, 0.510; and acetic acid, 0.004. The most consistent viricides for VSV were the phenolic types when a sufficient concentration was used, but the effective concentration was higher than the manufacturer's recommendations in some cases. Organic iodine (U) and sodium hypochlorite were both active viricides. The virus was inactivated in 10 min even with the presence of the chorioallantoic membrane and allantoic fluid in the virus mixture. However, halogens are more susceptible to inactivation by organic material than other disinfectants. The rate of inactivation was determined for three different types of disinfectants. Disinfectant L (2%) was rapidly viricidal by an apparent first-order reaction, but, when it was diluted to 0.5%, a diphasic inactivation curve was evident. The diphasic curve was also evident for acetic acid and NaOH, even though the pH of the NaOH remained stable at pH 12.2 over the time of exposure. It appeared that the survivor curve was diphasic when the concentration of disinfectant was below that which was rapidly viricidal. Disinfectants at viricidal concentrations produced logarithmic virus survival curves or curves with a slight change in slope. The survival of virulent virus particles may have been due to the size of the aggregate and degree of aggregation. When a sufficiently high concentration of phenolic and halogen type of disinfectants was present, the additional time required to inactivate the virus was minimal. Based on laboratory evidence, substituted phenolics, halogens, or cresylic acids are recommended for use when vesicular stomatitis virus is to be destroyed on an infected premise. These chemicals are in the proper concentration under clean conditions and can be used with greater personal safety than acids and bases.

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1970-03-01T00:00:00.000Z

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Thermal Inactivation of Newcastle Disease Virus The rate of destruction of hemagglutinins and infectivity of Newcastle disease virus was determined over a temperature range of 37.8 to 60 C. From the calculated values of δH and δS, it was concluded that inactivation of the hemagglutinating activity and viral infectivity was due to protein denaturation. The significance of virus-contaminated foods in human diets is not yet established. However, food, including milk, has been implicated epidemiologically with several outbreaks of viral diseases (1,6,7,8,12,17). Poliovirus and echovirus have been isolated from the soil of fields irrigated with sewage, and a few vegetables grown in these fields have been shown to contain cytopathic agents (4). Although it is known that viruses do not replicate in foods, echovirus, coxsackievirus, and poliovirus have been shown to survive on vegetables stored under household conditions for as long as 2 months (5). These findings seem to indicate that viruses pathogenic to man can gain entrance to foods during their production, processing, or preparation. Food processes designed for the elimination of harmful organisms from food cannot be assumed to eliminate foodborne viruses (13). Thus, it becomes a matter of practical importance to determine the thermal resistance of viruses. It is also of interest to study the kinetics of the thermal inactivation, since it may give some insight to the mechanism of inactivation. The present study reports experiments measuring energy of activation and entropy of activation of the thermal inactivation of hemagglutinin and infectivity properties of Newcastle disease virus (NDV). MATERIAL for 20 min, and stored at -40 C in 15-ml portions until used. Hemagglutinin titration. The virus in 0.5-ml amounts was serially diluted by factors of two in test tubes containing immunological buffer (NaH2PO4-H20, 27.6 g; NaCl, 29.25 g; NaOH, 6.05 g/liter). To each tube of virus, a 0.5-ml quantity of a 0.25% suspension of chicken red blood cells in immunological buffer was added. The virus titer was read after 1 hr at room temperature. The titer was determined from the highest dilution of virus which provided a visible pattern of agglutination. Agglutination patterns were read as ++++, +++, ++, +, 0 and were expressed as hemagglutinating units per ml. Virus infectivity titration. Tenfold dilutions of virus samples were made in phosphate-buffered saline containing 1% rabbit serum. One-tenth milliliter amounts of each dilution were injected into the allantoic cavity of six 10-day-old fertile hens' eggs and incubated for 1 to 5 days. Each day the eggs were candled, and the dilutions at which embryos died were recorded. The end point was taken as the highest dilution which resulted in 50% mortality of embryos. The 50% lethal end point (LDt,) was determined by plotting the logarithm of the per cent mortality of the dilution just above 50% and just below 50% mortality versus the dilution of virus. The curve was drawn through these two points, and the point where the curve intersected the 50% survival line was noted. The reciprocal of this value was regarded as the LD o per 0.1 ml. Thermal inactivation studies. A series of glass tubes (8 mm in diameter by 150 mm in length) containing 3-ml amounts of diluted infective allantoic fluid (pH 7.7) were heated for various times in a thermostatically controlled water bath which was constant to 0.1 C and were then immediately cooled in ice water and assayed for hemagglutinins and infectivity by the methods described previously. RESULTS Inactivation of hemagglutinin. The inactivation curves obtained for NDV hemagglutinin when allantoic fluids containing virus were heated at four different temperatures, ranging from 43.3 to 60 C, are shown in Fig. 1. The thermal inac-tivation curves are considered linear over the temperature region studied, indicating first-order 8 -kinetics. There was no reduction in hemagglutinin 6 _ activity after 75 min at 43. 3 C, but at the higher 4temperatures there was a loss which was a function of temperature. 3 The velocity constant (k) for the inactivation 2 at a given temperature was calculated from the equation k = (2.303 log V/Vo)/It, where Vo is the initial activity and V is the activity after heating Arrhenius plot, is presented in Fig. 2 An Arrhenius plot of the velocity constants is also presented in Fig. 2 Table 1. The calculated value of activation energy for inactivation of NDV infectivity falls within the range cited for denaturation of various proteins (18). DISCUSSION NDV is a ribonucleic acid (RNA) virus containing an inner coiled helix of ribonucleoprotein surrounded by an outer protein envelope. In addition, the virus contains some lipid material. The hemagglutination properties of the virus are associated principally with the protein of the outer coat. Denaturation of this outer protein should then result in a loss of hemagglutinin activity. The large values of AH (70.5 kcal/mole) and AS (143.4 cal per mole per degree) obtained in this study for inactivation of hemagglutinin activity are compatible with the requirement for protein denaturation (18). The two-component curve which characterized the thermal inactivation of NDV infectivity has been reported for other viruses (3,15,16,19). The shape of these curves has been attributed by some to an inherent heterogeneity of the virus, such that a small proportion of the thermally resistant particles account for the decreased slope of the latter segment of these curves. However, in the present study, it would appear that the fraction of resistant virus, which can be estimated by extrapolating the secondary portion of the inactivation curve to the ordinate, is not some fixed value but is dependent on the heating temperature and disappears at low temperatures. It is difficult to reconcile this result with the theory of a heterogeneous resistance. Others have postulated that the change in inactivation rate at high temperature is due to factors operating during the reaction, such as the formation of aggregates, adsorption to the walls of the vessel, or the presence of virus particles in aerosol droplets above the surface of the liquid (14), all of which may protect against inactivation. Polioviruses being treated with formaldehyde become progressively more resistant to inactivation by this compound, and it has been suggested that this is due to a hardening of the protein coat as the reaction proceeds (10). Nevertheless, it appears that the two-component inactivation curves for viruses will have to be accepted until the question is resolved as to whether this phenomenon is due to an artifact or to a particular inactivation mechanism. Inactivation of NDV infectivity can be caused by destruction of the RNA, denaturation of the nucleoprotein, or denaturation of the protein in the outer coat resulting in an inability of the virus to attach to host cells. Denaturation of protein is associated with a large value of AH and AS due to the rupture of a large number of hydrogen bonds which results in a collapse or unfolding of the secondary structure of the molecule. Smaller values of AH and AS are required for heat inactivation of viral RNA. Values of about 20 kcal/mole for AH and -19 cal per mole per degree for AS for thermal inactivation of tobacco mosaic virus RNA and 31 kcal/mole for AH and 4 cal per mole per degree for AS for thermal inactivation of a poliovirus RNA have been reported (9,11). Ginoza (11) postulated that inactivation of RNA is due to a rupture of the chain. The rather large values of AH and AS obtained in the present study for inactivation of NDV infectivity would indicate that loss of infectivity in the high temperature range was due to protein denaturation rather than destruction of RNA. In support of this conclusion is the fact that a poliovirus, a rhinovirus, and foot-and-mouth disease virus heated at high temperature (50 to 65 C) show a marked reduction in viral infectivity but only a slight loss in infectivity of the extracted RNA (2,9). It should be pointed out that it has been found with some viruses, at least, that at low heating temperatures (about 43 C or less) loss of infectivity is due to inactivation of RNA (9). It appears that this phenomenon may also apply to NDV. Note that there was no damage to protein (hemagglutinin) after heating for 75 min at 43.3 C (Fig. 1), whereas this same timetemperature treatment caused over 90% loss in infectivity (Fig. 2).

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2020-12-10T09:03:04.017Z

1970-02-01T00:00:00.000Z

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Spoilage Bacteria in Canned Foods Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established. MATERIALS AND METHODS Isolation of the organisms. Cans of mushrooms taken from a batch which showed heavy sulfide spoilage were incubated at 55 C, half the cans being sampled after 7 days and the remainder after a total of 14 days. In all cases, a sample of brine was withdrawn under aseptic conditions, and a 2-ml amount was inoculated into each of four tubes of sulfite agar (12). The tubes were incubated for 72 hr at 55 C, and the black colonies were transferred into "vanilla" tubes containing the same medium for purification and isolation (9). The isolated organisms were maintained in ATCC medium code No. 42 (1), and the biological characteristics were examined by the methods described in the Manual of Microbiological Methods (14) and Laboratory Methods in Microbiology (6). Organisms. Two strains of C. nigrificans were used for the comparative study of the media. The strain ATCC 7946 was obtained from the American Type Culture Collection and strain FPI 68102 was isolated from canned mushrooms opened at the Food Processing Institute. Stock cultures were maintained in ATCC medium code No. 42 (1). Spores of these two strains were prepared as follows. tubes of liver broth (12), kept at 55 C for 72 hr, and then transferred into 1 liter of the same medium in long-necked flasks for cultivation. The culture tubes and flasks were stratified with liquid paraffin. After incubation for 10 days at 55 C, the cultures were filtered through a sterile Toyo no. 5 filter paper to remove liquid paraffin and sediment. After heating at 100 C for 20 min to destroy vegetative cells, the spores were separated from the medium in a centrifuge (4,000 rev/min for 15 min), suspended in sterile normal 0.85% saline, shaken with a test tube mixer to facilitate washing, and centrifuged again (4,000 rev/min for 15 min). This procedure was repeated three times. Finally, the spores were suspended in sterile normal saline to give the stock suspension. Media. A versatile medium for determination and enumeration of C. nigrificans was a modification of a basal type (BETI medium). Seven liquid and 10 solid media were compared for outgrowth of C. nigrificans (Table 1). Decimal dilutions of both spore suspensions of strains ATCC 7946 and FPI 68102 [stcck spore concentrations: ATCC 7946, 49,000 spores/ml; FPI 68102, 24,000 spores/ml by most probable number (MPN) determination with liver broth] were prepared with sterile normal saline, and 1 ml of each dilution was inoculated into duplicate screw-capped test tubes (18 by 170 mm, outer diameter) which contained 15 ml of the test media. The liquid media were subsequently stratified with liquid paraffin, and a strip of lead acetate paper (6) was inserted with its lower end above the medium. All tubes were incubated at 55 C, and growth was observed daily for 7 days. Plate count of C. nigrificans. Each spore dilution (1 ml) was transferred into each of five tubes of BETI broth and a similar amount into each of two petri dishes (bottom: 90-mm inner diameter by 20-mm depth) and subsequently mixed with 15 ml of BETI agar. After setting, the agar was stratified with approximately 70 ml of hard BETI agar. All tubes and plates were kept at 55 C for 7 days in an ordinary incubator. The MPN values were read from Sharf's table (13), depending on the number of positive tubes APPL. MICROBIOL. in each dilution, and the total spore counts were also determined by counting colonies on the plates. Ten different spore concentrations were studied. These were randomly selected. (9) a L, liquid media; S, solid media. b BETI broth and BETI agar: basal type of BETI medium contained beef extract, 3 g; tryptone, 10 g; yeast extract, 1 g; soluble starch, 1 g; dipotassium phosphate, 1.25 g; Fe(NH4)2(SO4)2, 0.1 g; dextrose, 5 g; in 1 liter of distilled water as a liquid medium (BETI broth). The pH of the medium was adjusted to 7.0 before sterilization at 121 C for 20 min. When required in the form of a solid medium, two concentrations of agar were used: solid medium (15 g) and hard agar (20 g) in 1 liter of the above basal medium. BETI agar was used for the solid culture in tubes and on plates, and hard BETI agar was used as the agar for stratification on plate cultures. c Drained from commercial canned mushrooms with a pH of 6.3. d NORDISK, the committee for the methods of food examination, Norway. The agar contained tryptone (15 g), yeast extract (10 g), and agar (15 g) in 1 liter of distilled water. The pH was adjusted to 7.0, and the medium was sterilized for 15 min at 121 C. To 100 ml of this base medium, 1 ml of 5% ferric citrate solution, 1 ml of 5% aqueous solution of anhydrous sodium sulfite, and 1 ml of aqueous solution of potassium permanganate were added immediately before use. RESULTS Five strains were isolated from canned mushrooms, four from cans opened on the 7th day and one from a can opened on the 14th day of incubation. The isolates were rod-shaped, gramnegative, 0.4 to 0.5 by 3.0 to 6.0 Am in size, and moderately motile. They formed eliptical subterminal spores. The deep colonies in sulfite agar were surrounded by a blackened area of the medium. The color changed to black as a result of the fine black particles in BETI broth culture. Gelatin was not liquefied and no indole was produced. Hydrogen sulfide was produced from cystine, but acid was not produced from glucose, fructose, galactose, mannose, xylose, arabinose, rhamnose, sucrose, maltose, lactose, raffinose, starch, inulin, glycerol, mannitol, or salicin. Nitrate was not reduced to nitrite. The optimal temperature for growth was between 50 to 55 C, and the strains were obligate anaerobes. The comparative outgrowth of C. nigrificans in 7 liquid and 10 solid media is shown in Tables 2 and 3. The MPN values and the plate counts of 10 different spore concentrations of C. nigrificans were enumerated, and the results were shown in Table 4. 10-' a Incubated at 55 C; the same spore suspension was used in the comparative study of both liquid and solid media. There was no apparent change in the control tube. (3) except that they were gram-negative as reported by Campbell and Postgate for Desulfotomaculum nigrificans (5). The fact that four strains were isolated from cans incubated 7 days but only one from cans incubated 14 days might be the result of the inhibiting action of hydrogen sulfide (10) which accumulated in the contents of the can. The odor of sulfide was much more pronounced after 14 days than 7 days. The experimental results (Tables 2 and 3) showed that the modified media, BETI broth (media no. L6), and BETI agar (media no. S8) showed a slight improvement on the recovery and on the quicker growth of C. nigrificans over 7 liquid and 10 solid media which were commonly used. The recovery of spores from the broth was probably 102 times higher than that of the agar tube culture. The growth of C. nigrificans in liquid media could be detected by darkening of the lead acetate strips as a result of hydrogen sulfide production, and it was detectable only by this test when glucose liver broth (media no. L2) and canned mushroom brine (media no. L7) were used. It should be noted that this test can not be used as a method for detecting hydrogen sulfide production Baars' Medium (media no. L3) and Postgate's Medium B (media no. L4), since these media when incubated at 55 would show blackening of all strips, including the control, by chemically produced hydrogen sulfide. Bufton (4) examined several media and showed that none was quantitatively satisfactory for C. nigrificans. Postgate (11) published a procedure permitting colony counts on impure cultures and natural samples for which only MPN determinations were hitherto reliable. The experimental results, however, showed the mentioned method to be a proper procedure of simple plate culture for viable count of C. nigrificans. The points of the procedure are the following. (i) BETI agar should be used as the plate culture medium since it improves recovery (Table 3). (ii) Hard BETI agar is a suitable medium as a cover for the culture medium. Two per cent agar may be adequate for the deep colonies, but it cannot detect hydrogen sulfide which is produced by surface colonies on the plates. (iii) The depth of the covering medium must be more than 10 mm to make the conditions sufficiently anaerobic for the growth of C. nigrificans. VOL. 19, 1970 285

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Mechanical characterization of buckwheat noodles mixed with seaweed (fu-nori) The present study was conducted to clarify the effect of incorporation of seaweed, i.e. funori (Gloiopeltis tenax (Turner) J. Agardh) into buckwheat noodles on their mechanical characteristics. Mechanical analysis of buckwheat noodles with funori showed that incorporation of funori into buckwheat noodles enhanced breaking stress and energy. On the other hand, incorporation of funori into buckwheat noodles enhanced decreased solubility of the albumin plus globulin fraction. The present study findings suggest that the endogenous protein may be an important factor responsible for the mechanical characteristics of buckwheat noodles with seaweed. Assami et al., (2019): Mechanical characterization of buckwheat noodles with seaweed 6 (A) (B) (A) (B) called “hegi-soba”. These buckwheat noodles are prepared by incorporating into buckwheat dough a kind of seaweed, i.e. funori, (Gloiopeltis tenax (Turner) J. Agardh) as a dough-binder (Zen-men-kyo, 2014). Before ingestion, the noodles prepared with funori are usually put on a unique wooden-tray which is called “hegi”; so these buckwheat noodles are called “hegi-soba”. Although this buckwheat dish is traditionally utilized only in Niigata region, many Japanese people currently often enjoy these local buckwheat noodles. Hegi-soba noodles have a unique masticatory sense with refreshing sense on ingestion. Mechanical characterization of “hegi-soba” noodles is an interesting subject in view of buckwheat research. In this background, the present study was conducted to characterize noodles made from buckwheat flour with seaweed. MATERIALS AND METHODS Materials Buckwheat flour (Fagopyrum esculentum Moench, var. Kitawase-soba), which was harvested in Hokkaido (in 2017), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80oC until use. Ground seaweed, i.e. fu-nori in Japanese, Gloiopeltis tenax J. Agardh) used in this study was a commercial product (Oowaki-manzou-shoten Co., Fukui, Japan). Fig. 1. Buckwheat noodles. (A), non added seaweed; and (B) added seaweed (1.7% addition). INTRODUCTION Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003; Ikeda, 2002). Buckwheat flour contains various beneficial components for human health such as protein, polyphenolics, rutin and minerals at high levels (Ikeda 2002; Ikeda and Yamashita 1994). Thus, buckwheat can contribute as an important dietary source of such beneficial components. There is a large variety of buckwheat products produced on a global basis (Ikeda, 2002). Attention has been currently paid to the palatability and acceptability of buckwheat products from the perspective of their cooking and processing. However, there are still unanswered questions on the palatability and acceptability of buckwheat products. As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japanese yam flour, are often added in preparing buckwheat noodles (ZMCS, 2004). A variety of buckwheat noodles with various dough-binders has been traditionally available in Japan. We reported mechanical effects by addition of various dough-binders to common and Tartary buckwheat noodles in view of two analysis, i.e., tensile analysis and breaking analysis (Ikeda, et al., 2005). However, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buckwheat products. In Niigata district, located in the middle region of Japan, there is a famous buckwheat dish. This dish is Fagopyrum 36(1):5-9 (2019) 7 called "hegi-soba". These buckwheat noodles are prepared by incorporating into buckwheat dough a kind of seaweed, i.e. funori, (Gloiopeltis tenax (Turner) J. Agardh) as a dough-binder (Zen-men-kyo, 2014). Before ingestion, the noodles prepared with funori are usually put on a unique wooden-tray which is called "hegi"; so these buckwheat noodles are called "hegi-soba". Although this buckwheat dish is traditionally utilized only in Niigata region, many Japanese people currently often enjoy these local buckwheat noodles. Hegi-soba noodles have a unique masticatory sense with refreshing sense on ingestion. Mechanical characterization of "hegi-soba" noodles is an interesting subject in view of buckwheat research. In this background, the present study was conducted to characterize noodles made from buckwheat flour with seaweed. Materials Buckwheat flour (Fagopyrum esculentum Moench, var. Kitawase-soba), which was harvested in Hokkaido (in 2017), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80 o C until use. Ground seaweed, i.e. fu-nori in Japanese, Gloiopeltis tenax J. Agardh) used in this study was a commercial product (Oowaki-manzou-shoten Co., Fukui, Japan). Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003;Ikeda, 2002). Buckwheat flour contains various beneficial components for human health such as protein, polyphenolics, rutin and minerals at high levels (Ikeda 2002;Ikeda and Yamashita 1994). Thus, buckwheat can contribute as an important dietary source of such beneficial components. INTRODUCTION There is a large variety of buckwheat products produced on a global basis (Ikeda, 2002). Attention has been currently paid to the palatability and acceptability of buckwheat products from the perspective of their cooking and processing. However, there are still unanswered questions on the palatability and acceptability of buckwheat products. As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japanese yam flour, are often added in preparing buckwheat noodles (ZMCS, 2004). A variety of buckwheat noodles with various dough-binders has been traditionally available in Japan. We reported mechanical effects by addition of various dough-binders to common and Tartary buckwheat noodles in view of two analysis, i.e., tensile analysis and breaking analysis (Ikeda, et al., 2005). However, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buckwheat products. In Niigata district, located in the middle region of Japan, there is a famous buckwheat dish. This dish is Mechanical measurements For the study of the effects of the seaweed on the mechanical characteristics of buckwheat noodles, buckwheat noodles were prepared by hand. The mechanical characteristics of buckwheat noodles were evaluated by breaking analysis. Prior to the mechanical analysis, the buckwheat flour which had been stored at -80 o C was placed in a desiccator at room temperature until the flour exhibited a constant moisture content. The moisture of the flour was measured with a moisture analyzer (ML-50, A&D Co. Ltd., Japan). Seaweed was boiled, and sticky seaweed was added to buckwheat flour. The buckwheat dough was prepared just prior to mechanical analysis to have a moisture content of 42% by adding the appropriate amount of distilled water. Then the buckwheat noodles were made from the buckwheat dough using a handmade pasta machine (SP-150, Imperta Co., Torino, Italy). Figure 1 shows buckwheat noodles prepared in this study. The buckwheat noodles obtained were subjected to mechanical analysis. Before the mechanical analysis, buck-wheat noodles prepared were heated in boiling water for 150 seconds and subsequently were cooled for 150 seconds at 4 o C. Immediately after cooling, mechanical measurements of the noodles were performed. The breaking analysis of the buckwheat noodles was performed with Rheoner RE2-3305C (Yamaden Co. Ltd., Japan). Measurements of breaking analysis were performed with a load cell of 200N and measurement speed of 0.50 mm/ sec. A wedge-style plunger (No.49: W 13mm, D 30mm, H 25mm) was used in measurements with the Rheoner RE2-3305C. Mechanical measurements were replicated twenty times for each sample. Protein determination For chemical analysis of the combined fractions of buckwheat albumin plus globulin (AG) in the heated noodle samples which had been subjected to the mechanical measurements, the noodle samples were lyophilized and then ground into flour. The flours obtained were extracted with a ten-fold (v/w) volume of 0.2M NaCl for 1hr at 4oC. After extraction, the suspensions were centrifuged at 17,000 Xg for 20 min. Protein concentration was determined using the Bradford method with bovine serum albumin as a standard protein. Statistical analysis Statistical analysis was conducted using a personal computer with the program Excel (Microsoft Co., USA), Ekuseru-Toukei 2015 (Social Survey Research Information Co., Japan) and SPSS Ver.23.0 (IBM, USA). Figure 2 shows the breaking characteristics of hegi-soba buckwheat noodles prepared with funori-seaweed. The breaking stress and energy of the hegi-soba noodles gradually increased as the added concentration of funori seaweed increased (Fig. 2 (A and B)). A significant high breaking stress (Fig. 2 (A)) was found with hegi-soba buckwheat noodles with a concentration of funori seaweed with 1.4% or over as compared the buckwheat without funori seaweed (P<0.05). Similarly, a significant high breaking energy (Fig. 2 (B)) was found with buckwheat noodles with a concentration of funori seaweed with 1.7% as compared the buckwheat without funori seaweed (P<0.05). These findings characterize showed the unique mastication characteristics of hegi-soba noodles. Figure 3 shows the NaCl-soluble protein content of buckwheat noodles made with seaweed. The NaCl-soluble protein exhibits the combined fraction of the major buckwheat proteins, i.e., albumin plus globulin (Ikeda, 2002), designated as the AG fraction below. Changes by the addition of the seaweed in solubility of the AG fraction were found (Fig. 3). Incorporation of seaweed into buckwheat dough was found to reduce the solubility of the AG fraction in buckwheat dough as the funori seaweed added increased (Fig. 3). The seaweed contains dietary fiber at high levels (Ooishi, 1993). Judged from our previous findings (Ikeda and Kusano 1983), this phenomenon may be due to in-solubilization of proteins arisen by dietary fiber in seaweed. Interest in the nutritional function of dietary fiber for humans is currently increasing. Dietary fiber has many beneficial effects on human such as blood glucose increase suppression and antihypertensive (Mori and Tsuji, 1997). Considering in view of current nutritional science concerning the beneficial effects of dietary fiber, the intake of buckwheat noodles with seaweed with high level of dietary fiber, should be recommended as a key source of dietary fiber. Protein compositions of buckwheat noodles made with seaweed Relationships of the observed breaking characteristics (Fig. 2) to the protein components ( Fig. 3) was analyzed. The AG fraction content (Fig. 3) negatively correlated to their observed breaking stress (Fig. 2 (A)) with r = -0.934 (P<0.01), breaking energy ( Fig. 2 (B)) with r = -0.942 (P<0.01). These findings suggest that proteins in the AG fraction may be an important factor involved in the observed changes in mechanical characteristics arisen by the addition of funori seaweed. Finally, the present study shows changes in mechanical characteristics of buckwheat noodles made with seaweed. The present study suggests that changes in the protein of AG fraction in buckwheat noodles with seaweed may be an important factor affecting the mechanical characteristics of buckwheat noodles, although the exact mechanism remains uncertain. The present findings provide a scientific basis in the understanding of palatability and acceptability of buckwheat noodles.

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Changes of physicochemical properties and correlation analysis of common buckwheat starch during germination In order to clarify the physicochemical properties of starch during germination of common buckwheat, Xinong9976 was selected as the experimental material to study the main nutrients, particle structure, particle size distribution, transparency, aging value, pasting properties and the correlation between pasting properties and starch composition and main nutrients. The results showed that main nutrients were significantly different. The diameter of starch granules ranged from 2.36 to 8.89μm, and the shapes of starch granules were irregular with obvious holes and cracks on the surface. There were significant differences in starch transparency, aging value and pasting properties at different germination stages. Peak viscosity, through viscosity and final viscosity of germinated common buckwheat was significantly positive correlated with amylopectin content (P < 0.05) and breakdown, final viscosity and setback were significantly negatively correlated with amylose content (P < 0.05). The correlation analysis of starch pasting properties and main nutrients showed that breakdown, setback and crude fat content were significantly negatively correlated (P < 0.01), peak viscosity, through viscosity and final viscosity were significantly negatively correlated with crude fat content (P < 0.05), while the starch pasting properties had no significant correlation with other nutrients. Gao et al. (2019): Common buckwheat starch during germination 44 INTRODUCTION Common buckwheat (Fagopyrum esculentum) belongs to Polygonaceae, it is an important minor grain crop in China (Nam et al. 2018). Common buckwheat is rich in nutrients, containing 10.6% -15.5% of protein, 1.2% 2.8% of fat, 63% -71.2% of starch, as well as flavonoids, mineral elements and cysteine (Gimenez-Bastida and Zielinski 2015). Recently, with the improvement of people’s living standard, it has become fashionable to pursue a balanced diet with scientific based nutrition. Therefore, common buckwheat products with the reputation of “the same origin of food and medicine” will be widely welcomed by people. Besides, the development and research of common buckwheat healthy food have broad market prospects, high economic value and social value (Yoshimoto et al. 2004). There have been many studies on the cultivation methods, yield and protein content of common buckwheat in China and abroad (Salehi et al. 2018, Fang et al. 2018, Khan et al. 2012) and studies on the starch properties have also been reported. However, changes of physicochemical properties of germinated common buckwheat starch is not reported. Starch is the main nutritional component of common buckwheat. Its physicochemical properties affect the nutritional properties of related products and are also related to the development of new uses of common buckwheat starch (Stibilj et al. 2004). Germination is a dynamic process where plants come from resting state to the state with many physiological activities. Germination treatments can enhance the respiration of plants and significantly increase the species and number of enzymes (Mamilla and Mishra 2017). After germination, the starch content of mung bean decreased (Liu et al. 2014), while the amylase activity of kidney bean increased, leading to the changes in starch structure and composition (Yanli et al. 2018). Studies have showed that buckwheat has a high peak viscosity, hot paste stability and cold paste stability (Hara et al. 2007). Tartary buckwheat flavone content significantly increased after germination (Xiao-Peng et al. 2015). In addition, germination treatment can improve the edible and health value of buckwheat, enhance the resistance of starch paste and improve the stability of starch paste (Hara et al. 2007). Therefore, the study on the characteristics of germinated common buckwheat starch is of great significance to the development and utilization. In this experiment, the main nutrients, particle structure, physicochemical properties and the correlation between gelatinization characteristic value and main nutrients of germinated common buckwheat starch were studied by using cv. Xinong9976 as material to provide basis for the development and utilization of common buckwheat sprouts and bean flour and the deep processing of starch. MATERIALS AND METHODS 1. Experimental materials Cv. Xinong9976, a common buckwheat variety, provided by small grain laboratory of Northwest A&F University, was used for the experiment. Common buckwheat seeds with full grain and no disease were selected and were sterilized with 0.1% H2O2 for 10 min and soaked in the distilled water for 24 h. Then seeds were placed in a petri dish with two layers of filter paper, the cultivation of the sample under 25 ̊C in dark for germination, respectively taking sample after 2, 4, 6 d, removed shell, dried at 40 ̊C. The dried sample was grinded and passed through a 0.100 mm mesh. Added 80% ethanol, 50 ̊C, with the ultrasonic treatment (500 w) 30 min to remove flavonoids, then added the volume of distilled water, heated under the condition of 30 ̊C for 24 h. Centrifuged (4000 rpm, 10 min) three times, scraped off the grayish-brown soft layer. Finally, dried at 40 ̊C for 48 h and sieved with a 0.150 mm mesh. 2. Measurement of physicochemical properties The morphology of starch granules was observed by scanning electron microscope (JSM-6390, Jeol Ltd, Tokyo Japan) at 2000 x magnification, the particle size distribution was determined by a laser diffraction particle size analyzer and the pasting properties were measured using a rapid visco analyzer (RVA) (Newport Scientific, Pty Ltd, Warriewood, Australia). Starch transparency was determined using a modified version of (Zhou et al. 2014). 0.5g of the starch sample was blended with 50 mL distilled water and heated in a boiling water bath for 30 min. After the starch was completely gelatinized by stirring, the starch was removed and cooled to room temperature. Distilled water was used as a blank for zero adjustment and the transparency was measured at the wavelength of 620 nm with a visible-light spectrophotometer. The starch aging value was determined as follows: 0.5g of the starch sample was blended with 50 mL distilled waFagopyrum 36(2):43-50 (2019) 45 Germination time/d Water/% Ash/% Crude fat/% Crude protein/% Total starch/% Amylose/% Amylopectin/% 0 12.26 ± 0.09 a 1.12 ± 0.03 c 1.37 ± 0.01 a 9.22 ± 0.06 c 68.41 ± 0.11 a 24.11±0.28 a 44.30±0.39 a 2 11.21 ± 0.03 b 1.36 ± 0.02 b 1.04 ± 0.01 b 9.42 ± 0.10 c 58.77 ± 0.81 b 22.89±0.17 b 35.88±0.97 b 4 9.78 ± 0.01 c 1.44 ± 0.01 b 0.92 ± 0.01 c 10.83 ± 0.17 b 52.80 ± 0.09 c 21.85±0.25 c 30.95±0.32 c 6 7.73 ± 0.01 d 1.69 ± 0.03 a 0.84 ± 0.01 d 13.79 ± 0.20 a 47.66 ± 0.11 d 19.77±0.20 d 27.89±0.31 d ter and heated in a boiling water bath for 30 min and added distilled water to keep the total volume constant. Removed and cooled to room temperature, refrigerated for 24h, and defrosted at room temperature, and then centrifuged at 4000 rpm for 10 min, finally weighed the sediment quality. The starch aging value index was determined as follows: starch aging value = (weight of starch paste before centrifugation – weight of sediment quality) X 100. 3. Data analysis Three parallel tests were conducted in the experiment. SPSS 17.0 was used for statistical analysis, Origin 9.0 was used for drawing, and LSD minimum significant difference test was used for the determination of significance of differences. RESULTS AND DISCUSSION Changes of the concentration of main nutrients Main nutrients of common buckwheat were shown in table 1. The results showed that after germination, the contents of water, crude fat, total starch, amylose and amylopectin decreased significantly (P < 0.05), while the contents of ash and crude protein increased significantly (P < 0.05). Among them, the crude fat mass fraction decreased the most, from 1.37% to 0.84%, a decrease of 38.69% while the ash quality score increased the most, from 1.12% to 1.69%, increasing by 51%. In addition, the relative standard deviations of main nutrients in water, ash, crude fat, crude protein, total starch, amylose and amylopectin at different stages were 19.14%, 16.76%, 22.38%, 19.50%, 15.66%, 8.30% and 20.61%, respectively, indicating that the nutritional composition of the main nutrients in different stages of the germinated common buckwheat was significantly different. After germination, the crude protein mass fraction increased exponentially, which may be caused by the decreased protease activity in the seeds of common buckwheat during the germination process, which effectively weakened the hydrolysis of related proteins and thus promoted the protein accumulation (Ikeda et al. 1984). The decrease of total starch mass fraction may be due to the activation of α-amylase and β-amylase in the sprouting of common buckwheat, which could promote the degradation of starch and provide part of sugars needed for the germination (Mohan et al. 2010). The decrease of fat mass fraction might be due to the action of lipase, which could decompose part of the fat into the energy required for the germination and growth of common buckwheat seeds. Starch grain structure As can be seen on Fig. 1a, the starch particles of mature common buckwheat seeds were complete with clear gaps, mostly spherical and oval in shape, with smooth surface and no holes or cracks. After the germination of 2 d, most starch granules were irregular in shape, while a few were spherical in shape. In addition, some of the crystalline structures of starch were destroyed, and a few starch granules showed cracks on the surface (Fig. 1b). In 4 d, the starch granules were disordered. A small number of starch granules were spherical in shape, while some starch granules were deformed and condensed together with the surrounding granules (Fig. 1c). And in 6 d, the starch granules were polygonal in shape with few of them being spherical. The crystalline structures of most starch granules were destroyed, and obvious cracks and holes appeared on the surface of most granules (Fig. 1d). The table 2 showed that in the process of germination, starch granule size distribution was more dispersed, which indicated that the size of starch granules had obTable 1 Changes of main nutrients of common buckwheat before and after germination Note: different letters in the same column mean significant difference of P<0.05, the same as below. Gao et al. (2019): Common buckwheat starch during germination 46 Transparency can reflect the mutual solubility of starch and water, and the transparency of buckwheat starch is positively proportional to the absorbance value, the higher the absorbance value is, the higher the transparency of the starch is (Li et al. 1997). As can be seen from Fig. 2, the transparency of common buckwheat starch first increased and then decreased with the extension of time in the germination process. And the transparency in different stages was significantly different (P < 0.05), the most transparent stage was 2 d and the absorbance value was 1.75, indicating that starch particles were completely expanded at this time, and there was no mutual association among the starch molecules after gelatinization. While the lowest transparency period was 6 d and the absorbance value was 1.00, which decreased by 36.30% compared with that of mature seeds. The average absorbance of starch in different germination stages was 1.41. After germination of 2d, starch transparency decreased significantly (P < 0.05), which may be due to the starch retrogradation, rearrangement of starch molecules and Note: D10, D50 and D90 represented the critical particle size values when the minimum particle size was added up to 10%, 50% and 90% of the sample. Germination time/d D10 D50 D90 Average sphericity Average aspect ratio 0 4.97 7.86 8.89 0.84 1.45 2 4.68 6.64 7.98 0.76 1.43 4 3.29 5.17 6.97 0.68 1.40 6 2.36 4.05 5.84 0.57 1.37 Table 2 Starch particle size distribution during the germination of common buckwheat d. Starch granules in 6d (×2000). Fig.1. Scanning electron microscopy of starch granules of common buckwheat at different germination stages a. Starch granules in 0d (×2000); b. Starch granules in 2d (×2000); c. Starch granules in 4d (×2000); vious differences. Chang found that the corn starch size ranged from 5.76 to 8.64 μm (Chang et al. 2004). Common buckwheat starch particles between 4.00 to 9.00 microns in diameter, which was smaller than the corn starch granules. After germination, the diameter, average sphericity and average aspect ratio of starch granules decreased significantly with the increase of germination time. In 6 d, the average diameter of starch granules decreased from 7.24 μm to 4.08 μm, a decrease of 43.65%. The average sphericity and the average aspect ratio decreased by 32.14% and 5.52%, respectively. Starch transparency Transparency is one of the important external characteristics of starch, which is directly related to the appearance and use of starch products (Wang et al. 2017). Fig.2. Changes of the transparency of common buckwheat starch at different germination stages Fagopyrum 36(2):43-50 (2019) 47 scattering of light, thus reducing light transmission and starch transparency (Zhou et al. 2017). Starch aging value The essence of starch aging is that gelatinized starch molecules re-form hydrogen bonds during the cooling process(Jiamjariyatam et al. 2014). The aging process of starch can be regarded as the reverse process of gelatinization, but the degree of starch crystallization decreases after aging (Verma et al. 2018). It could be seen from Fig. 3 that there were significant differences in the aging value of common buckwheat starch in different germination stages (P < 0.05). The maximum aging value was 71.20% in mature grains. Subsequently, the aging value decreased gradually with the extension of germination time. Both 4d and 6d, the aging value decreased by 28.84%, 39.35%, respectively, which might be related to the weakened ability of buckwheat starch molecules to form hydrogen bonds again after germination (Liu et al. 2006). Starch aging not only makes food taste worse, but also reduces the digestibility (Verma et al. 2018). However, the aging value of common buckwheat gradually decreased during germination, indicating that common buckwheat sprouts were good in taste, easy to digest and had broad market development value. Pasting viscosity Starch granules rapidly absorb water in aqueous solution due to thermal expansion, resulting in the fracture of intramolecular and intermolecular hydrogen bonds, and the process of gradual diffusion of starch granules is called starch paste (Jane et al. 1992). The pasting temperature was different in different germination stages due to the size of starch granules. The starch pasting properties of common buckwheat in the process of germination were shown in table 3, the results showed that starch pasting viscosities gradually decreased and significantly different (P < 0.05) in different period. After germination, peak viscosity, through viscosity, breakdown, final viscosity, setback, peak time and pasting temperature were lower than those of the mature grain starch. Peak viscosity refers to the increase in the viscosity of starch paste caused by the friction between starch particles after full water absorption and expansion, which can reflect the expansion capacity of starch (Xiao-Li et al. 2008). As shown in table 3, the peak viscosity was 1394 2982 cp, the average peak viscosity was 2242 cp, and the difference was great. Through viscosity is caused by the sharp decrease in the viscosity of starch paste due to the fact that starch particles no longer friction with each other after they have expanded to the limit (Xiao-Li et al. 2008). The through viscosity was between 1335 and 2819 cp, and the average was 2131 cp. The breakdown is the difference between peak viscosity and through viscosity, which can reflect the thermal stability of starch paste. The smaller the breakdown is, the better the thermal stability is (Karim et al. 2000). The average breakdown was 111 cp, and the breakdown in mature grains was 2.76 times higher than that after germination, indicating that the starch Fig.3. Changes of the aging value of common buckwheat starch at different germination stages Germination time/d peak viscosity /cp Through viscosity /cp breakdown/cp final viscosity /cp setback/cp peak time /min pasting temperature /°C INTRODUCTION Common buckwheat (Fagopyrum esculentum) belongs to Polygonaceae, it is an important minor grain crop in China (Nam et al. 2018). Common buckwheat is rich in nutrients, containing 10.6% -15.5% of protein, 1.2% -2.8% of fat, 63% -71.2% of starch, as well as flavonoids, mineral elements and cysteine (Gimenez-Bastida and Zielinski 2015). Recently, with the improvement of people's living standard, it has become fashionable to pursue a balanced diet with scientific based nutrition. Therefore, common buckwheat products with the reputation of "the same origin of food and medicine" will be widely welcomed by people. Besides, the development and research of common buckwheat healthy food have broad market prospects, high economic value and social value (Yoshimoto et al. 2004). There have been many studies on the cultivation methods, yield and protein content of common buckwheat in China and abroad (Salehi et al. 2018, Fang et al. 2018, Khan et al. 2012) and studies on the starch properties have also been reported. However, changes of physicochemical properties of germinated common buckwheat starch is not reported. Starch is the main nutritional component of common buckwheat. Its physicochemical properties affect the nutritional properties of related products and are also related to the development of new uses of common buckwheat starch (Stibilj et al. 2004). Germination is a dynamic process where plants come from resting state to the state with many physiological activities. Germination treatments can enhance the respiration of plants and significantly increase the species and number of enzymes (Mamilla and Mishra 2017). After germination, the starch content of mung bean decreased (Liu et al. 2014), while the amylase activity of kidney bean increased, leading to the changes in starch structure and composition (Yanli et al. 2018). Studies have showed that buckwheat has a high peak viscosity, hot paste stability and cold paste stability (Hara et al. 2007). Tartary buckwheat flavone content significantly increased after germination (Xiao-Peng et al. 2015). In addition, germination treatment can improve the edible and health value of buckwheat, enhance the resistance of starch paste and improve the stability of starch paste (Hara et al. 2007). Therefore, the study on the characteristics of germinated common buckwheat starch is of great significance to the development and utilization. In this experiment, the main nutrients, particle structure, physicochemical properties and the correlation between gelatinization characteristic value and main nutrients of germinated common buckwheat starch were studied by using cv. Xinong9976 as material to provide basis for the development and utilization of common buckwheat sprouts and bean flour and the deep processing of starch. Experimental materials Cv. Xinong9976, a common buckwheat variety, provided by small grain laboratory of Northwest A&F University, was used for the experiment. Common buckwheat seeds with full grain and no disease were selected and were sterilized with 0.1% H 2 O 2 for 10 min and soaked in the distilled water for 24 h. Then seeds were placed in a petri dish with two layers of filter paper, the cultivation of the sample under 25˚C in dark for germination, respectively taking sample after 2, 4, 6 d, removed shell, dried at 40˚C. The dried sample was grinded and passed through a 0.100 mm mesh. Added 80% ethanol, 50˚C, with the ultrasonic treatment (500 w) 30 min to remove flavonoids, then added the volume of distilled water, heated under the condition of 30˚C for 24 h. Centrifuged (4000 rpm, 10 min) three times, scraped off the grayish-brown soft layer. Finally, dried at 40˚C for 48 h and sieved with a 0.150 mm mesh. Measurement of physicochemical properties The morphology of starch granules was observed by scanning electron microscope (JSM-6390, Jeol Ltd, Tokyo Japan) at 2000 x magnification, the particle size distribution was determined by a laser diffraction particle size analyzer and the pasting properties were measured using a rapid visco analyzer (RVA) (Newport Scientific, Pty Ltd, Warriewood, Australia). Starch transparency was determined using a modified version of (Zhou et al. 2014). 0.5g of the starch sample was blended with 50 mL distilled water and heated in a boiling water bath for 30 min. After the starch was completely gelatinized by stirring, the starch was removed and cooled to room temperature. Distilled water was used as a blank for zero adjustment and the transparency was measured at the wavelength of 620 nm with a visible-light spectrophotometer. The starch aging value was determined as follows: 0.5g of the starch sample was blended with 50 mL distilled wa- ter and heated in a boiling water bath for 30 min and added distilled water to keep the total volume constant. Removed and cooled to room temperature, refrigerated for 24h, and defrosted at room temperature, and then centrifuged at 4000 rpm for 10 min, finally weighed the sediment quality. The starch aging value index was determined as follows: starch aging value = (weight of starch paste before centrifugation -weight of sediment quality) X 100. Data analysis Three parallel tests were conducted in the experiment. SPSS 17.0 was used for statistical analysis, Origin 9.0 was used for drawing, and LSD minimum significant difference test was used for the determination of significance of differences. Changes of the concentration of main nutrients Main nutrients of common buckwheat were shown in table 1. The results showed that after germination, the contents of water, crude fat, total starch, amylose and amylopectin decreased significantly (P < 0.05), while the contents of ash and crude protein increased significantly (P < 0.05). Among them, the crude fat mass fraction decreased the most, from 1.37% to 0.84%, a decrease of 38.69% while the ash quality score increased the most, from 1.12% to 1.69%, increasing by 51%. In addition, the relative standard deviations of main nutrients in water, ash, crude fat, crude protein, total starch, amylose and amylopectin at different stages were 19.14%, 16.76%, 22.38%, 19.50%, 15.66%, 8.30% and 20.61%, respectively, indicating that the nutritional composition of the main nutrients in different stages of the germinated common buckwheat was significantly different. After germination, the crude protein mass fraction increased exponentially, which may be caused by the decreased protease activity in the seeds of common buckwheat during the germination process, which effectively weakened the hydrolysis of related proteins and thus promoted the protein accumulation (Ikeda et al. 1984). The decrease of total starch mass fraction may be due to the activation of α-amylase and β-amylase in the sprouting of common buckwheat, which could promote the degradation of starch and provide part of sugars needed for the germination (Mohan et al. 2010). The decrease of fat mass fraction might be due to the action of lipase, which could decompose part of the fat into the energy required for the germination and growth of common buckwheat seeds. Starch grain structure As can be seen on Fig. 1a, the starch particles of mature common buckwheat seeds were complete with clear gaps, mostly spherical and oval in shape, with smooth surface and no holes or cracks. After the germination of 2 d, most starch granules were irregular in shape, while a few were spherical in shape. In addition, some of the crystalline structures of starch were destroyed, and a few starch granules showed cracks on the surface (Fig. 1b). In 4 d, the starch granules were disordered. A small number of starch granules were spherical in shape, while some starch granules were deformed and condensed together with the surrounding granules (Fig. 1c). And in 6 d, the starch granules were polygonal in shape with few of them being spherical. The crystalline structures of most starch granules were destroyed, and obvious cracks and holes appeared on the surface of most granules (Fig. 1d). The table 2 showed that in the process of germination, starch granule size distribution was more dispersed, which indicated that the size of starch granules had ob- Transparency can reflect the mutual solubility of starch and water, and the transparency of buckwheat starch is positively proportional to the absorbance value, the higher the absorbance value is, the higher the transparency of the starch is (Li et al. 1997). As can be seen from Fig. 2, the transparency of common buckwheat starch first increased and then decreased with the extension of time in the germination process. And the transparency in different stages was significantly different (P < 0.05), the most transparent stage was 2 d and the absorbance value was 1.75, indicating that starch particles were completely expanded at this time, and there was no mutual association among the starch molecules after gelatinization. While the lowest transparency period was 6 d and the absorbance value was 1.00, which decreased by 36.30% compared with that of mature seeds. The average absorbance of starch in different germination stages was 1.41. After germination of 2d, starch transparency decreased significantly (P < 0.05), which may be due to the starch retrogradation, rearrangement of starch molecules and Note: D 10 , D 50 and D 90 represented the critical particle size values when the minimum particle size was added up to 10%, 50% and 90% of the sample. vious differences. Chang found that the corn starch size ranged from 5.76 to 8.64 μm (Chang et al. 2004). Common buckwheat starch particles between 4.00 to 9.00 microns in diameter, which was smaller than the corn starch granules. After germination, the diameter, average sphericity and average aspect ratio of starch granules decreased significantly with the increase of germination time. In 6 d, the average diameter of starch granules decreased from 7.24 μm to 4.08 μm, a decrease of 43.65%. The average sphericity and the average aspect ratio decreased by 32.14% and 5.52%, respectively. Starch transparency Transparency is one of the important external characteristics of starch, which is directly related to the appearance and use of starch products ). Fig.2. Changes of the transparency of common buckwheat starch at different germination stages scattering of light, thus reducing light transmission and starch transparency ). Starch aging value The essence of starch aging is that gelatinized starch molecules re-form hydrogen bonds during the cooling process (Jiamjariyatam et al. 2014). The aging process of starch can be regarded as the reverse process of gelatinization, but the degree of starch crystallization decreases after aging (Verma et al. 2018). It could be seen from Fig. 3 that there were significant differences in the aging value of common buckwheat starch in different germination stages (P < 0.05). The maximum aging value was 71.20% in mature grains. Subsequently, the aging value decreased gradually with the extension of germination time. Both 4d and 6d, the aging value decreased by 28.84%, 39.35%, respectively, which might be related to the weakened ability of buckwheat starch molecules to form hydrogen bonds again after germination (Liu et al. 2006). Starch aging not only makes food taste worse, but also reduces the digestibility (Verma et al. 2018). However, the aging value of common buckwheat gradually decreased during germination, indicating that common buckwheat sprouts were good in taste, easy to digest and had broad market development value. Pasting viscosity Starch granules rapidly absorb water in aqueous solution due to thermal expansion, resulting in the fracture of intramolecular and intermolecular hydrogen bonds, and the process of gradual diffusion of starch granules is called starch paste (Jane et al. 1992). The pasting temperature was different in different germination stages due to the size of starch granules. The starch pasting properties of common buckwheat in the process of germination were shown in table 3, the results showed that starch pasting viscosities gradually decreased and significantly different (P < 0.05) in different period. After germina-tion, peak viscosity, through viscosity, breakdown, final viscosity, setback, peak time and pasting temperature were lower than those of the mature grain starch. Peak viscosity refers to the increase in the viscosity of starch paste caused by the friction between starch particles after full water absorption and expansion, which can reflect the expansion capacity of starch (Xiao- Li et al. 2008). As shown in table 3, the peak viscosity was 1394 -2982 cp, the average peak viscosity was 2242 cp, and the difference was great. Through viscosity is caused by the sharp decrease in the viscosity of starch paste due to the fact that starch particles no longer friction with each other after they have expanded to the limit (Xiao- Li et al. 2008). The through viscosity was between 1335 and 2819 cp, and the average was 2131 cp. The breakdown is the difference between peak viscosity and through viscosity, which can reflect the thermal stability of starch paste. The smaller the breakdown is, the better the thermal stability is (Karim et al. 2000). The average breakdown was 111 cp, and the breakdown in mature grains was 2.76 times higher than that after germination, indicating that the starch Table 3 Characteristic values of gelatinization of common buckwheat starch during germination had good thermal stability after germination and was suitable for the development of noodles and thickening agents. The final viscosity can reflect the retrogradation property of starch (Xiao-Li et al. 2008). After germination, the final viscosity decreased significantly, reaching 58.28% in 6d. The setback is the difference between final viscosity and through viscosity, which can reflect the stability of cold paste of starch. It can be seen from table 3 that the starch was not easy to age after germination and was suitable for making food such as common buckwheat instant noodles. Correlation analysis of pasting properties and starch composition Starch is the main component of common buckwheat grain, accounting for 60 -75% of the grain. The contents, composition and properties of buckwheat starch directly affect the processing technology of buckwheat food (Xin-Hua et al. 2009). The table 4 showed that peak viscosity, through viscosity and final viscosity of germinated common buckwheat was significantly positive correlated with amylopectin content (P < 0.05), which was the same as the relationship between the pasting viscosity and starch composition of the rice starch or germinated brown rice starch reported in previous studies, that was, the higher the content of amylopectin was, the higher the peak viscosity, through viscosity and final viscosity were. Break-down, final viscosity and setback were significantly negatively correlated with amylose content (P < 0.05). Studies have shown that the short-term retrogradation of starch is mainly caused by the gelation order and dehydration crystallization of amylose molecules. However, setback was significantly negatively correlated with amylose content (P < 0.05), indicating that germinated common buckwheat with low amylose content was easy to retrogradate. Correlation analysis of pasting properties and other nutrients The correlation analysis of starch pasting properties and other nutrients was shown in table 5. In 6d, pasting properties was positively correlated and negatively correlated with the main nutrients. Breakdown and setback were significantly negatively correlated with crude fat content (P < 0.01), indicating that the thermal stability and cold paste stability gradually increased after germination with the decrease of crude fat content. Peak viscosity, through viscosity and final viscosity were negatively correlated with crude fat content (P < 0.05). There was no significant relationship between pasting viscosity and water content, which may be due to the fast growth rate and the need to consume more water for growth, resulting in low water content. Similarly, pasting viscosity had no significant relationship with ash content and crude protein content. Table 4 Correlation coefficient between pasting properties and starch composition (r/p) Note: Linear correlation coefficient between r. **: Significant correlation at the level of 0.01, *: Significant correlation at the level of 0.05, the same below. CONCLUSIONS The results showed that in comparison to non-germinated grain, after germination, the concentration of main nutrients of common buckwheat were significantly different, where the content of crude fat, total starch, amylose and amylopectin decreased significantly while the content of ash and crude protein increased significantly. Starch granules were arranged in a disorderly manner, most of which were irregular in shape, few of which were spherical in shape. Moreover, the crystal structure of most starch granules was destroyed, and obvious cracks and voids appeared on the surface. In addition, starch size of mature common buckwheat was 4-9 μm. Pasting properties were closely related to the starch composition, and peak viscosity, through viscosity, final viscosity and setback were sig-nificantly positively correlated with amylopectin content, while breakdown, final viscosity and setback were significantly negatively correlated with amylose content. In addition, breakdown, setback and fat content were significantly negatively correlated, and peak viscosity, through viscosity and final viscosity were significantly negatively correlated with fat content, while pasting properties were not significantly correlated with other nutrients.

v3-fos

2019-04-27T13:09:06.200Z

1970-01-01T00:00:00.000Z

135167625

s2

CASSAVA FORTIFICATION AND QUALITY EVALUATION The broad objective of this work is to improve the nutrient content of cassava flour by inclusion of cowpeas seed flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. Cassava tuber flour was fortified with cowpeas flour and from cassava leaves at 20% and 30% of dry weight. Standard methods were used for the determination of parameter such as protein and carbohydrates. All samples were analysed for potassium, phosphorus, calcium, magnesium, iron, and cyanide. Unfortified cassava had significantly lower (P<0.05) values (protein: 0.942%, P: 0.093%, K: 0.749 Mg: 0.052%, Fe: 5.008 ppm) than fortification with both cowpeas flour and cassava leaf flour. Fortification with cowpeas flour did not significantly (P>0.05) change the Ca content however they were significant (P<0.05) increases cassava leaf flour. Cyanide content increased significantly for Treatment LF 20 and LF 30 but remained unchanged for Treatment CW 20 and CW 30 . Both cowpeas and cassava leaves had significantly (P<0.05) lower carbohydrate content than cassava tuber flour. Both cowpeas and cassava leaves are excellent for fortification but cassava leaves have to be used with additional pre-treatments to reduce the cyanide content in them. Organoleptic qualities analysed indicate high acceptability of fortification of cassava tuber flour with cowpeas flour. cowpeas leaves. The broad objective of this to improve the nutrient content of cassava flour by inclusion of cowpeas flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. It is envisaged that this will enhance the protein content of cassava, decrease the incidence of protein malnutrition among the less privileged cassava in sub-tropical Africa and other countries. It is significant that the can be easily adopted domestically and at cottage level. of cowpeas and cassava leaves as cassava fortificant will improve its production and accessibility and also diversify the use of INTRODUCTION Cassava (Manihot esculenta Crantz) is one among the important cultivated woody shrub in the family Euphorbiaceae and is used for food and feed purpose. Throughout the world, in tropical and subtropical areas, this crop is cultivated as an annual crop. Cassava roots are rich in carbohydrates [1]. Dependence on cassava diets therefore may lead to serious protein deficiency problems. Such malnutrition problem has been reported among consumers that rely primarily on cassava flour and other cassava products as major food source with little or no high protein food sources as complements. Cassava usually not eaten alone most times as a full meal but is rather taken with vegetable stew/soup/sauce that can provide other nutrients like protein. The animal origin diet like meat, fish and egg are expensive items for people in low-income families in African region. The current exorbitant cost of animal protein especially for low income earners deters the inclusion of such animal protein source in the stew that cassava is eaten with. Improving the protein content of cassava may be an alternative and affordable option. The enriching of the cassava meal with high nutritional way can solve the problems of mal nutrition [2]. This makes the need to improve the protein quality of cassava imperative [3,4] and to search for cheaper but good quality protein sources that are readily available [5]. In SADC region, the research on cassava crop seems scanty when compared to rice, maize and wheat. Fortification of cassava flour with plant protein is a viable affordable alternative to tackle specifically the problem of protein energy malnutrition in those areas affected by malnutrition. The plant protein can be sourced from unexploited indigenous legumes with high protein content (18.1 to 25.8 %) like cowpeas and cassava leaves, soybeans, and Bambara nuts among others. Cowpeas and cassava leaves unlike other mentioned plant protein sources have not found used in many food formulations as soybean [6]. This study addresses the problem of protein deficiency in cassava, a major staple food, using food-to-food fortification approach with the use of cowpeas and cassava leaves. The broad objective of this work is to improve the nutrient content of cassava flour by inclusion of cowpeas seed flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. It is envisaged that this will enhance the protein content of cassava, decrease the incidence of protein malnutrition among the less privileged cassava consumers in sub-tropical Africa and other developing countries. It is significant that the technology involved can be easily adopted domestically and at cottage level. Utilization of cowpeas and cassava leaves as cassava fortificant will improve its production and accessibility and also diversify the use of cassava. Experimental details Cassava root (Manihot esculenta Crantz) (Variety M7) was procured from Chiredzi research station, Chiredzi, Zimbabwe. Cowpea seed (variety CBC 2) was procured from Chiredzi research station, Chiredzi Zimbabwe and cassava leaves will be procured from Africa University Research Block, Mutare Zimbabwe. Preparation of Cowpeas into flour (CW100): The cowpea seed is winnowed to remove any trash and milled using a hammer mill. The first 5 kg of the milled flour is discarded to make sure there is no contamination of the flour. The flour is put in a polythene bag until used for the study. Preparation of cassava flour (Control): Harvested cassava roots are washed to remove all the dirty, the washed roots are peeled care should be taken that all peeled roots are kept under water to avoid discolouration. After peeling the roots are cut into small chips and fermented in water for 60 h. After 60 h the fermented cassava is drained to remove the water and sun dried. When completely dry the cassava is milled using a hammer mill. First 10 kg of the milled cassava is discarded to avoid contamination. The flour is put in polythene bags until used for the study. Preparation of cassava leaves flour (LF100): Cassava shoots approximately 20 cm in length are harvested, the hard petioles are removed and the leaves are sun dried. When completely dry the leaves are milled using an electric miller. The flour is put in a polythene bag until used for the study. Preparation of the fortified cassava flour 20% cowpea concentration (CW20): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 300g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 30% cowpea concentration (CW30): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 450g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 20% cassava leaf concentration (LF20): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 300g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 30% cassava leaf concentration (LF30): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 450g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. Chemical analysis Protein and Carbohydrate were determined according to AOAC [7]. The micronutrients including magnesium, potassium, cyanide, calcium and iron were evaluated using an Atomic Absorption Spectrophotometer (Buck 210VGP) Germany according to AOAC [7]. Sensory analysis The organoleptic evaluation of the biscuits samples was carried out for consumer acceptance and preference. Samples of the biscuits prepared from the cassava tuber flour and the different cowpeas and cassava leaf composite flour. Consumers evaluated the treatments on overall appreciation, taste, color and odour of the flour. Scores were given against the choice and preferences of the respondents. Statistical analysis All data collected to be statistically analyzed using the GenSTAT Analysis of Variance (ANOVA) software. Differences between means were determined using the Least Significant Difference (LSD) test at 0.05 level. Nitrogen (N) Data regarding the fortification of cassava tuber flour (CTF) with cowpeas flour (CWF) and cassava leaf flour (CLF) showed significant (P<0.05) differences for the nitrogen content. Results show that CTF (Control) has the least amount of nitrogen (0.151%) in comparison to CLF (Treatment LF100) and when fortified with either CLF or CWF. Results show that CLF (Treatment LF100) have has higher nitrogen (4.780%) content than CWF (Treatment CW100) (3.445%). After fortifying the CTF, Treatment LF30 produced the highest amount of nitrogen (1.220%) while treatment CW20 had the least (0.727%). Treatment CW30 and Treatment LF20 produced result which were not significantly (P>0.05) different from each other (table 1). Treatment LF30 produced the highest P content (0.161%) and it was not significantly different from Treatment CW20 and CW30 which produced 0.136% and 0.143% respectively. Potassium (K) The Control treatment had the least (0.749%)) potassium (K) content with respect to CW100 and LF100 which had 1.304% and 1.873% respectively (table 1). Results from this investigation show that treatments from fortification of CTF with CLF produced significantly (P<0.05) higher K content than treatments from fortification with CWF. Treatment LF30 produced the highest K content (0.990%) followed by Treatment LF20 (0.943%) while Treatment CW20 and CW30 had 0.847 and 0.867% respectively. Magnesium (Mg) The Mg content from the Control treatment, Treatment CW100 and Treatment LF100 was significantly (P<0.05) differently from each other with treatment LF100 having the highest Mg content (0.302%). Similarly, fortification of CTF with CLF produced significantly more Mg content as observed in Treatment LF20 (0.090%) and LF30 (0.107%) compared to Treatment CW20 and CW30 with Mg content; 0.067% and 0.070% respectively (table 1). Iron (Fe) Results pertaining to Fe content show that the Control treatment has significantly the lowest (5.008 ppm) Fe content in comparison to CW100 and LF100 with 60.034 ppm and 80.517 ppm respectively ( fig. 1). Fortification of CTF with CWF from level treatment CW20 to level treatment CW30 did not significantly (P>0.05) increase the Fe content. However, Fe content increased significantly (P<0.05) from level treatment LF20 to level Treatment LF30. Calcium (Ca) Results from the investigation reveal that the calcium (Ca) content in the Control treatment and CW100 was not significantly (P>0.05) different from each other ( fig. 2). Similarly, fortification of CTF with CWF was not significant (P>0.05) at all levels. However, Ca in CLF was 8 times more than that in CTF such that the Treatments LF20 and LF30 produced Ca content which was significantly (P<0.05) more than Treatment CW20 and CW30. The more the proportion of CLF used in the fortification of the CTF the more the Ca content that was obtained and this is true for Treatment LF20 and LF30. Cyanide (CN) Data pertaining to the CN content in the Control treatment, CW100, LF100 and fortified CTF at different levels is shown in fig. 3. The CN content in the Control treatment and CW100 was not significantly (P>0.05) differently from each other. Similarly, the fortified CTF with both levels of CWF (CW20 and CW30) did not significantly influence any change to the CN content. However, fortification of CTF with CLF increased the CN content significantly (P<0.05) as observed from Treatment LF20 and LF30 ( fig. 3). Fig. 3: Shows the influence of different fortifying agents on CN content Carbohydrate Results from the investigation reveal that LF100 has significantly (P<0.05) lower carbohydrates compared to CW100 (36.044%) and CTF (73.225%). Similarly, fortification of CTF with CWF and CLF at different levels significantly (P<0.05) reduced the carbohydrate content ( fig. 4). Overall appreciation Results for the overall appreciation of the unfortified cassava and fortified cassava with either cowpeas or cassava leaves are shown in table 2. The fig. show that 36 out of the 50 respondents scored very good to the overall appreciation of the Treatment CW20 and 18 respondents also scored very good to Treatment CW30. Treatment CW30, LF20 and LF30 had each 2 respondents who scored excellent on the overall appreciation of the levels of cassava fortification. Treatment LF20 and LF30 had 4 respondents each who scored poor on the overall appreciation. Fig. 4: Shows the influence of different fortifying agents on carbohydrate content Overall taste Data pertaining to the overall taste preferences recorded from the survey is shown in table 3. Out of the 50 respondents 6respondnents scored excellent for the overall taste of cassava. Twenty-eight respondents scored very good for the overall taste of the Control treatment while only 2 respondents did not like the taste of cassava. After fortification of cassava, Treatment CW20 and Treatment CW30 recorded 12 and 4 respondents respectively who scored excellent for the overall taste. The preference for Treatment LF20 and LF30 was somewhat lower as there were more respondents who scored poor and fair compared to the fortification of cassava with cowpeas. Appearance of the flour Data regarding the opinions of the respondents towards the appearance of the treatments is shown in table 4. For the Control treatment, CW20 and CW30 all the respondents scored good, very good and excellent. However, for Treatment LF20 and LF30 the preferences of the respondents were showing that the fortification of cassava with different levels of cassava leaves was unpopular as the color of the flour became more colored than being white. There were fewer respondents who score very good and excellent than those who scored fair to poor preferences of appearance. Fortification with cowpeas were the most preferred treatments. Odour of the flour Data from the respondents regarding their preferences towards the odour of the flours under study is shown in table 5. The Control treatment, Treatment CW20 and Treatment CW30 reveal that they have a got moderate to no odour which is most preferred by the respondents. However, fortification of cassava tuber flour with flour from cassava leaves, as in Treatment LF20 and LF30 was relatively unpopular since the treatments somewhat produced a more moderate to strong odour. DISCUSSION Traditionally, the roots of cassava are harvested and processed by many methods and produce different food products for diverse purposes. Cassava which is an important staple in the Sub-Tropics is low in protein and deficient in essential amino acids. However, the protein content of all composite flours with different levels of both CWF and CLF increased as a result of the significantly more nitrogen that is in cowpeas and cassava leaves than cassava tubers. Therefore, the more the proportion of cowpea or cassava leaf flour is added the more the protein content is obtained in the composite flour. It has been reported that fortification of cassava with soybean or cowpea extract increased the protein content of cassava [6,8]. In a similar study using soya bean by Collins and Falasinnu, they observed that legumes are generally high in their protein content and proposed that they make an ideal source for protein supplementation [9]. This observation agrees with previous findings of several researchers [10][11][12]. The results of the P content of CTF, CWF and CLF are shown in table 1. Significantly (P<0.05) unfortified cassava tuber flour has got less phosphorus than CWF and CLF. The increase in the P content of the CTF was as a result of adding either CWF or CLF. These results agree with the work by Anuonye et al. [13] on fortifying cassava with yams. There was also a corresponding increase in most of the other elements with increase in either CWF or CLF. The K content increased significantly (P<0.05) with fortification of the cassava tuber flour, similarly Mg and Fe content from CWF and CLF is significantly (P<0.05) superior to CTF. The Ca content of cassava tuber flour and cowpea flour does not differ significantly (P>0.05) from each. As a result, fortification of CTF will not benefit in increased Ca content of the product. Similar results were observed in the work of Anuonye et al. [14]. However, the Ca content in cassava leaves is significantly more than that in the tubers [15]. Therefore, as expected, to improve the Ca content in CTF fortification with the flour from its leaves will benefit as expressively observed for Treatment LF20 and LF30. This is attributed to the replacement of the cassava tuber flour with calcium-rich flour from cassava leaves. Fig. 1 shows that cassava tubers have significantly much less Fe content than cowpeas and cassava leaves. The increase in the Fe content of the fortified CTF is as a result of adding the flour of cowpeas and/or cassava leaves. Treatment LF20 produced the same effect with Treatment CW30 revealing that cassava leaves are very rich in Fe content. Results obtained in this study show that CTF has significantly (P<0.05) more CHO content that the products from the fortification with cowpea flour or flour from cassava leaves. Correspondingly, there was a significant (P<0.05) decrease in the CHO content of the CTF with addition of flour from cowpeas and cassava leaves. This is attributed to the fact that there is poor CHO content in both cowpeas like any other legume and cassava leaves. As a result, there was no significant difference in the CHO content by increasing the level of either CWF or CLF. These results are similar to those obtained in the work of Obadina et al. [16] with soya beans. The total CN content of the simple cassava tuber-flour is very low and statistical not significantly different with the CN content in cowpeas flour ( fig. 3). Fortification of CTF with CWF at all levels did not result in any change of the CN content. However, fortification of CTF with CLF increased the CN content in the composite flour significantly (P<0.05). This is attributed to the very high CN content in CLF and the replacement of the low CN content flour with high CN content flour in the composite flour. The organoleptic Test investigated the overall consumers' appreciation as well as taste preferences, preferences in terms of color appearance and odour of the flour. Unlike those countries in central Africa were cassava is a staple and there are many dishes that have been developed from cassava, in Zimbabwe our staple is maize. An appreciable number of Zimbabweans do eat cassava and the numbers are increasing as more and more people are being to eat a wide range food. So, it was expected that the results from this investigation was likewise influenced by the fact that cassava is not a very popular dish in the region. For the overall appreciation however, the product from fortifying with cowpeas flour was rated better from good to excellent. Similarly, with regards to taste preferences and appearance fortified cassava tuber flour with cowpeas flour scored more from good to excellent as well. With regards to the odour of the composite flours, more respondents said that the composite flour with cowpeas had no odour to questionable odour. However, for the composite flour with cassava leaves more respondents said that it had a moderate to strong odour. These results are attributed to the fact that cowpeas are already an acceptable food unlike cassava leaves. CONCLUSION The results of this study have shown that substitution of both cowpeas flour and cassava leaves in cassava tuber flour is possible. Both cowpeas flour-fortified cassava tuber flour and cassava leaf flour-fortified cassava tuber flour could be used to fight macronutrient and micronutrient deficiencies. The mineral composition of the cassava tuber flour was enhanced as a result of the flour substitution. The composite flour could help in reducing protein energy and micronutrient deficiency prevalent in developing countries such as Zimbabwe. Cassava tuber flour can be fortified by adding flour from cassava leaves however, these results shows that the fortification with cassava leaf flour leads to increase in cyanide content and organoleptic challenges. It is concluded that protein, phosphorus, potassium, magnesium and iron content in cassava tuber flour could be enriched with up to 30% cowpeas flour without organoleptic challenges.

v3-fos

2020-12-10T09:01:31.857Z

1970-06-01T00:00:00.000Z

237230264

s2

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min. Although the degree of freshness or spoilage of meats is often evaluated by plate counts, it is known that spoilage is not the result of bacterial numbers per se but is caused by biochemical changes brought about by the growing flora. Investigations on the mechanism of spoilage of fresh refrigerated meats over the past several years have led to the proposal of a number of techniques for assessing its presence and extent. Among these are techniques based upon the phenomena of extract-release volume (ERV), water-holding capacity (WHC), meat swelling, and viscosity (1,2,5,6). All four of these are based primarily on changes in hydration capacity of meat proteins, which is lowest for fresh meat but gradually increases as spoilage occurs. Although the pH of fresh beef is around 5.6 to 5.8 and gradually increases to as much as 8.5 when beef becomes putrid, the increase from freshness to incipient spoilage generally does not exceed 0.3 to 0.5 of a pH unit. This, along with the fact that the change is usually not uniformly distributed in a meat sample, makes direct pH measurements unsuitable for the purpose of detecting incipient spoilage. Also, beef is often judged as spoiled without any noticeable pH changes The present report describes a more direct 1 technique for detecting incipient beef spoilage by accurate titration of the basic (alkaline) substances or functions produced in beef by the spoilage flora. The technique employs the measurement of the quantity of dilute acid required to bring beef homogenates to pH 5.00. The volumes of acid have been correlated with log bacterial numbers, ERV, and pH on beef from different sources and in different stages of spoilage. MATERIAL3 AND METHODS Titrations were carried out by blending 10-g samples of beef in 100 ml of deionized water for 2 min and filtering through cheesecloth to eliminate connective tissue. Duplicate samples of the homogenate containing 2 g of meat each were titrated with 0.02 N HCI by using an autotitrator (model iT-T and ABU1, Radiometer, Copenhagen). The amount of acid required to bring the homogenates to pH 5.00 was recorded. The initial pH of homogenates was read simultaneously on the titrator. The relationships between titration and spoilage, aging, and fat content were studied employing semitendinous (ST) muscle. To study the effect of spoilage on titration values, 15-g samples of ground muscle were stored at 5 C in small beakers covered with aluminum foil. Log bacterial numbers, pH, and titration values were determined on the stored meat at 2-day intervals. For the study of aging, 15-g samples of meat were stored at 5 C in gas-impermeable plastic bags as previously described (3). The effect of fat on titration values was determined by adding known quantities of beef fat to fat-free ST ground muscle followed by immediate titration. ERV values, log bacterial numbers, and percentage of fat were determined as previously described (1). RESULTS AND DISCUSSION The relationship between log bacterial numbers, pH, and titration values in fresh ground ST muscle undergoing spoilage at 5 C is presented in Fig. 1. During spoilage, log bacterial numbers, titration values, and pH showed a marked increase from the 2nd day of storage to the 10th; spoilage was detected on the 5th day, at which point the log bacterial count was 9.1 /g, the titra- FiG. 1. Relationship between bacterial nwnbers, pH, and titration values on ground semitendinosus muscle held at 5 C for 10 days. Beef stored in beakers and covered with aluminum foil underwent spoilage in the usual manner, whereas spoilage was delayed in beef stored in gas-impermeable plastic bags. 10 ui 9 z 0 0 8 7 tion volume was 2.9 ml, and the pH was 6.3. Beef stored in plastic bags, however, showed only a slight increase in both pH and titration values after 10 days of storage when the log bacterial count did not exceed 8.90/g. This meat was judged acceptable, even at the end of this 10day holding period of 5 C. With respect to titration volume of ground beef, the method of sampling may affect results. Since ground beef spoilage at refrigerator temperatures is largely due to surface growth, surface samples would yield higher titration volumes than those taken from the interior. To minimize this difference, the entire batch of ground beef [1 to 1.5 lb (453 to 679 g) portions] was thoroughly mixed by use of spatula, followed by the removal of test samples in a random manner. Replicate samples from ground beef treated in this manner gave results with low degrees of variation. When beef cuts are to be tested, both surface and interior portions should be mixed as for ground beef. The effect of mixing surface samples with interior samples, where there are generally fewer bacteria, is to dilute the generally higher level of titration substances present in surface samples and to neutralize any microbially produced organic acids that may be present in subsurface portions. A pattern similar to that presented in Fig. 1 is presented in Fig. 2, employing retail-store stew beef which was ground in the laboratory. Statistically, the correlation coefficients (r) between titration values and pH and between titration values and log bacterial numbers were significantly above the 1% level; however, between titration values and ERV, r was significantly above the 2% level. The effect of fat content on titration is presented in Table 1, from which it can be seen that mean values for four replicates decreased from 1.73 ml to 0.91 ml as the percentage of fat increased to 50. It was concluded from these findings that fat alone does not contribute to the acid-titratable groups in fresh beef, although there is some evidence that fat may affect titratable functions as beef undergoes spoilage. In an effort to determine the performance of titration on market meats, hamburger meat was obtained from 23 retail chain stores, and titration values, log bacterial numbers, ERV, pH, and fat content are presented in Table 2. Titration values, log bacterial numbers, ERV, and pH are given at freshness (day of purchase) and at onset of spoilage. The titration volumes of fresh hamburger meats ranged from 0.53 to 2.15 with a mean value of 1.32 i 0.36, whereas values at the onset of spoilage ranged from 1.34 to 3.41 with a mean of 2.58 i 0.56. The average time for the onset of spoilage was 5 days, with a range of 2 to 7 days at 5 C. With respect to its degree of sensitivity to the changes that occur when fresh beef undergoes spoilage, the titration volume increased by 95.4% from freshness to spoilage, based on mean values. The per cent increase with respect to bacterial numbers was 24.8, with the mean log number for fresh beef being 7.41 ± 0.61 and 9.25 i 0.56 at the first signs of detectable spoilage. With respect to ERV, the per cent increase was 44.1; the mean at freshness was 34, whereas the mean value at spoilage decreased 7.0 7.0 6.3 6.5 6.7 6.5 6.6 6.3 6.4 6.3 6.7 6.8 6.5 6.5 6.9 6.8 6.9 to 19. The r between values for titration, log bacterial numbers, and ERV at the two different times is very highly significant (P < 0.001). In an effort to determine the titration value of ground beef at the onset of spoilage, titration volumes were determined on the 23 samples of retail-store hamburger and related to the previously established ERV of 25 and log bacterial numbers of 8.50/g (1). These values are presented in Table 3. Employing an ERV of 25, the corresponding titration values ranged from 1.33 to 2.68 with a mean of 2.01 i 0.37, whereas the mean was 2.10 + 0.35 when a log bacterial number of 8.50/g was employed as reference. On the basis of these findings, ground beef that requires in excess of 2 ml of 0.02 N HC1 for titration of 1 g to pH 5.00 may be expected to be in some state of microbial spoilage. Of the 23 retailstore samples, only two required more than 2 ml of acid for titration at freshness (no. 13 and 21, Table 2). It may be noted further from Table 2 that sample 13 had a titration value of 2.15, a log bacterial count per gram of 7.91, but an ERV of 25 at freshness; whereas sample 21 had a titration value of 2.05, a log bacterial count of 8.32, and an ERV of 29 at freshness. Both of these samples of meat were apparently undergoing incipient spoilage at the time of purchase. A simplified technique for the rapid detection of spoilage in ground beef can be achieved by adding 2 ml of 0.02 N HCl/g of meat to the blended and filtered homogenate and checking the final pH of the homogenate. Using this method, when the pH is >5.0, the meat may be presumed to be in some state of incipient spoilage. Although the identification of the basic substances that are titrated by this technique is not known at this time, all available evidence suggests that they are microbially produced and their appearance is time-dependent. When fresh beef was inoculated with meat spoilage flora to log 8.5 to 9.0/g and tested immediately, titration values remained low and no signs of spoilage resulted. The homogenizing step, along with the constant stirring that accompanied titration, suggests that the substances in question are mainly nonvolatile. In a previous report from this laboratory (4), amino sugar complexes were shown to increase along with bacterial numbers and hydration capacity. The possibility exists that these compounds are at least partly responsible for the increased amounts of acid necessary to lower the pH of spoiling beef. Further research towards identification of the basic functions and their role in meat spoilage is in progress.

v3-fos

2020-12-10T09:04:12.914Z

1970-04-01T00:00:00.000Z

237233015

s2

Chemical States of Bacterial Spores: Heat Resistance and Its Kinetics at Intermediate Water Activity Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is prominently nonlogarithmic for both chemical forms of the spore. Log survival varies approximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, Ea, and Q10 cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed. Traditionally, bacterial spores have been considered to have two kinds of heat resistance, wet and dry. In general, wet has referred to test environments in which liquid-phase water was present, dry to measurements made in the absence of liquid water. Murrell and Scott (10) showed that in the absence of liquid water the water vapor activity (a,) or relative humidity of the test environment had a very large effect on spore heat resistance. They showed that heat resistance was maximal at environmental a, values in the 0.2 to 0.4 range. The heat resistance effects they obtained by variation of the relative humidity of the test environment were large, amounting to many-fold. Thus, those categories of spore heat resistance based on the water status of the test environment could no longer be confined to two, wet and dry, but must be increased to at least three general classes: wet, meaning in the presence of liquid water at a, values near 1; dry, meaning the total absence of water activity in the test environment; and third, resistance in the absence of liquid water but at an intermediate water vapor activity. In the last category, environmental water activity must be specified precisely because of the large variation of resistance with relative humidity. As a practical probability, most heat challenges of bacterial spores in the absence of liquid water would fall into the third category since quite rigorous precautions are required to insure the total absence of water activity (3). This inadequacy of the traditional term "dry heat" as a sufficient specification of a nonaqueous environment for spore heat resistance has also been pointed out by Angelotti et al. (5), Pflug and Schmidt (11), and others, but the term seems to persist for a description of environments with water activities from zero to near one so long as obvious liquid water is absent. Since Murrell and Scott's work (10), several recent studies of Angelotti (5), Fox and Pflug (7), Mullican and Hoffman (9), Hoffman, Gambill, and Buchanan (8), and Bruch and Smith (6) on so-called dry-heat resistance have included comments on the pertinence of these water activity effects to the results. Unfortunately, in none of these studies could the level of water activity at the lethal temperature be specified quantitatively. For ordinary aqueous heat resistance, we have shown that mature bacterial spores can exist in sensitive and resistant states (1,3,4). These different resistance states are prepared and interconverted by in vitro chemical pretreatments of the spores. The changes of aqueous heat resistance between the states can amount to about a thousand-fold. These changes or differences in heat resistance reside within the spores rather than being a response to environmental conditions during the heat resistance test since the changed resistance properties persist when the reagents used to effect the change of state are removed and the spore is transferred to a new environment for measurement of its heat resistance capacity. Thus, to the extent of three orders of magnitude, aqueous spore heat resistance for Bacillus stearothermophilus is an inducible property, and meaningful measurements of heat resistance potential are not possible without knowing the chemical state of a spore sample. Chemical events accompanying these changes of heat resistance state have been described (1,4). Later, we showed (3) that these same chemical states of the spore also have different resistances to strictly dry heat and we presented a method for dry-heat resistance measurements designed to avoid artifacts due to interference by unknown spore chemical state, water activity effects, and uncertain mechanical recovery of the dry-heat-challenged spores. Here we show that the sensitive and resistant states of spores also exist for heat resistance at intermediate environmental water activity. Characteristics of the nonlogarithmic survivor curves and their temperature dependence are given for each of the chemical states at an optimal environmental water activity. A method is presented for testing heat resistance at intermediate water activity. The method is designed to avoid the interfering factors mentioned above and also to furnish known and easily controlled environmental water activity at the test temperature. Some consequences of the existence of a maximum in the survivor versus a. relation are given for the temperature dependence of survival rates of spores encapsulated with a definite weight of water. MATERIALS AND METHODS Preparation of spore crop and the spore chemical states. Spores of B. stearothermophilus NCA 1518 were grown and cleaned as previously described (3). The preparation of the sensitive state, hydrogen form, spores, and the three preparations of the resistant state spores were the same as before (3). Method for measurement of heat resistance at controlled water activity. The general approach to water activity control was direct rather than through the use of humidity-controlling solutions. It was arranged to have a known weight of water in an otherwise evacuated sealed glass tube of known volume at a known temperature. The amounts of water required to give the desired a, at the lethal test temperature were taken from handbook tables of the properties of saturated steam. The ratio of space volume to spore weight should be chosen such that these amounts of water are large by comparison with any expected emission or uptake of water by the spores themselves. Borosilicate glass thermal death time (TDT) tubes (9 by 150 mm) were preconstricted to facilitate later flame sealing. The volume of the tubes up to the middle of the constriction (about 4 ml) was then determined by filling with water. The tubes were segregated into lots whose members had a volume variation range of less than 0.1 ml. The tubes were then filled and covered with distilled deionized water and autoclaved for 1 hr to leach out soluble alkali near the surface of the glass (2). The leaching process was repeated and the tubes were allowed to dry. Very small piles (200 Mg) of the "dry" (freeze dried) spores were weighed into the tubes. The amount (steam tables) of water required to give the desired water activity at the test temperature was then injected by a Hamilton microliter syringe as a drop on the inner wall of the tube. The area of the tube around the water drop was pressed against a piece of dry ice until the drop froze firmly. The open top of the tube was then quickly connected to a piece of gum-rubber tubing connected to, but sealed off by a spring clamp from, an oil pump vacuum. The TDT tube with the drop still frozen was then inserted up to the constriction into powdered dry ice in a Dewar flask. After about 1.5 min, the spring clamp to the preexisting oil pump vacuum was removed and vacuum was pulled for about 20 sec. The TDT tube still attached to the oil pump vacuum was then quickly sealed in a gas-oxygen flame and the seal was annealed in a smoky flame. The sealed TDT tube was then canted against the sharp edge of a slab of dry ice until needed for the lethal heating. The water drop can be moved quickly from place to place within the sealed tube by chilling a small area against dry ice. This serves as a vacuum test and was applied to each tube both before and after the lethal heating. Also, it was confirmed that no significant loss of water occurred during the above described vacuum sealing procedure. This was done by weighing, before and after drying, severed portions of previously prepared TDT tubes into which the water had been collected into one end by such spot chilling with dry ice. For the lethal heating step, the tubes were enclosed singly in flat wire cages (-1.5 by 10 by 12 cm) fabricated from 0.64-cm mesh wire (hardware wire cloth). Prior to insertion of the tubes into the cages, the chilled spot on the tube was thawed with fingers. The tubes were heated for measured times in an oil bath controlled to better than 0.01 C. After the elapsed heating time, the wire cages were quickly plunged into cool water for a few seconds. The cages were then set upright to a depth of about 2.54 cm in warm (-40 C) water for a few seconds to drive the condensed water away from the spores. The tubes were then removed from the cages, quickly wiped free of oil, and again slanted against the sharp edge of a slab of dry ice to collect all the condensed water into one spot away from the spores. If plating for survivor count was to be delayed to another day, the tubes at this point were stored in crushed dry ice. The tubes were then snapped open after scoring thoroughly to facilitate a clean, even break. The spores were washed out with four 1-ml rinses of Tryptone broth into a Teflon homogenizer cup (A. H. Thomas Co., Catalog #4288, size A) and homogenized thoroughly enough to disperse them singly, as judged by direct microscopic count on oil-cleared membrane filters (12). The homogenized spore suspension was then further diluted as required with Tryptone broth for plating (Tryptone, 1%-glucose 0.5%-soluble starch, 0.1%). Incubation was for 2 days at 53 C. Direct microscopic counts were made on the Tryptone broth dilutions by the method of Snell (12) for each tube. The number of spores in the original little pile of spores was based on this direct count. This avoided needing to know and control the moisture content of the original spore sample and the problems of accurate weighing in the microgram range as well as that of correcting for possible loss in opening the evacuated tubes. After rinsing the spores out of the opened tubes, the volume of the two halves of each of the tubes was measured by filling (level meniscus) with water. With some practice in flame sealing and the preselection for volume uniformity mentioned above, the standard deviation of the measured volumes of the opened tubes within a lot was about 0.4% of the mean. However, the volume of both halves of each tube was always checked because of the obvious possibility of serious volume variation from the flame sealing operation. In actual practice, such variations did not occur. RESULTS Variation of survivors with a,. In Fig. 1 are plots for a given heat treatment of log survivors versus water activity for each chemical state of the spores. Log survivors fall off very steeply on each side of the moderately broad maximum. Fortunately, this maximum is at about the same position for each chemical state. The optimal water activity (0.28) chosen for this study was taken from the central portion of these maxima. The position of the maximum was not significantly changed when the heating time for the resistant state (preparation 3) was heated for 50 min at 150 C rather than 30 min, although the height of the maximum was reduced by about 2.5 log units. The water activity at the maximum agrees closely with that reported by Murrell and Scott (10) for their bithermal method. Survivor curve characteristics for the sensitive and resistant states. It is obvious from Fig. 2 and 3 that the log survivors versus heating time curves are indeed curves without the possibility of reasonable approximation by straight lines (log death). This curvilinearity holds for both chemical states but is most prominent in the log survivor curves for the resistant-state spores. Since the log survivor versus time plots are not linear, the D value concept is not applicable to heat inactiva- tion of spores under these conditions of intermediate water activity. For describing heat inactivation at a, = 0.28 under these conditions, an empirical expression, [(log initial count/g) -(log survivors/g)]a = ktmin + C, was chosen which gave a reasonable fit to the log survivor curves. The exponent a for VOL. 19,1970 each chemical state was chosen from the least squares slope of plots of log [(log initial count/g) -(log survivors/g)] versus log tmin. For the sensitive-state spores, a value of the exponent a of 0.58 was selected from such slopes. For the resistant state, a was taken as 0.33. The log initial count/g was 11.47 for the original unheated resistant-state preparation and 11.85 for the sensitive-state preparation. Thus, the expressions used to describe heat inactivation at a, 0.28 under these experimental conditions were (11.47 -log 5)0 33 = k'tmin + C for resistant state (1) (11.85 -log S)o -18 = k'tmin + C for sensitive state (2) where S is the number of survivors per gram of spores at heating time t in minutes, and k' and C are constants. As shown in Fig. 4, plotting (log Soriginallog S)0 33 for the resistant state and (log Soriginallog S)0 58 for the sensitive state versus time gave reasonably straight lines for several test temperatures. The least squaresdetermined slopes of these lines yielded coefficients of t in equations 1 and 2 for various temperatures. These coefficients of t were used as reaction velocity constants (k') in determining the temperature dependence of heat inactivation ( Table 1). For convenience, the Arrhenius plots for both states have been included in Fig. 5, but it should be noted that the rate expressions (equations 1 and 2) for the two states are different and so equal values of the rate constants (k') between states do not mean equal numbers of survivors will result from equal reaction times. 5, It is apparent that the temperature dependence of spore heat survival for each of the chemical states at a, 0.28 is similar to that for strictly dry heat resistance (3), even though the survivor curve shapes and the general level of heat sensitivity are quite different. The activation energies for both chemical states at both environmental humidity conditions of heat challenge (strictly dry and a, 0.28) are all about 40,000 cal. However, the log survivor versus time relations for strictly dry heat resistance are straight, that is, follow "logarithmic death," whereas these same relations for heat challenge at intermediate water activity of 0.28 are prominently curved. The general level of heat sensitivity of the chemical forms under strictly dry conditions is about like that for ordinary aqueous heat resistance. That for the same chemical forms at the intermediate water activity is much higher. DISCUSSION It is evident from Fig. 2 and 3 that mature bacterial spores can be manipulated chemically into different states for the property of resistance to heat at intermediate water activity. Since, like heat resistance at both strictly dry and strictly wet environmental conditions, heat resistance at intermediate water activity is a spore property manipulatable by in vitro pretreatments, the same implications for experimental strategy which we have stressed previously (2, 3) for the a, = 0 and a, 1 conditions also hold for this intermediate humidity situation. The most important of these is that meaningful estimates of heat resistance potential cannot be made without knowing the chemical state of the spores. This knowledge of chemical state can be gained by converting deliberately a portion of the spore sample into each chemical state before measuring heat resistance at controlled water activity. As pointed out previously (2), if artifacts are to be avoided in the measurement of the aqueous heat resistance of the different spore chemical states, special care must be taken to use a testing medium inert with respect to its capacity to induce change of chemical state during the test. In fact, aqueous conditions can be arranged deliberately for the adaptation of spores to heat (4) by using a noninert suspending medium for heating. Nonaqueous heat resistance of spores, both dry and at intermediate water activity, is generally not subject to these changes of chemical state effects during the lethal heat challenge itself. However, we have pointed out how inadvertent changes of spore resistance state could occur by interaction of spores and their supporting surface in the preparatory phases of nonaqueous heat resistance measurement (3). Even in the case of aqueous heat resistance, it has been found possible to sensitize spores to heat in the presence of complex biological mixtures at their ordinary pH (2). It is not necessary to have acid conditions present during lethal heating of spores in such wet, complex biological mixtures, probably because free calcium ion is absent by virtue of the complexing action of the organic materials. Such sensitization to heat for facilitating spore killing in a practical situation should be straightforward in the case of nonaqueous heat resistance, providing the spores are or can be made physically available to the sensitizing reagent. Merely washing the sensitizing acid away before lethal heating would restore the original nonacid condition of an inert substrate on which the spores were supported. Conceivably, in some situations, the spores could be chemically sensitized before encapsulation in solid matrices. From the survivor versus a, plots of Fig. 1, some inferences may be drawn about the apparent temperature dependence of heat resistance of spores encapsulated within a given volume with a given weight (as contrasted with activity) of water which considerably exceeds the water-holding capacity of the enclosed spores, that is, where the encapsulating volume exceeds spore volume by several hundred-fold. In the absence of liquid water, specification of the water activity within an enclosed space requires knowledge of three terms: volume of space, weight of water, and temperature. Of course, if pure liquid water is known to be present at all temperatures of interest, a, is 1 by definition. For spores encapsulated in solid matrices such as plastics, crystals, tightly joined surfaces, etc., neither the volume of the encapsulating space nor the weight of water is likely to be known, leaving temperature as the only measurable variable, and water activity unknown. In Fig. 1 it may be seen that the depend-ence of survivors on water activity is quite sharp on either side of the maximum. This could make for an apparent unusual temperature dependence of heat resistance for spores encapsulated with a given weight of water since, when volume and weight of water (short of saturation) are fixed, a, varies inversely with temperature. If, for example, the particular tube used for the last point on Fig. 1 (Resistant State) had been heated at a temperature 100 higher, the spores would tend to be protected against the higher temperature. Instead of 0.50, the water activity at the 100 higher temperature would be only 0.39 for these encapsulating conditions. Thus, on the right side of the maximum, the lethal effect of an increase in heating temperature would tend to be compensated by the temperature-induced lowering of water activity. On the left side of the maximum in the a, versus survivor curve, the killing effect of raising the temperature would be reinforced by an a, change toward a less favorable (for survival) value. If, on the other hand, the tubes mentioned were heated at a 100 lower temperature the situation would be reversed with survival for tubes on the left side of the maximum being reinforced, whereas survival on the right side would be opposed by such a lowering of heating temperature. It is thus apparent that for such casually (with a given weight of water) encapsulated spores, it is not possible to determine temperature dependence of heat lethality in the usual sense. The temperature dependence of spore heat survival rate is commonly expressed as a z value, the temperature change required for a 10-fold change in survival rate. Such z values are considered to be a property of the spore in a given, presumably constant, environment. For example, in both aqueous and strictly dry heat environments water activity is essentially independent of temperature. However, as discussed above, when spores are encapsulated with a given weight of water, the kind of environment is also temperature dependent; water activity itself is a function of temperature. On the left side of the a, versus survivor maximum, apparent z values would be low, on the right side, high. Even in cases where the encapsulating fit around a spore is close with the resulting expected buffering of environmental water activity by the spore itself, the required lowering of equilibrium water content with increasing temperature would be expected to give some environmental disturbance. Only when volume and water content of the encapsulating space are known and manipulatable is it possible directly to determine a z value in the usual sense of its being a spore property. It appears that such considerations of environ-mental water activity temperature dependence can explain the anomalous z values reported by Angelotti et al. (5) for their paper system and possibly also for their highly torqued stainlesssteel-surface system. The cellulose moisture sorption isotherm and the geometry of the system used for drying the paper would indicate an appreciable final water content in the paper, probably at least 2%. Such a moisture content of 2%, when released by high temperature, would result in an a, of about 0.06 at 125 C, about 0.04 at 140 C. Such values lie on the steep, left side of the curves of Fig. 1 at which point lethality of heat is reinforced by temperature-induced lowering of aw, in other words, where apparent z values would be low. Doubling or halving, or more, the assumed value of 2% moisture content for the paper would not affect this qualitative argument for low z values since the a, would still be on the steep, left side of the a, versus survivor plot of Fig. 1. As we have reported (3), extremely low water activities still can elevate spore heat resistance over that at a strictly dry condition. Thus, the abnormally low z value operationally observed for the paper system appears to have been due to lack of environmental constancy during its measurement rather than being an expression of a temperature characteristic which could be interpreted as a "wet kill mechanism." Although sufficient information on environmental water activity is not available for the other unusual z value in the highly torqued stainless-steel system, its direction (high) would be expected for wetter-than-optimal conditions on the right side of the curves of Fig. 1. The cavities provided by the roughness of the no. 4 stainless finish of the washers should not have been large enough to accommodate spore size particles. One possible source for sufficient water in the environment would be water released from mechanical disintegration of most of the spores. Another recent report of an anomalously high (139 C) z value is that of Bruch and Smith (6) for spores on Teflon film. Here, the Kapton film interlayer was known to have an appreciable water-holding capacity and is a reasonable source for sufficient water to furnish a wetter-thanoptimal environment with the tendency to apparent high z values. Some inferences can be drawn on the question of efficient heating conditions for killing spores encapsulated under conditions in which environmental water activity is highly temperature dependent. For random encapsulating conditions in which the water activities of the cavities are distributed over the whole of a curve like that of Fig. 1, temperature cycling should bq useful in successively moving spores off the optimum (for survival) of the curve down to the steep sides where heat sensitivity is much greater. Murrell and Scott's comprehensive report (10) on the effect of water activity on spore heat resistance clearly established the large effect of environmental water activity on the heat resistance of several species of bacterial spores in the absence of liquid water. They showed that the optimal relative humidity for heat survival was in the range 0.2 to 0.4 a, and that in this optimum environmental a, region the great interspecies differences in heat resistance largely disappeared. There are several differences between our work here and that of Murrell and Scott (10). These differences fall in three principal categories: (i) experimental methods of water activity control, (ii) heat survivor curve shape, and (iii) knowledge and control of the chemical resistance state of the spore samples used. For the bulk of their data, Murrell and Scott used several conventional humidity-controlling solutions packaged with, but separated from, the spores by various two-container arrangements. In all these arrangements, the whole system was evacuated prior to final sealing so that equilibration of water activity would be speeded during lethal heating. They also made less extensive use of a bithermal method. Each of these variations of the methods was more than adequate to show the striking maximum in the curve relating a, and D value. However, the a, at the maximum was somewhat variable, and at particular a, values off the maximum, D value variations among the methods amounted to many-fold. It was recognized that the behavior of the controlling solutions at high temperature was somewhat uncertain and the water activity control was described as approximate. We have chosen a simpler, direct method of controlling water activity which insures that the gaseous water concentration in the spore-encapsulating environment is known unequivocally once equilibration to the lethal temperature has been achieved. The usable range is not limited to low temperatures. So long as the spore weight is kept sufficiently low that any water uptake or emission by the spores is negligible by comparison with the total water content of the whole encapsulating environment, there appears to be no reason to use humidity-controlling solutions. Both with our proposed method and that of Murrell and Scott, temperature lags will occur during the come-up and come-down periods of the lethal heating operation. Such temperature lags should be less with our method because of its smaller mass and the fact of two containers in the system of Murrell and Scott. We have made some measurements of the time required for disappearance of the water drop in our method and these times are measured in seconds. For example, in a 4-ml volume, 2.55 mg of water took 15 sec to disappear at 1480 from one spot. When the 2.55 mg of water was divided into three roughly equal spots on the wall of the evacuated tube, disappearance took about 5 sec. Water spread over the upper 1 to 2 cm of the tube was more difficult to observe precisely but disappeared very quickly. During the temperature come-up period with our method and also with arrangements 1 and 5 of Murrell and Scott (10), the spores temporarily would see a water activity lower than that intended. We did obtain increases of survival by spreading the water over the upper part of the tube as compared to letting it vaporize from a single spot, but the differences were not large. The second major difference between our work and that of Murrell and Scott lies in the radically different log survivor curve shapes. They reported a linear log survivor versus time relation and expressed survival in the usual D-value terms. Our log survivor curves, on the other hand, are, as pointed out above, definitely and prominently curvilinear and not susceptible to even rough approximation by the single D value concept. The reasons for this difference in the survival versus time relation are not definitely known. The experimental physical arrangements and means of water activity control are quite different for the two methods. Another possibility for the origin of the curve shape discrepancy may lie in the fact that Murrell and Scott determined resistance at temperatures which are, at least for the resistant form of spores like B. stearothermophilus, quite low. Under the conditions of the resultant long survival times such as curve # 2 of their Fig. 2, it would be difficult to distinguish between a curvilinear log survivor relation like our equation 1 and classic logarithmic death. Both expressions would have about the same appearance of linearity with low slope over the early portion of the log survivor drop. For example, in our Fig. 3, if only the data in the first three quarters of the time period were available, an interpretation of log death could easily be made. At such low temperatures, only in tests observing survival over several log units would the curvilinearity following the long lag period become evident. The third main category of difference between this work and that of Murrell and Scott lies in the control of the chemical state of the spore sample. Here, we converted the spores to a known heat-resistance state before measuring heat resistance. Murrell and Scott used naturally occur-ring spore crops which can be composed of various mixtures of the heat-resistance forms.

v3-fos

2020-04-16T09:09:14.417Z

2020-01-01T00:00:00.000Z

216654592

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Qualitative analysis of some bioactive components of methanolic leaf extract of M. citrifolia (Noni) Medicinal plants offer endless opportunities for new drugs discovery due to their supremacy for the possession of phytochemical compounds known for diverse antimicrobial activities. The world ever increasing demand for therapeutic drugs from natural products with particular interest in edible plants for safety purposes is now catching researchers’ attention. This study therefore aimed at determining the presence of some bioactive phytochemical components of methanolic leaf extract of M. citrifolia L. Qualitative screening of leaf extract has confirmed the existence of Tannins, steroids, saponins, flavonoids and alkaloids in the mixture. And these bioactive compounds correspond to phytochemicals with antimicrobial, nematicide, pesticidal, antioxidant, ant-inflammatory, cytotoxic, anti-allergy, and anti-carcinogenic properties (bioactive compounds) earlier documented by previous researchers. INTRODUCTION Voucher number SK 3255/17) (Appendix A). Thereafter leaves were brought to the Biocontrol Laboratory, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia (UPM), washed under running tap water to get rid of dust and debris and rinsed in sterile distilled water. Leaves were first allowed to stay for 6 hrs under the laminar air flow to dry up the wet leaves surface. And finally dried at 40-45 o C in a mechanical convection oven (Memmert, Germany). An electrical grinder Retsch SK100 standard Guβeisen, 2002 was used to gried the dried leaves into the powdered form for use [5]. Test for Tannins To determine the presence of Tannins, Braemer's test was performed. Following the method described by [6], where 2 g of the powdered M. citrifolia leaf was dissolved in 10 ml of methanol, then macerated and filtered by means of cotton wool and funnel. Thereafter, 2 ml of the filtrate was added to 2 ml of 10 % alcoholic ferric chloride (1:1). Formation of greenish grey coloration of the mixture indicates the presence of tannins. Test for Steroids To test for the presence of steroids, Lieberman Burchardt test was used. For the test, 2 g of the leaf powder was added to 20 ml of methanol in a 150 ml conical flask and covered for 30 min, mixture was filtered using cotton wool and funnel. Filtrate was poured into a 50 ml beaker and placed on a water bath until filtrate was completely evaporated. 6 ml of chloroform was added to the evaporated extract and mixed thoroughly. Then 2 ml of the chloroform mixture was transferred into a test tube where few drops of acetic anhydrite was added, followed by addition of few drops of H 2 SO 4 , which was added slowly to the wall of the test tube. Formation of dark green colour designated the presence of steroids [9]. Plants steroids are derivatives of cyclization of the triterpene squalene [10] and [11]. Test for Saponins To determine the presence of saponin in the phytochemical components of M. citrifolia leaf. 70 ml of sterile distilled water was placed in a beaker containing 3 g of plant powdered leaf, mixed then boiled for 2 min. Mixture was filtered into a new test tube using cotton wool and funnel to produce an aqueous extract. 2 ml of the aqueous extract was discharged into a graduated test tube, and vigorously agitated. The formation of 1cm form that persists for 3 minutes designated the presence of saponins [12]. Test for Flavonoid Ammonium test was employed to test for the presence of Flavonoid in leaf extract of M. citrifolia, following the method as described by [13] and [14]. To achieve this, 0.2 g of M. citrifolia leaf powder was added to 10 ml of ethyl acetate in a 100 ml conical flask. Mixture was then heated for 5 min in a water bath, allowed to cool and filtered. 4 ml of the filtrate was discharged into a test tube where 1 ml of diluted ammonia solution was added to the mixture, agitated and kept at room temperature for a few seconds then observed. Appearance of layer of yellow coloration at the bottom of the test tube indicates the presence of flavonoid Test for Alkaloids Dragendroff reagent test: For this test, 0.2 g of M. citrifolia leaf powder was added to 20 ml of diluted H 2 SO 4 in methanol in a conical flask. Mixture was boiled for 5 minutes in a water bath, cool and filtered. Three drops of dragendroff reagent were added to the filtrate. Formation of creamy, orange solution indicates the presence of alkaloids [15]. RESULTS AND DISCUSSION The qualitative analysis of phytochemical components of M. citrifolia leaf extract using the conventional phytochemical screening assay (chemical tests) detected the presence of tannins, steroids, saponins, flavonoids, and alkaloids (Table 1 and Figure 1) which is in agreement with the findings of [16] and [17]. During the Braemer's test, the M. citrifolia leaf extract turned greenish grey which was an indication for the presence of Tannins in the solution according to the reports of [18] and [8]. Tannins belong to the class polyphenol compounds with an astringent property, soluble in acetone, alcohol, and water. Similarly, Lieberman test (Burchardt test) of the leaf extract solution turned dark green in colour ( Figure 2) which was an indication for the presence of steroids. This finding is in line with the report of [8]. The Frothing test also showed the formation of I cm form height above the mixture which persisted for more than 3 min. According to [6], the appearance of up to 1 cm form height above mixture that lasts for up to 2-3 minutes is an indication for the presence of saponins (Figure 3). Saponins are naturally produce by many plants for defense against pest and pathogens, they are easily converted to drugs, cosmetics and taste modifiers and are therefore considered economically viable compounds [19] and [20]. Result for the Ammonium test (test for flavonoids) showed layer of yellow coloration at the bottom of the test tub, which is an indication for the presence of flavonoid in the leaf extract, according to [21] and [14] ( Figure 4). Flavonoids is another member of the compounds class polyphenols which are known for their Pesticidal, antimicrobial, antioxidant, chemotherapy activities, and their mechanism of action against microorganisms includes; complex activities with the cell wall, cell lysis, membrane disruption, inactivation of enzymes and death [22]. Findings of the Dragendroff test (test for Alkaloids) shows formation of layer of creamy-orange solution ( Figure 5). This result is in agreement with the report of [15], that by this test, the formation of creamy-orange colouration of the test solutions suggests the presence of alkaloids in the test solution. Alkaloids have a toxic effect on microbials, in human medication and or as biopesticides [22].

v3-fos

2020-12-10T09:04:16.882Z

1970-01-01T00:00:00.000Z

237229927

s2

Aflatoxigenic Isolates of Aspergillus flavus from Pecans Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production. While investigating a bakery mold problem involving pecans, it was noted that a number of pecan halves (meats) being cultured for internal fungi yielded colonies of Aspergillus flavus Link ex Fries. Inasmuch as the presence of aflatoxin in foods may constitute a health hazard, a study was made to determine the frequency and toxigenic potential of these strains as well as the suitability of pecans as a substrate for aflatoxin production. MATERIALS AND METHODS The presence of A. flavus in pecans was determined by plating the seed on either 6% malt-salt agar (1) or rose bengal-streptomycin agar (RBM-2) after surface sterilization by immersion for 2 min in a solution of sodium hypochlorite (10 ml of bleach, 10 ml of 95% ethyl alcohol, and 80 ml of water). The RBM-2 medium was prepared as described by Tsao (9), except that the streptomycin sulfate was added at the rate of 0.06 g/liter. The plates were incubated at room temperature and examined every other day for 3 weeks. Colonies of A. flavus were isolated onto malt extract agar slants (7) for identification and toxin analysis. A total of 2,061 pecan pieces were plated out during this study; the source and nature of each sample are shown in Table 1. Samples 1-10 were taken from pecan lots used in the production of commercial bakery products; samples 11-16 were packaged pecans purchased in local supermarkets. Screening for aflatoxin. For screening of mold isolates, 50 ml of 2% yeast extract plus 20% sucrose [YES medium (3)] in a 250-ml Erlenmeyer flask was inoculated with 106 spores, incubated at room temperature for 7 days, and extracted with two 100-ml portions of chloroform (CHCl,) on a gyratory shaker. 1 The pooled extracts were filtered and evaporated to dryness on a flash evaporator; the residue was cooled and resuspended in 5 ml of CHC13. Visual estimates of aflatoxin content were made by comparing thinlayer chromatograms of appropriate dilutions of the extracts with aflatoxin standard obtained from the Southern Utilization Research and Development Laboratory, USDA, New Orleans, La. Thin-layer chromatograms (20 by 20 cm, 0.25 mm thickness of silica gel G-HR) were developed in acetone-chloroform (1:9, v/v) in an unequilibrated tank. Chloroform extracts were purified by column chromatography and precipitation with hexane in the first part of this study. However, thin-layer chromatography (TLC) results indicated that visual estimates could be made easily and accurately without further purification. Consequently, column chromatography and hexane precipitation were discontinued in the latter part of this study. For screening batches of pecans, pecans were extracted directly by the method of Pons et al. (6) and extracts were examined by TLC to determine the aflatoxin content. For screening individual nuts, moldy pecans were extracted by the method of Cucullu et al. (2) for determining aflatoxins in individual peanuts and peanut sections. Presumptive TLC results were confirmed by spectrophotometric analysis and chick embryo bioassay. Spectrophotometric determinations of aflatoxin were made by the method of Nabney and Nesbitt (5) on a Shimadzu model MPS-50L recording spectrophotometer. The method of Verrett et al. (10) was used for chick embryo bioassays. Further confirmation of aflatoxins was obtained by administering extracts per os to 1-day-old Peking ducklings and examining for bile duct cell proliferation (8). Extracts for duckling bioassays and spectrophotometric analyses were purified further by preparative TLC. RESULTS AND DISCUSSION The level of A. flavus in the bakery pecans (lots 1-10) ranged from 2 to 21 % (average 9%), whereas in market pecans the level ranged from 0 to 85% (Table 1). Of the 120 colonies isolated and identified, 105 (87.5%) were A. flavus and 15 (12.5%) were A. parasiticus Speare. Table 2 shows the results of screening mold isolates for aflatoxin production in YES medium. Presumptive TLC results showed that 29.1% of the isolates were negative; 13.4% produced aflatoxins B1, B2, G1, and G2, and 57.5 % produced only aflatoxins B1 and B2. Diener and Davis (4) reported that 90% of their toxigenic isolates of A. flavus from agricultural commodities produced primarily aflatoxin B. In this study, 80% of the toxigenic isolates produced only aflatoxins B1 and B2. Almost all the negative isolates and those producing only the B toxins were A. flavus; most of the isolates producing all four aflatoxins were A. parasiticus. Isolates were considered to be negative when aflatoxin was not detected in 3 ,uliters of undiluted chloroform extract. In all but one instance, when four toxins were produced in YES medium there was more aflatoxin G produced than B. The one exception was A. parasiticus ( # PC101) which produced more aflatoxin B than G. Table 3 gives the number of isolates producing aflatoxin within several arbitrarily chosen ranges. All isolates of A. parasiticus produced aflatoxin in amounts of 10 ;g/ml or more, whereas the majority of A. flavus isolates produced aflatoxin in lower ranges (less than 10 lg/ml). Though A. parasiticus isolates seem to produce more total aflatoxin (B plus G) than do A. flavus isolates, this does not necessarily indicate a greater potential hazard. As has already been pointed out in Table 2, most of the A. parasiticus isolates produce more aflatoxin G than B and, to date, aflatoxin B1 is regarded as the most carcinogenic of the aflatoxins. Spectrophotometric determinations on chloroform extracts of 20 randomly selected samples confirmed preliminary positive TLC results. Maximum absorption typical of aflatoxins was obtained at 363 nm from samples that were positive on TLC but not from those that were negative. Toxigenicity of randomly selected strains of A. flavus and A. parasiticus was further confirmed by bioassay. Culture extracts from eight strains of A. flavus that were negative by TLC were also negative by the chick embryo bioassay. Two of these cultuies were shown to be negative by duckling bioassay. Extracts of six cultures that were suspected to produce four aflatoxins by TLC screening were shown to be toxic to chick embryos. Toxigenicity of three of these cultures was further confirmed by duckling bioassay in which administration per os resulted in bile duct cell proliferation typical of aflatoxin. Extracts of 18 randomly selected cultures that produced only aflatoxins B, and B2 were also toxic to chick embryos. Confirmation of the toxigenicity of 5 of these 18 isolates was obtained by duckling bioassay. LILLARD, HANL Eighty-five isolates were found to be aflatoxigenic when grown on YES medium. Since many of these isolates were obtained from pecans used commercially in bakery products, the ability of randomly selected isolates to produce aflatoxin on cooked pecans was also determined. Nine isolates of A. flavus and four of A. parasiticus were. grown on 25 g of crushed, autoclaved pecans. The five isolates that were negative in YES medium were also negative on autoclaved pecans. The eight isolates showing positive TLC results after growth on YES medium also gave positive TCL results after growth on autoclaved pecans. A. flavus and A. parasiticus were isolated from moldy, raw pecans obtained at retail commercial outlets. Results of direct extraction for aflatoxin of 22 of these pecan samples (50 to 100 g each) were inconclusive. Strong presumptive evidence of aflatoxin in commercial pecans was obtained by extracting individual moldy raw pecans by the method of Cucullur et al. (2). Fluorescent compounds with RF values identical to standard aflatoxin were extracted from several individual sections of moldy pecans. However, due to the small amount extracted, confirmation by bioassay or ultraviolet spectroscopy was not possible. In view of the known carcinogenic properties of aflatoxin, the detection of these compounds in market pecans poses a potential health hazard to the consumer. Consideration should be given to the conditions under which pecans are stored and processed. Additional research is needed to determine the conditions which would minimize ,Ip N, AND LILLARD APPL. MICROBIOL. mold growth and the threat of aflatoxin contamination of pecans.

v3-fos

2020-03-26T10:40:14.343Z

1970-01-01T00:00:00.000Z

223147932

s2

MGNREGA Farm Pond Works – Perspectives of People and Policy India is ranked thirteenth among the list of water stressed countries in the world (Bansal, 2019). In recent years India has witnessed acute water scarcity across states, resulting in a growing recognition that lives and livelihoods of all are intrinsically linked to water, and measures on a war footing are required to ensure that our basic needs of water is met. The Jal Shakti Abhiyan (JSA), set up in 2019, has reported that over the past few years 3.5 lakh water conservation measures were implemented, of which 1.54 lakh were direct water conservation and rain water harvesting measures. The criticality of monsoons and rainwater harvesting has been championed by the Prime Minister, who in June 2019 had sent personalised letters to all village chiefs and had made a national call to take measures to conserve rainwater (IANS, 2019). INTRODUCTION India is ranked thirteenth among the list of water stressed countries in the world (Bansal, 2019). In recent years India has witnessed acute water scarcity across states, resulting in a growing recognition that lives and livelihoods of all are intrinsically linked to water, and measures on a war footing are required to ensure that our basic needs of water is met. The Jal Shakti Abhiyan (JSA), set up in 2019, has reported that over the past few years 3.5 lakh water conservation measures were implemented, of which 1.54 lakh were direct water conservation and rain water harvesting measures. The criticality of monsoons and rainwater harvesting has been championed by the Prime Minister, who in June 2019 had sent personalised letters to all village chiefs and had made a national call to take measures to conserve rainwater (IANS, 2019). Excavation of farm ponds, trenches and other earthen works are popular measures for water harvesting and conservation, and many of these receive monetary support from the government departments and ministries concerned. Under the JSA and the flagship public works programme of the country, the Mahatma Gandhi National Rural Employment Guarantee Act (MGNREGA), emphasis is given to farm pond works. 1 If we look at MGNREGA, since its inception, over 50% of its works are related to natural resource management, with a focus on rejuvenation of traditional water bodies and building new ponds and tanks on common lands. In addition to works on common/public property, the MGNREGA also have a provision to develop irrigation facility on private lands of eligible households, particularly those from the weaker socioeconomic groups (SC/ST and BPL), and marginal and small farmers (those owning less than 2 hectare of land respectively). This provision aimed at creating sustainable livelihoods for individual households. The data indicate that the share of water harvesting and irrigation structures on private lands to total works have risen in recent years. 2 The notes from the field discussed here are based on insights from an extensive study 3 undertaken by CWDS in 2018 on MGNREGA private land works across the states of Karnataka, Madhya Pradesh (MP) and West Bengal. In addition, the notes also draw comparison to similar works in the state of Uttarakhand studied in 2019. All observations are limited to farm pond works alone. POLICY PUSH ON FARM PONDS Since the inception of MGNREGA, farm ponds are an integral part of its water conservation related works. But a big push came into the programme ecosystem when a commitment was made by the Prime Minister in 2016 towards creating 500,000 farm ponds (Varma, 2016). Excavation and re-excavation of these water harvesting and storage structures were common works on private/individual lands in the initial 1 It is referred commonly as the MANREGA or the NAREGA. But in published documents, including that by the Ministry of Rural Development, Government of India, it is written as the MGNREGA 2 The share of these individual land works out of total works has increased over time from 18.6% (out of the total 104.6 lakh works) in 2012-13 to 31.7% (out of the total 122.1 lakh works) in 2016-17. 3 CWDS (2018) study had covered over 3000 MGNREGA private land works, broadly categorized as category B works. These works were completed using MGNREGA funds but their maintenance is the responsibility of the individual beneficiary household. Beneficiaries who received completed income generating category B works from 2013-14 to 2015-16 FY as per the MGNREGA MIS (management information system) were selected in the sample. years. But later re-excavation works were removed from the list of ‗eligible works on private lands' (owing to increasing number of ghost works and inability to identify re-excavations from new pond works) as is clear from directives issued by the Ministry of Rural Development (GOI, 2016). Our research observed that policy on farm pond works and specifications related to size and other parameters varied across study states with MP and Karnataka pushing for these works with a vision for water harvesting for agriculture and ground water recharge, while in West Bengal its focus was to facilitate fish farming for home consumption and livelihoods. Officials in-charge of MGNREGA implementation across states championed for farm ponds works on private lands as these were labour intensive (over 90% of work expenditure was spent on labour/wage payments); hence helped to maintain the mandatory 60:40 labour to material ratio. 4 For instance, in Karnataka cattle/goat sheds were popular works among the beneficiaries. A ‗package of works' offer, to be completed within a 36 month time period (depending on number of applications received and budget for the year), was made to beneficiaries. There were three different packages, and in a popular option beneficiaries were provided with land development works (Rs.10,000/acre), a cattle/goat shed (Rs.35,000/unit) and farm pond works (Rs.19,500/unit). This ‗package' was policy strategy to ensure targets 5 for farm ponds and other ground water recharging works would also be achieved without much difficulty. However, policy changes were common and took place at frequent intervals. For instance, in the block of Rajgarh in MP, farm pond construction (of a certain minimum dimension) was made as a supplementary work with dug wells for all new sanctions from 2017-18. This meant that a potential beneficiary had to possess sizable land area to ensure a farm pond was also constructed along with a dug well (with funding support from MGNREGA), which in effect meant the beneficiary had to earmark about one-fifth of a hectare for the ‗well-farm pond combination' work. Officials stated that this condition was added to ensure that at least some, if not all, of the ground water drawn from the well would be replenished through the farm pond (through rainwater harvesting and further ground water recharging). In addition, combination works also helped to ensure farm ponds were built and targets were achieved (GOI, 2016), though this was never made in public statements. Rather officials maintained these works were critical to ecology. A personal communication from an official with regard to why farm ponds were made an accompaniment work with dug wells is given below: Wells are like bank ATMs while ponds are equivalent to saving bank (SB) accounts. If you have no money in the SB account, then the ATM will not dispense any cash. Similarly, you should not be allowed to dig wells and draw upon ground water if you do not make farm ponds alongside. As only then you are contributing to recharging the ground water which you are drawing up from the well. PERCEPTION OF PEOPLE AND FARM PONDS Across the states, the initial years saw resistance towards farm pond works with only a few households applying for it. The general perception was that ‗precious land would be lost', and the loss was not commensurate with the gains from improved crop yields as people felt there would hardly be any increase in cash incomes. The only exception was the case of those who were keen to venture into commercial fish farming, as was the case of some beneficiaries in blocks of West Bengal and Uttarakhand. While it was possible to undertake commercial fish farming even if the farm ponds retained water only for 8-9 months a year, beneficiaries hoped to farm fish round the year. Many of the beneficiaries in 24 Parganas South and Purba Medinipur district of West Bengal had farmed fish previously for home consumption, but with the new ponds they were able to grow fish for longer periods or about 9 months and earned higher incomes through fish sales. Most of the beneficiaries here abided with natural availability of water and stopped fish farming from March/April until monsoons. But beneficiaries of farm ponds in Dehradun and Haridwar districts of Uttarakhand, who incidentally were venturing into pisciculture for the first time, were keen to ensure availability of water round the year, for which they dug borewells alongside ponds. Installing bore wells and pumping up water to fill farm ponds was ecologically adverse for overall water economy of the area but no onevillagers, officials or elected representatives -took note of this paradox. In West Bengal, many households with farm ponds were raising fish for household consumption, while some were also earning regular income from sale of fish. Farm pond works were at times jointly executed for 3-4 beneficiaries in cases where individual land holdings were tiny 6 in size and hence land pooling by beneficiaries who owned adjacent plots helped to excavate larger ponds. This improved the scope for commercial fish farming. Beneficiaries in Laksmijanardhanpur (Pathar Pratima block of 24 Parganas South), T Majee and M Samal, who received farm ponds in 2015-16 were farming fish and earning Rs.500-1000/month. But J Bunia, a beneficiary who had ventured into betel leaf farming after completion of farm pond in 2013-14 had experienced manifold increase in income with the household earning close to Rs.1,00,000 from sale of betel leaves (0.08 ha) in 2017. In all cases, beneficiaries also reported that the ponds ensured plenty of fish for their home consumption, a staple in Bengali diet. In Karnataka, across study districts of Ramanagara and Tumkur farm ponds were individually provided, as unlike West Bengal, here average household land ownership was sizable. One aspect that was clear from discussions with officials and people was that the resistance towards excavation of farm ponds during initial years, had significantly reduced and many households were applying for these works as they were able to ‗witness' its benefit. In some of the villages in Ramanagara, particularly, there were many ‗new' applications as villagers saw how sericulture incomes of their neighbours had risen owing to these works. In Choodahalli GP, Balaramu who had received a farm pond in 2015-16 was able to harvest 425 kg of cocoon compared to about 375-390 kg previously. In I Gollahalli, another beneficiary, Kulliah who had received a farm pond in 2015-16, had benefitted from higher mulberry leaf production from his 1 acre plantation, which in turn led to increased cocoon production of 290kg (earlier the average was not above 250kg). Dairy farmer Sannegowda, also from Choodahalli, had always bought water spending close to Rs.10, 000 during the summer season for raising napier grass for his cows. But construction of a farm pond on his land in 2015 had helped to harvest rainwater and store it to tide over the summer months. After the farm pond was built on his land, he had stopped purchasing water. CONCLUSION The positive impact of farm ponds is substantial and for many there was decipherable improvement in crop yields improving cash incomes or household food consumption or both. In case of beneficiaries who were only practicing subsistence agriculture, households were able to grow crops over longer duration, and reduce market dependence for grains and vegetables. But it was also observed that in many GPs across the study states, there were beneficiaries whose farm ponds had shrunk in size and depth, owing to carelessness with regard to regular maintenance (periodic cleaning activities, including removal of accumulated silt). These beneficiaries hoped that some support would be extended towards maintaining the ponds to ensure sustainability over time.

v3-fos

2014-10-01T00:00:00.000Z

1970-05-01T00:00:00.000Z

15514105

s2

A study of sieve element starch using sequential enzymatic digestion and electron microscopy. The fine structure of plastids and their starch deposits in differentiating sieve elements was studied in bean (Phaseolus vulgaris L.). Ultrastructural cytochemistry employing two carbohydrases specific for different linkages was then used to compare the chemical nature of "sieve tube starch" (the starch deposited in sieve elements) with that of the ordinary starch of other cell types. Hypocotyl tissue from seedlings was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon-Araldite. Treatment of thin sections on uncoated copper grids with α-amylase or diastase at pH 6.8 to cleave α-(1 → 4) bonds resulted in digestion of ordinary starch grains but not sieve element grains, as determined by electron microscopy. Since α-(1 → 6) branch points in amylopectin-type starches make the adjacent α-(1 → 4) linkages somewhat resistant to hydrolysis by α-amylase, other sections mounted on bare copper or gold grids were treated with pullulanase (a bacterial α-[1 → 6] glucosidase) prior to digestion with diastase. Pullulanase did not digest sieve element starch, but rendered the starch digestible subsequently by α-amylase. Diastase followed by pullulanase did not result in digestion. The results provide evidence that sieve element starch is composed of highly branched molecules with numerous α-(1 → 6) linkages. INTRODUCTION The plastids of higher plant sieve elements are characterized by several distinctive types of inclusions . Among these, the starch deposits are the most widely distributed . It has been known for many years that these deposits differ from ordinary starch of other cell types in their reaction to the iodine stain (cf. 11) . Other inclusions peculiar to the plastids of sieve elements are the crystalline proteinaceous bodies found in monocots (5) and the ring-shaped bundles of proteinaceous filaments apparently confined to certain families of dicots (6,14,15) . The starch in sieve elements (known classically as "sieve tube starch") is distinguished primarily by its reddish color response to the iodine stain, in contrast to the blue-black reaction of the ordinary starch found in the amyloplasts of parenchyma cells and other cell types (11) . In this respect it resembles certain other polysaccharide deposits (12), including the waxy starch found in the endosperm of some members of the grass family, the floridean starch of red algae, and the glycogen of animal tissues . Ordinary starch contains two fractions, amylose and amylopectin . Amylose consists of a-(1 -> 4) linked glucose residues and only a few a-(1 -p 6) branches (21) . Amylopectin is much more extensively branched, since it contains numerous a-(1 -> 6) bonds that provide branch points. Waxy starch consists almost exclusively of amylopectin (37), and floridean starch and glyco-gen are quite similar in composition (28,30) . There is reason to suspect, therefore, that sieve element starch may also be related structurally to amylopectin . Although conjectures as to its nature have been made in the past by students of phloem tissue (12,36), its composition has remained obscure . The starch grains of sieve elements cannot be isolated for study in vitro, since the elements are embedded among other cells that also contain amyloplasts . However, recent advances in carbohydrate enzymology have made it appear more attractive to attempt to explore the structure of starch and other plant polysaccharides in position within the plant cell . In planning the present work, it appeared promising to try to use a-amylase to digest starch in thin sections, since this enzyme cleaves a-(1 -+ 4) bonds and converts starch into breakdown products of various lengths . It also appeared likely that pullulanase, a recently discovered a-(I --s 6) glucosidase (1,7), could be used in a similar way on thin sections . Since pullulanase is now being used in starch research to cleave the a-(1 --s 6) linkages of amylopectin (2), it seemed feasible to attempt to use it sequentially with a-amylase to compare the digestibility of starch grains in sieve elements with that in other cells . MATERIALS AND METHODS Seeds of Phaseolus rulgaris L . cultivars Kentucky Wonder and Dwarf Horticulture (Olds Seed Co ., Madison, Wis.) were germinated for 3 to 7 days in wet sand or vermiculite following an overnight presoaking . Segments about 1 mm long were cut from the seedling hypocotyl under 3% glutaraldehyde in 0 .05 M potassium phosphate buffer at pH 6 .8 and fixed for approximately 2 hr . The segments were then rinsed in several changes of 0 .05 M phosphate buffer and postfixed in 2 % OsO4 in the same buffer for 2 hr . This was followed by dehydration through an acetone series and propylene oxide, then embedment in a resin mixture consisting of Araldite, Epon, and dodecenyl succinic anhydride (DDSA) in a ratio of 3 :3 :8 by volume . Silver sections were cut on a Porter-Blum MT-2 ultra-microtome (Ivan Sorvall Inc., Norwalk, Conn .), mounted on uncoated copper grids, and poststained with uranyl acetate and lead citrate before viewing in a Hitachi HU-l IA electron microscope operating at 75 kv with a 30 µ objective aperture. The content of thin sections was monitored with the light microscope by observing sections cut at a thickness of 0 .5 u and stained with toluidine blue O . Polysaccharide was demonstrated with the periodic 384 THE JOURNAL OF CELL BIOLOGY • VOLUME 45,1970 acid-Schiff (PAS) reaction using plastic sections 0 .5-1 .0 s thick . Starch was identified with an 1 2KI solution (19), using both fresh material and tissue fixed in glutaraldehyde. The fixed tissue was frozen and sectioned at 16 µ in a model CTD cryostat (International Equipment Company, Needham Heights, Mass.) operating at -12 to -15°C . Protein was demonstrated in plastic sections 1 u thick using mercuric bromophenol blue. Silver sections mounted on uncoated copper, gold, or nickel grids were digested with bacterial a-amylase (Type IIA, Sigma Chemical Co ., St . Louis, Mo .) or malt diastase (type VA, Sigma Chemical Co.) at 37°C for 1 to 48 hr at concentrations varying from 0 .1 to 1 .07 in 0 .02 M phosphate buffer at pH 6 .8. Diastase and a-amylase were used interchangeably, as no differences in effect were observed between them . Attempts to employ ß-amylase on thin sections were unsuccessful, as described in the Results . Following digestion, grids were washed in a stream of distilled water and poststained in the usual manner . The pullulanase, a gift from Dr . K . L . Smiley of the USDA Northern Regional Research Laboratory, Peoria, Ill ., was a lyophilized preparation from Aerobacter aerogenes, 30 mg of which were capable of hydrolyzing the pullulan in 100-200 ml of a 1 % solution in 1-2 hr . Copper, nickel, or gold grids bearing silver sections of material prepared in the usual manner were immersed in a 0 .5-3 .0% solution of this preparation in buffer at 37°C for 3-24 hr . Buffers used included 0.02 M phosphate at pH 6 .8 and 0 .05 M citrate at pH 5.4 . Following digestion, grids were washed in a stream of distilled water and either poststained or subjected to further digestion by a-amylase or diastase for 1-24 hr. For enzyme exposure of short duration (1-6 hr), digestion was carried out in covered depression slides . For longer exposures (6-48 hr), grids were incubated in the center well of covered micro-Conway diffusion vessels containing a few drops of toluene in the outer well . A number of controls were run with different enzyme and buffer exposures to test alternative explanations for the observed results . Leaf material of timothy (Phleum pratense) was also examined in order to compare the histochemical properties and enzyme digestibility of the proteinaceous crystals in the plastids of monocot sieve elements with the same properties of starch grains in bean sieve elements . Changes in Amyloplast Fine Structure During Ontogeny of the Sieve Element The proplastids seen in the earliest recognizable sieve elements are spherical to ovoid and relatively small ( Fig . 1) . They are bounded by an envelope, the inner membrane of which bears a few cisternal invaginations . Their stroma is moderately electron opaque and contains clearer regions in which a fibrillar material, possible DNA, is sometimes seen . Plastid ribosomes are also present in the stroma . No starch deposits are present at this stage of plastid development . Changes in the plastids can be correlated readily with other events taking place during differentiation of the cytoplasm and walls of the sieve element . In the early stages of differentiation, the plastids enlarge and develop into irregular, elongate, and branching forms whose stroma contains one or more small starch grains (Fig . 2) . These grains enlarge rapidly as differentiation of the sieve element continues . During their growth they are surrounded by a thin shell of finely granular material located in the adjacent matrix of the plastid (Figs . 3 and 4) . Presumably this material is related to active synthesis and deposition taking place at the grain surface . As the grains approach mature size, the plastids containing them become roughly spherical (Fig . 4) . Degenerative changes in the plastids accompany the breakdown of other components of the cytoplasm in later stages of sieve element differentiation . The most obvious changes include the loss of granular material from the stroma, and the development of irregularities in the outline of the plastid envelope and in the spacing of its membranes (Figs. 5 and 6) . Concomitantly the starch grains become fissured and coarsely granular (Fig. 6) . Also frequently observed in the plastids are small clumps of granules that resemble the peripheral portions of the larger masses in appearance . These granules and granular clumps are similar in appearance to the glycogen inclusions observed in liver and muscle cells . The smallest granules are approximately 200 A in diameter, and thus are similar in size to the subunits of the glycogen (3-particles observed by Wanson and Drochmans (39) . In old sieve elements with perforated plates, the envelopes of many of the plastids have ruptured, and the starch bodies which were formerly in the plastids now lie free in the lumen (Fig . 7) . Whether release of these bodies from the plastids occurs naturally during aging of the sieve elements or results from cutting and fixing the tissue has not been established (9,10,12,24) . Fully developed starch grains in sieve element plastids (Figs. [3][4][5] commonly differ in appearance in several respects from those in parenchyma cell plastids (Fig . 8) . The sieve element starch grains are generally smaller and more nearly spherical, and exhibit radial rather than concentric striations . Also, as has been reported previously (13,24), their cores frequently appear electron lucent (Figs. 3 and 4) . Otherwise the sieve element grains appear highly electron opaque (Figs . 2-7), whereas ordinary grains are more variable in this respect . Poststaining with the lead salt contributes most importantly to the electron opacity of the grains, as can be demonstrated by omitting the poststaining entirely (Fig. 9), and by comparing the effects of staining with lead and uranium singly and in combination . Finally, the stroma of the sieve element plastids remains closely associated with the starch grains (Figs . 2-5), and does not become separated by an electron-lucent region like that seen between stroma and starch grains in parenchyma cell plastids fixed in glutaraldehyde ( Fig. 8) . Preliminary Histochemical Investigation of Sieve Element Starch Preliminary attempts to identify the type of starch in sieve element plastids were made with standard light microscopic cytochemical techniques . From the results summarized in Table I it is clear that in P. vulgaris the deposits in the plastids correspond to classical "sieve tube starch ." When the PAS test was applied to sections of plastic-embedded material cut at a thickness of 0 .5 u, the grains in the plastids gave a positive (red) response indicative of polysaccharide . In fresh and frozen sections treated with I2KI solution, the grains in sieve elements were stained reddish brown, while those of plastids in the cortical parenchyma were stained bluish black . Grains in the vascular parenchyma stained reddish brown and thus showed a greater affinity in this test to the starch of sieve elements than to that in the similar parenchymatous type of cell located in the cortex. Cuneate, crystalline inclusions are typical of the sieve element plastids of many monocots (5) . When tests for the presence of protein were made on these bodies, using mercuric bromophenol blue applied to plastic sections of Phleum pratense, a strongly positive staining reaction was obtained, in agreement with the previous results of Behnke (4) . Starch grains observed in Phleum guard cells and in Phaseolus parenchyma cells and sieve elements gave negative results in the same test . Plastid showing degenerative changes in a sieve element in a late stage of differentiation . The stroma of the plastid is relatively sparse and the starch grain shows incipient granulation . X 52,000 . Sieve element starch and ordinary starch were differentially digested from thin sections by appropriate treatment with a-amylase (or diastase) and pullulanase preparations (Figs. [10][11][12][13][14][15][16][17] . These enzymes acted in a highly selective manner without affecting other fine structural components . As the starch in a grain was digested, the grain first lost much of its electron opacity and developed a network of small holes (Figs. I 1 and 14) . After more complete digestion, the holes coalesced and the entire starch deposit was replaced by a single large hole (Figs. 10, 15, and 17) . Precautions were taken to minimize the effect of the electron beam of the microscope on enlargement and coalescence of the holes by reducing the exposure of the sections to the beam as much as possible during examination . The use of a-amylase and pullulanase readily distinguished between sieve element starch and the ordinary starch stored in plastids of other cell types. In experiments on tissue representing nu- [10][11][12] . Large starch grains in the numerous plastids of parenchyma cells were completely digested by treatment with 1 % a-amylase for 1 hr (Fig . 10) . Also, it was observed that 170 diastase for 1 hr removed the electron opacity of starch grains in the plastids of phloem parenchyma cells and destroyed the central portions of these grains, but did not affect noticeably the grains in adjacent sieve elements (Fig. 11) . Even when the exposure to 1% a-amylase was lengthened to 24 hr (Fig . 12) or to 48 hr, starch deposits in sieve elements remained unaffected . A 3 % solution of pullulanase alone was also without apparent effect on sieve element starch grains. This is shown in Fig. 13 for material mounted on copper grids and exposed to pullulanase for 24 hr . However, if the section was treated first with pullulanase and then with 1 % a-amylase or diastase, the sieve element starch was digested . hr followed by a-amylase for 1 hr is shown in Fig . 14 : the deposit appears electron lucent and contains numerous holes . After a more complete digestion (pullulanase for 24 hr followed by aamylase for 24 hr), little or none of the sieve element starch grains remained (Fig . 15) . Exposure to 31'/6 pullulanase for only 6 hr was inadequate, however, since subsequent treatment with amylase or diastase produced no digestion . These results indicated that treatment with pullulanase renders the sieve element starch susceptible to subsequent a-amylolysis . This conclusion was reinforced by the results obtained when the order of exposure to the two enzymes was reversed in some of the above experiments . When sections were treated with a-amylase followed by pullulanase, no digestion of the sieve element starch was observed (Fig . 16) . were required for starch grain digestion than with copper at pH 6 .8. For example, using gold grids, 0.5% pullulanase at pH 5 .4 for 3 hr followed by 0.1 to 1 .0% diastase at pH 6 .8 for 2 hr resulted in rather complete grain digestion (Fig. 17) . At these respective concentrations, however, neither pullulanase alone for 3 hr nor diastase alone for 5 hr resulted in digestion . In treatments of thin sections, the enzyme preparations proved to be highly specific for starch, as they did not noticeably digest other cellular struc-394 THE JOURNAL OF CELL BIOLOGY • VOLUME 45,1970 tures even when the treatment was continued for as long as 48 hr (Fig . 15) . Bounding membranes, organelles, ribosomes, and microtubules were not visibly altered . It is particularly noteworthy that the cell walls comprising a variety of polysaccharide components showed no digestion . This includes the developing sieve plate, whose prominent middle lamella and thickenings of callose remained intact (Figs . 11 and 15) . Proteinaceous deposits, Rinsing the sections following incubation in the preparations of a-amylase, diastase, or pullulanase was sufficient to free them of contaminating deposits (Figs . [10][11][12][13][14][15][16] . This was not true for the sections treated by ,Q-amylase, however, as these always showed a coating of obscuring material when examined subsequently in the electron microscope . Since the contaminant was not removed by centrifuging and filtering the enzyme preparation before use or by washing the grids more thoroughly, the trials with f3-amylase were discontinued . be hydrolyzed by a-amylase, which cleaves a-(1 -+ 4) bonds at random, resulting in an increase in reducing power and in formation of oligosaccharides of shorter and shorter chain lengths . It can also be degraded by 3-amylase, which cleaves a-(1 -+ 4) linkages in a sequential fashion from the nonreducing ends, yielding maltose . Amylopectin, on the other hand, is a more extensively branched glucose polymer . Like amylose, it possesses a-(1 -> 4) bonds that can be hydrolyzed by a-and ß-amylase, but it has in addition numerous a-(1 -+ 6) linkages that provide branch points and reduce the extent of hydrolysis by a-and ß-amylase (Fig . 16) . The a-(1 -> 6) bonds can be split by R enzyme, an enzyme commonly used in starch research, and by pullulanase, a recently discovered a-(l --+ 6) glucosidase . Pullulanase is an inducible extracellular enzyme produced by certain bacteria, including Aerobacter aerogenes . For activity the enzyme specifically requires the presence of at least one a-(l -+ 4) glucose bond on each side of the a-(I -6) branch points (1) (Fig . 18) . Largely on the basis of the iodine reaction, it was suggested some years ago that sieve element starch may be similar to an amylodextrin or dextrin (cf. 12) . However, these substances are known only as hydrolytic degradation products of starch, and should not be confused with intermediates or end products in starch biosynthesis (18) . It seems more likely that the reddish response to the iodine stain given by the starch of sieve elements is indicative of a branched, amylopectin type of molecule . Other starches that stain reddish with iodine are known to be of this type, including the starch in the endosperm of waxy maize, which consists entirely of amylopectin, and glycogen and floridean starch, both of which are quite similar to amylopectin . Furthermore, the iodine reaction has been studied for many years, and a correlation has been made between the observed color reactions and the molecular structure of the starch (16,20) . As the proportion of long straight chains of amylose molecules increases, more iodine molecules are bound inside these helically oriented glucose chains and the color response tends toward blue or black ; on the other hand, as the chain length decreases or the ratio of branched-chain amylopectin components in the structure increases, the proportion of chain units in a helical configuration is reduced, resulting in less bound iodine and shifting the response toward red or reddish brown . (28,29,40 (33) have emphasized that in order to show relative differences in the susceptibility of chemical bonds to digestion, it is necessary to control the conditions carefully so as to achieve a low level of hydrolysis . In It seems likely that interference from osmium has not proved to be a problem in our study because appreciable amounts of osmium are not bound by the starch grains . Most of the electron opacity of the grains results not from osmium fixation but from poststaining with lead . It also appears likely that the starch grains are digested readily because very little plastic penetrates into their compact structure during embedment. This view is supported by numerous micrographs indicating that digestion starts first in the center of the grain and that it proceeds faster in large grains than in small ones . The latter fact is interpretable if the reasonable assumption is made that the degree of infiltration increases with decreasing size of the grain . Since glycogen is composed of relatively small granules it may not be readily digestible for the same reason. Since amylose and amylopectin may be synthesized by separate metabolic pathways (3,27), it can be assumed that variations in the composition of starch grains depend on the relative amounts or activities of the pertinent enzyme systems in the plastids . The synthesis of amylose is probably governed by the starch synthetase system utilizing UDPG and ADPG (22), while the synthesis and degree of branching of amylopectin appear to depend on the balance between the biosynthetic and degradative activities of phosphorylase and the branching activity of the Q enzyme . Presumably both synthetic pathways are operative in the plastids of parenchyma cells, but only the branching system is functional in plastids of the sieve element. A parallel example is afforded by the waxy maize mutant . The red-staining starch of waxy maize is not found in all tissues, but is limited to the endosperm, pollen, and embryo sac, while normal blue-staining starch is found in the embryo, seed coat, and other tissues (8) . It appears that the amylopectin starch grains of the waxy maize endosperm have little or none of the starch synthetase system (17,25,26), although this system is present in the embryo and seed coat (26) . There is some evidence that there may not be an abrupt transition from synthesis of one type of starch in the sieve elements to another in other cell types, since the starch in the vascular parenchyma cells appears to be intermediate between ordinary starch and sieve element starch in its reactions . It stains red-brown rather than blueblack with iodine as was noted by McGivern (24) and confirmed in our study . It is also more slowly digested by a-amylase than is ordinary starch although since the grains of ordinary starch in cortical parenchyma cells are usually larger, a comparison of the relative rates of hydrolysis in the two tissue regions must be treated with caution . Whether the indicated structural difference of sieve element starch from ordinary starch is important in the metabolism of the developing sieve element or in solute transport in the phloem is unclear at the present time .

v3-fos

2019-06-01T13:16:59.284Z

1970-01-01T00:00:00.000Z

171906675

s2

Effect of Farm Yard Manure and Planting Densities on Growth, Yield and Quality of Okra under Natural Farming Among rapidly consumed vegetables, okra is one popular green crop in Pakistan grown during spring summer season. As the plant life cycle falls in short duration crops, proper plant nutrition for high yielding varieties influence greatly on the production and quality traits of okra. To regulate high quality fruiting and sufficient picking adequate minerals are essential for potential fruit yield required by okra plant. Trend of intense cultivation of agricultural crops resulted in the degradation of fertile land. Recently awareness of organic farming and sustainable agriculture is taking charge over intensive farming systems around the globe. Organic manure is one of the best choices to full fill the needs of plant essential mineral nutrition for enhanced quality and crop yielding, along with properties of improving soil structure, texture and moisture holding capacity (Maheswarappa et al., 1999). Although the amount of micro minerals is reasonable in organic manures as compared to inorganic fertilizers, existence of growth promoting components like hormones and enzymes besides mineral nutrition of plants makes them essential for improvement of soil fertility and productivity (Bhuma, 2001). It is well known fact that farm yard manure (FYM) definitely improves the soil to sustain the nutrient status and water holding capacity. INTRODUCTION Among rapidly consumed vegetables, okra is one popular green crop in Pakistan grown during spring summer season. As the plant life cycle falls in short duration crops, proper plant nutrition for high yielding varieties influence greatly on the production and quality traits of okra. To regulate high quality fruiting and sufficient picking adequate minerals are essential for potential fruit yield required by okra plant. Trend of intense cultivation of agricultural crops resulted in the degradation of fertile land. Recently awareness of organic farming and sustainable agriculture is taking charge over intensive farming systems around the globe. Organic manure is one of the best choices to full fill the needs of plant essential mineral nutrition for enhanced quality and crop yielding, along with properties of improving soil structure, texture and moisture holding capacity (Maheswarappa et al., 1999). Although the amount of micro minerals is reasonable in organic manures as compared to inorganic fertilizers, existence of growth promoting components like hormones and enzymes besides mineral nutrition of plants makes them essential for improvement of soil fertility and productivity (Bhuma, 2001). It is well known fact that farm yard manure (FYM) definitely improves the soil to sustain the nutrient status and water holding capacity. The significance of organic manuring in sustainable agriculture is acknowledged well. Gaur et al., 1972 andSubbarao et al., 2001 reported that the application of different organic manures showed a significant increase in plant height and number of fruits plant -1 of chilli (Dileep, 2005). Use of manure from animal and plant origin is most common practice for sustainable and organic farming around the ABSTRACT A field study was conducted to investigate the effect of farm yard manure and planting densities (P x P 10, 15, 20 and 25 cm and R x R 60 cm) under natural farming condition at National Agricultural Research Centre, Islamabad during 2016. The land was incorporated with well rotten farm yard manure (FYM) @ 25 t ha -1 in respective plots (4 x 8 meter) one week before bed preparation and mixed thoroughly in the soil. Seeds of okra (Abelmoschus esculentus L., var. Pusa Green) were sown according to the plan following split plot design with four replications. The crop was allowed to stand till maturity and data on growth traits like plant height, number of pods plant -1 and fresh pod yield (tender young pods) were recorded. The quality characters like crude fiber and moisture percentage were estimated. The mean data were analyzed statistically. Though all the FYM application treatments showed positive effect through growth and yield characters, however, among plant densities, 10 and 15 cm P x P and FYM incorporation @ 25 t ha -1 produced comparable fresh pod yield (11.22 and 10.97 t ha -1 , respectively) which was 25 and 24 % higher than that of without FYM application. Dense populated crop i.e., 10 and 15 cm P x P performed better than rest of planting densities through improved fresh pod yield with comparatively improved quality. Dense populated treatment (P x P 10 cm) and FYM application @ 25 t ha -1 produced better quality fruits with less crude fiber content (9.89 %) and higher moisture content (79.14 %) as compared to without FYM application (10. There is increasing demand for okra because of its importance for delicious cooking in farming systems in many regions. It is a good source of vitamins A, B, and C and iron, calcium, magnesium, phosphorus, and zinc (Asian Vegetable Research Development Council, 1991). The edible part of okra represents 90% of the total weight of fruit with an energy value of 31 kcal per 100 g (De Lannoy, 2001). Keeping in mind these facts, a field experiment was planned to assess the effects of farm yard manure and plant density on yield and quality of okra to determine the optimum population for maximum fruit yield production with better quality attributes of okra var. Pusa green under natural farming environment. MATERIALS AND METHOD A Field study was carried out to investigate the effect of farm yard manure (FYM) and planting densities (P x P = 10, 15, 20 and 25 cm and R x R = 60 cm) under natural farming condition during dry season 2016 at National Agricultural Research Centre, Islamabad. The land was incorporated with well rotten farm yard manure (FYM) @ 25 t ha -1 in respective plots (4 x 8 meter) one week before bed preparation and mixed thoroughly in the soil. The experiment was laid out according to split plot design with four replications. Four seeds of okra (Abelmoschus esculentus L., var. Pusa Green) were directly sown per hole at a depth of 2 cm. After germination, seedlings were thinned to one plant per stand three weeks after planting. Soil samples from (0-30 cm) were collected from 12 different spots in the study area and were composited, air-dried and sieved through a 5 mm sieve and their physical and chemical characteristics were determined before application of treatment (Table 1). The samples were analyzed for soil textural class by hydrometer method (Bouyoucos, 1962). Calcium carbonate was estimated by acid neutralization method and soil organic matter by oxidation with potassium dichromate in sulfuric acid medium under standardized conditions by Walkley and Black procedure (Nelson and Sommers, 1982). Soil pH was determined in water (soil water ratio 1:1). Electrical conductivity (ECe) of the soil suspension was measured using conductivity meter. The P, K and Zn were determined by using AB-DTPA method (Ryan et al., 2001). The crop was allowed to stand till maturity and data on growth characters like plant height, number of fruits plant -1 and yield (tender young pods) were recorded. The quality characters like crude fiber and moisture percentage were estimated. The data thus collected were subjected to statistical analysis and treatment differences were compared by using LSD (Gomez and Gomez, 1984). RESULTS AND DISCUSSION All the treatments of FYM application @ 25 t ha -1 along with dens plantation performed significantly in terms of plant height, pods plant -1, pod yield (t ha -1 ), crude fiber contents (%) and moisture contents (%) over non FYM treatments and less dense okra population. Data in Tables 2, 3 and 4 registered statistically significant increase in okra plant growth, yield and quality among all treatments receiving FYM over control treatments (without FYM) and dense population. Results listed in Table 2 Results mentioned in Table 3 indicate that FYM applied treatments produced significantly higher fresh pod yield (11.22 t ha -1 and 10.97 t ha -1 ) of okra plated with 10 and 15 cm planting distances, respectively against 10.97 t ha -1 and 8.81 t ha -1 under same planting densities without FYM application. Comparatively less fresh pod yield under no FYM application might be due to lack of nutrients resulting in reduced growth and yield. The positive effect of FYM on plant growth and yield could be due to the contribution made by manure to fertility status of the soil as the soils were low in organic matter (Table 1). Manure when decomposed increases both macro and micro nutrients as well as enhances the physico-chemical properties of the soil. This could have led to its high vegetative growth. The significant difference observed in the treatments supplied with FYM as compared to control treatment (without FYM) could be due to the presence of nutrients in the soil which satisfied plants requirements for their health growth. Ajari et al., (2003) reported that in okra production organic manure could increase plant height of crops when compared with other sources of manures or having no manures. Similar findings had also been reported by Premsekhar and Rajashree (2009). Furthermore, higher and statically significant values of fresh pod length (9.98 cm) and fresh pod weight (45.92 g) of okra under normal plant population (20 × 20 cm) were recorded. On an average, FYM @ 25 t ha -1 application also showed elevated pod length (9.52 cm) and weight of fresh pod (44.65 g pod -1 ), respectively as compared to non FYM treated plots. An opposite trend was observed egarding quality of okra pod fruits in terms of minimum crude fiber (8.87 %) and higher moisture (78.90 %) contents harvested from FYM treated (25 t ha -1 ) under dense populated okra plants (Table 4), as okra pods are found to be the best for cooking and consumption with lesser crude fiber contents. Previous findings of Dileep (2005), Gopalakrishnan (2007) and Tiamiyu et al., (2003) are in line with our results who reported that FYM increased yield and quality of vegetables providing healthy environment and good soil structure and texture. CONCLUSION Current experimentation has revealed that farm yard manure (FYM) application @ 25 t ha -1 actively enhanced vegetative growth, increased yield and improved pod quality traits of okra. In addition, dense plantation (P × P 10 cm) improved fresh pod yield per unit area of okra vegetable.For attaining a higher yield and maximum quality of okra FYM @ 25 t ha -1 application and plantation at higher density (P × P 10 cm) could be a very effective protocol for organic and sustainable farming under natural farming.

v3-fos

2020-12-10T09:04:11.587Z

1970-11-01T00:00:00.000Z

237230314

s2

Sugar Substrates for l-Lysine Fermentation by Ustilago maydis The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; KLa in the fermentors, 0.41 mmoles of O2 per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. Five auxotrophic mutants of Ustilago maydis, obtained by a combination of ultraviolet and ethyleneimine treatment, were previously studied (8) on more than 60 media with agave juice (aguamiel), corn-steep liquor, corn oil, and ammonium salts as the main constituents. Yields as high as 2.5 g per liter were reached in 300-liter fermentors by using mutant UV-ET-15. Since this is a high yield for this particular species, it was decided to study three other inexpensive substrates: commerical sucrose, sugar cane clarified juice ("clarified juice," International Society of Sugar Cane Technologists terminology), and blackstrap molasses and to continue working on the development of new high-yielding mutants. Among these, homoserine-, methionine-, and threonine-requiring mutants were found capable of producing acceptable amounts of L-lysine. The present report deals with studies carried out to gain maximum yields of free L-lysine by using such mutants. However, the yields here presented are far below any value that would warrant production of this amino acid by U. maydis on an industrial scale. Bacteria will probably continue to be the selected microorganisms for this purpose at the present time, but the possibility exists that by improving the yields of U. maydis through induced genetic changes in the strains, by selection of fermentation media, and by adequate bioengineering operations fungi may be used for the commercial production of L-lysine. MATERIALS AND METHODS Cultures. Mutants of U. maydis were kept in potato-dextrose-agar slants or on sterilized corn, and monthly transfers were made. Inoculum was prepared from 4-day cultures at 28 C as follows. A loopful was transferred to tubes containing 10 ml of a sucrose medium, composed of (per 100 ml) sucrose, 0.25 g; peptone, 1.0 g; NaCl, 0.5 g. After 38 hr of cultivation in a rotary shaker, 5% inoculum was used to seed Dulaney's medium (3) in Erlenmeyer flasks. After 48 to 72 hr of agitation at 250 rev/min and 28 C, sufficient seed was taken to inoculate the fermentation media at a level of 5%. Media. To compare the activity of the strains, Dulaney's glucose medium (3), B glucose medium (8), and agave juice media [26-a, 48, and 19 (8) ] were used. Theeffect of ammonia nitrogen and naturally occurring nitrogen sources on L-lysine formation by mutant UV-ET-15 was studied in Dulaney's glucose medium with 1% corn oil added. Fermentations were carried out in this medium with the following carbon sources: medium S-l (commercial sucrose as received); S-2 (invertase-treated sucrose); M-l (blackstrap molasses as received); M-2 (blackstrap molasses treated with 10 mg per liter of K4Fe (CN)6 at 80 C during 6 hr, centrifuged, and filtered); M-3 (ferrocyanide-treated molasses, hydrolyzed with invertase); J-l (clarified juice, as received); J-2 (clarified juice treated with ferrocyanide as indicated before), and J-3 (clarified juice, ferrocyanide-treated and invertase-treated). Commercial invertase (0.1%) was used for the treatment of these sugar substrates at 50 C for 48 hr. When CaCOs was used, it was sterilized separately and then incorporated in the medium. Media were autoclaved at 115 C for 20 min, cooled, and then inoculated with 5% seed. Conditions. The inoculated Erlenmeyer flasks were mechanically agitated for 120 hr at 250 rev/min in a 687 New Brunswick rotary shaker. The incubation temperature was 30 C; pH was adjusted at 5.8 to 6.2 in all fermentation media. The same pH and temperature conditions were established for the 7.5-, 20-, and 100liter fermentors. The 7.5-liter fermentors corresponded to model F-7 of New Brunswick Scientific Co., equipped with pH, temperature, and foam automatic controls, one disc sparger, two turbine impellers, and four baffles. The 20-liter fermentor was a French E.I.V.S.-672 glass model with three flat-blade impellers, one disc sparger, and no baffles. The 100-liter fermentor was included in a steel pilot plant designed by Olsa from Milan, Italy; this fermentor has one turbine impeller, a ring sparger, and four baffles. Aeration, agitation, and other operational conditions are indicated in the respective tables. Concentrate. A lysine concentrate was obtained by evaporation and spray-drying of the fermentation broth from the 100-liter fermentor, with medium J-3 as a substrate. The material was then passed through an electrical mill, and the brown powder obtained was submitted to chemical analysis. Determinations. Reducing sugar, pH, viscosity, oxygen-transfer rate, and mycelial weight were determined as indicated previously (8). The microbiological standard method with Pediococcus cerevisiae P-60, occasionally checked against paper chromatographic techniques, was used in the estimation of Llysine. For the chemical determinations of the amino acid concentrate, previous hydrolysis with 6 N HCl for 22 hr at 100 C was made, and then AOAC analytical methods were followed (1). The amino acid pattem was detected by means of a Beckman model B automatic analyzer. For the digestibility tests of the powdered concentrate, defatting was performed in a Goldfish apparatus; the AOAC method (1) was followed by using a buffer boric acid solution in place of the conventional acid solution to receive the distilled ammonia. Table 1 shows the L-lysine yields in five media by the IFSC 65-1 parent strain of Ustilago maydis and the four mutants obtained from it. The UV-ET-15 homoserine-requiring mutant had been previously studied (8) in the agave juice media 26-a, 48, and 19. Results indicate that this mutant gave the highest yields in most of the media tested, and it was therefore selected for all subsequent experiments. RESULTS The effect of three ammonium and two naturally occurring nitrogen sources on L-lysine formation by this particular strain on Dulaney's glucose medium with 1% corn oil is shown in Table 2. The three ammonium salts at their respective optimal concentrations gave almost identical results, and their lysine yields were higher than those obtained with corn steep liquor or yeast extract. Yeast extract appears to affect both growth and lysine synthesis, whereas corn steep liquor and yeast extract apparently are used for growth only. Some other naturally occurring nitrogen sources such as gelatin, peptone, corn meal, cotton seed meal, and soya flour did not consistently improve yields. The addition of some probable precursors such as aminoadipic and ketoadipic acids did not influence results in any way. When the effect of some commercial carbon sources was studied, the addition of corn steep liquor seemed favorable for good and reproducible lysine production. Results in shaker-flask experiments shown in Table 3 refer to medium 26-a inoculated with the UV-ET-15 mutant. Clarified juice appeared to be a better substrate than sucrose or blackstrap molasses. A slight increase in lysine yields or a shortening of the fermentation time were apparent when these substrates were previously hydrolyzed by means of commercial invertase, especially in the case of clarified juice. Growth and sugar consumption were affected in a similar manner. The best yields were approximately 2 g of lysine per liter. Ferrocyanide treatment was of no particular benefit. Fermentations carried out in 7.5-liter fermentors by using five selected commercial sugar media usually gave inconsistent results when no corn steep liquor was added to the media or when the aeration and agitation conditions were not properly adjusted. The yields given in Table 4 show the slight variations observed with the best operating conditions. An increase in yield to 2.81 g of lysine per liter in medium J-3 (clarified and inverted juice) was reached at the end of 72 to 120 hr at 30 C, 500-rev/min agitation, and 0.3-volume per volume per min aeration. To secure consistent results, it was estimated that a Kd X 102 value of 12 gram-molecules of 02 per liter per hr was apparently adequate for good lysine production in medium J-3 by the mutant under study ( Table 5). The mean viscosity and density values of these fermentation broths were lower than those of the agave juice media (8). Further experiments suggest that a KLa value of 0.41 mole of 02 per liter per min would permit more consistent results. Some metabolic data of L-lysine formation in a 20-liter fermentor operated at a KLa value of 0.41 mmole of 02 per liter per min are shown in Table 6 and Fig. 1. The highest yield, 2.58 g of lysine per liter, was reached at the end of 72 hr coincident with rapid mycelial growth and low sugar consumption. The pH decrease paralleled carbohydrate utilization. Similar results were obtained in 7.5-liter fermentors. The lysine yields, by using mutant UV-ET-15 in the J-3 medium, are higher than those reported in the scientific literature and in some patents (Table 7) and are similar to those reached in agave juice media (8). Finally, one of the fermentation broths obtained from a 100-liter fermentor with medium J-3 was evaporated and spray-dried; the powder obtained showed the chemical composition given in Table 8. As can be seen, the protein content is acceptable and the digestibility is good. Some of the main amino acids present in the concentrate are shown in Table 9. DISCUSSION Mutant UV-ET-15 of Ustilago maydis is a fair lysine producer. Yields of approximately 2.5 g per The ammonium salts studied (phosphate, sulfate, and acetate) are equally good nitrogen sources for lysine production in this medium, depending on the concentration used. Contrary to observations of Kurtz and Ericson (5), ammonium acetate slightly increased the lysine yields as pointed out by other investigators (7,8). The efficient utilization of the acetate would suggest a lysine biosynthetic pathway via aminoadipic acid (10). However, the addition of this acid or ketoadipic acid to the culture medium in concentrations as high as 10 mg/ml did not improve lysine production. We have no valid explanation for this fact. The addition of corn steep liquor has a slight effect upon metabolite production but, according to previous observations (6,8) and to our data ( Table 2), this seems to be an indirect result of growth. Clarified juice could be utilized as a cheap carbon source for lysine production, because its local price is below that of cane sugar. The unsuitability of blackstrap molasses has been reported by Tauro et al. (9). However, our mutant grows well in this substrate and produces more lysine than the strain studied by these workers. Hydrolysis of cane molasses does not improve yields but can reduce the fermentation time from 120 to 72 hr since glucose is better assimilated than sucrose (Table 2). By using the fermentation conditions recommended by several workers (4-7), unreproducible results, as they themselves have observed, were attained when commerical carbon sources were incorporated in the media. However, when the oxygen transfer was properly controlled, the variations in yield were not as wide as they appear from the results shown in Tables 4 to 6, which were obtained in fermentors of different sizes. Kd values around 0.12 gram-molecule of 02 per liter per hr or KLa of 0.41 mmole of 02 per liter per min allowed high and relatively constant results. Details of this study will be published elsewhere. The maximum lysine value is reached between 48 and 72 hr during the initial growth period when the pH is around 5.3 and the sugar consumption is low (Table 6; Fig. 1). At this time, sugar is apparently used for rapid mycelial growth and lysine reaches its highest value and remains constant, as does growth, until a period of 144 hr is reached. Afterwards, yields of lysine increase slightly as a probable result of mycelium lysis. Maximal lysine synthesis occurs when 11 to 20% sugar is consumed. Therefore, pH and carbohydrate utilization, as well as growth and lysine synthesis, seem to be related as it was observed in the case of agave juice media (8). Since preliminary experiments showed that the addition of specific minerals to the media (MgSO4, MnCl2, NaCl, FeSO4, ZnSO4, and CaCl2), independently or together, did not affect lysine production even at a concentration of 0.2 g per liter, they were not added to the fermentation media. Probably the small amounts present in corn steep liquor are sufficient. The chemical composition and the amino acid pattern of the lysine concentrate (Table 8,9) suggest its utilization as a feed supplement. The vitamin B content of this concentrate has been previously reported (6,8).

v3-fos

2014-10-01T00:00:00.000Z

1970-07-01T00:00:00.000Z

5374614

s2

Studies on the posterior silk gland of the silkworm Bombix mori. IV. Ultracentrifugal analyses of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm. Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.716, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 105, with a tendency to lie fairly close to 3.7 x 105. INTRODUCTION It is well known that the silk glands of the silkworm, tinctorial properties of the silk proteins in the Bombyx mori, synthesize at least two kinds of gland lumina (3, 4), and no extensive physicoprotein, fibroin and sericin ; the former is synthe-chemical analysis has been carried out on native sized in the posterior division of the silk gland silk proteins extracted from the silk glands . In (conventionally called posterior silk gland as used this report, the sedimentation properties of the here) and is transferred to the middle division of native silk proteins which were extracted directly the same gland or middle silk gland to be stored from various parts of the middle silk gland were there as a fibroin core, while the latter is secreted analyzed and compared. This study shows not in the middle silk gland and builds up three only how many kinds of protein exist in the gelatinous sericin layers round the fibroin core lumen of the middle silk gland but also suggests (1, 2) . This kind of conclusion, however, is based the site of biosynthesis for each protein . exclusively on microscopic observation of the Physicochemical properties, such as ultra-centrifugal heterogeneity, sedimentation, and diffusion coefficients, molecular weight, etc ., of the fibroin have been the subject of a number of investigators (2, 5-11) . Quite inconsistent and controversial results, however, have been reported . For example, the molecular weights for fibroin hitherto reported scatter from 33,000 to the order of 1,000,000 (2, 10) . The fibroin solutions used for those studies usually were prepared from cocoons or raw silk by rather violent procedures such as boiling in alkaline soap solution (1, 2) or dissolution in alkaline cupriethylene diamine II hydroxide solution (5) . It is possible, therefore, that the fibroin suffered from more or less extensive molecular degradation during these procedures as discussed in detail by Lucas et al . (2) . It appears desirable, therefore, to prepare native fibroin directly from the silk glands and to examine its physicochemical properties. We report here results of such experiments . Silkworms The strains of the silkworms used for the extraction of silk proteins and the seasons of rearing are the following : Nichi 124 x Shi 124 (spring), Shungyoku x Gunpo (spring), Nichi 122 x Shi 124 (summer), and Nichi 115 x Shi 124 (summer) . Such factors as strains and seasons of rearing do not seem to have serious effect on the physicochemical properties of the silk proteins . The fifth instar larvae were cultivated as described previously (12) and, when they arrived at full maturation, they were sacrificed for extraction of silk proteins . Extraction of Native Silk Proteins from Various Parts of the Middle Silk Gland Native silk proteins were extracted according to Shimizu et al . (1) . The middle silk glands of both sides were dissected out and washed briefly in glassdistilled water . Immediately they were cut into five parts as shown in Fig. 1, and each part was put into a small Petri dish containing a small amount of distilled water. After several minutes in distilled water the luminar silk protein columns were softened and partially extruded out of the glandular lumina, and the swollen glandular tissues became easily separable by fine forceps from the silk protein columns . These Petri dishes with silk protein columns were then shaken very gently overnight in the cold (2-3°C) . Vigorous shaking should be avoided strictly because it induced extensive surface denaturation and gelation of the silk proteins. The water extract was then col- lected by decantation, and a small amount of distilled water was added again to the dishes and the second extraction continued for several hours more . These procedures gave almost complete extraction of the silk proteins. The combined extracts were centrifuged for 60 min at 105,000 g, and the clear supernatant was collected by decantation and stored in the cold (2-3°C) until just before use. Extraction of Native Fibroin Only the most posterior part of the middle silk gland or part 5 of Fig . 1 was used to prepare native fibroin . The silk protein columns were prepared as described previously, and they were washed gently by distilled water several times . This washing serves to extract a very small amount of sericin which sticks to the surface of the fibroin column . The subsequent procedures are similar to those in the previous case . Extracts from the anterior and middle portions of the middle silk gland (parts 1-3) became turbid within 1 or 2 days, even in the cold, and then gradually separated into two layers : an upper turbid, and a lower transparent layer . In contrast to this, extracts from the posterior portion of the middle silk gland (parts 4, 5) and native fibroin solution are both much more stable in the cold and remain as clear solutions for as long as 2-3 wk . It is noted, however, that native fibroin solution became increasingly slightly opalescent on standing in the cold, suggesting gradual formation of aggregates, and it gelled suddenly 2-4 wk after preparation. These solutions should be treated very gently, because fibroin gels quite easily ; gelation is induced by gentle shaking, stirring, or even by pipetting. Sedimentation Analyses by the Schlieren Optical System Sedimentation analyses by the schlieren optical system were carried out at X20°C and at a rotor speed of 59,780 rpm with a Spinco Model E analytical centrifuge (Beckman Instruments, Inc ., Palo Alto, Calif.) . Either 0.1 M carbonate-bicarbonate buffer (CB buffer), pH 9.0, alone or the same buffer containing 0 .1 M NaCl (SCB buffer) was used as the solvent . Native fibroin gelled rapidly in pH 7 .0 0 .1 M phosphate buffer. Schlieren photographic plates were read on a Nikon comparator type 6CT (Nippon Kogaku K.K., Tokyo) . Conversion to a standard state (S40,,,) was accomplished by the following equation (1) according to Svedberg and Petersen (13) . The standard sedimentation coefficient at an infinite dilution (So2o,w) was estimated by extrapolation to zero concentration . Sedimentation Analyses by the Interference Optical System In order to examine in more detail the sedimentation properties of the native fibroin solution, sedimentation analyses were also carried out by the interference optical system . The latter optical system makes it possible to detect even a small amount of aggregates, if present, and to analyze more dilute solution than does the schlieren optical system . Interference photographic plates were read on the same Nikon comparator . Equilibrium Ultracentrifugation The meniscus depletion method of Yphantis (14) was used exclusively for determination of the molecular weight of fibroin. The equilibrium centrifugation was carried out either in the cold (2-5°C) or at X20°C for about 24 hr at a rotor speed of 9,945 rpm. Fibroin sometimes gelled during the run, especially when very dilute solution of fibroin was analyzed at 20°C, and was spun down to the bottom of the cell. Although fibroin is unstable in warm alkaline buffer as shown later, degradation of fibroin was not observable under the present experimental condi-tions except for some broadening of the sedimenting boundary . Apparent weight-average molecular weights were calculated according to equation ( (1 -Vp)w 2 d(r2) r where_R is the gas constant, T is the absolute temperature, V is the partial specific volume of the protein, p is the density of the solvent, w is the angular ve- (3) where Inj, 2 • • • lnj,-2 are five data points spaced at equal increments 1ßr . No fringe displacements less than 100,u were used. Determination of Partial Specific Volume (V) The aqueous solution of fibroin was dialyzed overnight against cold 0.01 M CB buffer, pH 9 .0, and then the density of fibroin solution at various concentration (0-14 mg/ml) was determined in this buffer by a pycnometer at a constant temperature of 20°C, and f was calculated . A trial to obtain V in distilled water was not successful, because the fibroin gelled during dialysis against cold distilled water. Determination of Chemical Composition and Concentration of the Fibroin Solution Fibroin solutions were dried by lyophilization and lipids were extracted and determined as described previously (12) . RNA was extracted by Schneider's procedures (15), and the amount of RNA was determined by orcinol reaction (16) . Determination of the lipids was kindly carried out by Dr. K . Saito, Department of Biochemistry of the Kansai Medical School . Since it was found that the fibroin solutions contain neither lipids nor RNA as described later, concentrations of fibroin solutions were determined by measurement of dry weight . An aliquot of a fibroin solution was treated with 5% (final) trichloroacetic acid (TCA), washed three times with 5% TCA, dehydrated by alcohol and by alcohol--ether (3 :1), and finally the fibroin was washed with ether . The fibroin was then dried at 110°C for at least 5 hr before weighing . Electrophoretic Analyses In order to examine the electrophoretic homogeneity of the fibroin, electrophoretic analyses were Y. TASHIRO AND E . OTsUât PostertiQr ffl Gland of B. mori . IV carried out by a Hitachi HTB-2A electrophoretic apparatus . The fibroin solutions were dialyzed against cold 0 .1 M Veronal-Veronal acetate buffer of pH 8 .6 containing 1 .0 M urea and were analyzed in the same solution. Addition of urea seems essential for successful electrophoretic analyses of the fibroin solution ; in the absence of urea, strange spikes or anomalies appeared frequently on the electrophoretic patterns . Probably gelation of fibroin during electrophoretic analyses is responsible for these anomalies . Ultracentrifugal Analyses of Native Silk Proteins Extracted from Various Parts of the Middle Silk Gland Fig . 2 a-e shows the sedimentation patterns of the native silk proteins that were observed at a concentration of -5 mg/ml extracted from parts 1-5 of the middle silk gland, respectively . It is apparent that Fig . 2 e shows only a single peak, which will be called component 1 . For comparison, the sedimentation pattern of the native fibroin run at the same concentration of 5 mg/ml is shown in (Fig. 2 a) . This component has S°20 ,,,, = 4 .4S as calculated byextrapolation of two series of sedimentation runs . 4 THE JOURNAL OF CELL BIOLOGY . VOLUME 46,1970 It is to be noted here that the present experimental conditions are far from ideal for components 1-3 ; several ultracentrifugal anomalies were observed, such as a marked concentration dependence of the sedimentation coefficients, hypersharpening of the boundaries, and the Johnston-Ogston effect, etc. Fig, 3 h . This effect is so marked in this system that the area of each component, even after correction for radial dilution, is not even qualitatively proportional to the concentration of each component . For example, although there is apparently almost as much component 3 as component 1 in Fig . 3 g, the sedimentation pattern of the same sample after a twofold dilution (Fig . 3 h) clearly shows that much more component 1 does exist in this extract than component 3 . In order to interpret these sedimentation patterns reasonably, therefore, it is essential to first correct for these ultracentrifugal anomalies . After correcting these effects by analyzing sedimentation patterns of serially diluted silk proteins, we suggest that component I in Fig . 2 a most probably corresponds to fibroin because the S 020,, value of this component was about 10S either in 0 .1 M CB or SCB buffer, pH 9 .0, and this component constitutes approximately 70% of the total silk proteins in the extract from the part 1 . In order to confirm this conclusion, a small amount of native fibroin was added to the extract from part 1 and the mixture was analyzed by ultracentrifugation . Comparison of Fig. 4 a with Fig . 4 b clearly shows that the amount of component 1 increased markedly after the addition of pure fibroin . In this experiment, the addition of a large amount of native fibroin caused an apparent increase of both components 1 and 3 . This is certainly owing to the Johnston-Ogston effect . The S°H,,,, value of component 2 was found to be 9-10S . As to the existence of component 2, there are two possibilities : one is that it is an independ-ent component, and the other is that it is identical, extract from part 4 but also from parts 3, 2, and 1 . at least ultracentrifugally, with component 3 . If Our experiments have failed to show such a peak the former possibility is correct, a peak of com-and, in this sense, the former possibility is not ponent 2 should be detected not only in the supported experimentally. It is possible, however, FIGURE 2 Sedimentation patterns of the silk proteins extracted from part 1 (2 a), part $ (2 b), part 3 (2 c), part 4 (2 d) and part 5 (2 e) of the middle silk gland, respectively. The concentration of silk proteins for Fig . 2 a, c, d, and e was 5 mg/ml, while that for Fig . 2 b was 6 mg/ml. For the purpose of comparison, native fibroin was also analyzed at a concentration of 5 mg/ml (Fig. 2 f) . Solvent : CB buffer, pH 9 .0 . All the sedimentation velocity analyses by the Schlieren optical system in this manuscript were carried out at a temperature of 20°C and at a speed of 59,780 rpm . Fig. 2 e -f were taken 51 min, and all the other pictures 57 min, after attaining full speed, respectively. FIGURE 3 Sedimentation patterns of the serially diluted silk proteins extracted from part 1 (3 a -f) and from part 3 (3 g-h), respectively. Initial concentrations of silk proteins in Fig . 3 a -f are 10, 7 .5, 5.0, 2 .5, 1 .25, and 0 .625 mg/ml, respectively, while those of silk proteins in Fig . 3 g-h are 5 .0 and 2 .5 mg/ml, respectively . Solvent : SCB buffer, pH 9 .0 . All the pictures were taken 56 min after attaining speed. FIGURE 4 a-b Sedimentation patterns of the silk proteins extracted from part 1 before (Fig . 4 a) and after (Fig. 4 b) addition of a small amount of fibroin. 4 a, silk protein extract from part 1 (5 mg/ml) . 4 b, a mixture of silk protein extract from part 1 (2.5 mg/ml) and fibroin (1 .25 mg/ml) . Solvent : SCA buffer, pH 9 .0 . Picture was taken 64 min after attaining speed . that component 2 does exist but was not detected simply because it was so small in amount, and furthermore its S value was so close to that of component 3, that the peak of component 2 overlapped with and, therefore, was concealed by component 3 . At the present time, therefore, there is no decisive evidence to support either of the two possibilities, and this manuscript was written assuming that the former possibility is correct . As a matter of course, this assumption should be confirmed by future experiment . From the Sa20 , value of component 3 (approximately 9S), it seems certain that component 3 of Fig . 2 a corresponds to component 3 of Fig . 2 b and c. Component 3 in the extract from part 2 is larger in amount than that in the extract from part 3 and, furthermore, the amount of component 3 in part 1 is approximately equal to that in part 2 . This suggests that component 3 is secreted mainly in the middle portion of the middle silk gland (part 2 + part 3) . Finally, since component 4 is observed only in the extract from part 1, it is certain that component 4 is secreted only in the anterior portion of the middle silk gland . We conclude, therefore, that components 2-4 are the other silk proteins, sericins, and they are called sericins 1-3, respectively . It is further suggested that sericins 1-3 are probably secreted in 6 THE JOURNAL OF CELL BIOLOGY . VOLUME 46, 1970 the posterior, middle, and anterior portion of the middle silk gland, respectively . The above conclusion is also supported by the differential water extraction of silk proteins from part 1 ; that is, silk protein columns prepared from part 1 were extracted with water for several hours by gentle shaking in the cold and the first extract was collected as described above . A small amount of water was added to the residues and a second extraction was continued overnight, until complete dissolution of the remaining silk proteins . Both extracts were analyzed by ultracentrifugation as shown in Fig. 4 c and d, respectively . The first extract (Fig. 4 c) clearly contains more component 4 and 3 than component 1, while the second extract (Fig . 4 d) contains quite a large amount of component 1 . This finding is consistent with the above conclusion because sericin is known to be more water soluble than fibroin . As the extracts from parts 1-3 gel rapidly even in the cold, the physicochemical properties of the sericins have not been studied further . Sedimentation Analyses of the Native Fibroin Solution by the Schlieren Optics Since it has been confirmed that the water extract from part 5 of the middle silk gland contains exclusively fibroin, a number of silkworms were sacrificed so as to prepare a large amount of fibroin by the methods described previously . The sedimentation analyses by the schlieren optical system showed only a single 10S peak, and usually neither any slower component nor any aggregate was observed under the present experimental conditions as illustrated in Fig. 5 . In the early stage of the present experiments, the lOS peak of the fibroin showed frequently s o skewness towards a smaller radius, suggesting the presence of smaller sedimenting species, either from heterogeneity or from dissociation . Such a skewness, however, disappeared as the method of preparation of the fibroin has been improved ; as described in Materials and Methods, the fibroin was prepared only from the most posterior part of the middle silk glands, and the silk protein columns were washed well with distilled water. Probably the fibroin solutions prepared in the early stage had been contaminated with a small amount of sericin 1 . Sedimentation analyses of much more dilute fibroin solution down to 0 .016% have shown essentially similar patterns, except for broadening of the peak (Fig. 6) . In order to calculate the S°2 of this fibroin, I /S20 ,,, was plotted against the concentration of fibroin as shown in Fig . 7 . These plots give a straight line and, in the three series presented here, extrapolation gave the average value of 9.9S for S°20 , x . Sedimentation Analyses of the Native Fibroin Solution by the Interference Optics (1), assuming that the equation is valid even in such concentrated urea and guanidine hydrochloride solutions, was 6 .1 and 12 .2S, respectively . Data published by Kawahara and Tanford (17) were used for the viscosity and density corrections in these solutions . Electrophoretic Analysis of the Native Fibroin The electrophoretic patterns of the native fibroin in 0 .1 M pH 8 .6 Veronal-Veronal acetate buffer containing 1 M urea show only a single peak as illustrated in Fig. 13 . The electrophoretic mobility of the fibroin calculated from ascending (Fig . 13 a) and descending boundaries (Fig . 13 b) was the same, -2 .8 X 10-5 cm2 per sec per volt at 5 .5 0C . Chemical Composition and Partial Specific Volume (V) of the Native Fibroin Solution The native fibroin solution contains neither detectable amounts of lipids nor RNA ; lipids and RNA content were less than 0.1 % . The amount of Y . TASsIRO AND E . OTSUKI Posterior Silk Gland of B . mori. IV FIGURE 13 Electrophoretic patterns of 0 .95% native fibroin in cold (5.5°C) 0.1 M Veronal-Veronal acetate buffer, pH 8 .6, containing 1 .0 as urea . The pictures were taken 130 min after the start of the run in a field of 6.7 v/cm . The arrows in the ascending (Fig . 13 a) and descending patterns (Fig . 13 b) show the direction of migration of fibroin from 8 and e boundaries, respectively. 10 THE JOURNAL OF CELL BIOLOGY . VOLUME 46,1970 protein in the native fibroin solution, therefore, could be determined reliably by the dry weight measurement as described previously . The partial specific volume of the fibroin at 20°C was 0 .71 6 in 0 .01 M CB buffer of pH 9 .0 . This value was used for the calculation of molecular weight of fibroin as described in the following . No correction was applied for the possible effect of temperature . It was further assumed that the V value in, 0 .1 M SCB buffer of pH 9 .0 is equal to that in 0 .01 M CB buffer of pH 9 .0 . Molecular Weight Determination by an Equilibrium Centrifugation The graph of the logarithm of net fringe displacement against square of the radius does not give a straight line, but rather a line curved upwards towards the bottom of the cell . This nonlinearity was more marked in the cold than at 20°C. M,,,(r), calculated according to equations (2) and (3), was plotted as a function of concentration of fibroin as shown in Fig . 14 Fig . 14 that the sedimentation equilibrium experiments yield the type of molecular weight vs . concentration relationships characteristic of a heterogeneous, partially reversible, aggregating system . The average molecular weight estimated by extrapolation to zero concentration of each series of the experiments is 3 .7 f 0.5 X 10 5 , which corresponds most probably to the molecular weight of the LOS component . Similar relationships were observed in the cold as shown in Fig . 15 . In this case, however, concentration dependence of M,o (r) is so marked in some series that it is difficult to estimate by extrapolation to zero concentration the molecular weight of the smallest species present . It is quite apparent, however, that in such samples there exist considerable amounts of molecular species smaller than the 10S main component, i .e ., smaller than 3 .7 X 10 5 in molecular weight, which are presumably in chemical equilibrium with the loS component . This problem will be discussed further in detail at the end of this paper . The sedimentation equilibrium experiments reported in Figs. 14 and 15 were carried out at rather high initial concentration of 0 .25-1 .5 mg/ ml . In order to calculate the molecular weights of the complete sample (M) by linear extrapolation to the base of column as shown by Yphantis (14), it is necessary to perform the equilibrium experiment at a lower concentration . A sedimentation equilibrium experiment run at an initial concentration of 0.01 % was not successful because fibroin gelled during the equilibrium run and was spun Y. TASnixo Sedimentation patterns of native fibroin (6 mg/ml) kept in cold CB buffer, pH 9 .0, for various periods . Fig. 16 a-b, 72 hr (16 a) and 96 hr (16 b) after dilution to 6 mg/ml . Fig. 16 c-d, kept in cold CB buffer at a concentration of 18 mg/ml for 96 hr (16 c) and 240 hr (16 d), respectively, and then diluted to 6 mg/ml just before the sedimentation analysis . The pictures shown were taken 56 min (16 a-b), 58 min (16 c), and 59 min (16 d) after attaining speed, respectively . FIGURE 17 Sedimentation patterns of native fibroin (2 .5 mg/ml) kept in cold SCB buffer, pH 9-11. Fig. 17 a-b, immediately after addition of SCB buffer, pH 9 .0 and 11 .0, respectively. Fig. 17 c-d, 18 hr after addition of SCB buffer, pH 10 .0 and 11 .0, respectively . Fig. 17 e-f, 10 days after addition of SCB buffer, pH 10.0 and 11 .0, respectively . The exposures presented were taken 64 min (17 a, b, e, f) and 66 min (17 c-d) after attaining speed, respectively . FIGURE 18 Sedimentation patterns of native fibroin (2 .5 mg/ml) kept at 25°C in SCB buffer, pH 10 and 11 . Fig . 18 a-b, 5 hr after addition of SCB buffer, pH 10 (18 a), and pH 11 (18 b), respectively. Fig . 18 c-d, 25 hr after addition of SCB buffer, pH 10 (18 c), and pH 11 (18 d), respectively. Fig. 18 e -f, 49 hr after addition of SCB buffer, pH 10 (18 e), and pH 11 (18 f), respectively . The patterns shown here were taken 58 min (18 a-b), 57 min (18 c-d) and 56 min (18 e-f) after attaining speed, respectively . In one series of experiments, shown in Fig . 16 a-d, an aliquot of native fibroin was diluted to 6 mg/ml and 18 mg/ml in the CB buffer, pH 9 .0, respectively, and these solutions were kept in the cold (-3°C) . The degradations of both solutions were followed by ultracentrifugal analyses at the same final concentration of 6 mg/ml . At 72 hr after dilution to 6 mg/ml, hardly any degradation of native fibroin was observed (Fig . 16 a) . At 96 hr after dilution, however, the solution became slightly turbid (gelation completed next day) and a slow heterogeneous component appeared in the sedimentation pattern (Fig . 16 b) . The more concentrated solution of fibroin (18 mg/ml) seems to be more stable because hardly any degradation was observable at 96 hr ( shown that there exist three kind of sericins which cover the fibroin core concentrically : the inner, middle, and outer sericin layers . Kikkawa (18) and Oba (19) have confirmed this finding and have further suggested that the sericins in the inner, middle, and outer layers are secreted exclusively in the posterior, middle, and anterior portion of the middle silk gland, respectively . The present sedimentation analyses have shown that there exist three sericins in the extracts from the middle silk gland ; sericins 1-3, though the existence of sericin 1 is still questionable . Further, it was suggested that sericins 1-3 are probably secreted from the anterior, middle, and posterior portion of the middle silk gland, respectively. This estimation is consistent with the above morphological suggestion . There have been also a number of lines of biochemical evidence which suggest heterogeneity of sericin molecules . For example, Shelton and Johnson (20) fractionated sericin into two parts by fractional precipitation with ammonium sulfate . Later, Mosher (21) and Bryant (22) concluded that there are three distinct sericins (A-C), of which sericin C is in the innermost layer, in intimate contact with the fibroin core . It is tempting to suppose that the sericins 1-3 correspond to sericin C, B, and A, respectively. Of course, since different methods of preparation of the sericins were used in these studies, this correspondence should be tested by future experiment . Sedimentation analyses of fibroin were reported first by Holmes and Smith (6) who examined a solution of fibroin prepared with cupriethylenediamine hydroxide solution by Coleman and Howitt's technique (5) . They obtained evidence for only one component whose mean sedimentation coefficient was 2 .6S. A similar value of 2.6S for the sedimentation coefficient of the main fraction in a solution of fibroin prepared by a similar technique was also obtained by Hayashi and Oda (8) . Mercer (7) has shown the existence of 1 .8, 1 .5, and 1 .OS components in alkaline extract (1% NaHCO3) of the posterior silk gland . The present experiments, however, have clearly shown that the main component of the native fibroin is an ultracentrifugally homogeneous molecule whose standard sedimentation coefficient at infinite dilution (S0nn ,,,,) is IOS . The fibroin solutions also contain considerable amounts of heavier aggregates probably formed by reversible and/or irreversible aggregation of the 10S component . The low values of the sedimentation coefficient of fibroin reported by previous authors (6-8) are probably due to the degradation of fibroin molecules during preparation procedures, as will be discussed later. It is interesting to examine whether the 10S component of the fibroin is a monomeric form of fibroin or is in a polymeric form bound by forces 1 4 THE JOURNAL OF CELL BIOLOGY . VoLumE 46, 1970 other than covalent bond. As described previously, extensive dilution of the solution, addition of monovalent salts such as LiBr and NaCl, or treatment with EDTA, urea, and guanidine hydrochloride has failed to dissociate the 10S component into smaller species . Therefore, we conclude tentatively that the IOS component is the monomeric species of the native fibroin molecules . The molecular weight of fibroin has been determined by various methods. The data hitherto reported scatter over quite a wide range : 33,000 (5) by osmotic pressure measurement ; 60,000 (6) by sedimentation-viscosity measurement; 250,000-450,000 (average 300,000) (10), 290,000 (11), and 1,000,000 (7) by light-scattering measurement ; 278,000 (23) by the low-angle x-ray-scattering method ; 84,000 (24) and 200,000-300,000 (25) by amino acid analysis. The fibroin solutions used for the previous studies were prepared by rather violent procedures, and it is probable that the fibroins may have suffered more or less extensive degradation during these procedures . This estimation is supported by our experiments which clearly showed the instability of native fibroin in warm alkaline buffer ; even at pH 9 .0, fibroin was degraded rapidly if the temperature was kept at 37°C . In this experiment, native fibroin was extracted directly from the middle silk gland by very gentle procedures as described previously . The molecular weight of the monomeric species present, which most probably corresponds to the 10S component, was found to be between 3 .2 and 4.2 X 10 5 , with a tendency to lie fairly close to 3 .7 X 10 1. This value is at the upper limit of the previous values of the fibroin which was prepared from raw silk or from cocoons without using cupriethylenediamine hydroxide (10, 11, 23,25) . From these results, it is suggested that silk fibroin is secreted as a macromolecule -3 .7 X 10 5 in molecular weight and is probably incorporated into silk fiber without a marked change in molecular weight . The present experiments have shown that native fibroin is unstable in warm alkaline buffer, and several signs of degradation appeared in the sedimentation patterns of the native fibroin solution when treated with warm alkaline buffer : (a) broadening of the 10S boundary (Fig. 16 d) ; (b) decrease in the area of the 10S boundary in proportion to the aggregation of the 10S component ( Fig. 18 e-f, Fig . 19 d) ; (c) slowing or decrease in the sedimentation velocity of the 10S compo-nent in the more alkaline solution (compare Fig . 17 a with 17 b, and Fig. 18 a with 18 b) ; and (d) appearance of a slower component as shown in Figs . 16 b, 17 e-f. The third sign, or the slowing of the J OS component, is probably due to the unfolding of the fibroin molecules in the warm alkaline buffer . The fourth sign, or the appearance of a slow component, is quite interesting and could be interpreted in two ways . One is that this phenomenon represents simply the conformational change of the native fibroin molecules, as suggested in the previous case . The other possibility is that the native fibroin molecules are dissociated into subunits in warm alkaline buffer . It has been reported that some disulfide bonds of certain protein molecules are unstable in warm alkaline solution (26, 27) . Recently we examined, therefore, the effects of sulfhydryl compounds such as ß-mercaptoethanol and dithiothreitol upon the IOS component of the native fibroin and found that the IOS component is dissociated reversibly to a 6 .8S component, -1 .7 X 10 5 in molecular weight (28) . This result suggests that each native fibroin molecule is composed of the two subunits, approximately equal in molecular weight, which are connected by one or by several disulfide bonds . As described previously, the presence of a small amount of molecular species that are significantly smaller than the J OS component and are probably in reversible equilibrium with the IOS component

v3-fos

2018-04-03T03:58:21.212Z

1970-01-01T00:00:00.000Z

37138198

s2

Effect of added moisture on the heat resistance of Salmonella anatum in milk chocolate. The heat resistance of Salmonella anatum in milk chocolate at a processing temperature (71 C) was greatly decreased by adding 1 to 4% moisture. the heat resistance of salmonellae in dried egg white is 600 to 700 times higher than in liquid egg whites. Ingredients such as sucrose can act to remove available water. Foster (1) reported at least a 10-fold increase in the heat resistance of Salmonella typhimurium when the sucrose concentration was increased from 5 to 40% at 60 C. Since one of the major differences between milk chocolate and other food products is the low moisture content, this study was made to determine the effect of additional moisture on the heat resistance of salmonellae in milk chocolate at a practicable processing temperature. At the temperature chosen for this study, 71 C, the physical properties of milk chocolate are not seriously altered. Salmonella cultures isolated from chocolate products were obtained from the Food and Drug Administration. S. anatum (FDA no. 3989) was found to be the most heat-resistant strain in milk chocolate of our collection; therefore, it was used in this study. To avoid altering the moisture content of the chocolate and to prevent osmotic shock of the cells upon inoculation, lyophilized cultures were used for inoculation. Lyophilization was accomplished by centrifuging 500 ml of a 24-hr nutrient broth culture, resuspending the cells in 40 ml of sterile skim milk, freezing, and drying the cells The log1O of the number of surviving cells was plotted as a function of time. The survivor curves were extrapolated to obtain the D value (time required to reduce the population by 1 log cycle). The D value was then plotted as a function of percentage of added moisture. The effect of added moisture on the D value of S. anatum in milk chocolate at 71 C is shown in Fig. 1. A dramatic decrease in the D value was evidenced with 2.0% added moisture, reducing the D value from 20.0 hr to 4.0 hr. D values decreased as the level of added moisture increased. However, as illustrated in Fig. 1, the change per increment of moisture was especially pronounced at the 2.0% level and below. In no instances were survivors detected after heating periods longer than the calculated D value with low initial inocula. With high initial inocula (105 cells per g of chocolate), when the chocolate was heated for periods of time greater than that calculated to assure a salmonellae-free product, viable cells were detected. A progressive loss of moisture occurred when the chocolate was heated, with the greatest loss occurring early in the heating period. For example, with 2.0% added moisture, the moisture was reduced from 3.72 to 2.50% during 2 hr of heating at 71 C, as shown in Table 1. This moisture loss may be offered as a possible explanation as to why survivor curves with large initial inocula showed a "tailing off" effect. Since only one strain of Salmonella was employed in this study, caution should be used in applying these results to all situations involving contamination of milk chocolate. With low levels of contamination, the results indicated that a short-time heating process with additional moisture could be used for recovering milk chocolate contaminated with salmonellae. By continuously replacing moisture, it might be possible to render chocolate salmonellae-free, even with relatively high levels of contamination. This investigation was sponsored by the Chocolate Manufacturers' Association of the US.A.

v3-fos

2020-12-10T09:04:17.712Z

1970-09-01T00:00:00.000Z

237233398

s2

Enumeration of Byssochlamys and Other Heat-Resistant Molds Methods for the detection of low numbers of heat-resistant molds on fruits were studied by using cultures of Byssochlamys and a number of unidentified mold isolates. Ascospore dormancy had a marked effect on viable recoveries, and the medium in which ascospores were heated influenced activation rates. Best results were obtained when fruit homogenates were heated for 60 min at 70 C in Concord grape juice, followed by culturing on acidified Potato Dextrose Agar. In recent years, there has been an increase in the spoilage of thermally processed fruit products caused by Byssochlamys and related molds (6). The problem is that their ascospores are able to survive the fill temperatures of 80 to 90 C commonly used as the process for these foods. Increasing the severity of the thermal treatment is not a solution because too often this results in a marked reduction in product quality. It appears that the best method for preventing spoilage is to control contamination. This, in turn, requires sensitive methods for the detection of viable spores on fruit and in processing lines. Numerous methods have been used in culturing for Byssochlamys. It has long been known that the ascospores exhibit a dormancy that can be broken with heat (3), and a variety of treatments, ranging from 5 min at 75 C to 35 min at 80 C, have been used (2,5,7,11). After this, the material usually has been cultured on a nutrient medium such as potato-sucrose or potato-dextrose-agar. Growth of bacterial spores that would survive the heat shock has been prevented by acidifying the agar (4) or by the inclusion of compounds such as chloramphenicol (9) When only small quantities of spores were needed, a typical trial consisted of inoculating 5 ml of broth in a 16by 150-mm culture tube (10). After incubation for 28 days at 32 C, the mat and media were transferred to a sterile 50-ml chamber for blending in a Sorvall Omni-mixer homogenizer. After a visually homogeneous suspension was obtained, the material was centrifuged and washed three times in sterile distilled water. Suspensions to be used in later experiments were frozen and stored at -23 C. Activation. In most trials, 0.5 ml of washed spores was added to 4.5 ml of the activation medium. The tube then was placed in a water bath for the desired time period. The media formulas represent concentrations after dilution by the spore suspensions. To assure that different responses were not artifacts resulting from clumping or the breaking up of asci, microscopic counts, determined with a hemocytorneter, of single spores and asci were conducted routinely before and after treatment. Viable counts. Appropriate dilutions of spore suspensions were cultured on Difco Potato Dextrose Agar acidified to pH 3.5 with tartaric acid. The incubation was at 32 C for 2 to 4 days depending upon the strain. RESULTS AND DISCUSSION Heat activation. The heat-labile structures present in culture homogenates, principally conidia and hyphal fragments, may mask much of the effect of heat on ascospores. Heating a homog-393 1 Approved by the Director of the New York State Agricultural Experiment Station for publication as journal paper no. 1817. enate at 70 C, for example, reduced the viable count by over 3 log cycles during the first 5 min, followed by a slight increase in count due to activation of dormant ascospores (10). The kinetics of activation during the initial 5 min could not, of course, be measured. Attempts to obtain pure suspensions for these studies by repeated centrifugation and by filtration (8) were not successful. Although asci could be concentrated by slow, short-time centrifugation, the method was tedious and the preparations were never completely free from conidia and hyphae. The filtration technique was not applicable because of the large numbers of conidia produced by certain strains. Fortunately, it was found that suspending homogenates in 85% ethanol for a short time destroyed the conidia and hyphae (Table 1). Heat and ethanol both reduced the viable count of NYS 1, composed mainly of conidia, by about 4 log cycles, indicating that the two treatments inactivated the same structures. With NRRL 2614, mainly asci, the counts obtained with the "ethanol only" and "washed only" (control) treatments reflected the low populations of active asci that were present. Heating these suspensions increased this population about 10-fold. It was concluded that ethanol did not activate the ascospores or affect their viability, and, therefore, the treatment was used routinely. Early studies on the effect of the heating menstruum on activation included grape juice because it represented one of the foods in which a methodology for spore detection was desired. The results of numerous trials showed an interaction between the medium and temperature. When spores in Concord grape juice were heated at 60 C, activation was completed in 60 min or less, whereas heating in water for as long as 3 hr produced no detectable increase in the viable count (Fig. 1). Spore activation also was enhanced by grape juice at 70 C, although at this temperature a significant number of the spores in water now were activated. These data indicated that grape juice accelerated the rate of spore activation. The spores from numerous strains have been activated at a variety of temperatures in grape juice. At 40 C, the lowest temperature studied, a slight increase in viable count was detected after heating for 3 hr, although 95% of the activatable spores remained dormant. At 80 C, the counts usually were lower than at 70 C, indicating that this temperature was lethal for the spores of many strains. It was concluded from this that 70 C was the optimal activation temperature for most strains. However, a lower temperature such as 60 C was more suitable for studying certain variables, because it emphasized differences in activation rates. The stimulatory effect of grape juice varied with the variety, with Concord being more active than juice from Riesling and Seibel 9549 grapes (Fig. 2). Other trials supported these data in that Concord juice could be diluted to a concentration as low as 20 Brix without reducing activation rates, whereas any dilution of Seibel 9549 juice below the original 160 Brix resulted in fewer spores being activated. Studies on the mechanism of grape juice stimulation revealed an interaction between pH and the active factor (Fig. 3). Adjusting Concord juice to pH 4 caused a marked reduction in the number of spores activated, whereas at pH 5 to 7, the viable counts were no higher than obtained in water. It is known that pH per se was not responsible for the grape juice effect, because heating spores in pH 3.5 solutions of malic and tartaric acids, the principle acids in grape juice, failed to enhance activation ( Table 2). The data ( Table 2) also show that, although glucose and malt extract broth had no effect, a 5% solution of yeast extract gave results comparable to Concord juice. It appears that the same principle was active in both menstrua since their activity responded similarly to changes in pH. Other trials have shown that the factor was still present after grape juice was dried at 100 C, autoclaved, or fermented. Work presently is underway to isolate and define the compound. Cultural conditions. At first, most-probablenumber (MPN) procedures employing broth cultures were used to enumerate ascospores on fruit samples. It was assumed that the incidence of heat-resistant spores would be low, and, therefore, the method would have the advantage that it permitted relatively large samples, 25 g or more, to be cultured. Unfortunately, the method was found to require a very long incubation period. In one trial, for example, 50-ml amounts of 5% malt broth in each of 10 milk dilution bottles were inoculated with 10 g of grape homogenate containing an average of two active asci. The bottles were incubated at 32 C, on their sides with caps loosened to facilitate aeration. The first growth, in one bottle only, was detected after 7 days, whereas 22 days was required before all of the potentially positive bottles, a total of nine, showed growth. Although the MPN recovery figures were in good agreement with the number of asci in the inoculum, the long incubation reduced the effectiveness of the method. When inoculated homogenates were cultured concurrently in broth and on agar media, the latter were found to yield maximal viable recoveries after an incubation of only 2 to 4 days at 32 C. More rapid growth because of better aeration plus the fact that colonies wvere more FIG. 4. Procedure adopted for the detection an2d eniumeration of heat-resistanit mold spores. PDA, Potato Dextrose Agar. easily seen on agar probably accounted for these results. The following agar media have been compared to determine which gave the higher viable counts when inoculated with activated ascospores: Potato Dextrose, Plate Count, 5 % yeast extract, 5% Malt Extract, Put's (9), and concentrations of Concord grape juice from 1 to 15°Brix. The results indicated that many heat-resistant molds were not particularly fastidious in that a number of strains gave comparable counts in all media. When differences were noted, acidified Potato Dextrose Agar usually provided the higher figures. For example, a study of 11 strains showed that 5 gave higher counts in this medium than in 7.5°-Brix grape juice-agar. In general, the differences were by a factor of twofold or less which indicated that the plating medium was not as important a variable as was the heat activation menstruum. Adopted methods. Figure 4 illustrates the procedures used for the detection and enumeration of low numbers of heat-resistant molds on a variety of fruit samples. To minimize the opportunity for chance contamination, a potential problem in laboratories in which Byssochiamys is routinely cultured, the sterile, screw-cap blendor jars were usually carried to the sampling site. Although the amounts varied, 50 to 100 g of fruit often was blended with 100 ml of Concord juice. A 5-min treatment usually produced a homogeneous mixture that could be readily poured into petri dishes. Heat activation was carried out in the blendor jars. The jars were enclosed in polyethylene bags before being placed in the water bath as a safeguard against leakage through the bottom bushing. A 2-hr hold assured that the contents were at the equilibrium temperature for about 1 hr. Overheating was not a problem since spores have been held for as long as 6 hr at 70 C without reducing the viable count. After heating, the entire contents of the jar were distributed into petri dishes, approximately 10 ml per dish. Equal volumes of double-strength Potato Dextrose Agar then were added to the plates. The culturing of such a large sample was required because usually the level of spore contamination was very low. The method underestimated spore populations to some extent because of material retained on the blendor walls. Most strains produced countable colonies by 48 hr at 32 C. Because a few required a longer incubation, negative plates were held for 96 hr before being discarded. The above method has been used for the examination of over 60 samples collected from orchards, vineyards, and processing lines. The procedure appears to be effective in that many contaminated samples, ranging in spore counts from under 1 to over 1,000 per 100 g of grapes, have been detected and some 40 different mold types have been isolated. Interference by other microorganisms has not been a problem. These microorganisms apparently were eliminated by heating at 70 C coupled with the low pH plating medium. LITERATURE CITED

v3-fos

2019-04-26T13:49:45.016Z

1970-01-01T00:00:00.000Z

132308121

s2

Comparison of different weed control methods in groundnut (Arachis hypogaea L.) under rainfed conditions The experiment was carried out at Adaptive Research Farm Bhaun Chakwal during Kharif 2014, 2015 and 2016 successive seasons to evaluate the most appropriate method of weed control in groundnut. Three weed control measures were evaluated i.e., pre emergence application of Ipiflour (trifluralin), Stomp (pendimethaline) and hoeing. A weedy check (Control) was also included in the experiment for comparison purpose. The results showed that both chemicals gave significant better yield than hoeing. Stomp as pre emergence weedicides gave significant yield (710.6 kg/ha) during all tested periods and gave maximum net return of Rs. 17919/with CBR 5.66 followed by ipiflour with net return of Rs.8773.2/and CBR 3.77. It is concluded from the experiment that the hand hoeing gave reasonable groundnut yield (680kg/ha) but it was uneconomical due to high labor wages. So it is concluded that weedicides application is most appropriate and economical method of weed control in groundnut. INTRODUCTION Peanut or groundnut (A. hypogaea L.) occupies a pre-dominant position in cash crops of Pakistan. It is one of the highest oil containing crop and produce good quality of edible oil. It contains about 44 -56% oil and 22-30% protein [1]. The oil is also very desirable for use in making ghee, margarine, shortening and salad oil. The resulting cake after oil extraction is a high protein (55%) meal that is very desirable for human consumption or livestock feed. The meal also contains several essential vitamins and minerals. Numerous weeds are found in peanut plants which is the main factor for its low yield per unit area. It is estimated that weeds reduce groundnut yield up to 75% [2]. Weeds not only compete with crops for water, light and nutrient but also impart physiological disorder to man and livestock and economic resources [3]. The critical period for weed control in groundnut is from three to six weeks after sowing. Generally weed control in groundnut is achieved through hand weeding and herbicide application. Hand weeding is often the expensive control measure to control weeds as it includes high labor cost. Therefore chemical control is an excellent alternative method to obtain better peanut yield [4]. Generally, control of annual grasses and small seeded broadleaf weeds can be achieved with a preplant incorporated (trifluralin, pendimethalin or ethalfluralin. Application of pendimethalin was most effective in checking most of the monocot as well as dicot weeds and ultimately yield was improved [5], [6] and [7]. It is stated that in greenhouse studies, lateral root development was inhibited within bands of 0.5 to 1.0 ppm concentration of soil [8] treated with trifluralin, benefin [N-butyl-Nethyl-2,6-dinitro-4-(trifluoromethyl) benzenamine], and nitralin [4-(methylsulfonyl)-2,6-dinitro-N,N-dipropyl amline]. This experiment was aimed to find out most economical weed control method to control weeds in groundnut and their effect on peanut yield. MATERIALS AND METHODS The experiment was carried out at Adaptive Research Farm Bhaun Chakwal during Kharif 2014, 2015 and 2016 successive seasons to evaluate the most economical method for weed Comparison of different weed control methods in groundnut (Arachis hypogaea L.) under rainfed conditions Kausar, et al control. The soil in the experimental area was clay loam textured. The experimental design was a randomized complete block with each treatment replicated thrice.The area was prepared and divided to plots, each measuring 22m 2 , groundnut variety BARI-2010 was drilled manually within rows at seed rate of 100kg/ha at 45cm inter row spacing. Fertilizer NPK at the rate of 30-62.5-30 kg/ha respectively was applied to fulfill the nutritional requirements of the crop. The following treatments were included: All the chemical treatments were applied at pre emergence stage mixed in soil before sowing by planking. Data for weed population were recorded from one square meter, randomly selected from each replication, 40 days after weedicides spray. Crop was harvested in first week of October in all the three seasons. The pod yields per square meter from each experimental plot were weight then the value of kg per ha was calculated. The data pertaining to number of weeds per square meter and yield in kg/ha were analyzed statistically using analyses of variance techniques [9]. RESULTS AND DISCUSSION Data (Table1) showed that Stomp (Pendimethaline) at recommended rate induced highest effect on number of weeds during all growing seasons and showed minimum weed population of 6.3,7.2 and 5.7 weeds/m 2 during 2014,2015 and 2016 respectively with mortality rate of 76.89%. This was followed by hand hoeing with 67.87% mortality rate. Ipiflour (Trifluraline) had the least effect on weed population and showed 55.09% mortality. Control treatment gave maximum number of weeds (27.7/m 2 , average of three seasons) the above findings are coinciding with the results of previous workers [10, 11, 12 and 13]. Table 2 revealed that all chemical and mechanical treatments increase ground nut yield over control. Stomp (Pendimethaline) gave significant yield during all tested periods and showed average yield of 710.6 kg/ha. These fndings are in full agreement to other scientists [10,6,7]. Applications of herbicides reduce competition of weeds with crop resulting in higher crop yield. It might be due to that the herbicidal application prevented the germination of weeds and also reduced their growth by inhibiting the process of photosynthesis as found by [14].The yield of groundnut plot subjected to hoeing though gave desirable yield of 680.6 kg/ha (mean of three years) but statically it showed non significant relation with both Stomp and control. Hoeing conserves the soil moisture by breaking the capillary action and softens the soil due to which peg penetration was more effcient resulting in increased yield. Similar results were found by [15]. Data in Ipiflour gave an average yield of 537.8kg/ha whereas control (unweeded check) showed minimum yield of 325.2kg/ha. The significant reduction in yield in control treatment was due to weed competition which affected various yield component of groundnut. The economic analysis (Table3) reveals that application of pre emergence weedicides seems to be economical over hand weeding in increasing the ground nut yield. Analysis showed that maximum net return was obtained with application of stomp Rs. 17919/-with CBR 5.66 whereas Ipiflour also showed good performance with CBR 3.77. Hoeing treatment showed minimum net return of Rs. 7418/. The lowest income and cost benefit ratio was recorded in weedy check. These findings are in accordance with the work of other scientists. [16,17]. CONCLUSION On the basis of this experiment it is found that hand hoeing treatment gave reasonable groundnut yield of 680kg/ha but it is uneconomical due to high labor charges. So it is concluded that pre emergence application of pendimethalin or trifluralin at recommended rate is most appropriate and economical method of weed control in groundnut.

v3-fos

2018-04-03T01:16:52.855Z

1970-10-01T00:00:00.000Z

28800758

s2

Enterotoxigenicity of Staphylococcus aureus Cultures Isolated from Acute Cases of Bovine Mastitis To determine whether staphylococci causing bovine mastitis are potential causes of human intoxications, 142 cultures identified as etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were obtained from investigators in the United States and Canada, examined microscopically, and tested for carbohydrate utilization, terminal pH, catalase, coagulase, egg yolk hydrolysis, gelatin hydrolysis, cytochrome oxidase, urease production, nitrate re-duction, micrococcal nuclease, phage type, and enterotoxin production. Three cultures were not confirmed as Staphylococcus aureus. Of the 157 S. aureus cultures, 23 produced staphylococcal enterotoxins. Although a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning was not proved, results indicated that staphylococcal infections of the bovine mammary gland represent a significant reservoir of enterotoxigenic strains of S. aureus. Staphylococcus aureus is frequently present in milk and manufactured dairy products. Such products have been involved in outbreaks of staphylococcal food poisoning (1,14,15,20). Since it has been established that S. aureus is one of the principal etiological agents of bovine mastitis (8), it might be assumed that some relationship exists between staphylococcal mastitis and food poisoning outbreaks attributed to manufactured dairy products. However, a direct relationship between these two conditions has not been established. In an attempt to explore the relationship between staphylococci causing bovine mastitis and the potential for human intoxications, S. aureus cultures from validated cases of acute staphylococcal mastitis were solicited from investigators in the United States and Canada. One hundred and forty-two cultures identified as the etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were received. MATERIALS AND METHODS Cultures were subjected to the following morphological, cultural, and serological examinations to verify their classification and to determine their enterotoxigenicity and phage type. Microscopic examination. A simple stain with 1% aqueous solution of crystal violet was used. Carbohydrate utilization. Utilization of glucose and mannitol was tested by the method of Mossel (16), except the tests were terminated after incubation for 5 days at 35 C. Terminal pH. The test was performed in 2% glucose broth (4); pH was determined electrometrically after incubation for 7 days at 35 C. Coagulase. Brain Heart Infusion cultures (18to 24-hr) were tested with rabbit plasma and rabbit plasma containing 0.1% ethylenediaminetetraacetic acid by the method of Baer (3). Egg yolk hydrolysis. Plates of tellurite polymyxin egg yolk agar (12) were examined after 48 hr at 35 C for zones of clearing or precipitation surrounding points of inoculum. Gelatin hydrolysis. Gelatin hydrolysis was tested by plate method (17) after 48 hr at 35 C. Cytochrome oxidase. The Steel modification (18) of Kovac's method was used to test for cytochrome oxidase. Urease production. Slants of Christensen's agar (11) were incubated as long as 7 days at 35 C. Nitrate reduction. Daily tests from indole-nitrite medium (Difco, prepared according to instructions of manufacturer), incubated for as long as 7 days at 35 C, were made with reagents specified in Manual of Microbiological Methods (17). Micrococcal nuclease. Cell-free extracts of boiled, Brain Heart Infusion cultures were assayed by the method of Chesbro and Auborn (10), modified by changing the CaCl2 concentration to 10 mM and adjusting the reaction mixture to pH 9.5 (14). Cul- tures were grown on a rotary shaker 18 to 24 hr at 37 C, and each extract was assayed in triplicate. Staphylophage typing. Cultures were typed with a set of 22 basic phages recommended by the Subcommittee on Phage Typing of Staphylococcus, Nomenclature Committee, International Association of Microbiological Societies (7), by the method of Blair and Carr (6). Enterotoxin testing. Culture filtrates were tested for identifiable S. aureus enterotoxins by the microslide gel diffusion method described by Casman and Bennett (9). RESULTS AND DISCUSSION Eighteen cultures from chronic mastitis infections and 142 cultures from acute infections were received and tested. Of the 160 cultures, 3 could not be confirmed as S. aureus. Two were identified as Sarcina sp. and one as S. epidermidis. The 157 S. aureus cultures were subdivided into eight types on the basis of their biochemical features. All cultures were catalase-positive, cytochrome oxidase-negative, coagulase-positive, utilized glucose anaerobically, reduced K2TeO3, and produced heat-stable nuclease. In addition, most cultures liquefied gelatin, utilized mannitol anaerobically, hydrolyzed egg yolk, produced urease, and reduced nitrate. All cultures attained a terminal pH in 2% glucose broth in the range pH 4.0 to 4.6; for most, the pH was 4.5. The features in which variants differed from typical S. aureus (Bergey's Manual, 7th ed.) and the frequency of strain occurrence in acute and chronic infections are shown in Table 1. From information received from those who Phage typing revealed that in 8 of the 22 herds and in 9 of the 12 cows from which multiple cultures were received infection was due to a single phage type; more than one phage type was found among the multiple cultures received from the other 14 herds and 3 cows. Susceptibility of the 157 S. aureus cultures to phages of the various lytic groups is shown in Table 2. Typable strains were distributed among 46 separate phage types. Ten cultures were nontypable at a maximum phage concentration of 1,000 times the routine test dilution, with the set of phages used. Although the majority of cultures from chronic infections were most susceptible to phages of lytic group III, the sum of features tested suggested that there was no consistent physiological difference between cultures causing acute and those causing chronic infections. Of the 157 cultures, 23 produced staphylococcal enterotoxins. Eleven produced type C, eleven produced type D, and one produced both types C and D enterotoxins; none produced A or B. Phage types of strains producing enterotoxin are shown in Table 3. Since types of S. aureus in lytic group III are said to be most frequently implicated in food poisoning outbreaks (2,5,19), it is interesting to note the high frequency of types in lytic group I among the enterotoxigenic cultures. Enterotoxigenic cultures were contributed by 14 of the 36 identified herds and 22 of the 72 identified cows. It is perhaps noteworthy that in only one case did all cultures of the same phage type from a herd produce enterotoxin. In three instances, one culture from a herd produced enterotoxin and a similar phage type culture from the same herd did not. In two cases, different phage type cultures from the same herd produced the same type enterotoxin. Nearly 15% of 157 S. aureus cultures identified as etiological agents of bovine mastitis were shown to produce enterotoxins. The hypothesis of a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning remains unproven, but it has been shown that a significant number of S. aureus strains causing bovine mastitis were able to produce enterotoxins. The potential for production of staphylococcal enterotoxins is obvious in those cases in which raw milk containing enterotoxigenic types is poorly held or receives sublethal heat treatment and processing conditions or manner of use of contaminated products allow subsequent growth. The continued high incidence of staphylococcal mastitis and the rather high percentage of enterotoxigenic cultures isolated from validated mastitis cases indicate that greater efforts should be directed toward the prevention of mastitis in the interest of, among other reasons, enhancing the control of foodborne Staphylococcus intoxication. The abnor-mal milk program of the National Conference of Interstate Milk Shipments is working toward this goal.

v3-fos

2020-12-10T09:04:17.047Z

1970-05-01T00:00:00.000Z

237234930

s2

Salmonella Contamination in a Poultry-Processing Plant Bacteriological examination of 1,427 samples from a poultry-processing plant over a 2-year period yielded 202 (14.2%) cultures positive for salmonellae. The results indicate that contamination is reduced by washing procedures within the plant but that recontamination of the carcasses occurred in at least two different stages of processing, i.e., during evisceration and chilling. There was evidence of spread of salmonellae from flock to flock during the serial processing of flocks, but the spread was usually not extensive. The serotypes of salmonellae isolated in this study were similar to those of chicken origin reported from other areas of the country. Bacteriological examination of 1,427 samples from a poultry-processing plant over a 2-year period yielded 202 (14.2%) cultures positive for salmonellae. The results indicate that contamination is reduced by washing procedures within the plant but that recontamination of the carcasses occurred in at least two different stages of processing, i.e., during evisceration and chilling. There was evidence of spread of salmonellae from flock to flock during the serial processing of flocks, but the spread was usually not extensive. The serotypes of salmonellae isolated in this study were similar to those of chicken origin reported from other areas of the country. Salmonella contamination of poultry has been the subject of many investigations and numerous reports (9). Since poultry is a major food source, its contamination with salmonellae may result in the development of human illness. This study of the Salmonella contamination of chickens in a single poultry-processing plant was conducted to determine whether the Salmonella contamination in a plant was consistent or varied with the flocks being processed, to determine whether spread of Salmonella from one flock to another during serial processing of poultry flocks existed and to determine the stages of processing in which contamination or decontamination of the carcasses occurred. A total of 1,427 samples, collected from the plant over a 2-year period from February 1966 to February 1968, were examined for salmonellae. MATERIALS AND METHODS The poultry-processing plant studied processed broilers from a vertically integrated poultry operation and was federally inspected. The plant consisted of five separate rooms as follows: entrance and hanging area, killing area, scalding and picking area, eviscerating and chilling area, and packing area (Fig. 1). Sampling consisted of collecting swab samples from the chicken carcasses, viscera, and materials and equipment used during processing operations, such as tables, tubs, conveyers, knives, saws, and gutter water. Prior to swabbing dry surfaces the cotton-tipped swab was dipped in saline solution. An untreated dry swab was used to swab wet surfaces. Carcasses were examined by rapidly swabbing the external surface for approximately 30 sec. Feces of entering chickens were examined by rotating a swab in newly passed excreta in the chicken crates on the delivery truck. Bacterial analyses were conducted by inserting the swabs immediately after collection into a plastic screw-cap tube containing 10 ml of tetrathionate broth (Difco). The cotton-wrapped end of the swab was snapped off and dropped into the tube. A 1:100,000 dilution of Brilliant Green was added to tetrathionate broth in this study. The tubes were returned to the laboratory, usually within 4 hr after collection. The broth cultures were incubated for 48 hr at 37 C, and then a large loopful of the broth culture was streaked on plating medium. Brilliant Green agar (Difco) containing 80 mg of sodium sulfadiazine per liter of agar was used as the plating medium. At least three suspect colonies were picked from each positive plate, and each of these was inoculated into a Triple Sugar Iron agar tube (Difco). After 24 hr of incubation, tubes that showed typical reactions for Salmonella were subjected to serological (and where indicated, biochemical) tests. Details of the procedures followed the techniques suggested by Galton, Morris, and Martin (4). The 0 and H serological grouping, as described by Edwards (1), was followed by definitive serological typing (2). The nomenclature used in this report is based upon the three-species concept (5). RESULTS AND DISCUSSION Salmonella isolations from the chicken fecal matter of 10 flocks collected at the plant entrance were compared with those from carcasses, viscera, and equipment during processing in various areas of the plant (Table 1). Salmonellae were isolated with similar frequency from feces at the plant entrance, from carcasses before evisceration, from carcasses after evisceration, from edible viscera (gizzards and livers), and from environmental samples within the plant. The various serotypes of salmonellae isolated from the feces of the were being repacked into the chicken carcasses and 6 (4.0%)], but carcasses were subsequently (station 9 in Table 3, and Fig. 1). recontaminated in an area of extensive handling Carcass contamination was reduced by the [ Table 3, station 5 (17.0 %)] and in an area in washing procedures [ Table 3, stations 3 (4.5%) which there was extensive contact among car- Table 1. 6 Isolated from feces of chickens entering plant. c Not isolated from feces of chickens entering plant. 1st 2nd 3rda 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Group B, nonmotile 6 a These 7 serotypes were also listed among the 10 most common serotypes from chicken origin reported to the National Communicable Disease Center during 1968. casses, i.e., the chilling tanks where the carcasses are rotated in an ice slush [ Table 3, station 7 (11.0%)]. This recontamination is depicted clearly in Fig. 2. Flocks yielding no salmonellae were excluded from the data in Fig. 2; hence, the bacteriologically percentages are higher than those shown in Table 3. Results of the examination of three consecutive flocks in each of five visits to the plant (Table 4) indicated that the frequency with which salmonellae were isolated varied from flock to flock. The results from the third visit show that flock B was less contaminated with salmonellae than flock A, which was processed before and after flock B. There was evidence of spread of contamination in flocks A, C, F, G, J, and M to the flocks processed next, but this spread was not extensive. Minimal spread of contamination was indicated by the fact that only 3% of the samples from flock E were positive, whereas 24% of the samples collected from the two preceding flocks yielded salmonellae. There were indications that salmonellae spread extensively from flock G to H and from J to K. However, the histories of the latter flocks (H and K) were not determined; therefore, it is possible (but not likely) that these birds were already contaminated with the same serotypes when they arrived at the plant. The 10 most commonly isolated Salmonella serotypes in this study (Table 5) were compared to those serotypes from chicken sources reported to the National Communicable Disease Center during 1968 (6). During this study, 1,427 samples collected from this processing plant were examined for salmonellae, and 202 (14.2%) were positive. However, 400 of these samples were collected from flocks selected for examination in the processing plant because they had been shown to be contaminated when examined on the farm. Therefore, 1,027 samples were collected in the processing plant from flocks with unknown history, and 126 (12.3%) were positive. Of the latter flocks, 113 were samples of carcasses in shipping crates, ready for distribution to retailers, and 14 (12.4%) were positive. The level of contamination in this plant is similar to that reported in other studies (3,7,8), but levels of contamination as high as 50% have been reported for market broilers (8).

v3-fos

2020-12-10T09:04:10.769Z

1970-11-01T00:00:00.000Z

237231375

s2

Dry-Heat Destruction of Bacillus subtilis Spores on Surfaces: Effect of Humidity in an Open System Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 × 104 for spores conditioned and treated at the low humidities to 2.00 × 105 for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of N0, the y-intercept appears to be lowered by low-humidity conditions. The statistic log y0/log N0 is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments. the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments. Dry heat has been chosen to sterilize planetary lander spacecraft (12). To preserve reliability and minimize degradation of spacecraft materials, the sterilization treatment should be the iinimum which will achieve the desired results (12). Therefore, it is necessary to evaluate carefully the factors which affect dry-heat sterilization. The important effect of water on the dry-heat destruction of bacterial spores has been reported by a number of authors (1, 6,7,9,10). Both the relative humidity of the environment prior to treatment (conditioning humidity) and the relative humidity during treatment (treatment humidity) affect the dry-heat destruction of bacterial spores. The present study was designed to separate and measure the effect of conditioning humidity and treatment humidity in an open system for one typical spore suspension. MATERIAL AND METHODS Bacillus subtilis var. niger spores were deposited on stainless-steel strips and hung in a gravity convection oven with the surfaces parallel to the air stream. The spores were grown in our laboratory from spores supplied by the Communicable Disease Center Field Station, Phoenix, Ariz. The spores were grown on Difco TAM agar (8) approximately -10 C. After several months of storage, the spores were rinsed and resuspended in sterile distilled water; the suspension was subdivided into many small bottles and stored at approximately -10 C. A bottle of spores was thawed for each test; unused spores were discarded. Thus all spore samples had the same handling history except for the length of frozen storage. After thawing, the bottle containing the spore suspension was agitated in an ultrasonic bath and 0.02 ml samples (2.34 X 106 spores) were deposited on the test surfaces with an Eppendorf micropipette. Each test surface was a stainless-steel strip (1 by 2 inch, ca. 2.54 by 5.08 cm) which had been washed; rinsed sequentially in distilled water, isopropyl alcohol, and ether; and then sterilized with dry heat (11). All preparation and analysis procedures were carried out in a class 100 laminar downflow clean room (5). The contamination rate under these conditions was indistinguishable from zero. After the spore suspension was deposited on the test surface, it was dried at 23 C for 18 hr in the clean room. The test strips were then placed in plastic glove boxes over night for water conditioning. In the glove box, the relative humidity was maintained at less than 2 or 89% by using wetted silica gel (3). humidity of the boxes was measured with a Honeywell model W611A relative humidity indicator. The treatment system used a modified gravity convection oven (Blue M model #OV-12A) shown in Fig. 1. The strips (1 by 2 inch) were hung on racks and inserted through the small magnetically held doors to avoid disturbing the temperature of the oven. A rack with strips is shown in Fig. 2. A diffuser plate inside the oven helped to minimize temperature variation across the oven. A variable transformer and thermistor controller (Honeywell model 3679) were added to the oven to decrease "overshoot" and to promote more accurate temperature control. To maintain the relative humidity of the treatment system, the oven was placed in a refrigerator or in a humidified incubator where the dew point was maintained at 5 or 21 C. This produced treatment relative humidities of 0.34 and 1.1%, respectively, at the treatment temperature of 125 C. To measure temperatures in the oven, 12 thermo-couples were located in a grid above the location of the samples. The temperature of each strip during treatment was measured by a thermocouple directly above it. All data presented here have been corrected for slight temperature variation by using the NASAadopted z-value of 21 C. (2). Four treatment times (10, 30, 50, 70 min) and three strips per treatment time were used. The data presented here were from two replicate experiments. Recovery of spores from the strips followed the NASA Standard Methods (11). Strips were placed in 125-ml flasks with the contaminated surface facing downward; 50 ml of phosphate buffer was added, and the sample was sonically treated for 2 min in the center of an ultrasonic bath filled with a 0.3%0 solution of Tween 80 in distilled water (11). Aliquots of 0.05, 0.1, 1.0, or 10 ml, or combinations of these amounts of fluid, were plated in duplicate on Trypticase Soy Agar. Plates were incubated at 32 C for 48 hr. To avoid systematic errors, samples were randomly assigned to treatments and the processing and counting of petri plates was carried out in random order. To the extent possible, consistent with the volume of processing, the number of a plate in the experimental scheme was concealed from the person who counted it. The data were analyzed by a combination of linear regression and analysis of variance. Linear regression gave estimates of the slopes and intercepts of the various survival curves. The analysis of variance distinguished more precisely between the effects of various treatments and indicated whether the effects of the treatments and their interactions were significant. The analysis of variance was a combination of factorial and split-plot approaches. Each whole plot was a rack, carrying three strips, which was exposed to a particular treatment time and process. The whole plots were analyzed by a factorial scheme which allowed the effects of conditioning and treatment humidity to be separated. For both analyses the data were transformed according to the formula x = logio (datum + 1.0). The transformation put the data in the standard semilogarithmic format for the linear regression. It also made the variance of the data independent of the mean which is a requirement for analysis of variance. The number 1.0 was added to all data values so that data values of zero could be included in the analysis. The error produced by the addition of 1.0 was not significant. RESULTS AND DISCUSSION Table 1 shows the data from which the analyses were performed. Figure 3 summarizes the results in graphical form. Each point represents the geometric mean (mean of logs) of six strips, three processed in each of two replicate experiments. The lines are the least squares regression lines. Table 2 gives the death parameters for the experiments. The statistic log yo/log No measures the displacement of the regression line intercept from the control (No) value. Fig. 3 and Tables 2 and 3, note that the initial numbers were ignored in the data analysis because, as has been found by others (4,13), the behavior of the thermal destruc- ing the intercept as well as the slope of thermal death curves. The difference between the low-low and low-high D-values is rather small, but, when combined with the difference in intercept, there is a more than 10-fold difference in survival after 100 min of treatment. Variation was attributed to the day on which the experiment was carried out. The reason for this is not known at this time, but the problem is under study. Despite the day-today variation, it has been repeatedly confirmed for this and other experimental systems that within a given day the relationships between the data are preserved. The humidity both before and during treatment affected the survival of B. subtilis var. niger spores in the experiments reported herein. The effects should be considered separately. To describe the results of a thermal kill treat-of freedom; MS, mean square; F, frequency. ment, both the slope (D-value) and the intercept must be specified. Small changes in treatment humidity caused rather large changes in the killing effectiveness of dry heat in the reported experiments. A combination of regression and analysis of variance approaches to data analysis increases the amount of information which can be obtained from a given experiment.

v3-fos

2020-12-10T09:04:10.948Z

1970-09-01T00:00:00.000Z

237231940

s2

Environmental and Nutritional Factors Affecting the Production of Rubratoxin B by Penicillium rubrum Stoll Rubratoxin B can be produced in a semisynthetic medium by Penicillium rubrum under varying environmental and nutritional conditions. Maximum production (552.0 mg/500 ml) was obtained with P. rubrum NRRL A-11785 grown in stationary cultures of Mosseray's simplified Raulin solution supplemented with 2.5% malt extract broth at ambient temperature. Zinc is required at levels of at least 0.4 mg per liter. In the absence of iron sulfate, there was a 50-fold reduction in rubratoxin B production but not in growth. No toxin was produced by this isolate in 5- or 7-liter fermentors. Rubratoxin B can be produced in a semisynthetic medium by Penicillium rubrum under varying environmental and nutritional conditions. Maximum production (552.0 mg/500 ml) was obtained with P. rubrum NRRL A-11785 grown in stationary cultures of Mosseray's simplified Raulin solution supplemented with 2.5% malt extract broth at ambient temperature. Zinc is required at levels of at least 0.4 mg per liter. In the absence of iron sulfate, there was a 50-fold reduction in rubratoxin B production but not in growth. No toxin was produced by this isolate in 5or 7-liter fermentors. Research on the mycotoxins has been extensively reviewed since the initial report in 1960 of "Turkey X" disease being caused by a toxin produced by Aspergillusflavus growing on peanuts (4,18). However, few reports dealing with mycotoxins other than the aflatoxins have been published. The number of confirmed mycotoxicoses for which the responsible fungus has been identified and its toxin characterized is increasing. This paper deals with rubratoxin B, one of the toxic mold metabolites which has not received widespread publication. The rubratoxins are secondary metabolites produced by Penicillium rubrum Stoll and P. purpurogenum Stoll and have been associated with toxicoses caused by moldy animal feeds (13,15 (7)(8)(9)(10) and are shown in Fig. 1. Natori et al. (11) have isolated rubratoxin B in crystalline form from P. purpurogenum and have reported pathological findings observed in HeLa cells and mice. The biochemical manifestations of rubratoxin B in mouse liver recently have been reported by Hayes and Wilson (6). It is desirable to have these compounds available in large quantities if studies on their chemistry and toxicology are to be undertaken. Since a chemical synthesis has not yet been achieved, the rubratoxins must be produced by biological means. Bioproduction in a synthetic or a semisynthetic liquid medium would be preferable to production on solid substrates because of ease in scale-up, simplicity of extraction, and the suitability of such media for studies on rubratoxin biosynthesis. The purpose of this report is to describe the effect of different growth and cultural conditions on the production of rubratoxin B in liquid culture. MATERIALS AND METHODS Organism. Unless otherwise stated, the P. rubrum used in this investigation is the original strain P-13 isolated by Burnside, received from the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., and designated NRRL A-11785. P. rubrwn MR 043 and MR 180 were supplied by M. 0. Moss of the Tropical Products Institute, London, England, and P. rubrun Stoll 2 was obtained from the Central Leather Research Institute, HAYES, WYATI, AND KING Adyar, Madras, India. Stock cultures were maintained at 5 C on moistened, cracked corn. Culture. Low-form culture flasks (no. 4422; 2,500 ml; Corning Glass Works, Corning, N.Y.) containing 200 to 500 ml of semidefined medium were sterilized at 120 psi for 15 min. The medium was inoculated with a heavy conidial suspension from a corn slant and incubated for approximately 14 to 15 days at ambient temperature in a stationary environment unless otherwise stated. Previous experiments had demonstrated that rubratoxin B production approached a plateau in 14 to 15 days (5). At least two flasks were run for each of the variables investigated. Fermentators (5-and 7-liters), containing 2 or 4 liters of semisynthetic medium, were sterilized 25 mi at 120 psi. The medium was inoculated with a conidial suspension of P. rubrun and incubated for 14 days at 25 C with constant stirring (0, 10, 20, 40, and 80 rev/min) and aerating (0, 100, and 1,000 ml/min) in a Micro Ferm laboratory fermentor (New Brunswick Scientific Co., New Brunswick, N.J.). Samples were taken periodically for measurement of rubratoxin concentration. The pH of the medium was monitored automatically. Differences in rubratoxin B production between duplicate flasks were generally less than 20%, although occasional pairs differed by as much as 40%. Assays. Dry weight of the filtered mycelium was determined after drying the mycelial mats at 70 C for 24 to 48 hr. Rubratoxin B was determined by the method of Hayes and Wilson (5) by extraction with diethyl ether. The diethyl ether extracts were either concentrated to dryness and redissolved in acetone or spotted directly onto Silica Gel HF254 chromatographic plates. The plates were developed in an unlined tank saturated with glacial acetic acid-methanolchloroform (2:20:80, v/v). Authentic rubratoxin B was spotted on these same plates, and, after development, the plates were observed under short-wave ultraviolet light (253 am). Toxin was measured gravimetrically. RESULTS The effect of sucrose inthebasal medium supplemented with 2.5 % maltextract broth on rubratoxin B production by P. rubrum is shown in Table 1. This table indicates that a plateau for rubratoxin production is reached at 10 to 30 g sucrose per 470 APPL. MICROBIOL. liter, with maximum production occurring at 25 g per liter. Table 2 indicates the effect of malt extract broth in the basal medium (50 g of sucrose/liter) on the production of rubratoxin B by P. rubrum. Relatively large quantities of rubratoxin were produced at all levels investigated with peak production occurring at 30 g per liter of malt extract broth. The production of the toxin was greatly reduced in the absence of malt extract broth. Because a number of mineral elements have been implicated as being involved in production of other mycotoxins, the effects of trace metals on rubratoxin B production were investigated ( Table 3). Since deletion of iron almost eliminated rubratoxin production, it appears that iron is needed for rubratoxin biosynthesis. The reduced yields of rubratoxin B obtained without iron were not due to an apparent reduction of growth. Zinc appears to be specifically required for production of the toxin, and this requirement is met by the addition of a minimum of 0.4 mg of zinc to the medium (Table 4). However, maximum yields of rubratoxin B were produced when 400 mg of ZnSO4 was added to the medium. Table 5 shows the effect of temperature on production of rubratoxin B. Toxin was detected at None detecteda aAfter 20 days placed at ambient temperature (22 to 25 C) for 14 days. Growth and toxin production in both cases: 5 C, 20 mg/500 ml; 45 C, 83.4 mg/500 ml. VOL. 20, 1970 471 20, 22, and 30 C; however, none was detected when the organism was grown at 5, 37, and 45 C. When these cultures were placed in ambient temperature (22 to 25 C) for 14 days, growth and toxin production were observed (20 mg/500 ml, 5 C; 83.4 mg/500 ml, 45 C). The initial pH of the medium (Table 6) did not significantly affect either toxin production or growth as long as it was in the range of pH 4.0 to 8.0. If cultures were grown below and above this range, there was a partial or complete reduction in toxin production. Several experiments were carried out in 5-and 7-liter fermentors. As previously reported for shaken cultures and for 14-liter fermentors (5), no toxin was obtained in either fermentor under the various regimens of aeration and agitation employed in this investigation. Higher yields of rubratoxin production were noted when P. rubrum was grown in total darkness than in light (184.8 and 147.0 mg, respectively, per 500 ml in 14 days), whereas the mycelial weights were the same (6.9 and 7.0 g, respectively). Table 7 lists yields of mycelium and rubratoxin B produced by five isolates of P. rubrum. DISCUSSION A medium composed of sucrose (25 g/liter), malt extract broth (20 g/liter), ZnSO4 (0.5 g/liter) and FeSO4 (0.5 g/liter) provided all the necessary ingredients for the production of rubratoxin B by P. rubrum P-13. Other chemical, cultural, and environmental conditions investigated had less effect on toxin production. The requirement for zinc may explain the difficulty of obtaining rubratoxin production on unsupplemented media such as Czapek-Dox medium. This requirement for zinc may be a factor in the apparent loss of toxin-producing ability of the fungus when maintained for prolonged periods on Czapek Agar or on Czapek-Dox Agar (5). The reports of Natori et al. (11) on the production of rubratoxin B by P. purpurogenum in a Czapek-Dox medium modified with ZnSO4 (0.01 g/liter) and by Wogan and Mateles (17) on submerged culture production by P. rubrum confirm this nutritional observation. The large reduction in toxin but not in fungal growth when iron sulfate was deleted from the medium also may help explain the loss of toxin biosynthesis by isolates maintained on artificial media. The Czapek-Dox medium used for production of rubratoxin B by P. purpurogenum (11) and the submerged culture medium (17) both contained FeSO4. At present, however, there is insufficient evidence to speculate whether a lack of zinc or APPL. MICROBIOL. iron is selecting for nonrubratoxin-producing mutants. During the past several years, fungi capable of producing aflatoxin, ochratoxin, and other mycotoxins have been isolated from a number of foodstuffs in this as well as other countries. These findings, along with the recent Japanese report of rubratoxin B production by P. purpurogenum (11), further indicate the importance of the mycotoxin problem throughout the world in food hygiene and in stored agricultural commodities. Such facts indicate a pressing need for screening of molds for detection of mycotoxins other than those commonly screened for at the present (i.e., aflatoxin and ochratoxin).

v3-fos

2014-10-01T00:00:00.000Z

1970-10-15T00:00:00.000Z

11272173

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Need and methods of gene conservation in animal breeding The problem of gene losses was considered topical at least in cattle and poultry, possibly also in pigs and sheep. The need of preventing them is determined by several factors: 1) Past experience of actual losses ; 2) probable effect of present breeding methods on the genetic variability (both selection and random drift); 3)changing demand for animal products because of new knowledge in food science, rising living standard, new fashions, increased quality requirement, need of lower production costs, need of bigger quantities, etc.; 4) changes in environment (feeding, housing, managements, disease); 5) experiences from plant breeding; 6) existence of unexamined breeds; 7) better utilization of land; 8) utilization of hybrid vigor; 9) difficulties in creating new useful variation. The available methods of gene conservation were discussed, the main attention being paid to the maintenance of small nuclei of most breeds and strains, to the establishment of gene pools, and to the establishment of frozen semen banks. Finally, organisation and principles of gene pools were briefly discussed, and the need of a coordinating international body was indicated. The present era of frozen semen in cattle A.I. has re-actualized the problem of gene losses, which was amply discussed a few decades ago when the A.I. breeding was at the stage of a final breakthrough (E DWARDS , 1959 ). The second species in which this problem has become topical is the poultry, where the exceptional reproductive rate makes it possible to disseminate selected material very rapidly, thus concentrating the actual breeding work into the hands of very few enterprisers. Without doubt the problem is of some importance even in pigs and sheep, and it may be well-founded to consider almost the entire animal kingdom, in order that no species which might be of value to mankind in the future, would unnecessarily be lost. The problem arises mainly from the fact that an effective utilization of the best animals of to-day automatically means setting aside the poorer animals, strains, breeds, and even species. The purpose of the present paper is to consider, ( I ) whether this elimination of genetic material will have undesirable consequences, and ( 2 ) if so, how these could be avoided. II. -THE NEED OF GENE CONSERVATION The need of adopting special measures for preventing loss of genes is determined by several groups of factors, e. g. theoccurrence of actual gene losses in the past, the probable effect of present breeding systems on the genetic variability, the possible changes in the demand of various human foods, and possible future changes in the environmental conditions of farm animals. A. -Losses or nearby losses of breeds and strains Before the beginning of rational animal breeding, i. e. 5 0 -100 years ago, there were numerous local native breeds of different species, obviously well adapted to the primitive conditions of that time. Many of these breeds have been replaced by other breeds, which were considered superior to them. It is difficult to avoid the impression that the choice of breeds was often based on rather superficial knowledge, biassed by many environmental factors, since even now objective knowledge of the merits of different breeds is scarce. The importance given to the purity of breeds favoured the complete discarding of breeds with slightly inferior total merits, and hence any genes of special merit were lost together with the breeds discarded. Later on, many internationally well known breeds have undergone the same fate. The use of up-grading has meant a little more cautious treatment of the gene pools of the inferior stocks, but not very decisively so, except in cases where cross-bred males have been accepted for extensive use. In a random sample of 50 registered Finnish Ayrshire cows born in 1955 the proportion of imported genes was 95 p. 100 . It would be interesting to prepare a list of breeds having disappeared during the last 100 years, but since this would go far beyond the time and energy available for me, I shall confine myself to mention some examples from my nearest vicinity. In Finland, the North-Finnish type of native cattle (Finncattle) has almost entirely disappeared, the frequency of the East-Finnish type has considerably decreased, and even the West-Finnish type is losing ground to the biggersized Ayrshi y e and F y iesian breeds, although it is not yet settled whether one should strive for bigger animals for the sake of beef production. Fortunately, some genes of the different types of Finncattle will be saved, thanks to the moderately liberal breeding policy of the respective breed society. An idea of the importance of a cautious displacement is given by fig. i, which shows that hardly 30 p. 100 of the alleles of the B blood group system occurring in West-Finnish and Ay y shi y e bulls were common to both groups of bulls, while about 1 8 p. 100 of the alleles occurred only in Ayrshi y e and 50 p. 100 only in the West-Finnish cattle. In case this dissimilarity of blood group alleles partly reflects differences in the genetic make-up affecting production traits, a total loss of the Finncattle genes would obviously mean a serious decay of genetic variability of cattle in Finland. The difference in milk yield between the two breeds is nearly 20 p. 100 , but partly this difference can be explained by the difference of nearly 10 p. 100 in the adult live weight. In the growth rate of performance tested young bulls up to 6 months, there were almost no difference (L IND -STROM and M AIJA I, A , ig6g). Thus it is possible that even a given quantitative trait may partly be determined by different sets of genes in two breeds. The average non-return rate of Finn!Clttle is 3 -4 p. 103 higher than that of Ayrshire. Another special advantage of the Finncattle is its polledness ( fig. 2 ). Recently the Finnish Lan!race sheep (Finnsheefi) ( fig. 3 ) was also approaching extinction, as the population decreased from over i mill. head in 1950 to 0 . 15 mill. in 19 6 7 , and sheep breeds with better muscling and growth rate were simultaneously being looked for. Recently it has been realized, however, that the fertility genes of Finnsheep might be useful in intensive production of lamb meat. A third Finnish example is the native hen, the good brooding instinct of which was no longer required when the artificial incubators became popular. It may have possessed also other capabilities, like resistance to some diseases, longevity and unpretentiousness, but these were not properly investigated before the breed was lost. In Sweden, the situation with regard to the Swedish Polled Cattle is very similar to that of Finncattle. In Norway, there were about 30 breeds of cattle in the 193 o's, but now hardly every tenth is left. A part of the genes of the vanished breeds has been saved by the liberal use of crossbred bulls in A.I.. At least half of the British cattle breeds listed by HousMnrr ( 1905 ) are approaching ectinction (MMB, 19 68). According to B OY E R ( 19 6 4 ) there are 19 local breeds of hens in France near extinction, and V AN Ar, BADA ( 19 64) reports about a similar number for the Netherlands. In general, the so-called &dquo; half-heavy poultry breeds &dquo; started to disappear after the specialization into broiler and egg-type breeds became popular. Of course, even a long list of breeds which have disappeared, does not mean much in case one could be sure that they didn't possess genes which could be useful in future animal breeding. The recent « discoveries n of the leukosis-resistant Fayoumi-hen from Egypt, the muscular Cornish game-cock from India, and the fast-growing Charolais cattle from France, as well as of some fertile or muscular sheep breeds form examples that changes in the environment or in the market may bring to light the usefulness of breeds which were previously considered to be of little commercial value. Even the recent creation of useful compound breeds as Beltsville no r, Minn,,sola no z and n) 2 , Lacombe, Santa Gertrudis etc. speak in favour of preserving breeds from extinction. In poultry breeding, there has been some decrease in the number of strains within breeds in the last two decades, because of the decrease in the number of breeders. According to VA N A!,BADA (1954) and O R O Z CO (zg6q.) the number of strains has not decreased as rapidly as that of breeders, as the remaining breeders have increased the numbers of their lines at the same time. It is probable, howe-ver, that the new strains in many cases are sub-strains of the previous strains of the surviving breeders, and not based on the strains of the unlucky competitors. Some American poultry breeders have claimed that it is difficult to find new strains which could successfully be used for further improvement of their hens, while J AAP ( 19 66) made the interesting observation that alleles carried by slow-growing egg-production strains may have value for improving the growth rate of broilers: This is apt to support the hypothesis that a given quantitative trait may be determined by different genes in different populations, and that combining two or several such strains would give a good basis for selection. On the other hand, L ERNER and DONA!,D ( 19 66) consider that the two extreme breeds, Friesian and jersey, might well include all the genes needed in the future cattle breeding. In any case, it is reasonable to think that a loss of a strain with exceptional gene material is more detrimental than a loss of a strain having an exceptional frequency of the genes occuring in the remaining strains (O ROZ C O , 19 6 4 ). It is also clear that the establishment of Random Sample Tests and other improvements in testing methods, as well as the introduction of more and more efficient propagation methods, are inclined to increase the risk of losing interesting strains. B. -Effect of breed improvement on the genetic variability Although the losses of genes caused by culling entire breeds or strains obviously are the most serious ones, it is necessary to consider also the within-population losses caused by selection and chance. i. Decay of variability due to selection A natural consequence of successful efforts to increase the frequency of &dquo; good &dquo; genes and to decrease that of &dquo; bad &dquo; genes is a decay of genetic variability, although the experiences obtained in this respect are conflicting. In many cases it has been difficult to notice much of decrease in the phenotypic variability, in spite of considerable changes in the mean performance (e.g. FALCONER, 1955 ). On the other hand, many workers with laboratory animals or poultry have experienced a &dquo; ceiling &dquo; or &dquo; plateau &dquo; in their selection attempts, within a number of generations, and this lack of response has sometimes come rather abruptly, without obvious signs of warning (FALCONER, 1955 , C I ,A Y T ON and RO B E R TSO N , 1957 ). However, negative genetic correlations between the trait selected for and fitness traits, or a continuously varying environment, have been considered as main causes rather than any decay in genetic variability (D ICK E RSON 1955 , ROBERT-SON 1955). I,!xN!R and DONALD ( 19 66) considered that &dquo; gains in selection are always achieved at the cost of reducing variance &dquo;, and that &dquo; sooner or later genetic variability which responds to a certain system will approach exhaustion &dquo;. In addition to selection experiments with laboratory animals, some simulation studies have given valuable information of the probable effects of selection. For example, YOUNG ( 19 66), working with an imagined population of i.ooo individuals, considered 3 levels of selection intensity, 3 levels of initial heritability, 3 levels of recombination probability and genetic models. It was assumed that the trait was controlled by 10 loci, and that the initial gene frequency at each locus was 0 . 5 . The additive model led to the changes of additive variance shown in fig. 4 in YourrG's paper. It appears that the decay of variance can be very rapid, if one selects strongly for a trait with a high heritability. Even a medium heritability of 0 . 4 led to a total loss of additive variance within 14 generations, when only 10 p. ioo of the individuals were selected. Considering all the genetic models tried, the full-lives of additive variance under the same conditions varied from 9 to over 30 generations, and the half-lives between 1 . 4 and q.. 3 generations. In the former case the dominance model gave the highest value, while in the latter case it showed the lowest. The importance of linkage in determining the rate of decay was comparatively small. As a whole, it appears that the losses of genes caused by directional selection may often be worth considering when plans for additional improvement of animals are being made. 2 . Gene losses due to random drift. t. In small populations, one can lose genes even by chance, due to the random fluctuation of gene frequency, and these losses can be enhanced by intentional mating of related individuals. These losses are undirectional, since they affect the favourable and unfavourable genes with equal probability. In the populations analyzed so far the average decrease of heterozygosity has been a good 0 . 5 % per generation (LUSH, 1947 ), and it has been difficult to show that A.I would have increased the figure in cattle populations (ROTT!NSTEN, 19 6 1 ). Because of the intentional avoidance of inbreeding in A.I. operations, the actual coefficient of imbreeding is not very suitable for measuring gene losses, but these should be measured by the expected inbreeding coefficient, based on the average coefficient of relationship (RE ND E L , 195 8 and 19 6 3 , M AIJ A LA and I, IND ST R O M 19 66). In the studies reviewed by R!ND!I, ( 19 6 7 ) the expected homozygosity of B blood group alleles varied between 4 .o and 24 .8 %, and the number of different B-alleles in different breeds from 20 to 132 . Future gene losses due to random drift can be predicted with the aid of the concept of effective population size (N e ) introduced by WRIGHT ( 1931 ). Considering that the loss of heterozygosity per generation is approximately proportional to 2 N e the critical values of Ne lie between 50 and 10 : 2 Ne It has now become possible to aprroach these values in Finland and many other countries where frozen semen is used exclusively. One half of the 200 young Ayrshire bulls on the performance testing station are sons of one bull, which still is fully active and from which over 50 00 o pellets already have been stored. According to a recent simulation study (E BBERSTEN et al. ig6g) the average number of generations required for fixing a gene with an initial frequency of 0 .5 was as follows: A comparison of these figures with the full-lives of additive variance obtained by YOUNG ( 19 66) reveals that the decay of variability caused by random drift is somewhat slower than that caused by directional selection, thus supporting the results obtained by Ros!xTSOrr (ig6o). C. -Changing demand for animal products In the past there have been so many changes in the demand for various products on the market of different countries, that it may not be difficult to agree as to the necessity of maintaining a reasonable amount of variability in our animal populations, in order to be able to cope with future changes. The changes may be caused by one or several of the following factors : i. New knowledge in the field of human nutrition, enhancing the value of some nutrients or dooming some products as unwholesome. 2 . Increasing standard of living, making possible an increased consumption of nutritionally valuable but expensive foods (e.g. meat) or of foods with special taste (game animals). 3 . New fashions in clothing (furs) or eating (brown eggs). 4 . Increased quality requirements with regard to conventional products (leaner meat, protein-rich milk, strong eggshells etc.). 5. Necessity to decrease the costs of production (feed conversion, ease-ofmilking, calm temper), in order to be able to compete with industrial products or substitutes. 6. Increased requirements with regard to quantity, in order to combat hunger (reproduction ability, growth capacity). 7 . Necessity to compensate the exhaustion of natural reserves of some materials (fuels, minerals). 8. Necessity of finding new ways of utilizing agricultural plant products in case of surplus problems. The need of breeding different kinds of animals for different environments depends on the existence and magnitude of non-linear interaction between heredity and environment. Although the experimental results bearing upon this problem have been rather conflicting, more and more positive evidence has been accumulating during recent years, especially in poultry (M ERAT , 19 68). The interaction seems to concern particularly mortality and egg production. In a study by N ORDSKOG and K EMPTHORNE ( 19 6 0 ) the strain x farm interaction accounted for no less than 3 i p. 100 of the total variance among pen means for mortality, and the genetic correlation between mortality figures of the same production stock kept in different locations was only 0 . 03 . It is obvious that the importance of these interactions will vary with the species, character and the diversity of environment. Taking into account that some of the studies showing no genotype-environment interaction were based on slightly varying or poorly defined environments, and that specialization of environments is a likely direction of development, it appears well-founded to maintain enough variability for fitting animals for special environments. This may in some cases be cheaper than to fit the environment for special animals. The possible changes of environment can be grouped as follows : i. Changes in feeding. New economic feeds can be found, which may be inadequate in some respect (lack of concentration, certain essential amino acids, fatty acids, minerals or other qualities). It has been shown that there are some variations between breeds, strains and individuals in the utilization of nutrients (NES H EIM, 1966). conventional conditions. Lack of proper housing facilities also requires, perhaps, special animals. 4 . Changes in management may increase the desirability of certain traits (suitability for machine milking, polled animals for loose housing, certain colour genes for autosexing, out-of-season lambing for intensive lamb meat production, special battery hens). E. -Miscellaneous aspects Several other aspects bearing upon the importance of conserving genes appear from the reports of some meetings discussing the problem (HonGSOrr, ig6i, S COSSIRO I, I I C)6 4 , V AN A LBADA 19 6 4 , Siis 19 65, M!NZi 19 66, F AO 19 66). These include also discussions on experiences obtained in plant breeding, the existence of numerous unexamined breeds in the developing countries, the need of improving the utilization of land, and the possibility of utilizing hybrid vigor. III. -METHODS OF GENE CONSERVATION Although the aspects speaking in favour of gene conservation may have received too much attention above, at the expense of opposite arguments, it is obviously valuable to search for and apply some cheap and efficient methods for preventing unnecessary or serious losses of genes. There are at least three diffeient methods to be considered seriously: (1) maintenance of several pure breeds or strains, (2) establishment of a common gene pool, and (3) establishment of banks for frozen semen, eggs or gonadal tissues. A fourth possibility open to groups of breeders would be bringing in genes from other breeds or countries, but this presupposes that somebody else has taken care of the conservation. A cautious selection would be a fifth alternative, but the slow progress resulting from it on thousands of farms makes it a very expensive method. The choice between the two first-mentioned methods obviously depends on the size and reproductive ability of the species, as the maintenance of large animals in numbers big enough to avoid random losses of genes is expensive, unless the particular breed has a good average performance. It may be necessary to apply both methods in most species, but relatively fewer breeds can be kept pure in large livestock species than in the small ones. Another factor determining the choice consists of the kind of traits each breed possesses, and of the purposes of its preservation. Breeds which have some special traits well developed, deserve to be kept as « pure elements on the shelves of the chemist &dquo;, while an amalgamation into a gene pool may be an appropriate method for a breed whose average peiformance is assumed to be caused by exceptional sets of genes. This is also the most likely method to be used for a breed, the value of which is not known. Preservation of valuable strains as independent populations is particularly important for the utilization of non-additive inheritance. In any case, there is a question of preserving genes rather than breeds. In cattle, maintenance of many breeds or of a gene pool can now be managed in the form of frozen semen. It is not yet completely known how long the frozen semen maintains its fertilizing capacity in liquid nitrogen, and hence one cannot yet base long-term plans upon frozen semen. However, it would be important to save some doses of semen from every breed or strain which is likely to become extinct within the next few years. As far as is known to me, freezing of eggs or gonadal tissues is not yet of practical value in this respect. The costs of maintaining pure breeds or gene pools could, perhaps, be decreased by combining the activities with those of zoological gardens or with other leisure time businesses. This might be a fertile field for international cooperation between nature-conserving zoologists and animal geneticists. It may also pay to encourage and subsidize so-called fancy breeders to keep animals of rare breeds. There is a wide range of opinions among animal geneticists: some workers don't consider it worthwhile to worry about the conservation of genes, while others don't dare to utilize the present possibilities for selection, in order not to endanger future possibilities. The former attitude is sometimes based on the belief that useful genetic variation can be created with the aid of mutagens whenever required, while the advocates of the latter view consider that most of the mutations to be brought about would be undesirable, and that the testing of their usefulness would take too long time. The truth probably lies somewhere between the two extreme opinions, but where ? According to the experiments performed so far, the importance of induced mutations for short-term progress in quantitative traits has been very small, but the frequency of lethals has increased considerably (R OBERTSON , 1955 b). LE RN E R and D ON Ar, D ( 19 66) consider that the abundance of undesirable mutations makes the creation of new variability too expensive. The same authors thought that every generation has a duty to look after the maintenance of genetic variation, but that economic reasons often tempt one to act against this duty. However, the slowness of developmental processes and the obstinacy of many individual breeders are of great help in fulfilling the duty, at least for the present. IV. -ORGANISATION AND PRINCIPLES OF GENE POOLS The principles to be applied in establishing and maintaining gene pools have been discussed by J A A P (i 9 6q.), BO Y ER (1964), OROZCO (1964) andFno (1966). The basic step is to make an inventory of all available stocks, including collected informations of the characteristics of each stock. Then the most useful or interesting stocks are tried in a common environment, after which the final choice of strains to be conserved is made. According to J AAP it is inadvisable to combine more than 2 -3 populations into a pool, in order to keep the frequencies of most alleles at a useful level. This determines the number of populations to be maintained. He recommended 40 pairs of parents per generation and at least 10 offspring from each pair for each population. Matings should be randomized and the effects of natural selection should be avoided. One central station is usually recommended, but in cases where each strain has been kept pure, some private farms have been utilized. From the global or continental point of view, it might be well-founded to encourage each country to take care of the preservation of its own national breeds and to keep the coordinating international body, e.g. F AO , well informed of the numbers and characteristics of the stocks. In case there is temptation to destroy a breed, it would be advisable to negotiate first with the coordinating body, which for this purpose should have a responsible official and an advisory council. In evaluating national stocks, some widely distributed international breeds could be used as controls. Re!u pour publication en septembre 1970.

v3-fos

2019-04-27T13:12:28.135Z

1970-01-01T00:00:00.000Z

134213826

s2

Air layering in cinnamon ( Cinnamomum verum L . ) under wet humid tropical conditions Cinnamon is a cross pollinated species and seed propagation has resulted in development of considerable variability in growth, yield and quality among the populations. Air layering has been considered as one of the efficient methods of multiplication in cinnamon. However, the success of layering varies greatly depending on the local environmental conditions. In the present investigation, air layering was performed at 20 days interval during rainy season (July 3rd to October 11th) of two consecutive years in Bay islands. Result revealed that first week of July was the most suitable time for air layering in cinnamon as it supported better rooting percentage (87.5%). Cinnamon (Cinnamomum verum L.) or dalchini is one of the ancient tree spices grown in India. Though both bark as well as leaves are known to possess aromatic components mainly cinnamaldehyde and eugenol, the bark of this species is valued as a spice. It has been used in the form of dried bark, bark powder, oil and oleoresins. Cultivation of this spice in India is mainly confined to States of Andaman and Nicobar Islands, Kerala, Karnataka, North Eastern India and parts of Tamil Nadu and Maharashtra. Combined estimate of cinnamon and tejpat suggests that in India, it's being grown on 2,770 ha area with about 5,050 t production (Indian Horticulture Database 2014). However, the production is not enough to meet the domestic demand and hence, cinnamon is being imported from other countries of the world (Indian Spices 2016). Increasing the productivity through development and adoption of improved technologies is a key factor in reducing the dependence on import. Soil and climatic conditions of the Andaman and Nicobar Islands are well suited for the cultivation of cinnamon (Parthasarathy et al. 2009) and presently it is cultivated in about 150 ha yielding 40 t annually. Quality of cinnamon is assumed to be the finest in the islands compared with other parts of the country (Singh & Sankaran 2012). Large availability of interspaces in the coconut and arecanut plantations in the Andaman and Nicobar 72 Islands could be successfully utilized for its cultivation (Waman et al. 2016). However, existing plantations in the islands are of seedling origin. One can easily notice variations in cinnamon seedlings for leaf size, shape and colour of new flush, apart from the distinct chemotypes (Krishnamoorthy et al. 1988). Ergo, considerable variability is noticed in the yields and quality of the final produce and hence, vegetative propagation is of interest (Rema et al. 1997). The present report concerned an effort to identify the most appropriate time for carrying out air layering under island condition. The present investigation was carried out in the Division of Horticulture and Forestry of ICAR-Central Island Agricultural Research Institute, Port Blair, Andaman and Nicobar Islands during 2015 and 2016. The islands exhibit typical tropical climate with average temperature of 18-31°C and annual rainfall of 3,100 mm distributed over May to December. Further, the relative humidity ranges between 60-90% in a year, while the average lies near to 70-80%. For layering, healthy shoots of 25-30 cm length and 1.0-1.5 cm thickness were selected. Leaves and small branches near the ringing area on selected shoots were removed and two circular cuts were given to the shoots for removing a ring of bark of about an inch width. Commercial formulation of rooting hormone (Lipsa, Kolkata) was used for root induction and ringed portion was covered with soil: farmyard manure (1:1) before wrapping with polythene (20 cm × 20 cm). Experiment consisted of six treatments i.e. T 1 : layering on July 3, T 2 : layering on July 23, T 3 : layering on August 12, T 4 : layering on September 1, T 5 : layering on September 21 and T 6 : layering on October 11. Experiment was laid out in completely randomized design with 20 layers in each treatment. Various parameters were recorded at the time of separation (90 days after layering) and data was subjected to analysis of variance using Web Agri Statistical Package 2.0 (WASP 2.0, ICAR-RC for Goa, Ela, India). Air layering was performed for six times during rainy season under island condition. Generally, rooting process is facilitated by the rains (Ranaware et al. 1995) and hence, the dry periods in the islands were avoided during the experimentation. Pooled analysis of two years data revealed that percentage root induction varied between 35.0% and 87.5% amongst the treatments studied (Table 1). Maximum rooting percentage was obtained in layers done on 3 rd July (87.5%) followed by those done on 23 rd July, while it was the lowest in layering performed on 21 st September. Though rooting response varied considerably during different seasons, the mean number of primary roots per layer did not vary significantly (Table 1). Length of longest root varied significantly amongst the studied treatments (Table 1). Maximum length of primary root was observed in propagules obtained from cinnamon layered on 1 st September (8.5 cm), which remained on par with layers of 23 rd July. Similar to rooting percentage, length of root was found to be the lowest (3.6 cm) in layers of 21 st September batch. Thickness of root ranged from 2.04 to 2.74 mm; however, the differences were non-significant amongst the treatments studied. Waman & Bohra Similar to present communication, significant variations for rooting response and growth parameters have been reported from different agro-ecological regions of the country viz., West Bengal (Banerjee et al. 1982), Karnataka (Hegde et al. 1989) and Maharashtra (Ranaware et al. 1995). These reports have suggested the positive effects of rainy period on layering success; however, the raining season and pattern vary significantly in different regions and hence, location specific studies are required. As no reports are available for island conditions, present study was conducted. The inter-and intrarow spacing was kept at 45 cm and 20 cm, respectively. The recommended package of cultural practices was followed to raise a good crop. In each plot, five competitive plants were identified randomly for recording data on days to 50% flowering, plant height (cm), primary branches plant -1 , umbels plant -1 , umblets umbel -1 and seed yield plot -1 (kg). The data recorded during three years were subjected to stability analysis according to the model proposed by Eberhart & Russel (1966) and three stability parameters mean (m), regression coefficient (bi) and the deviation from linearity (S 2 di) were estimated. (Table 2). For days to flower, umbelets /umbel and umbels plant -1 FNL 69 had recorded wider stability (above average mean, bi = 1 and S 2 di = 0). The genotype FNL 70 was stable for plant height, primary branches plant -1 , umbelets umbel -1 and umbels plant -1 but had low seed yield, indicating its adaptation to stress environments (Lal 2008) while genotype FNL 71 recorded stability for plant height and umbels plant -1 . For umbels umbel -1 FNL 72 had high stability across the environments of Raigarh. The genotypes FNL 69, FNL 70 and FNL 71 were among the top entries which had their mean umbels plant -1 greater than the average of all the genotypes with regression coefficient (bi =1) and non-significant deviation for regression (S 2 di =0). This indicated their high stability over the different environments of years (Lal 2008). The genotype FNL 68 and RF 101 had above average mean for umbels plant -1 , S 2 di = 0 but the value of bi #1, indicating their adaptation to high input conditions. For umbels plant -1 genotype RF 205 and FNL 67 had bi = 1 and S 2 di = 0, but their mean was low, indicating their adaptation to stress environments (Table 3). The genotypic differences were found to be highly significant for all the traits in each environment (year). The mean genotypic values from different year were subjected to pooled analysis. The mean sum of squares (MSS) due to genotypes (G) and environments (E) were significant for all the traits except for primary branches plant -1 when tested against MSS due to genotype x environments. It revealed the non significant differential response of the varieties to the changing environments. The results were in close conformity to the findings of Lal (2008), Verma & Solanki (2015). The MSS due to G × E when tested against pooled error, were found highly significant for all the characters. Thus stability analysis was carried out for all the traits. The variance due to G × E were divided in to G × E (Linear) and due to pooled deviation (Non-linear). The G × E (Linear) mean squares were found significant for all the traits except primary branches plant -1 indicating the presence of predictable components where as significance of pooled deviation for seed yield plot -1 , days to 50% flowering, plant height (cm), primary branches plant -1 and umbels plant -1 showed the presence of non-predictable components. These observations indicated that some reliable predictions about G × E interaction as well as its unpredictable components can be made for these traits. Hence, both components contributed significantly in determining the stability of genotypes (Lal 2008; Verma & It was observed that national check (RF 205) showed wider stability for seed yield, days to flower, primary branches plant -1 , umbels plant -1 and umbelets umbel -1 while local fennel had wider stability for seed yield (kg ha -1 ), plant height, umbels plant -1 and umbelets umbel -1 . While none of the test entries showed superior stable performance over the national and local checks during all the three years. This appears to be evidence for much greater genotype × environment interaction for the entries to be evaluated than for the pure lines. This is somewhat unexpected in view of the greater homeostasis in unfavorable environments usually found in heterozygous genotypes. This needs further study. Acknowledgements Authors are thankful to Project Coordinator of AICRP on Spies, Kozhikode for support and constant encouragement. Authors are also thankful to Directorate of Research, Indira Gandhi Krishi Vishwavidyalaya (IGKV) for providing facility for conducting trial successfully. Abstract The research work was carried out to study the impact of various irrigation methods and mulching on plant growth, production and profitability of chilli cv. R.Ch. 1 at Agricultural Research Station, Mandor, Jodhpur during July, 2016 to February, 2017. The results of surface irrigation were compared with drip irrigation system under no mulch and in conjunction with plastic mulch. The results revealed that the crop was irrigated by drip irrigation on raise bed with 100 micron Linear Low Density Poly Ethylene plastic mulch (T 8 treatment) exhibited significantly higher seedling survival at 15 and 30 days after transplanting (95.16% and 91.70%), highest plant height (47.10 cm at 45 DAT and 54.60 cm at harvest), highest number of branches (14.93) plant -1 , maximum stem girth (2.32 cm) number of roots plant -1 (138.5), highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm) and fresh weight of fruit -1 (8.42g) was observed. The maximum number of fruits plant -1 (125), highest yield plant -1 (1052.5g), yield ha -1 (337.63q) and premier fruit quality score (9.11) with maximum net return (Rs.326407.28) and benefit: cost ratio (3.41) was also reported in same treatment. Comparatively minimum time (15 hours) required for one hectare irrigation was also reported in drip irrigation on raise bed with plastic mulch. This led to lower population of white fly plant -1 (4.53), minimum weed infestation (1.53 weed m -2 ), leaf curl (5.50%) and fruit rot (5.0%) incidence than other treatment combinations. The minimum growth, yield and profitability were reported in check basin method of irrigation without mulch (T 1 treatment). Chilli (Capsicum annuum L.) is an important commercial vegetable cum spice crop of India belongs to the family Solanaceae. The production of chilli crop is affected adversely by moisture deficit. Productivity of the crop can be increased by adopting improved package of practices, particularly in-situ moisture conservation by mulching as well as high-tech irrigation especially drip irrigation with appropriate irrigation scheduling. Use of soil cover and mulching is also known to be beneficial chiefly through their influence on soil moisture conservation, solarization and control of weeds. Beneficial response of plants to mulch includes early production, more yield and reduced insect and disease problems (Pattanaik et al. 2003). Linear Low Density Poly Ethylene (LLDPE) plastic films have been proved as better mulch because of their puncture resistance quality, thinness and lower cost (Panda 2004). Numerous experiments have reported the benefits of LLDPE mulch in several crops, but research is limited on response of chilli production in western Rajasthan by this method. Keeping this in background, the present study was undertaken to study the effect of different irrigation methods and mulching on chilli crop and compare the result with the conventional method of growing the crop under surface irrigation without mulch. A field experiment was conducted at Agricultural Research Station, Mandor, Jodhpur (Rajasthan), India during kharif seasons of year 2016-17. The soil of experimental plot was of sandy loam texture with average pH range 8.5, having organic carbon 0.55%, available N 180 kg ha -1 , P 27.5 kg ha -1 and K 250.0 kg ha -1 during experimentation. The experiment was conducted in a completely randomized design having nine treatments comprising by different irrigation methods and mulching viz., T 1 = Check basin method, T 2 = furrow irrigation method, T 3 = Raise bed with trench method, T 4 = Flat bed with drip irrigation, T 5 = Flat bed + plastic mulch + drip irrigation, T 6 = Raise bed with drip irrigation, T 7 = Raise bed + organic mulch + drip irrigation, T 8 = Raise bed + plastic mulch + drip irrigation, T 9 = Sprinkler irrigation method. In well prepared field, transplanting of Chilli seedlings variety RCh 1 of 35-40 days old were planted in pair row method with a spacing of 45cm x 45 cm/90 cm (33,333 plant ha -1 ) during last week of June. In check basin and sprinkler system of irrigation the bed size is 2 x 2 meter and in all other methods is 1 × 4 meters. The cultural practices of the crop were followed as per the recommendations. The organic material and LLDPE silver colour film of 100-micron thickness was used for mulching around the plant. The lateral lines of 12 mm diameter LLDPE pipes were laid along with crop rows. The laterals were provided with inlet drippers of 8 litre hr -1 discharge capacity. All the observations were taken from five randomly selected plant of each replication throughout the investigation period at appropriate time by adopting standard method for growth, development, fruiting behavior and yield. Seedling survival per cent (after transplanting in main field at 15 DAT and 30 DAT) was recorded by following formula; Survival percent = [Total survival transplanted plants / Total transplanted plants] × 100 Plant height (at 45 DAT and at harvesting) was measured from soil surface upto the highest shoot tip by straightening all branches. Stem girth was measured 1 cm from the base of the stem using vernier calliper. Observation of number of branches, days taken to first flower initiation, duration of fruiting period and number of fruit plant -1 was recorded by standard counting method. Number of roots, root length was measured by destructive method of uprooting the plants and taking measurement by standard method. Length of fresh fruits measured by scale and fruit diameter using vernier calliper and expressed in centimeter. Fruit set per cent was recorded by following formula; Fruit set per cent = [Total number of fruit set plant -1 / Total number of flowers plant -1 ] × 100 Fruit weight was determined by weighing method at the time of harvesting and expressed in gram fruit -1 . The total fruit yield plant -1 and hectare -1 was calculated by weighing total marketable fruits and has been expressed in gram and quiantal respectively. Further, the net return was calculated by subtracting cost of each treatment from gross return. The gross return was calculated from yield multiplied by average market rate during the period of investigation. The benefit cost ratio was calculated by dividing net return to total cost of cultivation. Benefit-Cost ratio and net profit were carried out to determine the economic feasibility of the crop using surface and drip irrigation as suggested by Tiwari et al. (1998a). 83 The seasonal system cost of drip irrigation system included depreciation, prevailing bank interest rate, and repair and maintenance cost of the system. The fixed cost of drip irrigation system was determined to be Rs 112,000 ha -1 . The useful life of drip system was considered to be 10 years. The system cost was evaluated by distributing the fixed cost of system over life period of drip irrigation set. For calculating depreciation, the life of the drip irrigation set and 10% junk value was considered. The interest was calculated on the average of investment of the drip irrigation set taking into consideration the value of the set in the first and last year @10% per annum. Cost of repairs and maintenance of set is @2% of initial cost of the drip irrigation set per year. The cost of cultivation includes expenses incurred in land preparation, interculture operation, fertilizer, crop protection measures, irrigation water and harvesting with labour charges. Therefore, total seasonal cost was worked as: depreciation, interest, repairs and maintenance cost of set + cost of cultivation + cost of mulch. The income from produce was calculated using prevailing average market price of capsicum @ Rs 1250 q -1 . Disease incidence (leaf curl and fruit rot) and quality of fruits was measured by visual inspection (Five member team of crop experts and plant pathologist). White fly population plant -1 and weed infestation meter -2 was calculate by simple counting method. The time required for irrigation was calculated as per actual required time of irrigation of specified area by different methods of irrigation. To test the significance of variance of data obtained from crop growth, yield and economics of variance technique for completely randomized design was done by standard procedure prescribed by Panse & Sukhatme (1985). Significance of difference among the treatments effect was tested by 'F' test and critical difference (CD) was calculated, wherever the results were significant. The results revealed that, the irrigation methods and mulching are significantly influenced growth attributes at all the growth stages ( Table 1). The maximum seedling survival per cent at 15 DAT (95.10%) and 30 DAT (91.70%) was recorded in T 8 treatment, which was significantly superior to other treatment but at par with T 6, and T 7 treatments. The maximum survival per cent of seedling in T 8 treatment might be due to more favourable moisture condition for seedling transplanting and reestablishment of roots than others. The height of plant under treatment T8 (47.10 cm at 45 DAT) and treatment T6 (62.60 cm at harvest) was found highest among all other treatments and is 67.19% and 13.40% higher than the T 1 treatment. About to number of branch plant -1 , maximum value was recorded in treatment T8 (14.93) followed by treatment T7 (12.53) and the lowest value was in treatment T1 (7.17). Maximum stem girth at harvest (2.36 cm) and highest number of roots plant -1 (138.50) were observed in T 8 treatment whereas longest root system (10.50 cm) was observed in T 3 treatment. The minimum stem girth (1.68 cm) and the number of roots plant -1 (53.57) were observed in T 1 treatment whereas shortest root system (7.97 cm) was observed in T 9 treatment. The higher available moisture status in soil favourably influences the uptake of nutrients which maintains the cell turgidity, cell elongation, photosynthesis and respiration at optimum level, leading to favourable growth and development of plant in terms of plant height, number of branches plant -1 , stem girth and number of root plant -1 in the present study. The highest increase in vegetative growth in drip irrigation with mulching might be due to the availability of soil moisture as well as favourable temperature at optimum level for plant growth development (Pattanaik et al. 2003;Paul et. al. 2013). The lowest value of vegetative growth in T1 might be because of unfavourable moisture regime (moisture stress or excess moisture) in the soil through surface irrigation and competition of weeds for nutrients (Pattanaik et al. 2003;Agrawal & Agrawal 2005). The increased growth attributes might have supplied water and nutrients in adequate proportion, which resulted in triggering the production of plant growth hormone, viz., indole acetic acid (IAA) and higher number of leaves and roots throughout the cropping period (Sankar et al. 2008). The drip irrigation in combination with mulch significantly increased the yield of chilli as compared to drip irrigation without mulch (Table 2) and surface irrigation methods. The minimum days (42.38) required for first flower initiation was reported in T 9 treatment whereas the maximum days (51.39) was required in T 5 treatment. Among various treatments, highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm), duration of fruiting (71.38 days), fresh weight of fruit -1 (8.42 g), maximum number of fruits plant -1 (125), highest yield plant -1 (1052.5 g) and yield ha -1 (337.63 q) was recorded under T 8 treatment, whereas lowest yield (153.45 q ha -1 ) was recorded under T 1 treatment. This might be due to water stress during the critical growth period and fruit development stage coupled with aeration problem in first few days immediately after irrigation. Another reason to get low yield by surface irrigation without mulch might be due to less availability of nutrients for crop growth due to leaching and high weed competition between the crops (Pattanaik et al. 2003). In drip irrigation system on raise bed with plastic mulch the water is applied at a low rate for a longer period at frequent intervals near the plant root zone through lower pressure delivery system, which increases the availability of nutrients near the root zone with a reduction in leaching losses and minimum weed competition. More nutrient availability, especially near the root zone might have increased the translocation of photosynthetes to storage organ of chilli resulting in an increased weight of fruits. This result corroborated the findings of Singh (2007), Sankar et al. (2008), Paul et al. (2013) and Kumar et al. (2016). Irrigation methods and mulching also significantly influenced the gross return, net return and benefit cost ratio in chill (Table 3). Maximum net profit of Rs. 326407.28 ha -1 with B: C ratio of 3.41 was recorded in T 8 treatment followed by Rs 296192.61 ha -1 with B: C ratio of 3.11 in T 5 treatment and lowest net profit of Rs 119007.80 ha -1 with a B: C ratio of 1.63 in T 1 treatment (Table 3). It is observed that, the drip irrigation with mulched treatments T 5 , T 7 and T 8 gave better net return with higher B: C ratio ha -1 than their corresponding treatments without mulching in conventional irrigation method. The highest net return (US$ 7098 ha -1 ), incremental net return (US$ 1556 ha -1 ), and incremental benefit-cost ratio (7.03) were found for 50% water application with straw mulch (Biswas et al. 2015). The results are in conformity with the findings of Singh (2007), Sankar et al. (2008) and Kumar et al. (2016). Apart from reducing water consumption, drip irrigation with mulching also helps in reducing cost of cultivation and improving productivity of crops as compared to the same crops cultivated under flood method of irrigation (Paul et al. 2013). Irrigation time significantly pretentious by different irrigation methods. The minimum time required in irrigation (14.50 hours) in T 5 treatment which closely followed by T 8 treatment. Drip irrigation method with or without mulching required less irrigation time than without mulching in conventional irrigation method. There was significant effect of LLDPE mulch over drip irrigation system alone. Drip irrigation with LLDPE mulching (T 5 & T 8 treatment) saving irrigation time (21.40 hour ha -1 and 21.35 hour ha -1 ) upto 60 per cent by reducing water losses and increased irrigation efficiency. The increase in water saving per cent in trench method (T 2 ), drip irrigation system alone (T 4 ), drip irrigation system with LLDPE mulch (T 5 & T 8 ), drip irrigation with organic mulch (T 7 ) and sprinkler system (T 9 ) over conventional surface irrigation by check basin method (T 1 ) was 38.8%, 46.6%, 60.0%, 54.4% and 41.9% respectively. The highest water use efficiency of 592 kg ha -1 mm -1 was obtained with 50 per cent water application under polyethylene mulch (Biswas et al. 2015). Drip irrigation with mulching helps to achieve yield gains of upto 100 per cent, water savings of upto 40-80 per cent, and associated fertilizer, pesticide, and labour savings over conventional irrigation systems in capsicum crop (Paul et al. 2013). Similar trend has been reported in water use efficiency for okra crop by Tiwari et al. (1998a) and for tomato crop by Singh (2007). Occurrence of chilli leaf curling and fruit rot was detected throughout the investigation period. The best performance, with a marked reduction in leaf curling and fruit rot and improve fruit quality was observed in drip irrigation with LLDPE mulching (Table 3). The minimum incidence of fruit rot (5.0%), leaf curing (5.5%) and highest quality score of fruit (9.11) was observed in T 8 treatment which was closely followed by T 7 and T 5 treatments where as maximum incidence of fruit rot (20.02%) and leaf curling (15.84%) was reported in check basin method of irrigation (T 1 treatment) whereas minimum quality score (6.50) was observed in T 9 treatment. Presence of white fly and weed was observed throughout the investigation period. The minimum population of white flies (4.53 plant -1 ) and weed infestation (1.53 weed m -2 ) was observed in T 8 treatment whereas highest incidence of white flies (17.43 plant -1 ) and weed infestation (30.03 weed m -2 ) was observed in T 1 treatment. This is due to the fact that in drip irrigation with mulching significantly reduced additional moisture level in field environment which in turn increase quality of fruits and reduce disease infestation, white fly population as well as it also trim down weed seed germination, growth and development. The increase in quality of fruits was due to the effective utilization of applied nutrients, water and significantly reduced weed growth; disease incidence and increased rate of photosynthesis, sink capacity and accumulated more amounts of dry matter and finally increased quality of fruits and yield. Conventional surface irrigation methods without mulching provide favorable environmental condition for increase insect population and development of disease as well as germinate and develop high density weed plants. The beneficial effect of drip irrigation and black LLDPE mulch in capsicum, tomato and okra was also reported earlier by Horo et al. (2003); Singh (2007) The combination of raised bed + drip irrigation system with LLDPE mulching is observed to be economical and cost effective as compared with conventional surface irrigation without mulching. Thus, the use of drip irrigation system either alone or in combination with mulching, could increase the chilli yield quality of fruits and profitability. It also reduces white fly population, disease incidence (root rot and leaf curling) and minimise with crop weed competition. Drip irrigation with mulching increase water use efficiency by significant reduction in irrigation time ha -1 . It is concluded that the drip irrigation method with LLDPE mulching is suitable for chilli production in arid and semi arid condition of western Rajasthan. Isabgol (Plantago ovata Forsk) is a short duration, more remunerative and medicinally important crop of arid and semiarid regions. In India, it is largely grown in Gujarat, Rajasthan, Madhya Pradesh and Haryana. The area under Isabgol in India during 2014-15 is 1.09 lakh hectares. The production recorded 72 thousand MT with productivity of 660 kg ha -1 . In Gujarat, area mostly falls in Banaskantha, Kachchh and Patan districts with acreage of nine thousand hectare with production and productivity 5000 MT and 556 kg ha -1 , respectively during 2015-16 (Anonymous 2016a). During the last decade, area and production of isabgol has decreased to the tune of 343% and 281%, respectively, mainly due to problems of seed shattering. At the time of maturity, unseasonal rain or heavy dew leads to failure of the crop which is the fact for reduction of area under Isabgol (Anonymous 2016b). The objective of the study was to evolve non-shattering isabgol cultivars. The stability analysis of variance and stability parameters viz., linear regression coefficient (bi) and deviation from regression (S 2 di) of genotype means over environment were computed as suggested by Eberhart & Russell (1966). 89 plants in each replication were selected at maturity stage. Entire spike were dipped in water and then observed for seed shattering from selected plants. The percentage of seed shattering calculated by using a following formula. Gujarat Isabgol 4 recorded high mean with regression coefficient (bi) near unity and deviation from regression (S 2 di) around zero for seed yield, indicating GI-4 has average responsiveness and are highly stable over environments ( Table 2). The new culture has compact spike and did not separate easily even after dipping in water. Only 7.25% seeds shattered after dipping in water. Swelling factor (cc g -1 ) 11.4 9.1 Patel et al. GI-4 recorded 89.67% less shattering than that of check variety, which revealed that Gujarat Isabgol 4 was non shattering in habit as compared to Gujarat Isabgol 3 which is prone to high seed shattering. The post dipped seed weight of GI 4, was 0.115 kg threshed seeds, which was 96% higher than that of the post dipped seed yield of GI-3 (0.037 kg threshed seeds) (Table 4). Hence, Gujarat Isabgol 4 recommended for cultivation.

v3-fos

2020-12-10T09:04:11.456Z

1970-11-01T00:00:00.000Z

237235090

s2

Metabolic Fate of Cysteine and Methionine in Rumen Digesta Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35S and 14C, it was observed that a large proportion of the 35S appeared in the sulfide pool and of the 14C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14C to 35S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 35S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources. Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35S and 14C, it was observed that a large proportion of the s5S appeared in the sulfide pool and of the 14C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14C to 35S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1 % and for methionine, at most 11 % of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 36S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources. Several reports have appeared (2-5, 9, 11, 15) indicating that inorganic sulfur compounds in the rumen are used in the synthesis of sulfur amino acids and that the pathway involves reduction to sulfide prior to upgrading into the protein of rumen microorganisms. Evidence cited by Allison (1) supports the idea that microorganisms of the rumen obtain most of their amino acid requirement by de novo synthesis. There seems good reason (2,11) to apply the same concept to the sulfur amino acids, and this has been made the basis of a method for measurement of proteinsynthesis rates of rumen microbes (22). The possibility remains that some direct incorporation ofsulfamino acid may occur and there is a good deal of qualitative work (6,7,12,20,24) dealing with this. It seems, however with one exception (14), that no attempt has been made to determine quantitatively the extent to which this may occur under normal conditions in rumen contents. We report here such a quantitative appraisal based upon change of the 14C to 35S ratio of added, doubly labeled methionine and cysteine. MATERIALS AND METHODS Labeled sulfamino acids. 35Sand 14C-labeled cysteine and methionine were obtained from the Radiochemical Centre, Amersham, England. Cysteine-U-4C of specific activity 18 Methionine-1,2,3,4-14C of specific activity 39 mCi/ mmole, containing a total of 100,pCi, was made to a volume of 5 ml (solution C). 3S-methionine of specific activity 270 mCi/mmole, containing a total of 500 pCi, was made to a volume of 16 ml (solution D). A 1.2-ml amount of solution C was mixed with 0.8 ml of solution D. To maintain concentrations of sulfur amino acids in rumen contents as close to normal as possible during the experiments, no carrier amino acid was added to either mixture. Incubation of labeled amino acids with rumen content. Rumen-content samples were obtained via a rumen fistula from a merino ewe which had been fed daily a diet of 500 g of wheaten hay chaff and 500 g of luceme hay chaff. On the day of each experiment, approximately 200 g of rumen content was taken as described by Walker and Forrest (21). A 10-g amount of this material was introduced into each of two preweighed Clin-Britic vaccine bottles (Britton-Malcolm and Co. Ltd., London, England), and the space above the rumen contents was ifiled with a gas mixture of 60%o N2-40% CO2. To each vessel was added 350 ,uliters of the prepared mixture of cysteine U14C: 8aS by injection through the rubber seal. For the methionine experiment, the design was identical except that 250 ,uliters of the prepared mixture of methionine-1,2, 3,4-14C: 5S was added. The unused portion of both radioisotope solutions was deep frozen and kept for measurement of the precise amount of each radioactive species present. Treatment of reaction mixtures containing labeled sufamino acids. After incubation at 39 C in a shaking bath for 105 min, the rumen contents were acidified with 10 ml of 6 N HCI, the evolved H2S was collected into 10% H202-1.5% NH3 mixture, and the 3sS present 677 in this solution was measured as BaSO4 (22). The solids after washing and extraction with 0.05 M Na2S were hydrolyzed in 6 N HCI for 16 hr after the addition of 10,umoles of the appropriate unlabeled amino acid as carrier. When methionine was the amino acid studied, 2-mercaptoethanol (1:2,000, v/v; reference 13) was added to the samples to minimize oxidative losses during the hydrolysis. HCI was removed from hydrolysates by rotary evaporation with two additions of distilled H20. Humin was removed from the samples by treatment with activated carbon and filtration. Isolation and purity testing of cystine and methionine. Cystine was purified on the basis of its insolubility at neutral pH. The hydrolysate from the rumen-content sample incubated with labeled cysteine was made to a volume of 20 ml, 2 ml was absorbed to a column of cation exchange resin (H+ form), and the amino acids were eluted from the washed column with 2 N NH40H. Cystine was precipitated from the eluate, after concentration, by adjustment of the pH to neutrality in a centrifuge tube. The precipitated cystine, after repeated water washing to remove traces of other amino acids, was redissolved in 3 ml of 0.1 N HCI. The sample The hydrolysate from the methionine experiment was made to a volume of 20 ml, and samples of this were used for methionine preparation by two independent methods. First, 2 ml was absorbed to a cation exchange resin and the amino acids were eluted with 2 N NH40H. The eluate was dried and the redissolved amino acids (10% ethanol-water), after streaking on several 3-mm Whatman papers, separated into neutral, basic, and acidic fractions by electrophoresis in pyridine-acetic acid-water (2.5:1.5:96) buffer (pH 5.8) at 20 v/cm for 75 min. The neutral bands were eluted with 10% aqueous ethanol; the eluates were pooled, dried in a rotary evaporator, and redissolved in 1 ml of the same solvent. The neutral fraction was chromatographed in n-butanol-acetic acid-water (25:6:25, v/v; reference 18). A band corresponding in position to a marker methionine spot was eluted and rechromatographed in n-butanol-pyridine-water (1:1 :1, v/v; reference 18). The band eluted from the first solvent system was found to contain valine as well as methionine which were sufficiently well separated in the second solvent system to allow elution of a pure sample of methionine. This material was further tested for purity by electrophoresis in 1.5 M formic acid solution (8). In the second method, 200juliters of hydrolysate was separated into component amino acids by chromatography in a Beckman amino acid analyzer (model 120C) on a 150-cm column eluting with 0.2 M citrate buffer (pH 3.25 initially) and changing to 0.2 M citrate buffer of pH 4.30 (16,17). Buffer change occurred just prior to the position of the methionine peak which was determined from a reference mixture of amino acids. The five fractions comprising the methionine peak were pooled, evaporated to dryness, and redissolved in 300,uliters of water. Purity of the fraction was established by using 60 jliters for electrophoresis in 1.5 M formic acid on Whatman no. 4 paper at 21 v/cm for 90 min, by using a reference solution of methionine containing the same final concentration of citrate as the unknown. Radioactivity in amino acids. Total 35S plus 14C was determined in added and purified samples of cysteine and methionine by counting small portions of sample dissolved in 2.5 ml of ethyl cellosolve with the addition of 5 ml of diphenyloxazole-toluene (0.3%, w/v) phosphor. A Packard 3375 scintillation counter was used throughout. Settings used were those optimal for '4C-window 50-500, amplifier gain 8%. To count 35S, the same volume of sample as for 14C counting was first wet-ashed in HNO3-HC104 mixture after the addition of 1 ml of N H2SO4 as carrier. Total sulfate was precipitated as BaSO4 and counted as a suspension (22) at conditions optimal for 35S (window 50-1000, amplifier gain 20%) as well as at 14C settings. Corrections for minor quenching differences measured by using automatic external standardization were applied, and the 14(2 to 3S ratio was calculated from: (counts per minute of undigested sample at 14C settingscounts per minute of wet-ashed sample at 14C settings)/counts per minute of wet-ashed sample at 35S settings. To eliminate errors due to isotope decay, 35S activity in the purified amino acid was determined at the same time as the 3nS activity of the appropriate added amino acid. For convenience of presentation of results, however, 85S activities were adjusted back to the day of each experiment. 14C in volatile fatty acids. The volumes of the aqueous washings obtained from acid-treated rumen contents before Na2S washings were measured, 2 ml samples were steam-distilled, and pH of the distillate was adjusted to 10 with NaOH. After concentration over a microburner to a volume of about 20 ml, 500 ,uliters was counted in 10 ml of Bray's phosphor. RESULTS AND DISCUSSION From analysis of I'S present in the sulfide pool and of 14C present in the VFA pool after incubation of 14C: 35S amino acid with rumen contents, it is clear that both cysteine and methionine were extensively degraded ( Table 1). The fact that a greater proportion of the added 35S than the 14C was recovered in these pools is undoubtedly due to formation of 14CO2 and 14C(H4 which were not recovered for assay. The size of the H2S pool during the incubations (about 10 zg of S per ml) was such that the amount of sulfur added as amino acid was low by comparison, being about 3% in the cysteine experiment and 0.3% in the methionine experiment. Consequently, there was a large trap for sulfide arising by degradation of the added amino acid. Thus, the finding of almost 90% of the added cysteine 35Sand over 50% of the added 35S-methionine in the sulfide pool gives a rough indication of the extent of degradation. Table 2 shows the '4C to 35S ratios in the amino acids as added to rumen contents and as isolated from microbial protein. The dramatic change in the ratio for microbial cysteine indicates an enrichment of 17.5-fold of 35S over 14C. Consequently, it can be stated that over the incubation period a maximum of about 6% of the added radioactive cysteine was directly incorporated into microbial protein. In the case of methionine, the dilution of 'IC compared to I'S in the amino acid isolated from microbial protein is substantially lower than the dilution obtained with cysteine. As a result, the calculated maximum direct incorporation for added radioactive methionine is 44%. In both experiments, the incubation period (105 min) was long to allow substantial total incorporation of radioactivity. This, together with the fact that very small quantities of amino acid were added as labeled species, allows us to assume that the behavior of the labeled amino acids represented that of the corresponding pool, and that virtually all of the labeled amino acid would have been metabolized. From Table 1 it is seen that 86% of the added 85S of cysteine was present in the H2S pool after 105 min, leaving 14% of the label incorporated into protein. Of the 14%, it is evident from the methionine were used for change in 35S to 14C ratio ( Table 2) that only 0.06 arose directly from added cysteine. If all of the incorporated 35S was present in the cysteine of protein then 14 X 0.06 = 0.8% of the cysteine pool was directly used for protein synthesis and 99% was metabolized via H2S. Since some 35S must have been incorporated into methionine in protein, the extent of direct incorporation of free cysteine into protein is overestimated. In the case of methionine, the proportion of the added 35S finally recovered in the H2S pool was 51%, leaving 49% incorporated into protein. From the change in 35S to '4C ratio, 0.44 of the label incorporated into methionine arose directly from the methionine pool. Thus, if all of the 35S present in the protein finally were in methionine, 49 X 0.44 = 22% of the methionine pool would be directed unchanged into protein. However, as we have just seen, virtually all of the cysteine sulfur entering the free amino acid pool is metabolized via H2S, requiring de novo synthesis of cysteine for protein synthesis. Since there are approximately equal amounts of cysteineand methionine-S in rumen microbial protein (23) and since the larger proportion of methionine-S is obviously metabolized via H2S, roughly half of the label incorporated in the methionine experiment would have been in cysteine. Consequently, only about 11% of the methionine would be directly incorporated into protein and 89% would be degraded and the sulfur metabolized via H2S. Extensive degradation of added cysteine is compatible with results published by Landis (14), in that he found that a large proportion of 85S label from cysteine appeared in methionine of the rumen microbes of the goat. However, his conclusion that 75 to 83% of methionine is directly incorporated into microbial protein is at variance with our work. Since Landis did not isolate and purify the amino acids from the microbes, we consider that he was estimating "bound-but-not incorporated" 35S-sulfide (22) as well as 35S-methionine, thus obtaining falsely high results. Emery, Smith, and Huffman (6,7) in studies with rumen liquor incubated with 35SO4 found that the label was incorporated into cystine, methionine, and glutathione of the microorganism protein, but, by using the relatively insensitive technique of microradioautography, suggested that the majority of the organisms did not take up the label. They also reported on the ability of 10 different strains of ruminal organisms, in pure culture, to utilize SO4--S and found that only three were able to do so. This is not surprising since it is most unlikely that all strains of organisms in the rumen have the ability to use S04as a terminal oxidant, converting it to sulfide which is required for de novo sulfamino acid synthesis (2, 11). Emery et al. (6,7) also found that Lachnospira multiparus uses cysteine-sulfur in preference to inorganic SO4-in pure culture. Such a finding tells nothing of the pathway of utilization of cysteine, that is whether it was first desulfhydrated to H2S, pyruvate, and NH3 prior to cysteine synthesis de novo. Consequently, the findings of these workers do not have direct bearing on sulfamino acid metabolism by the mixed rumen microflora. Wright (24) has demonstrated the incorporation into microbial protein of 14C from radioactive peptides incubated with rumen contents, the peptide apparently being utilized slightly faster than the corresponding amino acids. However, from the figures given, the concentration of peptide amino acid added was about 2 ,umoles/g of rumen content which is about 30 times the concentration of total amino acids generally found by Wright and Hungate (25). Since there are no reports of the concentration of peptides as such in rumen fluid, it is not possible to assess the direct incorporation of sulfamino acids into microbial protein by peptide utilization. The finding that a small proportion of the APPL. MIcRoBIoL. methionine entering the free amino acid pool is reutilized without prior degradation means that the method for determining microbial protein synthesis based upon incorporation of "S from the sulfide pool (22) yields a slight underestimate. From the amino acid analyses given for plant and microbial protein by Weller (23), it can be calculated that 0.58 g of cyst(e)ine-sulfur and 0.55 g of methionine-sulfur are present in 100 g of crude plant and microbial protein, so that approximately equal amounts of sulfur are presented to and incorporated by the organisms as methionine and cyst(e)ine. But since calculations presented here show that 89% of methionine-sulfur turnover and 99% of cysteinesulfur turnover is directed through H2S, the method for measuring microbial protein synthesis based upon "5S incorporation from the H2S pool (22) would account for (89 + 99) /2 or 94% of total sulfur incorporation, an underestimate of 6%, under conditions where all of the sulfur available for protein synthesis arises from dietary amino acids. Since normally other sources such as inorganic salts would contribute to the sulfur available, the above underestimate must be considered maximal. LITERATURE CITED

v3-fos

2018-04-03T05:41:56.669Z

1970-11-01T00:00:00.000Z

19231959

s2

Dry-Heat Destruction of Bacillus subtilis Spores on Surfaces: Effect of Humidity in an Open System Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 X 104 for spores conditioned and tre ated at the low humidities to 2.00 X 105 for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of No, the y-intercept appears to be lowered by low-humidity conditions. The statistic log yo/log No is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2 % RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments. the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments. Dry heat has been chosen to sterilize planetary lander spacecraft (12). To preserve reliability and minimize degradation of spacecraft materials, the sterilization treatment should be the iinimum which will achieve the desired results (12). Therefore, it is necessary to evaluate carefully the factors which affect dry-heat sterilization. The important effect of water on the dry-heat destruction of bacterial spores has been reported by a number of authors (1, 6,7,9,10). Both the relative humidity of the environment prior to treatment (conditioning humidity) and the relative humidity during treatment (treatment humidity) affect the dry-heat destruction of bacterial spores. The present study was designed to separate and measure the effect of conditioning humidity and treatment humidity in an open system for one typical spore suspension. MATERIAL AND METHODS Bacillus subtilis var. niger spores were deposited on stainless-steel strips and hung in a gravity convection oven with the surfaces parallel to the air stream. The spores were grown in our laboratory from spores supplied by the Communicable Disease Center Field Station, Phoenix, Ariz. The spores were grown on Difco TAM agar (8) approximately -10 C. After several months of storage, the spores were rinsed and resuspended in sterile distilled water; the suspension was subdivided into many small bottles and stored at approximately -10 C. A bottle of spores was thawed for each test; unused spores were discarded. Thus all spore samples had the same handling history except for the length of frozen storage. After thawing, the bottle containing the spore suspension was agitated in an ultrasonic bath and 0.02 ml samples (2.34 X 106 spores) were deposited on the test surfaces with an Eppendorf micropipette. Each test surface was a stainless-steel strip (1 by 2 inch, ca. 2.54 by 5.08 cm) which had been washed; rinsed sequentially in distilled water, isopropyl alcohol, and ether; and then sterilized with dry heat (11). All preparation and analysis procedures were carried out in a class 100 laminar downflow clean room (5). The contamination rate under these conditions was indistinguishable from zero. After the spore suspension was deposited on the test surface, it was dried at 23 C for 18 hr in the clean room. The test strips were then placed in plastic glove boxes over night for water conditioning. In the glove box, the relative humidity was maintained at less than 2 or 89% by using wetted silica gel (3). humidity of the boxes was measured with a Honeywell model W611A relative humidity indicator. The treatment system used a modified gravity convection oven (Blue M model #OV-12A) shown in Fig. 1. The strips (1 by 2 inch) were hung on racks and inserted through the small magnetically held doors to avoid disturbing the temperature of the oven. A rack with strips is shown in Fig. 2. A diffuser plate inside the oven helped to minimize temperature variation across the oven. A variable transformer and thermistor controller (Honeywell model 3679) were added to the oven to decrease "overshoot" and to promote more accurate temperature control. To maintain the relative humidity of the treatment system, the oven was placed in a refrigerator or in a humidified incubator where the dew point was maintained at 5 or 21 C. This produced treatment relative humidities of 0.34 and 1.1%, respectively, at the treatment temperature of 125 C. To measure temperatures in the oven, 12 thermo-couples were located in a grid above the location of the samples. The temperature of each strip during treatment was measured by a thermocouple directly above it. All data presented here have been corrected for slight temperature variation by using the NASAadopted z-value of 21 C. (2). Four treatment times (10, 30, 50, 70 min) and three strips per treatment time were used. The data presented here were from two replicate experiments. Recovery of spores from the strips followed the NASA Standard Methods (11). Strips were placed in 125-ml flasks with the contaminated surface facing downward; 50 ml of phosphate buffer was added, and the sample was sonically treated for 2 min in the center of an ultrasonic bath filled with a 0.3%0 solution of Tween 80 in distilled water (11). Aliquots of 0.05, 0.1, 1.0, or 10 ml, or combinations of these amounts of fluid, were plated in duplicate on Trypticase Soy Agar. Plates were incubated at 32 C for 48 hr. To avoid systematic errors, samples were randomly assigned to treatments and the processing and counting of petri plates was carried out in random order. To the extent possible, consistent with the volume of processing, the number of a plate in the experimental scheme was concealed from the person who counted it. The data were analyzed by a combination of linear regression and analysis of variance. Linear regression gave estimates of the slopes and intercepts of the various survival curves. The analysis of variance distinguished more precisely between the effects of various treatments and indicated whether the effects of the treatments and their interactions were significant. The analysis of variance was a combination of factorial and split-plot approaches. Each whole plot was a rack, carrying three strips, which was exposed to a particular treatment time and process. The whole plots were analyzed by a factorial scheme which allowed the effects of conditioning and treatment humidity to be separated. For both analyses the data were transformed according to the formula x = logio (datum + 1.0). The transformation put the data in the standard semilogarithmic format for the linear regression. It also made the variance of the data independent of the mean which is a requirement for analysis of variance. The number 1.0 was added to all data values so that data values of zero could be included in the analysis. The error produced by the addition of 1.0 was not significant. RESULTS AND DISCUSSION Table 1 shows the data from which the analyses were performed. Figure 3 summarizes the results in graphical form. Each point represents the geometric mean (mean of logs) of six strips, three processed in each of two replicate experiments. The lines are the least squares regression lines. Fig. 3 and Tables 2 and 3, note that the initial numbers were ignored in the data analysis because, as has been found by others (4,13), the behavior of the thermal destruc- ing the intercept as well as the slope of thermal death curves. The difference between the low-low and low-high D-values is rather small, but, when combined with the difference in intercept, there is a more than 10-fold difference in survival after 100 min of treatment. Variation was attributed to the day on which the experiment was carried out. The reason for this is not known at this time, but the problem is under study. Despite the day-today variation, it has been repeatedly confirmed for this and other experimental systems that within a given day the relationships between the data are preserved. The humidity both before and during treatment affected the survival of B. subtilis var. niger spores in the experiments reported herein. The effects should be considered separately. To describe the results of a thermal kill treat-of freedom; MS, mean square; F, frequency. ment, both the slope (D-value) and the intercept must be specified. Small changes in treatment humidity caused rather large changes in the killing effectiveness of dry heat in the reported experiments. A combination of regression and analysis of variance approaches to data analysis increases the amount of information which can be obtained from a given experiment.

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2020-12-10T09:04:11.457Z

1970-10-01T00:00:00.000Z

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Dry-Heat Inactivation Kinetics of Naturally Occurring Spore Populations Twenty-three soil samples were collected from areas of the United States where major spacecraft assembly and launch facilities are in operation. Soil samples were treated with ethyl alcohol, ultrasonic energy, and gross filtration. The resultant suspensions consisted of viable, naturally occurring bacterial spores and were used to inoculate stainless-steel strips. The strips were suspended in a forced air oven and assays were made at 5-min intervals for the number of viable spores. Most survivor curves were nonlinear. Subsequently, spore crops of heat-sensitive and heat-resistant soil isolates were found to have linear survivor curves at 125 C which were unaffected by the presence or absence of sterile soil particles from the parent sample. When two spore crops, one of which was heat-resistant and the other heat-sensitive, were mixed, the resultant nonlinear curves were unaffected by the presence or absence of sterile parent soil. Therefore, the survivor curves obtained originally with the soils were the result of heterogeneous spore populations rather than of protection afforded by soil particles in our test system. These results question the rationale both of assuming logarithmic death and of using decimal-reduction values obtained with subcultured standard reference spores in the derivation of dry-heat sterilization cycles for items contaminated with naturally occurring spore populations. were mixed, the resultant nonlinear curves were unaffected by the presence or absence of sterile parent soil. Therefore, the survivor curves obtained originally with the soils were the result of heterogeneous spore populations rather than of protection afforded by soil particles in our test system. These results question the rationale both of assuming logarithmic death and of using decimal-reduction values obtained with subcultured standard reference spores in the derivation of dry-heat sterilization cycles for items contaminated with naturally occurring spore populations. The National Aeronautics and Space Administration (NASA) requires that spacecraft which may impact Mars or other planets of biological interest be sterilized (NASA, Outbound Planetary Biological Contamination Controi, NASA Policy Directive 8020. 10, 7 September 1967). The problems associated with the delivery of a sterile capsule to a particular planet are manifest and cannot be fully covered in this paper so that reference is made to the more detailed reports of Bruch (2, 3), Craven et al. (5), Favero (8), Hall (11), and Light et al. (13). The basic approach that will be used to produce a sterile spacecraft involves manufacturing, testing, and assembling the hardware under rigid environmental controls, enclosing it in a hermetically sealed canister, and subjecting this unit to a terminal dry-heat sterilization cycle. Once sealed and heated, the biological barrier (canister) cannot be broken to determine that the spacecraft is in fact sterile. Therefore, sterility of the spacecraft is expressed as a low probability which must be established entirely by inference. The candidates most likely to survive the terminal sterilization cycle would be heterogeneous populations of bacterial spores. Rosebury (16) states that members of the genus Bacillus are widely distributed in nature, and, although they are frequently recovered from surfaces of the healthy human body, they are not indigenous to humans but are merely transient organisms whose ultimate origin is soil. Therefore, as a contaminated spacecraft enters its terminal sterilization cycle, the organisms of major concern with regard to thermal resistance will be bacterial spores of soil origin. Presently, dry-heat sterilization cycles for spacecraft are based upon the thermal inactivation characteristics of B. subtilis var. niger (B. globigii) spores. Craven et al. (5) have stated that many workers in the field of spacecraft sterilization technology accept the spores of this microorganism to be representative of the more heat-resistant organisms likely to be found on spacecraft. They further pointed out that a representative organism is necessary because of the magnitude in variations of thermal resistance among spore populations found in and on space hardware. In addition, the "logarithmic death rate model" of spore populations is assumed in subsequent calculations (12), since survivor curves of the standard reference organism are essentially straight-line functions on a semilog scale. Rather than employ a standard reference or-ganism, it was thought that the use of naturally occurring spore populations (i.e., mixed populations of spores employed directly without intermediate isolation and subculture on conventional laboratory media) might yield more representative and therefore pertinent information regarding the dry-heat inactivation kinetics to be encountered in actual practice. Even though methods are available for efficient removal and recovery of surface contamination (8,10), collection of spores directly from spacecraft surfaces in numbers sufficient for dry-heat resistance tests to be conducted is impossible. Assays on several unmanned spacecraft have shown the bacterial spore level to be approximately 104 to 10 per spacecraft (J. R. Puleo, personal communication). Since spores, including those found on spacecraft, are mainly of soil origin, soils collected in the vicinities of manufacture and assembly of spacecraft would be the next most logical source of naturally occurring spore populations for heat studies. The objectives of these studies were to formulate a method for obtaining spore populations from soil without the use of heat shock (4,7) or subculture and to observe the inactivation kinetics of these populations found in soil samples collected from various areas of the United States where spacecraft components are manufactured or assembled. MATERIALS AND METHODS Soil samples. One-hundred-gram amounts of each soil sample were dried in loosely capped 1-liter Erlenmeyer flasks at 50 C for 48 hr. A 100-ml amount of 95% ethyl alcohol was added to each dried soil, and the suspensions were insonated at maximum power (25 kHertz) for 30 min in an ultrasonic bath (Sonogen LTH60-3 transducerized tank; Sonogen A-300 generator; Branson Instruments, Inc., Stamford, Conn.). A single flask was placed on the center of the tank bottom with the level of the bath fluid, 0.3% Tween 80 (Hilltop Research, Inc., Miamiville, Ohio) in distilled water, slightly above the level of ethyl alcohol in the flask to ensure optimum transfer of ultrasonic energy. The temperature of the bath fluid was maintained from 4 to 20 C during the insonation period. After insonation, each suspension was filtered through a sterile linen towel to remove large particles and then stored at 4 C in a tightly capped bottle. These treatments yielded suspensions of finely dispersed soil and bacterial spores, free from viable fungi, actinomycetes, and vegetative bacteria. No isolations have been made from these suspensions other than gram-positive, sporeforming rods. Also, extended storage of bacterial spore crops in ethyl alcohol, as opposed to distilled water, has been shown to have no effect on viability or heat resistance (7). Assay system. Dry-heat inactivation kinetics of pure and mixed spore populations were determined by inoculating sterile stainless-steel strips [0.5 by 0.5 inch (1.27 by 1.27 cm); coldroll, type 302, no. 4 finish, 22 gauge] with 0.05 ml of an ethyl alcohol suspension. The strips were then dried under vacuum for 16 hr over silica gel. For each heating interval, three strips were suspended in a forced-air dry-heat oven (model no. 625, Precision Scientific Co., Chicago, Ill.) at 125 C (40.5 C). The time required to heat a strip to 125 C was 2.5 min as determined by a copper-constantan thermocouple attached to a control strip. This amount of time was added to each exposure interval. One set of samples was placed in the oven at each interval, and the temperature was monitored constantly with a recording thermometer. Immediately after removal from the oven, each strip was placed in a tube containing 10 ml of chilled (4 C), phosphatebuffered distilled water (BDW; 1) with several 3-mm glass beads and was insonated for 12 min in an ultrasonic bath. The suspensions were diluted appropriately with BDW and plated in triplicate with Trypticase Soy Agar (TSA; BBL). After the medium had solidified, 10 to 15 ml of TSA was overlaid on each plate to lessen spreading growth. One triplicate set of strips was used as a control (no heat), and six or more sets were heated for the desired times. Three uninoculated strips were processed as sterility controls, and counts of survivors were made after 24 and 48 hr of incubation at 32 C. All manipulations of sterile items, with the exception of transferring triplicate sets of strips from sterile petri plates into the oven and vice versa, were performed in class 100 horizontal laminar flow clean benches to eliminate background contamination (9). D125c values were determined from a best-fit regression line of the data points by using a least squares method. The variation of the data points around each regression line was measured by calculating a standard error of the estimate. Isolations and spore preparations. Colonies were picked randomly from pour plates of heated and unheated strips of selected soils and streaked for isolation on TSA. Pure cultures were maintained on TSA slants at 4 C. Spore preparations were made of selected isolates in the following manner. A turbid suspension of cells in sterile BDW was heat-shocked in a water bath at 80 C for 15 min. The suspension was then lightly swabbed onto TAM Sporulation Agar (Difco) supplemented with 20 ,g of MgSO4 per ml and 80 jg of CaCl2 per ml. After 2.5 hr of incubation at 32 C, growth was swabbed onto a fresh plate of medium. This procedure was performed twice. After the last incubation period, growth from the final plate was swabbed onto three plates of fresh medium and incubated at 32 C for 24 to 48 hr. Most isolates exhibited maximum sporulation at these times with only a few requiring up to 96 hr of incubation. Growth from the three final plates was harvested in approximately 20 ml of chilled BDW and insonated for 90 sec with a Biosonic III Ultrasonic Probe (Bronwill Scientific, Rochester, N.Y.) at 60% maximum intensity. The suspension was then centrifuged for 15 min at 4 C (8,590 X g; Servall SS-1, Ivan Sorvall, Inc., Norwalk, Conn.). The supernatant fluid was discarded, and the cellular debris layer of the pellet was removed by gentle washing with 5 ml of chilled BDW. The remainder of the pellet, consisting mainly of spores, was resuspended and washed twice more in the same man-ner. Simple staining with crystal violet and microscopic examination were used to determine if more washings were necessary. The pellet was then washed three times with 95% ethyl alcohol and resuspended for storage at 4 C. Subsequent heat inactivation studies were performed in the same manner as described for the ethyl alcohol-soil suspensions. Model spore population. A system to simulate the survivor curve of a mixed, naturally occurring spore population was developed by using one heat-sensitive and one heat-resistant aerobic mesophilic isolate from soil sample X (Phoenix). Clean spore crops were prepared, and D12s c values of each ethyl alcohol suspension were determined in the presence and absence of sterile soil from the parent sample to determine the characteristics of the respective survivor curves. The two isolate suspensions were then mixed in a proportion similar to the heat-sensitive and heat-resistant populations in the original soil. Sterile parent soil was added to a portion of this mixture to approximate the spore per unit volume of the original soil sample (0.3 g/ml). Dry-heat assays were performed in the same manner as the comparative DI25 c survey of soils. RESULTS During preliminary testing of the heating system, the soil sample collected in Phoenix (sample X) consistently produced a nonlinear survivor curve (Fig. 1), indicating a decrease in inactivation rate with time. In general, the survival data from the 23 soil samples tested subsequently did not appear linear, and the shapes of the survivor curves were similar to that of sample X. The sample containing the most resistant spores was the one collected in Phoenix. To examine the possibility that the nonlinear survivor curves of this sample could have been due to protective effects offered by the small amount of soil particles present, various subcultured spore suspensions of heat-sensitive and heat-resistant isolates suspended in ethyl alcohol were tested in the presence and absence of sterile parent soil. Survivor curves (Table 1). Consequently, the soil per se did not seem to alter dry-heat resistance by physical protection. Also, the nonlinear survivor curves of the soil samples did not appear to result from technique-induced error since pure spore cultures of B. subtilis vat. niger, B. cereus T, B. subtilis 5230, and several soil isolates have consistently yielded linear survivor curves at 125 C in our test system. By expressing the standard error of the estimate for each line as a coefficient of variation, it was possible to compare the variation from a straight line in the soil suspension tests with the variation measured in like tests on spore isolates. These data are presented in Tables 1 and 2. It is evident that the coefficients of variation for the soil suspension tests are markedly higher than those for spore isolates, the mean value of the suspensions being more than five times the mean value of the isolates. The consistency in the shape of the 24 survivor curves for the soil suspensions coupled with the large coefficients of variation for straight lines fit to these data indicated that the survivor curves were not linear. In an effort to define more clearly the nature of the nonlinear survivor curves observed with the soil samples, a model system described above was developed by using heat-sensitive and heat-resistant aerobic mesophilic isolates from soil sample X. Clean spore crops were prepared, and survivor curves of both suspensions were found to be linear and unaffected by the presence of added soil (isolates X-1 and XA, Table 1). When the two suspensions were mixed in the presence VOL. 20, 1970 and absence of soil and tested in parallel, the survivor curves were shown to be nonlinear and unaffected by the presence of soil (Fig. 2). DISCUSSION During comparison of D125 c values of naturally occurring spore populations in soils and respective subcultured spore isolates, another factor was noted which may significantly affect the current rationale behind calculation of spacecraft sterilization cycles. No spore isolates from heated soils were obtained which exhibited resistances equal to or greater than the portion of the survivor curves from which they were isolated. Consequently, it appeared that the subculture of the naturally occurring spores significantly lowered their dry-heat resistances. Whether these losses in dry-heat resistance were due to unsatisfied nutritional requirements or differences in environmental conditions or both is not known. However, studies by many other investigators have shown that the heat stability of spores may be altered significantly by manipulation of cultural conditions (14). Lowering of dry-heat resistance by subculture has also been noted with gramnegative organisms such as salmonellae (15). It must be reemphasized here that current space-craft sterilization cycles are calculated on the basis of a standard reference spore population, i.e., B. subtilis var. niger. However, there is no evidence available to suggest that this organism or its thermal inactivation characteristics are in any way associated with actual microbial contaminants on spacecraft. Qualitative and quantitative microbiological assays conducted on many spacecraft while in residence at Cape Kennedy (Apollo and Mariner) have failed to reveal a single instance of contamination by B. subtilis var. niger. Ernst (6) reviewed the thermal inactivation kinetics of bacterial spores and pointed out several pitfalls which may be encountered in the interpretation and subsequent extrapolation of death rate constants (D values). When studying the nonlogarithmic survival of mixed spore populations in soils, he found, as our observations also indicate, that isolates from the later phases of the survivor curves were less heat-resistant than the original tests would have indicated. From this observation, he assumed a mechanistic approach by stating that the more heat-resistant spores were "undoubtedly protected in some way in the soil menstruum." Based on this assumption, he concluded that the slope of a straight line passing through the point of extinction (obtained by end-point determinations) of a population would represent the largest possible D value of the unprotected spores. The findings in our study, however, indicate that when end-point data are used and logarithmic death of a naturally occurring spore population is assumed, there can be a significant error in estimating the time required to achieve a high probability of sterility ( Fig. 3). This figure was adapted from a hypothetical curve presented by Ernst (6) in his illustration of the protective effects by soil. The controversy regarding calculation of sterilization cycles in general can be appropriately described as a classical argument between two basic schools of thought. One school explains deviations from logarithmic survival in terms of inherent heterogeneity within a population, whereas the other school attributes these deviations to factors in operation within the lethal period. With particular regard to dry-heat inactivation of bacterial spores, members of either school would expect to observe nonlinear survival when dealing with mixed populations in soil. However, it remains that such nonlinear survival has been and is today explained entirely in terms of protective effects by the soil menstruum. This philosophy continues by reasoning that, when dealing with relatively low levels of surface contamination such as encountered with spacecraft, the protective effects of soil may be neglected with concomi- (6): calculated heating times necessary to effect a 7-log reduction. tant assumption of logarithmic death. Data presented in this report indicate such an assumption to be workable provided that the probability of contamination by a particular population is not extrapolated below 100. However, in extrapolation below measurable range, for example, to 10-, the greatest possible D value of the population becomes critical and may not be accurately reflected by an end-point determination. Therefore, in dealing with sterilization of naturally occurring spore populations, it is recommended that assumption of logarithmic death in conjunction with a standard reference organism may well constitute an invalid model in certain applied situations, particularly in the field of spacecraft sterilization. ACKNOWLEDGMENT Services were provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration under contract W-13,062. VOL. 20, 1970

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2020-12-10T09:04:17.261Z

1970-05-01T00:00:00.000Z

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Effects of Moisture Content and Temperature on Aflatoxin Production in Corn Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B1 and B2. The discovery that aflatoxin is a potent hepatotoxin produced by Aspergillus flavus (20) has led to intensive studies on the presence of mycotoxins in foodstuffs (1,6,9,27). Because this ubiquitous fungus is capable of growing over wide temperature and moisture ranges, many food materials are susceptible to aflatoxin contamination. High levels of aflatoxin (greater than 250 ng/g) have occurred in some batches of peanut and cottonseed meals (9). The problem of control of aflatoxin production in foodstuffs has been compounded by the implication of other aspergilli and penicillia as aflatoxin producers (7,10,11,22), although these results have recently been questioned (15,25). Some of the factors have been determined for the protection of stored grains against aflatoxin production. Schindler et al. (21) studied temperature effects on production of aflatoxins in wort media. Diener, Davis, and co-workers (8,12,19) determined the limiting temperatures, relative humidities, and effects of gaseous environment on aflatoxin production in sterilized, inoculated peanuts. Similar studies have been performed on stored barley (16). CoM has been shown to be a satisfactory substrate for aflatoxin production (9), and many workers are involved in screening (4) and standardizing (3) methods of analysis in corn. The significance of aflatoxins in corn was indicated by Wilson et al. (26) when samples of moldy corn were implicated in toxic hepatitis in swine and dogs; addition of crystalline aflatoxins to animal I Journal paper no. J-6463 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Projects no. 1348 and 1763. Presented in part at the 68th Annual Meeting of the American Society for Microbiology, Detroit, Mich., 6 May 1968. feeds simulated the symptoms of this disease. Lopez and Christensen (14) determined the moisture and temperature requirements for invasion of corn by A. flavus. A moisture content of 17.5% was minimal for growth of the mold, but A. flavus became the predominant species of the mycoflora at 18.5% moisture and temperatures of 25 or 35 C. Since aflatoxin-producing fungi and aflatoxins have been found present in corn and since moldy corn has been implicated in a variety of toxicoses of farm animals, this study was undertaken to determine the environmental conditions necessary for aflatoxin formation in corn. The aflatoxinproducing fungi from toxin-containing samples were isolated and further characterized. MATERIALS AND METHODS The corn used for this study was harvested with a mechanical picker and field-shelled. The moisture levels were adjusted to 18 to 28% (wet-weight basis), and the corn was held in 600-g quantities in water baths maintained at 2, 13, or 24 C. The corn was aerated with air scrubbed of carbon dioxide by passage through a 40% sodium hydroxide solution and adjusted to the proper relative humidity by passage through salt solutions. The quantity of carbon dioxide produced in the stored grain was measured and used as an index of deterioration in the corn sample. The apparatus used for corn storage is described by Steele and Saul (23). When a sample had lost 1.5% of its weight as carbon dioxide, it was analyzed for aflatoxins. A second series of experiments used corn previously stored at 13% moisture. This corn was remoistened to moisture contents of 17 to 26%, stored at 24, 30, and 35 C, and handled as previously described. Aflatoxins were determined by using the aqueous acetone extraction method of Pons et al. (17). The 781 Vol. 19,No. 5 extracts were analyzed by thin-layer chromatography (TLC) with a benzene, ethanol, and water solvent (46:35:19, v/v). The fluorescence of the aflatoxins on the TLC plate was compared with a quantitative standard of aflatoxins B1 and G1 under long-ray ultraviolet light. The fungi were isolated from the corn samples by grinding 10 g of corn in 60 ml of 0.1% peptone water in a Waring Blendor for 2 min and plating serial dilutions on Potato Dextrose Agar (Difco) and Malt Extract Agar (5). The plates were incubated for 1 to 2 weeks at either 30 C or room temperature. Colonies representative of the fungal population were picked and purified; then they were stored on Sabouraud Dextrose Agar (Difco) slants under refrigeration. From these isolates, 232 cultures were screened for the production of aflatoxins. For ease of extraction, a liquid medium, SMKY (8), was chosen. A 25-ml amount of the medium was placed into a 125-ml Erlenmeyer flask and autoclaved. The flask was inoculated with a loopful of spores from a slant culture and incubated for 1 week at 30 C in stationary culture. For extraction of aflatoxins, 50 ml of chloroform was added to the flask; the flask was shaken for 30 min on a wrist-action shaker, the mixture was filtered, and the chloroform layer was removed in a separatory funnel. The sample was concentrated on a steam bath, and the extract was spotted on a TLC plate with known aflatoxin standards and viewed under ultraviolet light for the characteristic colors and RF values of aflatoxins. The cultures producing aflatoxins in the screening medium were selected for further testing. They were grown on Potato Dextrose Agar plates and were identified by using the criteria of Raper and Fennell (18). The ability of these isolates to produce aflatoxins on sterilized corn was determined. A 100-g amount of shelled corn was rewet to approximately 40% moisture in a 1-liter flask, allowed to equilibrate for 12 to 24 hr, and autoclaved. The corn was inoculated with a spore suspension, incubated for 1 week at 30 C, and extracted by using the procedure of Pons et al. (17). The amounts of aflatoxins were determined by TLC. Purified aflatoxin B1 was prepared by streaking 250 uliters of the extract on a TLC plate, developing the plate, and scraping the fluorescent band of aflatoxin from the plate. The silica gel containing the toxin was added to a sintered-glass filter, and the aflatoxins were eluted with 100 ml of chloroform. The purified B1 was dissolved in propylene glycol and used for chick embryo bioassay by use of the procedure of Verrett et al. (24). RESULTS AND DISCUSSION Aflatoxins were detected in only two samples in the first series of freshly harvested corn samples (Table 1). Both samples were stored at 24 C, and the levels of aflatoxins were very low (50 ng per g of B1). An apparent discrepancy is seen in the 24 C data in that the two positive samples had moisture contents of 20 and 25%, but similar samples stored at comparable moisture levels did not contain aflatoxins. This is probably because the low levels of aflatoxin observed approach the limits of resolution of the test system. A second series of experiments was performed by using higher temperatures of incubation. The corn for these experiments was rewet from 13% moisture and stored at 24, 30, and 35 C. The levels of aflatoxins found in this series of samples were much higher than those found in the first series ( Table 1). The rewet corn contained higher levels of aflatoxins than the freshly harvested corn, even when stored under similar conditions. Of all the samples tested, only the corn held at 35 C and 26% moisture contained aflatoxin G1; all others contained only aflatoxins B1 and B2. In the rewet corn, higher aflatoxin levels were found at 30 and 35 C than at 24 C. This optimal temperature range (30 to 35 C) was somewhat higher than that found by Schindler et al. (21), who obtained best production of aflatoxins between 25 and 35 C. These workers also demonstrated toxin production at 13 C; however, we were unable to detect aflatoxins in corn incubated at this temperature. This is most readily explained by the natural competition between the mycoflora in our samples, whereas Schindler et al. The limiting moisture level appeared similar to that demonstrated by Lopez and Christensen (14) as the minimal level for growth of A. flavus (17.5%). This was inadequately tested, but the absence of aflatoxins in the rewet sample stored at 18% moisture indicates that this is at least the lower practical limit for toxin formation. The most striking difference was between the aflatoxin contents in freshly harvested and remoistened corn. In freshly harvested samples stored at 24 C, the aflatoxin content ranged from 0 to 50 ng/g, but, in rewet corn stored under similar conditions, the toxin content ranged from 100 to 500 ng/g. The rewet corn was found contaminated with large numbers of A. flavus and other aspergilli. Although only 10 cultures of A. flavus were among the 518 isolates from freshly harvested corn, 22 of 89 isolates from the remoistened corn were A. flavus. Thus, it appears that there is a selective advantage for A. flavus during drying of the shelled corn and subsequent rewetting. This can best be explained by selective survival of the xerophytic aspergilli while the corn was stored at 13% moisture and changes in the kernel after rewetting, allowing the fungi to invade and grow more readily. This phenomenon of increased aflatoxin formation and greater numbers of A. flavus in remoistened corn may be of considerable importance in the storage of corn. Lichtwardt et al. (13) found A. flavus frequently while studying the mycoflora of stored corn in Iowa, and Zeleny (28) described the condition of deterioration of grain stored at low moisture levels, caused by rewetting due to convection of warm, moist air through the storage structure. Our results indicate that the potential hazard of aflatoxin production increased greatly during rewetting. Similar conditions could occur when leakage of a storage structure allows moisture to enter. Reports of aflatoxin production by fungi other than A. flavus and A. parasiticus (10,11,22) prompted us to screen our diverse assortment of fungal isolates for aflatoxin production ( Table 2). Aspergilli and penicillia composed the majority of fungi screened (197 of 232), but only A. flavus was found to produce aflatoxins; 10 of the 32 A. flavus isolates produced aflatoxins. Similar results found by other investigators (2, 15,25) indicate that the primary source of aflatoxins in feedstuffs is A. flavus or the closely related A. parasiticus. Table 3 presents additional data on the 10 aflatoxin-producing isolates from corn. Only aflatoxins B, and B2 were produced; this is the same pattern found in the extracts from aflatoxin-containing corn samples. The cultures were very similar morphologically and appeared rather homogeneous in the levels of aflatoxins produced in sterilized corn. The B, component from these cultures showed a dose response in chicken embryos; the minimum doses that killed the chick embryos within 5 days of incubation are shown in Table 3.

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2020-12-10T09:04:16.975Z

1970-10-01T00:00:00.000Z

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Enterotoxigenicity of Staphylococcus aureus Cultures Isolated from Acute Cases of Bovine Mastitis To determine whether staphylococci causing bovine mastitis are potential causes of human intoxications, 142 cultures identified as etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were obtained from investigators in the United States and Canada, examined microscopically, and tested for carbohydrate utilization, terminal pH, catalase, coagulase, egg yolk hydrolysis, gelatin hydrolysis, cytochrome oxidase, urease production, nitrate reduction, micrococcal nuclease, phage type, and enterotoxin production. Three cultures were not confirmed as Staphylococcus aureus. Of the 157 S. aureus cultures, 23 produced staphylococcal enterotoxins. Although a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning was not proved, results indicated that staphylococcal infections of the bovine mammary gland represent a significant reservoir of enterotoxigenic strains of S. aureus. Staphylococcus aureus is frequently present in milk and manufactured dairy products. Such products have been involved in outbreaks of staphylococcal food poisoning (1,14,15,20). Since it has been established that S. aureus is one of the principal etiological agents of bovine mastitis (8), it might be assumed that some relationship exists between staphylococcal mastitis and food poisoning outbreaks attributed to manufactured dairy products. However, a direct relationship between these two conditions has not been established. In an attempt to explore the relationship between staphylococci causing bovine mastitis and the potential for human intoxications, S. aureus cultures from validated cases of acute staphylococcal mastitis were solicited from investigators in the United States and Canada. One hundred and forty-two cultures identified as the etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were received. MATERIALS AND METHODS Cultures were subjected to the following morphological, cultural, and serological examinations to verify their classification and to determine their enterotoxigenicity and phage type. Microscopic examination. A simple stain with 1% aqueous solution of crystal violet was used. Carbohydrate utilization. Utilization of glucose and mannitol was tested by the method of Mossel (16), except the tests were terminated after incubation for 5 days at 35 C. Terminal pH. The test was performed in 2% glucose broth (4); pH was determined electrometrically after incubation for 7 days at 35 C. Coagulase. Brain Heart Infusion cultures (18to 24-hr) were tested with rabbit plasma and rabbit plasma containing 0.1% ethylenediaminetetraacetic acid by the method of Baer (3). Egg yolk hydrolysis. Plates of tellurite polymyxin egg yolk agar (12) were examined after 48 hr at 35 C for zones of clearing or precipitation surrounding points of inoculum. Gelatin hydrolysis. Gelatin hydrolysis was tested by plate method (17) after 48 hr at 35 C. Cytochrome oxidase. The Steel modification (18) of Kovac's method was used to test for cytochrome oxidase. Urease production. Slants of Christensen's agar (11) were incubated as long as 7 days at 35 C. Nitrate reduction. Daily tests from indole-nitrite medium (Difco, prepared according to instructions of manufacturer), incubated for as long as 7 days at 35 C, were made with reagents specified in Manual of Microbiological Methods (17). Micrococcal nuclease. Cell-free extracts of boiled, Brain Heart Infusion cultures were assayed by the method of Chesbro and Auborn (10), modified by changing the CaCl2 concentration to 10 mM and adjusting the reaction mixture to pH 9.5 (14). Cul-tures were grown on a rotary shaker 18 to 24 hr at 37 C, and each extract was assayed in triplicate. Staphylophage typing. Cultures were typed with a set of 22 basic phages recommended by the Subcommittee on Phage Typing of Staphylococcus, Nomenclature Committee, International Association of Microbiological Societies (7), by the method of Blair and Carr (6). Enterotoxin testing. Culture filtrates were tested for identifiable S. aureus enterotoxins by the microslide gel diffusion method described by Casman and Bennett (9). RESULTS AND DISCUSSION Eighteen cultures from chronic mastitis infections and 142 cultures from acute infections were received and tested. Of the 160 cultures, 3 could not be confirmed as S. aureus. Two were identified as Sarcina sp. and one as S. epidermidis. The 157 S. aureus cultures were subdivided into eight types on the basis of their biochemical features. All cultures were catalase-positive, cytochrome oxidase-negative, coagulase-positive, utilized glucose anaerobically, reduced K2TeO3, and produced heat-stable nuclease. In addition, most cultures liquefied gelatin, utilized mannitol anaerobically, hydrolyzed egg yolk, produced urease, and reduced nitrate. All cultures attained a terminal pH in 2% glucose broth in the range pH 4.0 to 4.6; for most, the pH was 4.5. The features in which variants differed from typical S. aureus (Bergey's Manual, 7th ed.) and the frequency of strain occurrence in acute and chronic infections are shown in Table 1. From information received from those who Phage typing revealed that in 8 of the 22 herds and in 9 of the 12 cows from which multiple cultures were received infection was due to a single phage type; more than one phage type was found among the multiple cultures received from the other 14 herds and 3 cows. Susceptibility of the 157 S. aureus cultures to phages of the various lytic groups is shown in Table 2. Typable strains were distributed among 606 APPL. MICROBIOL. 46 separate phage types. Ten cultures were nontypable at a maximum phage concentration of 1,000 times the routine test dilution, with the set of phages used. Although the majority of cultures from chronic infections were most susceptible to phages of lytic group III, the sum of features tested suggested that there was no consistent physiological difference between cultures causing acute and those causing chronic infections. Of the 157 cultures, 23 produced staphylococcal enterotoxins. Eleven produced type C, eleven produced type D, and one produced both types C and D enterotoxins; none produced A or B. Phage types of strains producing enterotoxin are shown in Table 3. Since types of S. aureus in lytic group III are said to be most frequently implicated in food poisoning outbreaks (2,5,19), it is interesting to note the high frequency of types in lytic group I among the enterotoxigenic cultures. Enterotoxigenic cultures were contributed by 14 of the 36 identified herds and 22 of the 72 identified cows. It is perhaps noteworthy that in only one case did all cultures of the same phage type from a herd produce enterotoxin. In three instances, one culture from a herd produced enterotoxin and a similar phage type culture from the same herd did not. In two cases, different phage type cultures from the same herd produced the same type enterotoxin. Nearly 15% of 157 S. aureus cultures identified as etiological agents of bovine mastitis were shown to produce enterotoxins. The hypothesis of a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning remains unproven, but it has been shown that a significant number of S. aureus strains causing bovine mastitis were able to produce enterotoxins. The potential for production of staphylococcal enterotoxins is obvious in those cases in which raw milk containing enterotoxigenic types is poorly held or receives sublethal heat treatment and processing conditions or manner of use of contaminated products allow subsequent growth. The continued high incidence of staphylococcal mastitis and the rather high percentage of enterotoxigenic cultures isolated from validated mastitis cases indicate that greater efforts should be directed toward the prevention of mastitis in the interest of, among other reasons, enhancing the control of foodborne Staphylococcus intoxication. The abnor-mal milk program of the National Conference of Interstate Milk Shipments is working toward this goal.

v3-fos

2020-12-10T09:04:17.665Z

1970-11-01T00:00:00.000Z

237233499

s2

Identification of a Stimulant for Lactobacillus casei Produced by Streptococcus lactis A compound stimulatory to the growth of Lactobacillus casei was isolated from cell extracts of Streptococcus lactis, purified, and characterized. The stimulant was identified as a small peptide with a molecular weight of approximately 4,500 daltons. The purified peptide gave negative tests for nucleic acids, phosphorus, glucosamine, and carbohydrates. Sixteen amino acids were detected in acid hydrolysates of this peptide. Serine, proline, glycine, alanine, leucine, and glutamic acid were present in hydrolysates in greatest abundance. In an early report, Hansen (6) demonstrated that sonically prepared extracts of Streptococcus lactis and S. cremoris were stimulatory to the growth of Lactobacillus casei and concluded that this effect could play an important role in the ripening of cheese. We have previously confirmed and extended those observations (1). Cell extracts of several Streptococcus species were observed to stimulate growth of those lactobacilli prevalent in ripening Cheddar cheese. The stimulatory principle was dialyzable and partially inactivated by heat. In the present communication, we report the isolation and identification of a compound produced by S. lactis which is stimulatory to growth of L. casei. MATERIALS AND METHODS Organisms. S. lactis C2-F was used as the source of stimulant, and L. casei 393 was employed as the assay organism. The source of these organisms and methods for routine propagation and preparation of cell-free extracts have been described previously (1). For preparation of cell-free extracts, cells were harvested by centrifugation at 5 C and washed twice with amounts of sterile 0.85% saline equivalent to the volume of original culture (1). Growth assay. A modification of the bioautographic method of Cogan et al. (3) was employed to follow the isolation of the stimulant. The method was modified by pouring the milk-agar medium into sterile petri plates and allowing it to harden. Sterile assay discs were dipped into sterile solutions to be tested and placed on the hardened medium; the plate was incubated at 40 C. Stimulatory zones were observed after 6 to 12 hr as areas of red color around and under the disc. To confirm the presence of the stimulant in isolated fractions, growth curves were determined for the test organism in the presence and absence of isolated fractions as described previously (1). Isolation of stimulant. Cell-free extracts were pre-pared and dialyzed (1), and the dialysate was dried in vacuo, dissolved in minimal volumes of water, and applied to a Sephadex G-25 column ( active fraction obtained was applied to a column of Sephadex G-10 and eluted with distilled water. Fractions containing stimulant were combined, concentrated to a few milliliters, and applied to a column of diethylaminoethyl (DEAE) Sephadex A-50 (3 by 15 cm); elution was accomplished with a gradient of 0.02 to 0.1 M ammonium carbonate buffer. Active fractions were combined, concentrated, and applied to a column of carboxymethylcellulose. The stimulant was eluted with a gradient of 0.1 to 0.2 M NaCl in 0.01 phosphate buffer (pH 7.1). After concentration and desalting of the active fraction over Sephadex G-10, ethanol was added to achieve final concentrations of 30, 60, and 90%. The precipitate formed at each step was recovered by centrifugation, dried in vacuo to remove the last traces of ethanol, and dissolved in distilled water. The stimulant, recovered in the 90% ethanol fraction, was finally purified by chromatography on 500-pm layers of Silica Gel G orcellulose MN 300 (Brinkman Instruments, Inc., Great Neck, N.Y.). Chromatoplates were developed with butanol-acetic acid-water (5:1:4, by volume), and a strip along one edge of the plate was exposed and sprayed with ninhydrin. Ninhydrin-positive zones were marked on the unstained portion of the chromatoplate, scraped off, eluted with distilled water, concentrated, sterilized, and tested for stimulation of the test organism. At this point, the stimulant appeared homogeneous on paper electrophoresis in a Durrum-type cell (Spinco) at both pH 3.6 (acetate buffer) and 8.6 (barbiturate buffer). Amino acid analysis. Samples were dissolved in an approximately 200-fold excess of 6 N HCl, refluxed for 15 hr, dried in vacuo, and redissolved in a small volume of 0.2 M sodium citrate buffer, pH 2.75. A portion of the hydrolysate was placed on the column of a 757 Technicon amino acid analyzer with 0.2 ,umole of norleucine as an internal standard. Separation was accomplished according to standard procedures, and the relative molar concentrations of the amino acids were calculated. Chemical analyses. Phosphorus content of both hydrolyzed and unhydrolyzed fractions was determined by the method of Rouser et al. (12); ninhydrinpositive material, by the method of Moore and Stein (9); ribonucleic acid (RNA), by the method of Von Euler and Hahn (15); and deoxyribonucleic acid (DNA) by the method of Ceriotti (2). Carbohydrate was determined by reaction with anthrone (14), protein by the biuret method (5), and glucosamine by the method of Elson and Morgan (4). RESULTS Analysis of the concentrated cell-free dialysate of S. lactis revealed the following composition: protein, 1.3 mg/ml; RNA, 1.4 mg/ml; and DNA, 0.36 mg/ml. Three major fractions were obtained on separation of the dialysate on Sephadex G-25, with peak 2 containing the majority of the stimulatory material (Fig. 1). The active fraction was excluded as one major peak from Sephadex G-10 and yielded only one major peak when chromatographed on both DEAE-Sephadex and carboxymethylcellulose (Fig. 2). Thin-layer chromatography of the carboxymethylcellulose eluate showed a number of ninhydrin-positive zones; however, the 90%7, ethanol precipitate, which con- The active fraction recovered from chromatoplates gave negative tests for RNA, DNA, carbohydrate, organic phosphorus, and glucosamine and was homogeneous by electrophoresis. The final traces of RNA were removed from the stimulatory fraction after passage over carboxymethylcellulose, and the final traces of DNA were removed by thin-layer chromatography. The absence of nucleic acid in the final fractions was confirmed by the absence of absorbance maxima at 260 nm. The stimulant appeared to be peptidyl in nature since it was precipitated by ethanol and yielded a ninhydrin-positive reaction, the magnitude of which was increased by hydrolysis. Sixteen amino acids were detected in this peptide, those present in greatest quantity included serine, glutamic acid, proline, glycine, alanine, and leucine. Sulfur amino acids were not detected. It should be noted that hydrolysis may have destroyed certain amino acids present in the original peptide. Assuming that the number of residues in Table 1 is correct and rounding off these values to the nearest whole residue, the approximate molecular weight of the peptide is calculated to be 4,700. This is in agreement with its behavior on Sephadex columns, at which point the peptide was included by Sephadex G-25 (exclusion = 5,000) and excluded by Sephadex G-10 (exclusion = 700). The same pattern of stimulation of growth of L. casei was observed when either the isolated 758 APPL. MICROBIOL. peptide or the original dialysate was added to broth cultures of this organism (Fig. 3). This suggests that the isolated peptide is responsible for the major portion of the stimulation of the test organism by cell-free extracts of S. lactis. DISCUSSION Peptides have long been known to stimulate the growth of bacteria, particularly the lactic acid bacteria (13). Kihara and Snell (7) outlined several conditions which would reduce the avail-ability of an essential amino acid for bacterial growth, thereby enhancing the ability of appropriate peptides to promote growth. These authors were of the opinion that peptides play no special role in metabolism. Moss and Speck (10,11) found that peptides present in Trypticase and in frozen Escherichia coli cell extracts enhanced the recovery of E. coli cells injured by freezing. They suggested that these peptides manifested their activity by stimulating cell repair and growth during the lag phase. The stimulatory peptide characterized in the present study is similar to a series of peptides isolated by Moss and Speck (10, 11) from Trypticase and E. coli cells in Sephadex G-25 elution pattern, molecular weight, and amino acid composition. Stimulatory peptides isolated from pancreas also have an amino acid composition very similar to the peptide we isolated from S. lactis (8,13). These similarities suggest that stimulatory peptides of rather constant amino acid composition are widespread in nature. Since stimulation of the test organism by an amino acid mixture approximating the composition of the peptide was not evaluated, we cannot conclude that the integrity of the peptide is essential for activity. However, in view of the work discussed above, it seems logical that the peptide itself, and not its component amino acids, is necessary. Since exhaustively washed whole S. lactis cells, cell extracts (1), and the purified peptide were observed to stimulate growth of L. casei in milk culture, it can be concluded that this peptide is produced by the organism itself. Stimulation in milk culture implicates the peptide as a possible factor stimulating the development of lactobacilli in Cheddar cheese during ripening.

v3-fos

2019-03-20T13:05:13.588Z

1970-01-01T00:00:00.000Z

83772976

s2

ON THE CARBOHYDRATE COMPOSITION AND NUTRITIVE VALUE OF SOME CEREALS A cereal grain is an one-seeded fruit. The fruit coat (pericarp) envelopes the seed and is fused to the thin seed coat (testa). Together they form a protective coating around the endosperm and the germ (embryo). In barley and oats the kernel is furthermore covered by hulls, which are composed of floral bracts. In barley they are attached to the kernel, but are loose in oats. by hulls, which are composed of floral bracts. In barley they are attached to the kernel, but are loose in oats. The relative proportions of the different grain-parts vary in different species, and therefore differences also occur in the whole meal composition and nutritive value. Table 1 presents information in respect of four cereal grains. The cell walls of the endosperm and germ are chiefly composed of hemicellulose material, which can be divided into water-soluble and water-insoluble substances (Montgomery & Smith 1956). The water-soluble substances, known as cereal gums, contain araboxylans, glucosans and proteins and yield viscous solutions in water (Gilles & Smith 1954, Meredith & Anderson 1955, Pomeranz 1961. The wheat flour gums amount to 1-1.5% of the flour (Montgomery & Smith 1956). In oat this type of glucosan, called oat lichenin, has been known to make up 2.6-2.9 % of the oat kernel (Letzig 1960). Investigations of the cereal carbohydrates have usually been concerned with the purely chemical aspects, viz. clarification of the structure and building-units of the endosperm and the germ part. On the other hand, with the exception of starch, little information is available in respect of several important items, for instance, of the total amounts of the different polysaccharides present, and of the composition and digestibility of the husk parts. In this investigation attempts have been made to elucidate the nutritive aspects of the carbohydrate composition of cereals. The total amounts of the main carbohydrates or carbohydrate groups have been determined, and the distribution of the hemicellulose, cellulose and lignin between the hull and kernel has been investigated. Furthermore, the relative proportions of the different structural units in the hemicellulose have been determined. A method is also proposed for the determination of the cell-wall complex. Material and methods Materials. The analysed cereal samples came from the 1968 harvest of the Viik Experimental Farm. In addition to the whole meal analyses, the compositions of the hulls as well as the dehulled grains, are given for oat and barley. Furthermore, the composition is presented for barley flour, which corresponds to white wheat flour, except that the germ is included in the flour. The content ofhulls in the oat and barley was 21 and 9 %, respectively. Thus the grain quality was better than the average. The wheat bran investigated was of commercial origin. The samples were ground in a Wiley mill, using sieve No 40. Methods. The carbohydrate compositions and the related amounts of crude lignin, presented in Table 2, have been determined according to the method of Salo (1965a). In the case of starch, however, the determination plan has been improved by an amyloglucosidase method, developed later (Salo & Salmi 1968). The cell-wall complex given in Table 3 is the residue after starch digestion, corrected for ash and protein. The sample was extracted with 80 % ethanol, the starch was hydrolysed at pH 4.8 with amyloglucosidase (for 22 hours at 60°C), filtered through a Gooch asbestos crucible, washed with water, dried, and the loss of weight on ignition determined. The protein content was determined separately from another similarly treated sample. Results and discussion Carbohydrate composition of cereals. The results of the carbohydrate, crude protein and ash determinations are given in Table 2 was not determined, since it was known from previous experience that the cereal grains contain only about 1 % of sugar. According to Montgomery and Smith (1956), cereal sugar is mainly composed of glucofructosan, which is as easily hydrolysable as saccharose. Wheat bran contains 5-6 per cent of sugar (Salo 1965 a) indicating that the sugar content of the coats and germ exceeds that of the endosperm. The lipids were not determined; however, to supplement the data, the following contents of crude fat can be given (Myasnikova et al. 1969): oat 4 -6 %, barley, 2.4 %, wheat 1.7-2.3 %, and rye 1.7-2.2 %. The data listed in Table 2 indicate that the relative proportions of the hulls and coats, and to some extent also the proportion of the aleurone layer, affect the starch content of the grains. The interpretation of the results is facilitated by concurrent inspection of the data given in Table 1. Hemicellulose is the main compound of the oat and barley hulls and the wheat bran. To some extent it also occurs in the endosperm and germ as is observable from the barley flour sample (Table 2). According to published papers (Gilles et al. 1952, Meredith & Anderson 1955), araboxylan and glucosan have been found in the cell-walls of these grain parts, and the results obtained are thus in conformity with previous findings. The main component of the kernel hemicellulose are xylose and arabinose, which occur in approximately the same proportions. Also glucose and uronic acid units constitute significant proportions of the hemicellulose. According to paper chromatographic investigation, also traces of galactose were present in all the samples. In addition, traces of mannose were found in barley and oat. The hemicellulose composition of wheat bran closely corresponds to that noted by Wolf et al. (1953) for the pericarp of corn. The uronic acid present in bran is glucuronic acid (Montgomery & Smith 1956). Structurally, the hulled oat and barley grains correspond to the grain of wheat and rye. The composition of the hulled barley grain has been calculated on the composition of hulls (9 %) and whole meal. The results obtained indicate that the barley sample contained strawlike hulls only to the extent of the 9 per cent mentioned. On the other hand, in comparison with oat, barley contains a larger proportion of the fruit and seed coat part, the bran. This result is in agreement with data in the literature (Myasnikova et al. 1969). The last three rows of Table 2 present the compositions of leat-stage timothy, blooming timothy and rye straw, respectively, which serve as a comparison-basis for the oat and barley hull data. It can be noted that the compositions of rye straw, oat hulls and barley hulls bear a close resemblance. In fact the hulls are composed of a material which is more wood-like than the straw, because the hulls contain about 10 percentage units more xylose and glucuronic acid polymers than the rye straw. In Table 2 the relative proportions of the hemicellulose components have been rounded off to the nearest five percentage-unit figures; however, it should be mentioned that the uronic acid components in rye straw, oat hulls and barley hulls amounted to 3.6, 4.6 and 5.0 per cent of the dry matter, respectively. The composition of the hulls of oat and barley are very much alike. Lignin and cellulose are typical hull-compounds, whereas in the kernel they occur to a minor extent only. It can be concluded from the above data that the feed value of the hulls of oat and barley resembles that of the straw. A high content of lignin and xylose units in Gramineaeplants and clover is an indication of their poor digestibility (Salo 1965 a). It thus seems that the hulls of oat and barley would constitute comparatively poor ruminant forage, and be entirely worthless as fodder for swine and poultry. On the other hand, the feed value of wheat bran is rather high, 0.74-0.76 feed units/kg according to the feed-value Table. Compared with the hulls, the bran contains only a minor proportion of cellulose and lignin (Table 2). The data in Table 2 do not, however, disclose that the pentosans of wheat bran are exceptionally soluble in analytical processes (Salo 1965 a), their solubility being even higher than that of the pentosans of young grass. Moreover, they are rather well digested by swine. According to the previous investigation of the author (Salo 1965 a), the swine is able to digest nearly 50 per cent of the wheat bran hemicellulose, but only about 15 per cent of its cellulose. Probably the digestibility of the oat and barley bran is of the same category. Investigations into this field cannot be found in the literature. Determination of the cell-wall complex as the residue after starch digestion. In Table 3 the sum of the hemicellulose, cellulose and lignin content is compared with the quantity of the cell-wall complex remaining after starch digestion. The complex has been determined by the same method as the first part of the carbohydrate determination scheme. It is noticeable that the protein-corrected residue agrees well with the sum of the fractions. In the determination of starch, extra work is required only for the drying, ashing and weighing, in order to determine the insoluble residue. However, the establishment of the protein correction involves considerably more additional work. Attempts were made to make the protein correction unnecessary by using papain in the amyloglucosidase treatment. The papain decreased the protein content of the residue considerably, though not sufficiently for this purpose. However, the protein correction might possibly be calculated as a percentage-value based upon the total protein content of the grains. From the values presented in Table 3 it can be calculated that the residue contained 72 -80 per cent of the total protein. By analysing a larger series of samples, obviously rather exact correction coefficients would be arrived at, since the treatment involves only extraction with ethanol and enzymatic hydrolysis. It is unlikely that large variations would occur on dissolution of protein from the same grain species. The value of the so-called cell-wall complex determination is reduced by the fact that the term includes the sum of hemicellulose, cellulose and lignin. Of these three, the hemicellulose and cellulose are rather useful in the nutrition of herbivory animals, the lignin, on the other hand, is a worthless substance. Additional information should therefore be obtained regarding the approximate percentages of hemicellulose, cellulose and lignin in the complex; at least the proportion of lignin should be reported. The respective percentages for cereals, as calculated from the figures given in Table 2, are given in Table 4. Percentage-ratios of this kind may well be valid for different samples of the same cereal species. For example, in this investigation no noteworthy differences were detected between the analysed spring and winter wheat samples. However, in respect of grass or clover, for instance, no generally valid ratios can be put forward, as the composition of the cell-wall complex varies depending upon the stage of growth (Salo 1965 b). An inaccuracy exists even in specific work in the determination of hemicellulose for oat and barley whole meal; the figure obtained includes the hemicellulose material of both the poorly digestible hulls and the rather well digestible kernel. The composition of the hemicellulose does not sufficiently well explain these facts since the digestibility of the hemicellulose polysaccharides is influenced by the lignin content of the cellular tissue (in the case of swine forage also by the cellulose content) to a larger extent than by the hemicellulose composition proper. From the analytical data presented above it can be calculated that the valuable proportion of the barley whole meal hemicellulose amounts to 64 % or roughly two thirds, whereas in oat this proportion is 33 % or 1/3. In this respect knowledge of the hull-percentage makes a useful pointer. Results obtained by some fibre determination methods. The van Soest »neutral detergent fiber» values are also reported in Table 3. The determination was carried out according to the recommended procedure, by boiling for one hour 1 g of forage with 100 ml of a buffered solution (pH 7.0) of 3 % sodium lauryl sulphate containing 1.86 % of disodium ethylenediamine tetraacetate. The materials were filtered in sintered glass crucibles of coarse porosity, washed with hot water and acetone, dried overnight at 105°C, and weighed (van Soest 1963 a, van Soest & Marcus 1964). Since in the above treatment the starch tends to form a suspension, a sintered glass funnel of G 1 porosity was used. The boiled residue could not be filtered through a sinter of finer porosity. For this reason the finest particles were not quantitatively retained in the crucible, which accounted for the poor repeatability of the results. The figures represent the mean of three determinations. It appears that the result is influenced by the hull content of the sample. In oat the »detergent fiber» is about 5 % higher than the sum of hemicellulose, cellulose and lignin, whereas for barley the figures are about 10 % lower and for rye and wheat 20-25 % lower. Nevertheless, the »detergent fiber» -values include a small amount of both protein and ash. However, van Soest (1966) himself has remarked that the method has not been successfully applied to concentrates and feedstuffs containing starch because of interference in filtration. Several methods have been proposed for the determination of part of the cell-wall substances, for example the »normal-acid fibre» (NAF) of Walker and his collaborators (e.g. Walker & Hepburn 1955, Griffith & Jones 1963, the »lignocellulose» of Jarrige (1960), the »acid detergent fiber» of van Soest (1963b) and the »membrane substances» of Paloheimo and Paloheimo (1949). In the present investigation these methods were not applied, since judging by the author's previous findings (Salo 1965 a), at the acid concentration used the first three methods result in a slightly higher figure than the sum of cellulose and lignin presented in Table 2, because in the treatment these two substances and a small part of the hemicellulose remain undissolved. Paloheimo's method, on the other hand, yields somewhat higher values as the acid concentration is lower. The conventional crude fibre percentages are also reported in Table 3. The figures are lower than those obtainable by the above-mentioned fibre methods, due to the fact that the major part of the lignin dissolves during the treatment. Depending upon the plant material, the crude fibre corresponds to I-2 times the cellulose content (Salo 1965 b). For the grain samples listed in Table 2, the ratio of crude fibre to cellulose ranges from 1.3 to 1.7. Finally it should be noted that the purpose of the present investigation has not been to propose an analytical scheme for routine work, but instead to offer a general idea of the composition of cereal grains, especially their main components, the carbohydrates. General knowledge of the grain composition, along with even limited analyses, aids in the evaluation of the quality of a certain cereal lot. Three simple determinations already yield a rather good practical idea of the feed-value of oat and barley, and only the two latter are required for wheat and rye. These determinations are: 1) percentage of hull, 2) percentage of crude protein, and 3) percentage of starch. If the starch is determined by enzymatic hydrolysis, very little extra work is required for the determination of the cell-wall complex provided that the correction for protein is made on the basis of a percentage-factor. The conventional crude fibre determination is more or less pointless as a measure of cereal quality. Summary The contents and the distribution between the different grain parts of starch, hemicellulose, cellulose, crude lignin, crude protein and ash have been determined for oat, barley, rye and two wheat species. The relative proportions of the different structural units of the hemicellulose have also been determined. Furthermore, a method is proposed for the determination of the cell-wall complex in cereals. Oat and barley differ from wheat and rye to an appreciable extent only in respect of the hull. The hulls consist of strawlike material rich in lignin, cellulose and xylan; they are richer in xylan than the straw proper. The fruit and seed coat enveloping the seed (viz. the bran) contains relatively small amounts of lignin and cellulose. The bran is chiefly composed of hemicellulose with xylose and arabinose as the main components. The main compound of the endosperm is starch, although some hemicellulose and protein are present, whereas only minor amounts of cellulose and lignin can be found. In the samples investigated only small differences were noted between the compositions of wheat, rye, dehulled oat and dehulled barley; wheat had the highest starch content, rye the highest hemicellulose content, and oat the highest contents of crude protein and lignin. The cell-wall complex determined as the residue after enzymatic hydrolysis of the starch agreed well with that obtained by fractionation. On the other hand, the detergentfibre values differed from the fractionation results considerably, and the conventional crude fibre analysis yielded results which were about 1.5 times that of cellulose. The feed-values of the different grain parts are discussed on the basis of the analysis results obtained.

v3-fos

2020-12-10T09:04:17.284Z

1970-04-01T00:00:00.000Z

237233293

s2

Naphthylamidase Activity of Leptospira Extracts of 18 serotypes of the genus Leptospira were found to possess naphthylamidase activity, and differences in the pathogenic and saprophytic strains were noted. The former exhibited a preference for the leucyl naphthylamide substrate, whereas the latter demonstrated greater hydrolysis of alanyl naphthylamide. With the leucyl naphthylamide as substrate, pathogenic strains showed 10 to 20 times higher naphthylamidase activity than saprophytic strains. Optimal temperature and pH for enzymatic hydrolysis also differed between pathogenic and saprophytic strains. Maximal enzymatic activities for pathogenic and saprophytic naphthylamidases were 41 and 37 C, respectively, at pH 8.0 to 8.5. The pH and temperature optima suggested that the leptospiral enzyme activity was not leucine aminopeptidase. Many criteria have been used in attempts to distinguish the pathogenic from the nonpathogenic leptospirae in an attempt to elucidate the mechanism of pathogenesis. Some of the more recent identifying characteristics have been the resistance to copper sulfate (2), egg yolk decomposition (3), susceptibility to 8-azaguanine (7), and range of growth temperatures (6). In a study of leptospiral enzyme patterns determined by starch-gel electrophoresis, Green,Goldberg,and Blenden (5) found that pathogenic strains possessed naphthylamidase activity, whereas the nonpathogens tested were devoid of such activity. This paper is an extension of those observations. MATERIALS AND METHODS Leptospira serotypes were cultured, and intracellular enzymes were extracted as described previously (5). The method of Goldbarg and Rutenberg (4) was employed to determine naphthylamidase activity for each of the strains used, since this technique is more sensitive than the electrophoretic procedure used in the earlier work. A 1-ml amount of the cell-free extract and 1 ml of L-leucyl-fl-naphthylamide hydrochloride (Nutritional Biochemicals Corp., Cleveland, Ohio), 2.74 mm in 0.1 M phosphate buffer (pH 8.0), were incubated at 37 C for 2 hr, whereupon enzymatic hydrolysis was terminated by the addition of 1 ml of 40% trichloroacetic acid. The mixture was then centrifuged at 1,640 X g for 15 min to sediment the precipitated I Work done by G. B The amount of protein in each 1-ml sample of extract was determined by the method of Lowry et al. (8). Enzymatic activity was expressed as micrograms of j3-naphthylamine liberated per hour per milligram of protein. Appropriate controls substituting distilled water for the crude enzyme extract were employed in each case. Extracts were stored at -67 C and assayed within 1 week after preparation. Leucine aminopeptidase, a leucyl-jl-naphthylamidehydrolyzing enzyme from hog kidney (Nutritional Biochemicals Corp.), was used to provide a comparison with the crude leptospiral enzyme preparations. Five micrograms of this enzyme was allowed to react with the substrate under the same conditions as above. In all experiments with leucine aminopeptidase, 6 mm magnesium ion was required for enzymatic activity. Optimal temperature and pH range of enzymatic activity for leucine aminopeptidase and the leptospiral enzymes were also determined by the above methods. Use of additional substrate. DL-Alanyl-,8-naphthylamide (Nutritional Biochemicals Corp.) was employed in the same manner as the leucyl-,s-naphthylamide to ascertain the substrate specificity of both the leptospiral extracts and the commercial leucine aminopeptidase, but the assays were carried out at pH 7.0, the reported pH optimum for hydrolysis of the alanyl substrate (9). RESULTS AND DISCUSSION The colorimetric method of Goldbarg and Rutenberg (4) employed to determine hydrolysis of L-leucyl-/3-naphthylamide proved to be more sensitive than the starch-gel electrophoretic technique of Green et al. (5). All leptospiral strains tested were found to possess some leucyl naphthylamide-hydrolyzing ability. The relative activities of the strains tested are listed in Table 1, in which it can be seen that more than 100-fold variation in activity exists among certain saprophytic and pathogenic strains of Leptospira. The nonpathogenic biflexa serotypes exhibited the least leucyl-hydrolyzing ability. L. andamana, which previous investigators (2,3,11) showed to be related to the saprophytic biflexa strains, demonstrated a low activity which was similar to the nonpathogens. This indicates once more that L. andamana is more closely related to the biflexa group and should be classified as a nonpathogen. Three representive serotypes were selected for determination of the effect of temperature on the activity of their respective naphthylamidases. The optimal enzymatic activity of the leptospiral enzyme was at 37 C for saprophytes and at 41 C for pathogens. In contrast, leucine aminopeptidase showed its optimal hydrolytic activity at 50 C (Fig. 1). The optimal pH for enzymatic hydrolysis of the leucyl-f-naphthylamide was found to range between 7.5 and 8.5 for the leptospiral strains tested. No clear-cut difference was noted between pathogens and nonpathogens (Fig. 2). Pathogenic leptospiral enzymes and leucine aminopeptidase exhibited a preferential cleavage of the leucyl over the alanyl naphthylamide. In contrast, L. biflexa showed a definite preference for the alanyl substrate. This strain hydrolyzed the alanyl naphthylamide 2 to 10 times as effectively as the leucyl substrate ( Table 2). The differences in amino acid naphthylamide preference among the pathogenic and saprophytic enzymes would seem to indicate that at least two different enzymes (or isozymes) are involved in this hydrolysis. The divergence in optimal temperature of pathogenic and saprophytic enzymes supports this conclusion. Previous researchers have postulated the existence of more than one naphthylamidase (1,10). The leptospiral enzymes and leucine aminopeptidase differed markedly in optimal temperature and pH values for hydrolysis of the leucyl-fnaphthylamide, indicating that the leptospiral enzyme responsible for hydrolyzing amino acid naphthylamides is not leucine aminopeptidase. The enzyme or enzymes in Leptospira are most likely true naphthylamidases. The modifying influence of the amino acid side chain is evident from the different enzymatic activities with the alanyl and leucyl naphthylamide substrates. It would be valuable to use a greater variety of amino acid naphthylamides to determine the hydrolysis patterns for the various serotypes of Leptospira. APPL. MICROBIOL. No additional metal ion was required for enzymatic activity of the leptospiral enzyme. Leucine aminopetidase and some of the reported naphthylamidas have been found to require such ions as zinc, cobalt, manganese, and magnesium. The in vivo role of naphthylamidase remains obscure and its role in pathogenicity cannot yet be determined. However, it should be noted that investigators have used elevation of serum naphthylamidase levels as an indication of cancer and other diseases of the liver or bile ducts (4). Often in the more severe manifestations of leptospirosis liver involvement is seen. It is therefore possible that naphthylamidase plays a role in the virulence of the Leptospira.

v3-fos

2019-03-20T13:04:45.582Z

1970-12-01T00:00:00.000Z

83622290

s2

INFLUENCE OF IRRIGATION AND SUPPLY OF AVAILABLE NITROGEN ON GROWTH AND NUTRIENT CONTENT OF SPRING WHEAT The Law of Minimum states that the amount of plant growth is regulated by the factor present in minimum amount. In southern Finland this factor is often water, particularly, when spring cereals are in question, and an improvement in the water supply during the critical period in June will increase the yield, provided no other factor will start to limit the growth. According to recent experience, on mineral soils this second factor may be nitrogen, if not more than the normal amount of fertilizer nitrogen is applied. It is obvious that an increase in the amounts of the minimum factors will have an influence on the metabolism of the plant and on the uptake of other nutrients. Thus, effects both on the quantity and on the quality of the yield may be detected. In order to study the dependence of growth and the nutrient content of cereals on the supply of water and nitrogen, samples of the aerial parts of spring wheat were collected at various stages of development from a field trial on irrigation and placement of nitrogen fertilizers. A part of the results of this trial have been treated from the point of view of the effect of irrigation on the uptake of nitrogen (Kaila and Elonen 1970); in the present study attention is paid to the effect of irrigation and supply of available nitrogen on the growth and the content of nutrients in the wheat plants. dry 1969 start to limit the growth. According to recent experience, on mineral soils this second factor may be nitrogen, if not more than the normal amount of fertilizer nitrogen is applied. It is obvious that an increase in the amounts of the minimum factors will have an influence on the metabolism of the plant and on the uptake of other nutrients. Thus, effects both on the quantity and on the quality of the yield may be detected. In order to study the dependence of growth and the nutrient content of cereals on the supply of water and nitrogen, samples of the aerial parts of spring wheat were collected at various stages of development from a field trial on irrigation and placement of nitrogen fertilizers. A part of the results of this trial have been treated from the point of view of the effect of irrigation on the uptake of nitrogen (Kaila and Elonen 1970); in the present study attention is paid to the effect of irrigation and supply of available nitrogen on the growth and the content of nutrients in the wheat plants. Experimental The field trial was carried out in the dry summer 1969 in cooperation with the Finnish Research Institute of Agricultural Engineering. The experimental field was in the neighbourhood of Helsinki, on silty clay soil of about pH 6 (in 0.01 M CaCl 2 ) with 5 per cent of organic carbon and a satisfactory content of exchangeable potassium and »available» phosphorus. ears were cut from the samples and analysed separately. The grain samples were taken from the winnowed material. The samples were air-dried at room temperature, and ground in a Wiley mill. Total nitrogen was determined by the common Kjeldahl procedure. The total content of phosphorus, potassium, magnesium and calcium was measured from acid ash solution: phosphorus was determined by the ammonium vanadate molybdate method, potassium by an EEL-flame photometer, and magnesium and calcium by a Perkin-Elmer atomic absorption spectrophotometer. The results were treated with Duncan's new multiple range test. Values of each sampling date marked by the same letter in the table do not differ at P = 0.05. Results The growth of spring wheat under different treatments was estimated on the basis of the amount of dry matter in the plant samples collected. These results are reported in Table 1 calculated to correspond to the amount of dry matter as kg/ha. It is of interest to note that on June 16, there was not yet any response to the 30 mm of irrigation water applied one week before this sampling date. At the end of the month, or about two weeks since the application of the second 30 mm of irrigation water, the yields of the irrigated plots contained 750 to 1000 kg/ha more dry matter than those of the nonirrigated plots, except when the supply of available nitrogen was very low. The positive effect of nitrogen on the production of plant matter was apparent already on June 16. It tended to increase at the later stages of development, particularly on the irrigated plots. On these plots, the amount of dry matter produced when the nitrogen upply was very good was on June 16, June 30, July 21, and August 18 about 20, 36, 53, and 61 per cent higher, respectively, than the corresponding yields on the plots with very low nitrogen supply. The positive effect of irrigation appears to be markedly higher with good or very good supply of available nitrogen than with low or very low supply. On July 21, e.g., the increase in the total dry matter yield by irrigation was less than 20 per cent when the supply of nitrogen was very low or low, but 37 per cent when the nitrogen supply was good, and more than 50 per cent when it was very good. On the basis of the results of August 18, the response to the placement of 120 kg/ha of soluble fertilizer nitrogen was 2200 kg/ha of dry matter, the effect of irrigation alone corresponded to 2140 kg/ha, and the combined effect of both these treatments resulted in an increase of 6460 kg/ha in the dry matter production. The positive interaction of water and nitrogen supply is obvious. The nitrogen content of the plant samples (Table 2) gives a slightly different picture. The nitrogen percentage tended, of course, to rise with the improvement in the supply of available nitrogen, but the effect of irrigation was more complicated on the nitrogen content than on the dry matter yield. In June, the samples collected from the irrigated plots contained, in most cases, significantly more nitrogen than the corresponding samples of nonirrigated plots. In the July samples, there is no more any significant difference between the nitrogen content of the corresponding samples. In the ears, there is some tendency to lower nitrogen content in the irrigated samples, and this is statistically significant in the samples of August 18, except when the nitrogen supply was very low. In the straw samples this difference is significant only with very good nitrogen supply. The phosphorus content of the plant samples is reported in Table 3. In the June samples, irrigation significantly increased the phosphorus content, provided the nitrogen supply was not very low. A similar tendency is also found in the straw samples of July 21. This means that irrigation markedly improved the uptake of phosphorus by plants, since also the dry matter yields were increased. This increase in the accumulation of phosphorus in the shoots corresponded to 60 to 80 per cent in the samples of June 30. Later, the higher production of dry matter on the irrigated plots diluted the phosphorus concentration, but this is statistically significant only in the ears of August 18. The effect of nitrogen on the phosphorus content of the plant samples was less distinct than that of irrigation. Though there usually was a tendency to a slightly lower phosphorus content in the higher dry matter yields produced by the better supply of nitrogen, also some exceptions may be found. The potassium content (Table 4) appears to be more closely connected with the supply of nitrogen than the phosphorus content was. The potassium content of shoots and straw increased with improving nitrogen supply both in the irrigated and nonirrigated samples. In the potassium content of the ears, the differences between the nitrogen treatments were low, and there was in August a distinct tendency to a lower content of potassium in the higher dry matter yields produced by better nitrogen supply. Irrigation increased markedly the potassium content of the samples in June. In July it decreased the potassium content of straw, but increased that of ears. In August, there is again a distinct tendency to a higher potassium content in the straw samples of the irrigated plots, but no difference between the corresponding samples of ears is detectable. When the magnesium content of the plants are examined, the question arises, whether attention must be paid to the dolomitic magnesium (3 %) in Oulunsalpietari. In this trial only in the irrigated samples of June 16 there was a somewhat higher magnesium content in the shoots from the plots treated with Oulunsalpietari as compared with the corresponding samples treated with urea. In no other cases any significant difference was detected between the effects of these two fertilizers on the magnesium content of the samples, not even any average tendency to a higher magnesium content with Oulunsalpietari. Both irrigation and a better supply of nitrogen increased the magnesium content of the shoots in June (Table 5). The positive effect of nitrogen is apparent even in the magnesium content of the straw samples in July and August. The higher yields of irrigated plots resulted in July and August in somewhat lower content of magnesium in the ears. Table 6 show that the effect of irrigation and nitrogen supply on the calcium content of the plant samples is similar to their influence on the magnesium content. Only, in July, both straw and ears tended to be richer in calcium on the irrigated plots. The grain yields were winnowed, and thus the material analysed represents pure mature grains. Data in Table 7 indicate that dry matter yields were significantly increased by irrigation at all levels of available nitrogen supply. This increase in the dry matter yield was more than 1400 kg/ha when the supply of nitrogen was good or very good, but only about 600 kg/ha, when it was low or very low. The response to nitrogen was marked: the good supply of available nitrogen produced about 60 per cent more grains without irrigation, and about 80 per cent more with irrigation than did the very low supply of nitrogen. Yet, even the latter yields were by no means low. Irrigation decreased the nitrogen content of grains by 7 to 15 per cent. Since the nitrogen content of grains from the nonirrigated plots was high or very high, this drop is not serious. Means of dry matter yield and means of the content of respective nutrients followed by the same letter do not differ at P = 0.05. The grains were not poor in phosphorus, and only with good or very good supply of nitrogen irrigation decreased this content significantly. The potassium content of grain dry matter seems to decrease with improving supply of nitrogen, but irrigation had no significant effect. The magnesium content, on the other hand, did not markedly depend on the nitrogen supply, and it was distinctly decreased by irrigation, except when the nitrogen supply was very low. The calcium content was slightly increased by irrigation when the nitrogen supply was low or very low, but no difference was found when the supply was good or very good. It is alluring to use the results obtained from the samples collected on August 18, or two days before harvest, to estimate the amount of these nutrients in the mature wheat crop. These figures are recorded in Table 8. The amounts of nutrients in the irrigated crops are markedly higher than those in the corresponding nonirrigated ones, particularly, when the nitrogen supply was good or very good. Also the increase in the yields due to better supply of nitrogen resulted in higher uptake of potassium, phosphorus, magnesium, and calcium by the crop. Means of each nutrient followed by the same letter do not differ at P = 0.05. When the nitrogen supply was good or very good, irrigation increased the nutrients in the crop by almost 50 kg/ha of nitrogen, about 60 kg/ha of potassium, 5 to 6 kg/ha of phosphorus, 3 to 4 kg/ha of magnesium, and 6 to 7 kg/ha of calcium. The highest yield, produced by irrigation and very good supply of nitrogen, contained three times as much nitrogen and potassium, about 2.4 times as much magnesium and calcium, and almost twice as much phosphorus as the lowest yield with very low supply of available nitrogen and without irrigation. Discussion In this field trial in a dry summer the yield of spring wheat was markedly increased by the application of 60 mm irrigation water during the critical period before the development of ears. In order to get the best results, also 136 kg/ha of soluble fertilizer nitrogen placed in rows was necessary. The high production of grain by irrigation and nitrogen dressing was possible, partly, because both treatments, particularly the irrigation, allowed a more intensive uptake of essential nutrients during the most vigorous vegetative growth. Thus the shoots in June had a high content of at least nitrogen, phosphorus, potassium, magnesium, and calcium, all of them necessary for the photosynthesis and building up of plant material. It is easy to understand that in a dry soil the nutrients of fertilizers are not readily dissolved. Also, the availability of soil sources may be decreased. Thus, moistening of the soil is likely to enhance the uptake of nutrients by roots. On the other hand, it has been found that irrigation is likely to produce a larger root system (Kähäri and Elonen 1969), and even in this way it may improve the uptake of nutrients from soil and fertilizers. It is possible that most of the nitrogen was nitrified before the plants absorbed it. Therefore, the effect of nitrogen in increasing the potassium, magnesium and calcium content of the aerial vegetative parts of wheat may be partly connected with a higher uptake of cations to balance the more intensive absorption of nitrate anion. The phosphorus content, the uptake of phosphate anion, was to a far lower degree affected by the nitrogen supply. Probably, also the root system tended to be more vigorous, and the plant metabolism in general intensified, when the nitrogen content of the cells was not a minimum factor. The present results emphasize the importance of providing also other nutrients in sufficient amounts, when the usual minimum factors, water and nitrogen, are no more limiting the growth. In this trial a good yield was produced without irrigation and with a heavy treatment with soluble nitrogen applied as surface dressing which corresponds to the common practice in Finland. The top yield produced by irrigation and placement of the nitrogen fertilizer contained, according to the plant samples collected two days before harvest, more than twice as much potassium as the »normal»yield. The increase in the phosphorus requirement was about 50 per cent of the phosphorus content in the »normal» yield. The uptake of magnesium and calcium were increased 70 and 90 per cent, respectively. The top yield contained more nitrogen and potassium than was applied in the fertilizers. In this soil rich in organic matter and clay, the deficit could be made up by the soil sources, particularly, since irrigation was likely to enhance their mobilization. There is no doubt that the improvement in the efficiency ofplant production by irrigation and fertilizer placement will mean great demands even on soil sources. Particular attention must be paid to magnesium, sulfur, and the trace elements. The marked increase in the nutrient content of young wheat plants brought about by irrigation may point to a noteworthy possibility to improve the quality of forage from pasture and ley. Summary In the dry summer 1969 the effect of irrigation and supply of available nitrogen on the production of dry matter and on the N, P, K, Mg, and Ca content of the aerial parts of spring wheat was studied on the basis of samples collected at various stages of development from a field trial. Shoots collected from the irrigated plots (30 mm water on June 9 and 30 mm water on June 17) in the middle and at the end of June had a higher content of all the nutrients studied than those from the nonirrigated plots although at the latter date also the dry matter yield was markedly increased by irrigation. Later, the larger production of dry matter on the irrigated plots decreased this difference and resulted in equal or even somewhat lower contens of N, P, K, Mg, and Ca in the ears and straw, and also in the grains. An improvement in the supply of available nitrogen tended to increase the dry matter yield and the content ofK, Mg, and Ca in the vegetative parts of the plant; the N content was increased also in the ears and grains. The positive interaction of water and nitrogen supply was distinct in the production of dry matter and in the uptake of nutrients. The high grain yields produced by irrigation and good or very good nitrogen supply were attributed, at least partly, to the more intensive uptake of nutrients during the period of vigorous growth in June. It was emphasized that the improvement of the efficiency of plant production by irrigation and placement of fertilizers may result in an impoverishment of the soil of other nutrients. ainesato oli kasvanut sadetuksen vaikutuksesta. Myöhemmin sadetus lisäsi kuiva-ainesatoa siinä määrin, että erot vastaavien sadetettujen ja sadettamattomien koejäsenten näytteiden ravinteiden pitoisuuksissa pienenivät ja jopa muuttuivat päinvastaisiksi.

v3-fos

2018-04-03T00:11:22.254Z

1970-11-01T00:00:00.000Z

10551840

s2

Sugar substrates for L-lysine fermentation by Ustilago maydis. The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; K(La) in the fermentors, 0.41 mmoles of O(2) per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. Five auxotrophic mutants of Ustilago maydis, obtained by a combination of ultraviolet and ethyleneimine treatment, were previously studied (8) on more than 60 media with agave juice (aguamiel), corn-steep liquor, corn oil, and ammonium salts as the main constituents. Yields as high as 2.5 g per liter were reached in 300-liter fermentors by using mutant UV-ET-15. Since this is a high yield for this particular species, it was decided to study three other inexpensive substrates: commerical sucrose, sugar cane clarified juice ("clarified juice," International Society of Sugar Cane Technologists terminology), and blackstrap molasses and to continue working on the development of new high-yielding mutants. Among these, homoserine-, methionine-, and threonine-requiring mutants were found capable of producing acceptable amounts of L-lysine. The present report deals with studies carried out to gain maximum yields of free L-lysine by using such mutants. However, the yields here presented are far below any value that would warrant production of this amino acid by U. maydis on an industrial scale. Bacteria will probably continue to be the selected microorganisms for this purpose at the present time, but the possibility exists that by improving the yields of U. maydis through induced genetic changes in the strains, by selection of fermentation media, and by adequate bioengineering operations fungi may be used for the commercial production of L-lysine. MATERIALS AND METHODS Cultures. Mutants of U. maydis were kept in potato-dextrose-agar slants or on sterilized corn, and monthly transfers were made. Inoculum was prepared from 4-day cultures at 28 C as follows. A loopful was transferred to tubes containing 10 ml of a sucrose medium, composed of (per 100 ml) sucrose, 0.25 g; peptone, 1.0 g; NaCl, 0.5 g. After 38 hr of cultivation in a rotary shaker, 5% inoculum was used to seed Dulaney's medium (3) in Erlenmeyer flasks. After 48 to 72 hr of agitation at 250 rev/min and 28 C, sufficient seed was taken to inoculate the fermentation media at a level of 5%. Media. To compare the activity of the strains, Dulaney's glucose medium (3), B glucose medium (8), and agave juice media [26-a, 48, and 19 (8) ] were used. Theeffect of ammonia nitrogen and naturally occurring nitrogen sources on L-lysine formation by mutant UV-ET-15 was studied in Dulaney's glucose medium with 1% corn oil added. Fermentations were carried out in this medium with the following carbon sources: medium S-l (commercial sucrose as received); S-2 (invertase-treated sucrose); M-l (blackstrap molasses as received); M-2 (blackstrap molasses treated with 10 mg per liter of K4Fe (CN)6 at 80 C during 6 hr, centrifuged, and filtered); M-3 (ferrocyanide-treated molasses, hydrolyzed with invertase); J-l (clarified juice, as received); J-2 (clarified juice treated with ferrocyanide as indicated before), and J-3 (clarified juice, ferrocyanide-treated and invertase-treated). Commercial invertase (0.1%) was used for the treatment of these sugar substrates at 50 C for 48 hr. When CaCOs was used, it was sterilized separately and then incorporated in the medium. Media were autoclaved at 115 C for 20 min, cooled, and then inoculated with 5% seed. Conditions. The inoculated Erlenmeyer flasks were mechanically agitated for 120 hr at 250 rev/min in a 687 SANCHEZ-MARROQUIN, LEDEZMA, AND CARRENO APPL. MICROBIOL. New Brunswick rotary shaker. The incubation temperature was 30 C; pH was adjusted at 5.8 to 6.2 in all fermentation media. The same pH and temperature conditions were established for the 7.5-, 20-, and 100liter fermentors. The 7.5-liter fermentors corresponded to model F-7 of New Brunswick Scientific Co., equipped with pH, temperature, and foam automatic controls, one disc sparger, two turbine impellers, and four baffles. The 20-liter fermentor was a French E.I.V.S.-672 glass model with three flat-blade impellers, one disc sparger, and no baffles. The 100-liter fermentor was included in a steel pilot plant designed by Olsa from Milan, Italy; this fermentor has one turbine impeller, a ring sparger, and four baffles. Aeration, agitation, and other operational conditions are indicated in the respective tables. Concentrate. A lysine concentrate was obtained by evaporation and spray-drying of the fermentation broth from the 100-liter fermentor, with medium J-3 as a substrate. The material was then passed through an electrical mill, and the brown powder obtained was submitted to chemical analysis. Determinations. Reducing sugar, pH, viscosity, oxygen-transfer rate, and mycelial weight were determined as indicated previously (8). The microbiological standard method with Pediococcus cerevisiae P-60, occasionally checked against paper chromatographic techniques, was used in the estimation of Llysine. For the chemical determinations of the amino acid concentrate, previous hydrolysis with 6 N HCl for 22 hr at 100 C was made, and then AOAC analytical methods were followed (1). The amino acid pattem was detected by means of a Beckman model B automatic analyzer. For the digestibility tests of the powdered concentrate, defatting was performed in a Goldfish apparatus; the AOAC method (1) was followed by using a buffer boric acid solution in place of the conventional acid solution to receive the distilled ammonia. Table 1 shows the L-lysine yields in five media by the IFSC 65-1 parent strain of Ustilago maydis and the four mutants obtained from it. The UV-ET-15 homoserine-requiring mutant had been previously studied (8) in the agave juice media 26-a, 48, and 19. Results indicate that this mutant gave the highest yields in most of the media tested, and it was therefore selected for all subsequent experiments. RESULTS The effect of three ammonium and two naturally occurring nitrogen sources on L-lysine formation by this particular strain on Dulaney's glucose medium with 1% corn oil is shown in Table 2. The three ammonium salts at their respective optimal concentrations gave almost identical results, and their lysine yields were higher than those obtained with corn steep liquor or yeast extract. Yeast extract appears to affect both growth and lysine synthesis, whereas corn steep liquor and yeast extract apparently are used for growth only. Some other naturally occurring nitrogen sources such as gelatin, peptone, corn meal, cotton seed meal, and soya flour did not consistently improve yields. The addition of some probable precursors such as aminoadipic and ketoadipic acids did not influence results in any way. When the effect of some commercial carbon sources was studied, the addition of corn steep liquor seemed favorable for good and reproducible lysine production. Results in shaker-flask experiments shown in Table 3 refer to medium 26-a inoculated with the UV-ET-15 mutant. Clarified juice appeared to be a better substrate than sucrose or blackstrap molasses. A slight increase in lysine yields or a shortening of the fermentation time were apparent when these substrates were previously hydrolyzed by means of commercial invertase, especially in the case of clarified juice. Growth and sugar consumption were affected in a similar manner. The best yields were approximately 2 g of lysine per liter. Ferrocyanide treatment was of no particular benefit. Fermentations carried out in 7.5-liter fermentors by using five selected commercial sugar media usually gave inconsistent results when no corn steep liquor was added to the media or when the aeration and agitation conditions were not properly adjusted. The yields given in Table 4 show the slight variations observed with the best operating conditions. An increase in yield to 2.81 g of lysine per liter in medium J-3 (clarified and inverted juice) was reached at the end of 72 to 120 hr at 30 C, 500-rev/min agitation, and 0.3-volume per volume per min aeration. To secure consistent results, it was estimated that a Kd X 102 value of 12 gram-molecules of 02 per liter per hr was apparently adequate for good lysine production in medium J-3 by the mutant under study ( Table 5). The mean viscosity and density values of these fermentation broths were lower than those of the agave juice media (8). Further experiments suggest that a KLa value of 0.41 mole of 02 per liter per min would permit more consistent results. Some metabolic data of L-lysine formation in a 20-liter fermentor operated at a KLa value of 0.41 mmole of 02 per liter per min are shown in Table 6 and Fig. 1. The highest yield, 2.58 g of lysine per liter, was reached at the end of 72 hr coincident with rapid mycelial growth and low sugar consumption. The pH decrease paralleled carbohydrate utilization. Similar results were obtained in 7.5-liter fermentors. The lysine yields, by using mutant UV-ET-15 in the J-3 medium, are higher than those reported in the scientific literature and in some patents (Table 7) and are similar to those reached in agave juice media (8). Finally, one of the fermentation broths obtained from a 100-liter fermentor with medium J-3 was evaporated and spray-dried; the powder obtained showed the chemical composition given in Table 8. As can be seen, the protein content is acceptable and the digestibility is good. Some of the main amino acids present in the concentrate are shown in Table 9. DISCUSSION Mutant UV-ET-15 of Ustilago maydis is a fair lysine producer. Yields of approximately 2.5 g per The ammonium salts studied (phosphate, sulfate, and acetate) are equally good nitrogen sources for lysine production in this medium, depending on the concentration used. Contrary to observations of Kurtz and Ericson (5), ammonium acetate slightly increased the lysine yields as pointed out by other investigators (7,8). The efficient utilization of the acetate would suggest a lysine biosynthetic pathway via aminoadipic acid (10). However, the addition of this acid or ketoadipic acid to the culture medium in concentrations as high as 10 mg/ml did not improve lysine production. We have no valid explanation for this fact. The addition of corn steep liquor has a slight effect upon metabolite production but, according to previous observations (6,8) and to our data ( Table 2), this seems to be an indirect result of growth. Clarified juice could be utilized as a cheap carbon source for lysine production, because its local price is below that of cane sugar. The unsuitability of blackstrap molasses has been reported by Tauro et al. (9). However, our mutant grows well in this substrate and produces more lysine than the strain studied by these workers. Hydrolysis of cane molasses does not improve yields but can reduce the fermentation time from 120 to 72 hr since glucose is better assimilated than sucrose (Table 2). By using the fermentation conditions recommended by several workers (4-7), unreproducible results, as they themselves have observed, were attained when commerical carbon sources were incorporated in the media. However, when the oxygen transfer was properly controlled, the variations in yield were not as wide as they appear from the results shown in Tables 4 to 6, which were obtained in fermentors of different sizes. Kd values around 0.12 gram-molecule of 02 per liter per hr or KLa of 0.41 mmole of 02 per liter per min allowed high and relatively constant results. Details of this study will be published elsewhere. The maximum lysine value is reached between 48 and 72 hr during the initial growth period when the pH is around 5.3 and the sugar consumption is low (Table 6; Fig. 1). At this time, sugar is apparently used for rapid mycelial growth and lysine reaches its highest value and remains constant, as does growth, until a period of 144 hr is reached. Afterwards, yields of lysine increase slightly as a probable result of mycelium lysis. Maximal lysine synthesis occurs when 11 to 20% sugar is consumed. Therefore, pH and carbohydrate utilization, as well as growth and lysine synthesis, seem to be related as it was observed in the case of agave juice media (8). Since preliminary experiments showed that the addition of specific minerals to the media (MgSO4, MnCl2, NaCl, FeSO4, ZnSO4, and CaCl2), independently or together, did not affect lysine production even at a concentration of 0.2 g per liter, they were not added to the fermentation media. Probably the small amounts present in corn steep liquor are sufficient. The chemical composition and the amino acid pattern of the lysine concentrate (Table 8,9) suggest its utilization as a feed supplement. The vitamin B content of this concentrate has been previously reported (6,8).

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2019-05-31T13:07:11.034Z

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Encapsulación de sabores de d-limoneno mediante secado the quality properties of the product obtained by spray drying and on the performance of the process variables. The established, through optimization, that the best formulation is achieved with a mixture of 0.4% low-methoxyl pectin and 0.1% tween 60. The spray drying process was evaluated by varying the inlet air temperature (150-200ºC), outlet air temperature (90-110ºC) and speed of the atomizer disk (20000-30000 rpm). The optimal conditions for the drying process were achieved with inlet and outlet air temperature of 156.7 and 90ºC, respectively, and an atomizer disc speed of 30000 rpm. With these parameters, the following results were obtained: solids recovery (90.9%), percent of deposits (0%), solubility (98.4%), moisture content (2.22% w.b), hygroscopicity (9.2%), water activity (0.112) Introduction Encapsulation is a technology that allows the coating of solid, liquid or gaseous products with polymeric membranes to preserve the products. This technology has been widely used in the food industry because it protects sensitive material from processing conditions or storage and provides products with a longer shelf life. This technology also allows the active ingredients to be released in a controlled manner at the right time (Barbosa et al., 2005;Parra, 2010). When the technique of spray drying is used in emulsions, it is necessary to add emulsifiers to increase the emulsion stability and to assure that the emulsion can endure the drying temperatures without deterioration. This is possible because these molecules possess polar and nonpolar components, which allow the molecule to be adsorbed at the particle interface, forming aggregates that confer enhanced stability to the emulsion (McClements, 2004;Aranberri et al., 2006). During the encapsulation process, the stability of the emulsion is essential because of this depends that the properties of encapsulated product are appropriate, and for the particular case of flavors, to avoid oxidation, which increases the shelf life of the product for different industrial applications (Gharsallaoui et al., 2007). D-limonene is a terpene that is extracted from the peels of citrus fruits and plays an important role in the manufacture of foods, medicines and perfumes. The flavoring may be liquid or solid, and the latter when interacts with other components of different nature, such as in the manufacture of beverages (Lappas & Lappas, 2012). However, d-limonene easily reacts with oxygen and has a high sensitivity to heat, so at high temperatures, it degrades (Li & Chiang, 2012). The aim of this study was to encapsulate d-limonene using a spray drying technique to evaluate the influence of different agents with surface active properties (tween 20, tween 60, sodium caseinate and low methoxyl pectin) on some of the quality features of the product obtained and to assess the overall performance of the drying process for the purpose of industrialization. Emulsion optimization Using response surface methodology, the optimum region for composition/formulation of the emulsion was found by evaluating the stability and the rheological characterization using the zeta potential and the viscosity. The experimental data were fitted to a linear model, Eq. (1), and a cubic model, Eq. (2). (1) ( 2) where n is the number of components in the mixture, ßi is the expected response to the mixture with one component, βij and βijk are the synergistic interaction effects on the response values between components, δij is the cubic interactions of two components, X is the component in the mixture and Y is the response variable analyzed. Encapsulation by spray drying For the optimum emulsion, the effect of drying variables on the product quality and the overall performance of the process was evaluated. The drying was performed using a pilot spray dryer (Vibrasec) with an evaporative capacity of 1.5 Lh 1 . This dryer had an automatic control that allowed for varying the temperature of the drying air (inlet and outlet) and the speed of atomizer disk. The product (powder) was collected in a cyclone. A rotatable and orthogonal central composite design was established with the following factors: the inlet air temperature (150-200°C), the outlet air temperature (90-110°C) and the atomization speed (20000 -30000 rpm) (Gharsallaoui et al., 2007). Performance of the drying process The dependent variables for the performance of the drying process were the yield and percentage of material that adhered to the surface of the dryer. For the yield, the amount of product obtained during the drying was weighed and divided by the total solids in the emulsion. The material that adhered to the dryer was calculated by mass balance on the basis of the input material, the amount of dry product stuck in the dryer chamber and the product collected in the cyclone. Characterization of encapsulated Characterization of the encapsulated powder product was determined as follows: The encapsulation efficiency was determined by quantifying the total and surface d-limonene in triplicate. For the total d-limonene, 0.5 ml of distilled water was added to 10 mg of the powder, methanol was added and the mixture was centrifuged at 3000 rpm for 10 min. For the surface d-limonene, 10 mg of powder was washed with hexane and centrifuged at 3000 rpm for 5 min (Soottitantawat et al., 2005). Each sample was analyzed by gas chromatography coupled to mass spectroscopy by injecting 2 mL into the chromatograph (TRACE Ultra Gas Chromatography, Thermo Scientific, USA), which was equipped with a packed column (PEG-20M). The chromatographic conditions were the following: a flame ionization detector (FID) of 230°C with N 2 as the carrier gas. The column temperature was controlled at 120°C. The encapsulation efficiency was calculated using Eq. For the solubility, 1 g of the sample was homogenized in 50 ml of distilled H 2 O and centrifuged at 3000 rpm for 5 min at 25°C. Then, 25 ml of the supernatant was dried in oven at 105°C for 5 h. The solubility was calculated as the difference of the initial weight and the final weight divided by the initial weight (Cano-Chauca et al., 2005), and this measurement was performed in triplicate The hygroscopicity was determined using gravimetric methodology by placing samples (2 g) inside an oven with a controlled atmosphere at 20°C and 65% relative humidity. The samples (triplicate) were weighed until a constant weight was achieved (Δweight≅±0.001 g/g sample), and the result is expressed in terms of percent moisture (wet basis) (Arrazola et al., 2014). Optimization of the encapsulation process The central composite design was optimized using the response surface methodology to minimize the moisture content, the hygroscopicity and the amount of material adhering to the surface of the dryer and to maximize the yield (Laine et al., 2011). The experimental data were fitted to the second-order model given in Eq. (4): (4) where β0, ßi, βii and βij are the regression coefficients for the intercept, linear interaction terms and quadratic, respectively; X is the independent variable; and Y is the response variable. Particle size distribution For the product encapsulated under optimum conditions, the particle size distribution was determined using a Mastersizer 2000 light scattering system (Malvern Instruments, Malvern). The particle size of the product was expressed as the D 32 mean diameter weighted by the volume/ surface area ratio (Carneiro et al., 2013.). Morphological characterization The morphological characterization was performed using scanning electron microscopy (SEM) in which the product is placed on the slide of the SEM using double-sided adhesive tape (Nisshin EM, Tokyo, Japan) and was analyzed at an accelerating voltage of 20 kV after Pt-Pd sputtering using a MSP-1S magnetron sputter coater (Soottitantawat et al., 2005). Statistical analysis The results were analyzed using analysis of variance and a test for a lack of fit, by determining regression coefficients and by generating response surfaces using the Statgraphics Centurion VI software. Table 1 shows the mean values and standard deviations for the dependent variables in the emulsion characterization. The particle diameter varied between 0.471 and 2.586 µm, which favors the stability of the colloidal system (Soottitantawat et al., 2005). Paramita et al. (2010) found that d-limonene emulsions are stable when they had a drop size of less than 3 µm, and in turn, Soottitantawat et al. (2005) found particle sizes between 0.84 and 3.37 µm, which are similar to the results obtained in this work. The zeta potential ranged from -22.3 to -42.6 mV; values of approximately -30 mV are a suitable value that guarantees repulsive forces that promote emulsion stability (Oh et al., 2011). With respect to the rheological properties (viscosity), the analysis of rheograms showed a Newtonian behavior for all formulations where the viscosity ranged between 142.1 and 288.95 mPa.s and, according to Gharsallaoui et al. (2007), viscosity values of less than 300 mPa.s are advisable for this type of spray drying processes because greater viscosities interfere with the process due to the formation of large drops, which negatively affects the drying rate. Emulsion characterization The results of the statistical analysis showed significant differences (p <0.05) for the viscosity with respect to all of the factors and their interactions. A similar situation occurred with respect to the zeta potential. The regression coefficients for the models are given in Table 2. For viscosity, a cubic model, as it fits the data best, was established with an R 2 of 99.09% and no significant lack of fit (p> 0.05), which is shown in Figure 1A. The viscosity decreased when the emulsion was made using tween 60, sodium caseinate or pectin and was maximized with greater concentrations of caseinate and pectin. Surh et al. (2006) and Matia-Merino et al. (2004) reported similar results for the viscosity behavior in colloidal systems in which the viscosity was greater by adding sodium caseinate and pectin, which resulted in a viscous system that generates stability. By optimizing this response, a lower viscosity was achieved with a mixture of 0.1% tween 60 and 0.4% low methoxyl pectin. Analysis of variance (ANOVA) for the zeta potential showed significant differences when using a linear model, which established a relationship with the factors. It is evident from Figure 1B that the stability is lower when using 0.5% Tween 20, which may be due to the concentration used because Lee et al. (2013) reported a high microencapsulation performance and high stability in emulsions with the addition of 1% tween 20 in peanut extract microencapsulation. However, the concentrations evaluated in this study were lower (0.5%). Furthermore, the zeta potential increased with the addition of sodium caseinate ( Figure 1B), which is consistent with Surh et al. (2006), who stated that caseinate can form an interfacial layer up to 10 nm thick in oil droplets, which prevents flocculation and coalescence of the drops and increases stability. (1) and (2) for the emulsions and by equation (4) for the drying process. Figure 2A shows the response surface for the yield, and it is evident that by increasing the speed of the atomizer disk there is a slight increase in the solids recovery with respect to the different combinations of inlet and outlet air temperatures because with high atomization speeds, smaller droplets are formed that facilitate the spray drying process (Gharsallaoui et al., 2007). Additionally, by having low inlet drying air temperatures (approximately 150ºC) and high outlet temperatures (110°C), the product is in contact with the drying air longer, which allows for a solids recovery of greater than 80%. The behavior of this variable was similar to the behavior obtained by Kha et al. (2014) in Gac oil microencapsulation, where the optimum conditions for drying were 154°C and 80°C for the temperature of the drying air at the inlet and the outlet, respectively. At these temperatures, the yield was maximized, and the results of this study showed that yield was also maximized using similar temperatures. Moreover, the yield was influenced by the components present in the feed and the amount of adhered material to the dryer surface, but the feed was constant and included gum arabic and maltodextrin, which allow high yields (Bhusari et al., 2014). Figure 2B represents the response surface for the adhered material to the dryer, which indicates that by increasing the speed of the atomizer disk, the adhered material decreases. This result is because the vacuum imposed into the drying chamber easily drags the particles, which prevents material from contacting the wall and adhering to the surface because smaller particles are formed. This variable increases primarily due to high drying air inlet temperatures and low outlet temperatures. This may be caused by increasing the inlet air temperature, as the dry solids have a first order transition (melting), which causes the particles to adhere (Saffari & Langrish, 2014). Furthermore, the drying temperature also influences the state of the particle according to its glass transition temperature (Tg); when the drying temperature is greater than Tg, the particles are in a rubbery The zeta potential optimization demonstrated that greater stability was achieved by adding 0.5% sodium caseinate as an emulsifier. However, the standard model presented a low regression coefficient (R 2 of 35.55%), and therefore, the data do not fit the model appropriately. Therefore, due to the results obtained and the good fit of the model, the formulation established during the viscosity optimizing was selected as the optimum formulation, which was composed of 0.1% Tween 60 and 0.4% low methoxyl pectin and had a viscosity of 130.02 mPa.s and a zeta potential of -33.80 mV. From the results, the formulated emulsions had properties that favor stability, such as particle size and zeta potential (Soottitantawat et al., 2005;Oh et al., 2011). Accordingly, the positive effect of the emulsifiers that allows for increased stability due to their properties was demonstrated and produced smaller particle sizes and an adequate zeta potential. Additionally, it is worth noting that there was a combination of emulsifiers in which greater stability was obtained, and this result is consistent with Peng et al. (2010), who found that the addition of a second emulsifier in the formulation provided nanoemulsions (W/O) that were more stable. Stabilization with a combination of emulsifiers occurs due to the hydrophilic/ lipophilic balance (HLB) of the molecules, which describes the relationship between the hydrophilic and lipophilic parts of the emulsifier, and for O/W emulsions, an appropriate value is between 8 -18, and thus, mixing two or more substances results in proper balance and better stabilization properties because each emulsifier provides particular characteristics (Jin et al., 2008). Table 3 shows the results of the performance variables of the drying process: yield (92.87 -40.2%) and adhered material to the dryer (0 -22.62%). Analysis of variance showed significant differences (p <0.05) in the yield regarding the disk speed and for the adhered material relative to the inlet temperature and disk speed. state and thus stick more easily to the dryer walls (Keshani et al., 2015), and therefore, the high inlet temperatures increase the adhered material. Table 3 presents the results for the quality variables of the encapsulated product: encapsulation efficiency (92.18 -99.56%), solubility (98.12 -98.55%), moisture (2.13 -3.5%), water activity (0.173 -0.605) and hygroscopicity (6.56 -11.62%). The statistical analysis showed significant differences (p <0.05) for solubility, moisture and hygroscopicity with respect to all of the factors; however, for the solubility, the minimum differences that are not important on a practical level were presented. The regression coefficients for the models of the significant variables are shown in Table 2. For the encapsulation efficiency and the water activity, no significant differences (p> 0.05) were found. It has been found that the encapsulation efficiency depends on the conditions of emulsion preparation and particle size (Gharsallaoui et al., 2007;Paramita et al., 2010), which were constant in the spray drying process. Similarly, the water activity was not influenced by the drying temperatures or the atomization speed. Characterization of encapsulated For the moisture of the encapsulated product, the results showed that for a low atomization speed, the moisture decreased when a low inlet air temperature and a high outlet temperature were used, which diminishes the thermal gradient and results in a slow drying rate, resulting in a product with a lower moisture content. Furthermore, when high atomization speeds were used, the moisture content of the powder decreased when combining a high inlet drying temperature and low outlet temperatures, which increased the amount of water that evaporated from the product (Tonon et al., 2008). However, when comparing the behavior at high and low atomization speeds, it is evident that increasing the speed affects the moisture in the product. If the atomization speed is greater, the moisture decreases to less than 2% because a smaller droplet size forms ( Figure 2C). Similar results have been reported by Goula & Adamopoulos (2010) when drying orange juice in which the moisture decreased with an increase in the inlet air temperature; however, this behavior, in this research, is valid when using high atomization speeds. For the hygroscopicity, changing the atomization speed did not change the trend: the hygroscopicity was less when lower inlet and outlet air drying temperatures were used ( Figure 2D). Tonon et al. (2008) found similar results in the development of acai powder by spray drying, where the lowest hygroscopicity values were obtained by decreasing the temperatures; similarly, decreasing the moisture content of the particles increased the hygroscopicity, which agrees with the results of this work. It should be noted that the particle size is also important for the hygroscopicity because the surface area of small particles is greater and promotes the adsorption of water at the powder surface (Ghosal et al., 2010). The spray drying process optimization was performed and maximized the yield and minimized the moisture, hygroscopicity and adhered material to the dryer. The solubility and other variables that were not statistically significant were not considered in the analysis. The optimum conditions of the drying process were achieved with an inlet and outlet air drying temperature of 156.7°C and 90ºC, respectively, and the speed of the atomizer disk was 30000 rpm. At these conditions, the solids recovery was 90.9%, the material adhered to the dryer was 0%, the solubility was 98.4%, the powder moisture was 2.22% (w.b.), the hygroscopicity was 9.2%, the water activity was 0.112 and the encapsulation efficiency was 98%. The particle size distribution for the powder obtained under optimal conditions presented a bimodal distribution with two distinct peaks, each representing a predominant size. The analysis showed a D 32 size of 26.531 μm; the presence of larger particles can be attributed to agglomeration (Figure 3). Similar results have been reported in other studies (Garcia et al., 2012;Tonon et al., 2008). Furthermore, a low drying rate and a low inlet air temperature results in particles with flavoring was achieved with process conditions and quality characteristics that are suitable for industrialization. Acknowledgements The authors express their gratitude to the Research Directorate headquarters Medellin (DIME) at the National University of Colombia, to COLCIENCIAS and to the company Tecnas S.A. for financial support and infrastructure received. (Tonon et al., 2008), and the optimum conditions for drying resulted in small particles because of the low inlet air temperature. The external morphology of the encapsulated powder product created using the optimal conditions presented spherical particles without cracks or fissures ( Figure 4). Carneiro et al. (2013) stated that this morphology increases the protection of the active material because the capsules have a lower permeability to gases. Particles of different sizes and with rough surfaces were also observed, which is typical in a spray drying process. Similar features have been found in other studies of limonene and flaxseed oil (Soottitantawat et al., 2005;Carneiro et al., 2013). Conclusions The addition of a mixture of emulsifiers allows for the generation of stable emulsions that were suitable for the drying process. For d-limonene, a mixture of 0.4% low methoxyl pectin and 0.1% Tween 60 resulted in the best results in terms of colloidal stability and rheological characteristics, resulting in a viscosity of 130.02 mPa.s and a zeta potential of -33.80 mV. The optimal conditions for the d-limonene spray drying process are given for an inlet and outlet air temperature of 156.7°C and 90ºC, respectively, and the speed of the atomizer disk was 30000 rpm, whereby

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2020-12-10T09:04:11.129Z

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Detection and Incidence of Escherichia coli on Storage Pen Surfaces of Fishing Trawlers Six methods for the detection and enumeration of Escherichia coli on the storage pen surfaces of commercial fishing trawlers and in harbor wash water were evaluated. E. coli was found consistently present in Boston harbor water used for washing vessel holds and was detected either in small numbers or not at all on storage pen surfaces. Violet Red Bile Agar as a primary enumeration medium was found ineffective for detection of coliforms because of the nonselective development of large numbers of other gram-negative organisms. The use of E. coli broth at 44.5 C for primary most-probable-number determinations, followed by confirmation of E. coli on Levine Eosine Methylene Blue Agar, appears to offer numerous advantages over more conventional methods of detecting E. coli for survey studies of the fishing industry, where coliform-like organisms result in many false-positive presumptives with other methods. The common practice in the New England fishing industry of using raw, untreated harbor water for washing trawler fish storage pens is of sanitary and public health significance. Such water is usually associated with sewage pollution arising locally or from large-volume municipal sewage disposal in the same locale. The industry practice of briefly hand-scrubbing pen surfaces and rinsing with untreated harbor water was previously shown (4) to achieve no detectable reduction in the bacterial load. This study is concerned with the enumeration of Escherichia coli on storage pen surfaces after washing with unchlorinated harbor water and with the examination of the various schemes available for detection and confirmation. MATERIALS AND METHODS Sampling. Alginate wool swabs were used with stainless-steel templates to obtain 1-square inch swab samples (ca. 2.54 by 2.54 cm). Immediately after all of the fish in the storage hold of a vessel were unloaded, three swab samples were obtained from different panels of the same pen and dissolved in 20 ml of 1% Calgon. Three swab samples were again obtained adjacent to the first areas sampled after the storage surfaces of the hold and pens had been hosed down and scrubbed with unchlorinated harbor water in the usual manner by the cleanup crew. Water samples were also collected from the hose used on board the vessels for washing the storage pens and from the water source itself, the harbor water adjacent to the vessels. Bacteriological tests were performed within 24 hr after samples were obtained. Bacteriological methods. Decimal dilutions of dissolved swabs and water samples were made in Nutrient Broth containing 0.5% NaCl. The detection procedures were separated into six groups, each designated with a Roman numeral (Fig. 1). Difco media were used throughout. In method I, Lauryl Tryptose (LT) Broth was used for five-tube most-probable-number (MPN) presumptive determinations for coliforms. Tubes showing gas after 48 hr of incubation at 35 C were used to inoculate tubes of Brilliant Green Lactose Bile (BGB) Broth, followed by the transfer of resulting growth onto Levine Eosine Methylene Blue (EMB) Agar and incubation at 35 C for 24 hr; the combinations of these procedures served as the confirmed test. Typical colonies of E. coli were picked and transferred onto slants of Nutrient Agar (NA), from which the indole, methyl red, Voges-Proskauer, and citrate (IMViC) tests were performed. Method II made use of five-tube MPN determinations by using tubes of BGB incubated for 48 hr at 35 C, followed by confirmation with EMB and the IMViC tests. In method III, plates of EMB Agar were inoculated directly with 0.1 ml of diluted samples which were spread with a sterile glass rod and then incubated at 35 C for 24 hr. Colonies having the typical appearance of E. coli were picked for IMViC tests. Method IV consisted first in transferring growth from all positive LT MPN tubes from method I to tubes of Escherichia coli (EC) Broth followed by incubation at 44.5 C for 48 hr. The growth from positive tubes was then streaked onto EMB Agar plates, and colonies typical of E. coli were transferred to NA slants from which the IMViC tests were performed. In method V, pour plates of Violet Red Bile (VRB) Agar were used for initial enumeration. Coliform Counts are given as number of bacteria per square inch of surface or as number per milliliter of water sample. I Counts from pen surfaces represent the mean of three 1-square inch areas obtained from different locations of the same pen. colonies were picked after 18 to 24 hr of incubation, purified, and transferred to NA slants from which growth was transferred to BGB Broth tubes; resulting growth from positive tubes was then streaked onto EMB Agar plates. All coliform-like colonies picked from the original VRB plates were subjected to the IMViC tests. In method VI, EC Broth was used for primary MPN enumeration of E. coli. Growth from all tubes showing gas within 48 hr at 44.5 C was streaked onto EMB plates, and colonies typical of E. coli were transferred to NA slants from which the IMViC tests were performed. The IMViC tests were performed by the standard methods of the American Public Health Association (1). Preliminary results from these six methods indicated that all cultures presumptivelypositive regardless of the nature of confirmatory results yielded only gram-negative organisms, and the Gram stain was eliminated from later studies. Where solid media were used for initial enumeration, at least five typical coliform colonies per plate were picked where possible for confirming studies. RESULTS Comparison of five methods for detection and enumeration of E. coli from pen surfaces and wash water. Two vessels were used for this study ( Table 1). Method IV was found to be the method of choice for enumeration of E. coli in wash water. Only methods I, II, and IV successfully recovered E. coli from pen surfaces. The use of VRB Agar as the primary medium in method V proved highly unsatisfactory for detection of E. coli in both wash water and on pen surfaces because of the development of large numbers of red coliform-like colonies, some of which exhibited zones of precipitation typical of E. coli which failed to grow in BGB Broth or EMB Agar. Considerable bifficulty was encountered with the use of EMB Agar as the primary medium in method III because of the extensive growth of organisms other than coliforms. With all five methods used, E. coli was detected in only low numbers on pen surfaces or not at all, which is most likely accounted for by the low, though consistent, incidence of E. coli in hose and harbor water ( Table 1). Incidence of coliforms and E. coli on pen surfaces and in wash water. Eleven vessels were examined between October 1967 and October 1968 by using method IV. E. coli was detected on only two vessels before washing and on three after washing. The highest number of E. coli detected was 2.6 per square inch. The samples of hose and harbor water all yielded the consistent presence of E. coli and were highest in harbor water (harbor water, 1 to 35 per ml; hose water, 0.5 to 22 per ml). Effectiveness of EC Broth as a primary enumeration medium for E. coi. The effectiveness of EC Broth incubated at 44.5 C as the primary medium for MPN determinations for E. coli was compared to the conventional method with LT Broth at 35 C. Four trawlers were examined in this study ( Table 2). E. coli was detected on two vessels before washing and on three after washing (before, 0.3 to 1 per square inch; after, 1 to 2.7 per square inch). The samples of hose and harbor water all yielded E. coli (harbor water, 5 to 35 per ml; hose water, 4 to 21 per ml). No significant loss in the final number of confirmed E. coli was encountered with the use of EC Broth at 44.5 C, which offered the advantages of (i) eliminating many false-positive presumptives otherwise obtained with LT Broth, (ii) eliminating 1 additional day of incubation, and (iii) reducing considerably the amount of media and effort involved in obtaining final confirmation and in determining the identity of organisms producing gas in positive presumptive tubes. DISCUSSION Various methods and media are presently in use for enumerating coliforms and E. coli in food products and water. To determine the MPN values of coliforms in water, the Canadian Department of Fisheries (2) recommends Lactose Broth followed by confirmation with BGB Broth; for estimating the density of E. coli on fish fillets, the suggested method is the direct inoculation of dilutions into MPN tubes of BGB Broth, followed by confirmation in EC Broth at 45 C. The American Public Health Association (1) recommends the enumeration of E. coli and coliforms in water by performing a presumptive test in LT Broth, followed by confirmation with BGB Broth or EMB Agar and completing the test with the Gram stain and observing for gas production in LT Broth. Our results indicate that the direct inoculation of diluted samples into EC Broth incubated at 45 C appears to be the method of choice for the enumeration of E. coli in fishery products and untreated harbor water used for washing vessels. VRB Agar is recommended by some workers for selectively detecting coliforms from food products of nonmarine origin. Lewis and Angelloti (5) recommend VRB Agar as a primary solid enumeration medium for detecting coliforms in foods, followed by confirmation in BGB Broth; for E. coli, they recommend MPN determinations in LT Broth, followed by confirmation in EC Broth incubated at 44.5 C. Hartman (3) observed a great variation in the percentage of confirmed coliforms when various foods were tested by use of VRB Agar as the primary detection medium. Our results indicate that VRB Agar is ineffective as a selective medium for the direct enumeration of coliforms from fishery sources and that the term coliform has no significance in the fishing industry since many gram-negative organisms gave rise to APPL. MICROBIOL. false-positive presumptive tubes of LT Broth and BGB Broth and mimicked coliforms on VRB Agar.

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2018-04-03T06:21:35.772Z

1970-01-01T00:00:00.000Z

19932924

s2

Nutrition Requirements of Pleurotus flabellatus. The mycelium of Pleurotus flabellatus was grown in a synthetic medium to obtain accurate information on its nutritional requirements. Among various carbon sources tried, the organism was found to utilize hexose sugars more readily than other sugars. Ammonium citrate was found to be the best source of nitrogen. The yield of dry matter increased as the concentration of nitrogen was increased up to a certain stage beyond which there was no increase in the yield, but the crude protein content of the mycelium increased. Detailed studies on the effect of varying the concentrations of other major nutrients, i.e., potassium, phosphorus, calcium, and magnesium, on the growth and crude protein content of the mycelium were also carried out. Optimal pH range was fairly broad, lying between 4.5 to 7.5. The possibility of growing mycelium of Agaricus campestris in submerged culture by an aeration and agitation method was first demonstrated by Humfeld (7). Block (5) has suggested that such a process would offer a new and revolutionary method of food production. Detailed investigations were carried out on the nutritional requirements of A. campestris by Humfeld and Sugihara (8,9), who grew the fungus on a chemically defined medium containing suitable sources of carbohydrate and nitrogen with some mineral salts. Whitaker (17) also studied in detail the growth requirements of some mushrooms. The medium defined of Humfeld and Sugihara (8) was later tried by others (10) for the submerged culture of a few species of Psalliota and Boletus, and satisfactory results were achieved. In 1954, Sugihara and Humfeld (15) tried to grow selected stock of various species of mushrooms and tested their ability for growth in liquid medium. Mycelium, like the fruiting body, has been shown to be valuable nutritionally as a source of amino acids and B complex vitamins (4,8). Eddy (6) mentioned that higher fungi grown in submerged culture were capable of producing materials with good nutritive value. They can, therefore, serve as a protein supplement for human beings or as an animal feed. Mushrooms should be considered as one of the world's greatest natural resources, since they have the ability to transform nutritionally valueless substances into high fat and protein foods (13). 1 Present address: H. C. Srivastava, Hindustan Levers Ltd., Gaziabad, U.P. No literature seems to be available on the submerged propagation of Pleurotusflabellatus (Berk. & Br.) Sacc. Since this species of Pleurotus is eaten by the people in Mysore State (India), a detailed investigation was undertaken to obtain accurate information on its nutritional requirements, with the idea that it might be grown on a cheap, natural medium on a large scale for food. MATERIALS AND METHODS P. flabellatus grows on dead and decaying wooden logs under natural conditions. This mushroom is cooked like a vegetable and is eaten by the people in Mysore. The culture of P. flabellatus was obtained from the tissue of the sporophore. By use of standard aseptic methods, small pieces of sporophore tissue were placed on potato-dextrose-agar slants (potato infusion from 200 g of potatoes; dextrose, 20 g/liter; agar, 20 g/liter) and incubated at room temperature (21 to 35 C). Subsequent subculturing produced a pure culture of mushroom mycelium which was maintained on potato-dextrose-agar. Inoculum was produced in 500-ml Erlenmeyer flasks containing 50 ml of potato-dextrose-broth. Bits of mycelium planted on this medium were allowed to grow at room temperature (21 to 35 C) under stationary conditions for 15 days. The mycelium was then washed with sterile distilled water and transferred to 250-ml glass-stoppered flasks containing 50 ml of sterile distilled water and glass beads. The mycelium was broken up by vigorous shaking, and the resultant suspension was used as inoculum-about 4 ml per flask. Flasks were incubated on a rotary shaker having an eccentricity of 1 in and were operated at 200 rev/min until the mycelium had attained its maximum growth with complete utilization of substrate (approximately 8 days). 166 NUTRITION REQUIREMENTS OF P. FLABELLATUS The basal medium constituents per liter were as follows: KH2PO4, 1 g; MgSOO47H20, 0.5 g; CaCl,2 2H20, 0.35 g; MnSO4.H20, 3 mg; ZnSO4-7H20, 3 mg; FeSO4-7H2O, 3 mg; Na2MoO4, 3 mg. A mixture of vitamins containing thiamine, niacin, riboflavine, pantothenic acid, p-aminobenzoic acid, each at the rate of 100 ,ug/liter, and cyanocobalamin, biotin, pyridoxine, and folic acid, each at the rate of 50 jug/liter, wete added. The basal medium plus 50 g of glucose was used for the study of nitrogen utilization. Nitrogenous compounds were added at the rate of 0.424 g of nitrogen per liter (11). Other nutrients were added to the medium as shown (Tables 1-4). To obtain a clear indication of the effect of increasing the concentration of each major element, i.e., phosphorus, potassium, magnesium, and calcium, on the yield and the crude protein content of the mycelium, some experiments were conducted in which the nutrition was uniform except for the element under study. The quantities of major elements supplied per liter of the basal medium were as follows: phosphorus, 0.22 g; potassium, 0.287 g; magnesium, 0.049 g; and calcium, 0.098 g. Phosphorus was added in the basal medium at four different levels (varying from 0 to 0.33 g/liter) and was supplied in the form of potassium dihydrogen phosphate. A complete reduction of phosphorus in treatment 1 ( Table 5) was achieved by not adding potassium dihydrogen phosphate, and substituting for it sufficient potassium citrate to maintain the level of potassium. In treatment 2, in which the phosphorus content was reduced to 0.11 g/liter, 0.5 g of potassium dihydrogen phosphate per liter was added, and it was supplemented with potassium citrate so as to supply an equivalent quantity of potassium. In treatment 3, since 0.22 g of phosphorus was the basal dose required, no manipulation was needed. In treatment 4, a higher dose of phosphorus was added by supplementing the medium with ammonium phosphate and adjusting the nitrogen content of the medium with ammonium citrate. Potassium was added in different concentrations (varying from 0 to 0.42 g/liter) in the basal medium ( Table 5). The other nutrient elements were kept at a uniform level in all the treatments. Potassium was supplied in the form of potassium dihydrogen phosphate. A complete reduction of potassium in treatment 1 ( Table 5) was achieved by withholding potassium dihydrogen phosphate and substituting for it sufficient ammonium phosphate and ammonium citrate as to maintain the same level of both phosphorus and nitrogen. In treatment 2, in which the potassium content was reduced to 0.14 g/liter of the medium, only 0.5 g of potassium dihydrogen phosphate was added, and it was supplemented again with ammonium phosphate and ammonium citrate, so as to supply an equivalent quantity of phosphorus and nitrogen. In treatment 3, since 0.28 g of potassium was the basal dose required, no manipulation was needed. In treatment 4, a higher dose of potassium was added by supplementing the medium with potassium citrate. Calcium and magnesium were added to the basal medium in the form of calcium chloride and magnesium sulfate, respectively. Initial pH was adjusted in all the experiments to 5.5 with lactic acid and sodium hydroxide. Analytical methods. The mycelial weights were obtained by filtering the culture fluid containing the mycelial pellets through a fine sieve, washing the mycelium with distilled water, and drying it in an oven at 60 C for 15 hr before weighing. The total nitrogen in the mycelium was estimated by the micro-Kjeldahl method (1), and the formula, protein = total nitrogen X 6.25, was used to calculate crude protein. Reducing sugars were determined colorimetrically with the alkaline copper reagent of Somogyi (16) and the arsenomolybdate reagent of Nelson (12). All the data represent averages of four flasks. (Table 1), the mycelium of P. flabellatus grew fast and gave maximal yield of dry matter when D( +)mannose D-fructose, or D-glucose was used. The sugar alcohols like glycerol and mannitol were not utilized by the mycelium. Other carbon sources, i.e., starch (soluble), maltose, and sucrose, gave SRIVASTAVA AND BANO yields of dry matter in a decreasing order. Mycelium failed to grow on D-xylose, L(+)arabinose, D( +)galactose, L( -)sorbose, lactose, or cellulose. Among various carbohydrates tried The maximal yield of mycelium was obtained with 50 g of glucose (Table 2). Yield, as well as the crude protein concentration of the mycelium, was influenced mainly by the nitrogen source used in the medium ( Table 3). The utilization of substrate was maximal when ammonium citrate was used as nitrogen source. Efficiency of protein formation was also maximal for ammonium citrate. In the case of asparagine, the substrate utilization was 62%, with a crude protein content of 25 %. Yield of the mycelium was low when ammonium sulfate was used in the medium and the final pH was quite low. Traces of mycelial growth were observed when ammonium nitrate was used in the medium and the final pH was 3.4. Ammonium chloride, like the other ammonium salts of inorganic acids, was not suitable as a source of nitrogen due to low pH. Addition of ammonium tartrate to the medium brought down the final pH to 3.2; however, the mycelium could grow. Concentration of nitrogen had considerable influence on yield as well as on the protein production by P. flabellatus. Yield of the mycelium increased from 5.75 to 12.87 g/liter of the medium as the ammonium citrate concentration was increased from 0.25 to 2 g/liter (Table 4). On further increasing the concentration of ammonium citrate, yield of the mycelium decreased progressively, although the crude protein concentration of the mycelium increased. In other words, the C/N ratio was suboptimal, which affected the final yield of the mycelium. Maximal yield was obtained when ammonium citrate was added at a level of 2 g/liter of the medium. Higher concentration of phosphorus did not have much effect on the crude protein content or on the yield of P. flabellatus mycelium (Table 5). Omission of phosphorus from the medium reduced the yield to 0.25 g of mycelium per liter of the medium, and the crude protein content was 5 %. Maximal yield of the mycelium was obtained when phosphorus content was 0.22 g/liter. Higher concentration of phosphorus did not significantly improve yield, nor was there any increase in the crude protein concentration of the mycelium. Similarly, in the case of potassium, calcium, and magnesium, mycelial growth was slow and the protein content was low when these elements were omitted from the medium. Again, higher concentration of these elements did not improve the crude protein content of the mycelium nor was there any increase in the yield. No growth occurred after 8 days at an initial pH of 3.0 (Table 6). Highest yields were obtained when the initial pH was 5.5. However, growth was observed when the initial pH was between 4.0 and 9.0 and the corresponding final pH was 3.9 to 6.0. DISCUSSION The results obtained (Table 1) by growing the mycelium of P. flabellatus on D( +)mannose, D-glucose, and D-fructose are in agreement with previous reports (2, 14) on other fungi. Mannitol and glycerol were not utilized by the mycelium at all, which compares favorably with nutritional requirements of Morchella esculenta studied by Brock (3). Xylose and arabinose, normally present in wood, were not utilized, even though P. flabellatus normally grows on dead or decaying wood. It has been observed that, when ammonium salts of inorganic acids were used as sole source of nitrogen, the final pH of the medium went down to 3.0. Probably there was an accumulation of inorganic acids in the medium which are strongly dissociated as compared to organic acids. Consequently, poor growth, of mycelium occurred. The results obtained for urea do not confirm the earlier report on Psalliota campestris (8). P. flabellatus forms firm mycelial pellets which can be easily recovered from the culture fluid by filtration. However, the pellets are not fibrous or tough and hence are palatable.

v3-fos

2020-12-10T09:04:12.350Z

1970-01-01T00:00:00.000Z

237232950

s2

Microbiological Aspects of Ethylene Oxide Sterilization Spores of Bacillus subtilis var. niger, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in one of four thicknesses of low-density polyethylene film (2, 4, 6, and 20 mils). The surfaces were then placed in a specially designed thermochemical death rate apparatus and exposed to an ethylene oxide concentration of 600 mg/liter (at 54.4 C) and 50% relative humidity. Survival data, including both spore survivor curves and decimal reduction values (expressed as D values at 54.4 C-600 mg of ethylene oxide per liter), demonstrated similar survivor patterns when the B. subtilis var. niger spores were enclosed in low-density polyethylene films 2, 4, and 6 mils thick. A comparison of these patterns with those of spores enclosed in glassine and subjected to identical exposure conditions revealed only slight variations. The use of 20-mil polyethylene film greatly increased the time required to kill B. subtilis var. niger spores under the exposure conditions. Spores of Bacillus subtilis var. niger, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in one of four thicknesses of low-density polyethylene film (2,4,6, and 20 mils). The surfaces were then placed in a specially designed thermochemical death rate apparatus and exposed to an ethylene oxide concentration of 600 mg/liter (at 54.4 C) and 50% relative humidity. Survival data, including both spore survivor curves and decimal reduction values (expressed as D values at 54.4 C-600 mg of ethylene oxide per liter), demonstrated similar survivor patterns when the B. subtilis var. niger spores were enclosed in low-density polyethylene films 2, 4, and 6 mils thick. A comparison of these patterns with those of spores enclosed in glassine and subjected to identical exposure conditions revealed only slight variations. The use of 20-mil polyethylene film greatly increased the time required to kill B. sublilis var. niger spores under the exposure conditions. Items to be sterilized by ethylene oxide are usually wrapped or packaged to maintain sterility after sterilization. It is important, however, that the material selected meet a major requirement of being permeable to ethylene oxide and moisture (7). Plastic films are widely used for packaging such items as foods, hardware, clothing, toys, and many disposable medical and surgical supplies (3,10,12). Many manufacturers of these films have tested them for gas permeability and moisture transmission. However, very few manufacturers test the films with ethylene oxide and, for this reason, it is difficult to learn which films are satisfactory for gas sterilization. Only limited information has been published on the permeability of the more commonly used plastic films to ethylene oxide, moisture, and to other gases (2,8,9,11). Of the commercially available plastic films for wrapping or packaging items to be gas sterilized, polyethylene and (more recently) nylon are the most widely used (10,12). Polyethylene meets almost all requirements for an ideal wrapper and is especially desirable because of its transparency. There are three density grades of polyethylene film: low, medium, and high. For gaseous sterilization, the low density, 1 to 4 mils (0.001 to 0.004 inches) thick grade is recommended because of its higher permeability to ethylene oxide and moisture (1). Because of the increased usage of plastic films for packaging articles to be gas sterilized, we present this report of spore-inoculated carriers enclosed in polyethylene film of various thicknesses and exposed to ethylene oxide. MATERLAL AND METHODS Spore preparation and carriers. Bacillus subtilis var. niger spores dried on hygroscopic and nonhygroscopic carriers were used. Preparation of the spore suspension and inoculated carriers was described in the preceding papers (4-6). Polyethylene film. Four thicknesses of polyethylene film, 2, 4, 6, and 20 mils (Zendel Polyethylene Film, Union Carbide Corp., Cartersville, Ga.), were used. Two and 1.27-cm squares of the plastic film were cut and folded to form envelopes. These were heat sealed on three sides. The envelopes were placed in open glass jars and sterilized with ethylene oxide. After sterilization, the glass jars were loosely capped and the plastic envelopes were allowed to degas for at least 48 hr. The spore-inoculated carriers were then aseptically transferred to the sterile plastic envelopes and the envelope openings were heat sealed. Ethylene oxide exposure. The thermochemical death rate apparatus and exposure procedures previously described (4) were employed. The exposure conditions were as follows: ethylene oxide concentration, 600 mg/liter; relative humidity, 50%; temperature, 54.4 ± 3 C. The exposure periods varied. Recovery and enumeration of survivors. The recovery and plating procedures were described in previous publications (4,5). gas as a result of the plastic film barrier was found. This was demonstrated by similar survival patterns for B. subtilis var. niger spores with and without (6) the film enclosure. However, if a thick polyethylene film (20 mils) is used, its thickness must be compensated for by a longer exposure period to allow complete diffusion of the sterilant. Information concerning the suitability of a packaging material for gas sterilization (whether it is permeable to ethylene oxide and moisture RESULTS AND DISCUSSION When B. subtilis var. niger spores, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in polyethylene films 2, 4, and 6 mils thick and were exposed to the aforementioned conditions, the spore survival patterns (Fig. 1) varied little from one another. This was also reflected in the decimal reduction values (expressed as D at 54.4 C-600 mg of ethylene oxide per liter) shown in Table 1. If one compares the survival patterns of B. subtilis var. niger spores dried on the same surface types but enclosed in glassine envelopes and exposed to the same conditions (6), only slight variations will be noted. This supports the findings (1) that the low-density grade film does not appreciably affect the diffusion or sporicidal effect of ethylene oxide. The recommendations of other authors (1, 2) to use polyethylene film, approximately 1 to 4 mils thick, are readily appreciated when one notes the extended survival times (Fig. 2) and higher decimal reduction values for B. subtilis var. niger spores sterilized in 20mil polyethylene film. This investigation confirmed the permeability of polyethylene film to ethylene oxide, and no radical alteration in the sterilizing ability of the and whether it can be employed as a wrapping material) should be sought from the manufacturer. Such information should be readily available from all manufacturers of packaging materials for sterilization applications.

v3-fos

2018-04-03T03:38:16.651Z

1970-06-01T00:00:00.000Z

7221271

s2

Influence of water activity on the growth of Clostridium perfringens. Each of four strains of Clostridium perfringens was grown in modified fluid thioglycolate medium which was adjusted to yield selected water activity (a(w)) levels. The adjustments to secure the desired a(w) levels were made with NaCl, KCl, or glucose. At each a(w) level, further modification was effected to produce four pH values. Cultures were incubated at either 37 or 46 C. The solute used to achieve the reduced a(w) levels appeared to have a definite effect on the magnitude of growth achieved, the rate of growth, and the limiting a(w) at which growth would occur. Use of glucose as the controlling solute permitted growth at the lowest a(w) level tested, 0.960, and yielded the greatest magnitude of growth as measured by turbidity values, at all of the a(w) levels investigated. Cultures grown in the medium with added KCl generally demonstrated the longest lag times and the least amount of growth. Regardless of specific solute used, as the a(w) level was lowered and the pH value decreased within each a(w) level, the rate and amount of growth were lessened. It appeared, however, that low pH values had less effect on inhibiting growth at low a(w) levels than at higher a(w) levels. Those cultures incubated at 46 C generally exhibited shorter lag periods than those at 37 C, although the maximal growth attained was somewhat less than that achieved at 37 C. The response to all of the investigated conditions was similar for each of the four strains tested. The concept of water activity (a,) as a measure of the moisture requirements of microorganisms has been emphasized by Scott (12,13). In these review articles, he defined awv as specifying the chemical potential of water in a solution relative to that of pure water at the same temperature and pressure and pointed out that it is numerically equal to the corresponding relative humidity expressed as a fraction. Wodzinski and Frazier (15) concurred in recommending the expression of the available moisture of the medium, in terms of aw, as the most accurate means of describing the quantitation of substrate water accessible to the organism for growth or metabolic processes. In a study in which two strains of Clostridium perfringens were employed, Kim (Thesis, Purdue University, Lafayette, Ind., 1965) reported the limiting a, values for growth to vary from 0.93 to 0.97, depending upon the solute employed to control the a, of the medium. She further reported that the a, demands for the various cell functions of an organism were not necessarily the same. For example, she indicated that C. perfringens required a higher a, level for spore production than for vegetative growth. These results were more recently reported in a paper by Kang et al. (8). Baird-Parker and Freame (1), in a study dealing with C. botulinum, also noted a variation in a, requirements predicated on cell function. The present study had as its objectives: (i) determination of the limiting a, for growth for four strains of C. perfringens type A in a complex medium, (ii) comparison of the effect on growth EFFECT OF WATER ACTIVITY ON C. PERFRINGENS course of these investigations. (i) Cooked Meat Medium (CMM; Difco) was routinely employed to maintain the frozen stock cultures in the laboratory. (ii) Fluid Thioglycolate Medium (FTG; BBL) was utilized in growing vegetative cells from a stock spore inoculum. (iii) Sporulation medium (DS) of Duncan and Strong (5) was utilized for preparing spore suspensions basic to the experimental work. (iv) A modification of Fluid Thioglycolate Medium (FTG-1) served as the basal medium for the cells grown in media adjusted to selected a, levels. FTG-1 was prepared from the same formula as FTG, omitting resazurin, agar, and cystine. Preparation of inocula. Spore suspensions were prepared in DS medium from the stock CMM cultures, as follows: I ml -10mlFTG--*0.1 ml-10mlFTG CMM 16 hr 16-24 hr 1 ml -* 100 ml DS 24 hr In those cases in which a strain apparently did not sporulate when the sequence just outlined was followed, an additional passage through DS medium, with an incubation period of 3 hr, was made before the final 24-hr incubation. All cultures were incubated at 37 C, and the resultant suspensions were checked microscopically for the presence of spores. The spores were stored in the spent DS medium at 2 C with no further treatment. A 1 -ml amount of the spore suspension was inoculated into 10 ml of FTG and heat-shocked for 20 min at 75 C. After incubating this FTG culture for 12 to 16 hr at 37 C, a transfer of 1 ml was made into 10 ml of fresh FTG, followed by incubation for 16 hr at 37 C. The latter FTG culture was centrifuged in a clinical centrifuge to obtain cells which were sufficiently packed so as to permit the supernatant liquid to be decanted. The cells were then resuspended in fresh FTG-1 that had been previously adjusted to yield the desired a, level, and 0.15 ml of this suspension served as the inoculum for 150 ml of similarly adjusted FTG-1. Adjustment of a,. Three series of experiments were designed to follow the growth of each of the four strains of C. perfringens in media of progressively reduced a, levels. For each series, the a, adjustment was achieved with a different solute: NaCl, KCl, or glucose. Media were routinely prepared within 24 hr of use. No attempt was made to maintain a constant aw level after inoculation of the medium, except that screw-capped glassware was routinely employed. In series I, the medium was prepared by adding reagent grade NaCl to FfG-1 in predetermined amounts (grams of solute per 100 ml of FTG-1) prior to pH adjustment and autoclaving. FfG-1 with the added NaCl will be referred to in the remainder of this paper as FTG-N. Series II utilized a similar procedure. Reagent grade KCl was added to FfG-1. This medium will be identified in this paper as FTG-K. In series III, a two-step preparation was employed to adjust FTG-1 with glucose. The FTG-1 was made double strength by use of a half volume of water. This double strength FTG-1 was adjusted for the specified pH values, allowing for a pH change during subsequent autoclaving and glucose addition. The predetermined amount of reagent grade glucose was dissolved in the remaining half volume of water. This glucose solution was sterilized by filtration using a Millipore filter (0.45 jM pore size), and stored in sterile bottles. Prior to inoculation of the medium, equal volumes of the double strength F0G-1 and the glucose solution were aseptically combined. This medium will hereafter be designated as FTG-G. Determination of a, . A modified method of Daniels et al. (4) for freezing point determination was used to calculate the amount of solute necessary to achieve the desired a, level in basal FTG-1. Two modifications were made: an alcohol bath was substituted for the air jacket, and crystallization was induced by addition of an ice crystal to the supercooled solution. The "effective" gram moles of the solutes present in basal FfG-1 were determined from the freezing point of the basal medium established experimentally by use of the following equations: n2 = g/M2 = G0/1,00 kf (1) aw = n1/(n, + n2) = P/PO = N(solvent) (2) where M2 = the "effective" molecular weight of solute; this value represents the effect that the total solutes present have on the solvent in terms of the freezing point of the solution; g = the number of grams of solute added to 1,000 g of water in preparing the medium; G = grams of solvent used in medium preparation; Kf = molal freezing point depression constant, taken as 1.86 for water; 0 = freezing point depression measured in degrees centigrade; ni = number of moles of solvent in the medium; n2 = number of moles of solute, from equation (1); P = vapor pressure of the solvent in solution; Po = vapor pressure of pure solvent; N(solvent) = mole fraction of solvent in solution. The validity of using freezing point determination as an index of a, lies in the relationship of the colligative properties of solutions, specifically between vapor pressure and freezing point. One form of Raoult's law states that the vapor pressure of the solvent in solution, P, is equal to the vapor pressure of pure solvent, PO, multiplied by the mole fraction of the solvent in solution, N (solvent). Thus, P = Po N (solvent) or N (solvent) = P/PO. By definition (equation 2), aw = PIP°; N (solvent) then is the only term needed to determine a, by this procedure. Again, from equation 2, N (solvent) = n1/(n1 + n2). But from equation 1, n2 is found with the freezing point depression value, and n1 is known; i.e., the solvent is water whose molecular weight is 18, and, since G is known, n, can be determined from the relationship n1 = G/18. The fact that both equations are based on numbers of particles present and degree of particle interaction with the solvent establishes a valid relationship between freezing point depression and a, . Adjustment of pH. In determing the effect of interaction of variation in degree of acidity and a, on the growth of C. perfringens, the media were adjusted as VOL. 19, 1970 follows. We added 2 N NaOH or 2 N HCl in appropriate amounts to the respective unautoclaved FTG-1 media at predetermined a, levels to produce the desired pH value (d0.1 pH unit deviation) after autoclaving. A Corning (model 10) pH meter was used to measure the pH of the media. Duplicate bottles of medium in 150-ml amounts were prepared. One bottle of medium provided samples used in checking for correct pH adjustment and for blanks in reading turbidity of the growing cultures; the second bottle served as the growth medium for the respective inocula. During the growth of the organism, no attempt was made to maintain a constant pH level by continuous buffering. Experimental design. For all experiments, the growth of C. perfringenis in the adjusted media was determined by turbidity readings for incubation periods to a maximum of 72 hr. A Klett Summerson photoelectric colorimeter (model 800-3) was used to determine turbidity, and the magnitude of growth was expressed in Klett units. Turbidity readings were made of the cultures at such intervals as to demonstrate best the end of lag, logarithmic growth, and beginning of death phases. Lag time in this paper refers to the time in hours for a culture to achieve a turbidity of 40 Klett units. Maximal growth is defined as the greatest turbidity reading recorded for a culture during the 72-hr growth period. Each of the four strains of C. perfringents was grown in media adjusted to each of six a, levels: 0.995 (basal), 0.98, 0.975, 0.970, 0.965, and 0.960 ( Table 1). The a, values 0.995 to 0.965 were calculated from equations 1 and 2 given above by using experimentally determined freezing point depressions; the solute concentration necessary to achieve an av level of 0.960 was extrapolated from a curve of a, versus grams of solute added to achieve the freezing point depression corresponding to the a,, level. The 0.960 a, level was approximate because the plot of a, versus solute concentration did not produce a linear curve because of the high concentration of solute and resultant lack of an "ideal" solution. During the course of the experiments, to permit the study of the possible relationships of a,, pH, and temperature on the growth response of the organism, two incubation temperatures were tested (37 and 46 C). At the incubation temperature of 37 C, each of four pH values was established at each a,, level; these were 7.0, 6.5, 6.0, and 5.5. Each experiment consisted of a strain being grown in FTG-1 adjusted to a specific a, by one solute and at each of four pH values. For the second incubation temperature, 46 C, only two strains of C. perfrintgens, one solute, and two pH values were tested. The two strains used were NCTC 8238 and IUI 168, and they were each grown in FTG-G atpH 6.5 or 5.5. For all experiments conducted during this investigation, a minimum of two replications was made. RESULTS Except where noted, data presented are based upon average values expressed as turbidity readings for growth response for the four strains of C. perfringens employed throughout these experiments. Incubation of cultures was at 37 C except for one series of experiments which is delineated. Limiting a,. Growth curves for cultures of C. perfringens in FTG-1 adjusted to pH 6.5 at each of six a,, levels for each of three solutes are presented in Fig. 1 (a-f). The basal medium (a,5 of 0.995, Fig. la) permitted the greatest degree of growth to develop with the shortest lag period. The limiting a,5 for C. perfringens appeared to be 0.970 when FTG-N or FTG-K served as the medium for growth; growth continued to occur at approximately 0.960 when FTG-G was similarly employed. As demonstrated by turbidity values, decreasing the a, level to 0.98 with each of three solutes resulted in a decrease in maximal growth attained in each case, as well as an increased lag period. Further lowering of the a, level with each of the solutes progressively increased the lag periods and decreased the level of maximal growth attained. These findings were further substantiated by the growth curves resulting from the data compiled when the other pH values were tested. Thus, the response of this organism under these conditions reaffirms the observations made for many other species, that growth is influenced by the levels of a,5 and tends to be proportional to the moisture available in the immediate environment. b-f). For example, at the aw level of 0.98 maximal growth attained by the cultures grown in FTG-G was greater than that in FTG-N, but the cultures in FTG-N exhibited shorter lag times than those in FTG-G. The effect of solute used to reduce the aw became more apparent as the aw level was decreased and was especially evident in the range of limiting aw for growth (Fig. le). No multiplication of the inocula was observed in the FTG-1 adjusted with either of the two electrolyte solutes at an aw of 0.965, whereas growth of inocula continued to be evident in FTG-G at an aw of 0.960. Once initiated, the rate and degree of growth achieved by cell inocula in FTG-G at at 0.960, the lowest aw level tested, were greater than the rate and magnitude exhibited by cultures grown in FTG-N or FTG-K at a, 0.970. Figure 2 (a-c) shows the effect of varying the degree of acidity on maximal growth attained by C. perjringens, when each of three solutes was used to adjust the aw levels. As the pH value of the growth medium was lowered, growth response of C. perfringens continued, but at a level which seemingly was influenced by the degree of acidity in the environment. At any aw level at which growth occurred in media adjusted to pH 7.0, growth also occurred in media of pH 5.5. There was some indication that, as aw was lowered, the consequence of the interaction between aw and the variously adjusted pH values on maximal growth of the organism was reduced. The curves obtained when NaCl was used as a controlling solute (Fig. 3) suggest that, in media of high aw levels, the effect of decreasing the pH level seemed more influential on limiting growth than in media of lower aw levels; that is, at lower a, levels the a, level seemed to be more important in determining the amount of growth than was the low pH value. This decreased effect of pH value occurred with each of the three solutes. The fact that the acid or alkali added in adjusting the pH value would have some effect on the precise a, value attained cannot be ignored, but the quantities involved were such as to suggest that any change would be negligible. Although the growth response to different pH values within each a, level, and to the a, levels adjusted by each solute, varied with respect to maximal growth attained, the pattern was essentially the same. It may be stated generally that pH 6.5 permitted as great or a greater magnitude of growth as did pH 7.0. Cultures grown in media adjusted to pH 5.5 produced the lowest levels of growth. In those cases in which great differences in growth response to the four pH values were observed, it was between pH 5.5 and the other three levels. When response to concurrent adjustments in aw levels and pH values was examined in terms of lag time (Table 2), it was observed that, in FTG-N, as the pH value was decreased from 7.0 to 5.5 and as the a, level was decreased at each Growth comparisons were made for two strains of C. perfringens (NCTC 8238 and IU1168) grown in FTG-G adjusted to pH 6.5 or 5.5 and incubated at either 37 or 46 C. Figure 4 270- observed fairly consistently as the aw level was decreased at all pH values for all strains, although strain ATCC 3624 produced a slightly more variable pattern than the other strains. Furthermore, the effect of decreasing the pH value at each a, level did not result in definitive difference in performance among the strains although there were some variations. Under the conditions of these experiments, with minor exceptions, the performance of the four strains was relatively uniform in terms of pattern of maximal growth. Also, when lag times of the respective strains were examined in terms of the effect of a, and pH, the four strains responded similarly. When the response of the individual strains to different temperature incubations is considered, IU1168 cultures grown at 46 C generally had shorter lag times than NCTC 8238 at the same temperature (Table 3). Similar comparisons at 37 C indicated that, in most cases, IU1168 again demonstrated shorter lag times than NCTC 8238. It is interesting to note that, when incubated at 46 C in media adjusted to pH 6.5, cultures of NCTC 8238 showed no growth at an a, of 0.965 or 0.960, although growth occurred at these a, levels in media adjusted to pH 5.5. At the lowest aw levels tested, lag times were greater for IU1168 cultures incubated at 46 C than at 37 C; the NCTC 8238 cultures did not grow at 46 C in media adjusted to pH 6.5, but they did grow at 37 C. DISCUSSION The a, levels of 0.995 to 0.96, which permitted various degrees of multiplication of cells for the four strains of C. perfringens tested here, fall within the range determined by Kim (Thesis, Purdue University, 1965) and reported also by Kang et al. (8). In the instance in which the most direct comparison between the two studies is possible, i.e., control of a, by use of NaCl, Kim observed growth at an a, of 0.97 as did we; however, the subsequent a, level tested by her was 0.95 and she found no growth; indeed, a rather rapid decrease in cell counts occurred. Our results indicate an a, of 0.965 to be limiting for growth under similar conditions. The strains of C. perfringens utilized in the experiments were not the same in the two laboratories. A second difference was some variation in the amount of NaCl per volume of water employed in achieving the desired a, control, and, third, the growth media to which the adjusting solute was added were not identical. The question of the influence on growth of an organism of the solute used to adjust the a, of the medium, other than an effect directly attributable to the alteration in aw, is moot. Scott (12, p. 106) stated "...the biological response to a particular a, was, at least, for some organisms largely independent of the type of solutes and the total water contents of the substrate." Support has been given to this view by other workers (6,11,(15)(16)(17), although review of the evidence offered does in some cases suggest differences in minimal a, levels, depending upon whether the solute added to the medium for control of a, was salts or sugar. Kim (Thesis, Purdue, University, 1965) concluded that the growth response of C. perfringens to various degrees of a, was not independent of the kind of solute. Her findings were supported by the results reported by Baird-Parker and Freame (1) for C. botulinum. The evidence accumulated in the present study would suggest that the specific solute used to reduce the a, of the medium has a bearing on the minimal a, level required for the growth of the organism (Fig. 1). It would seem quite reasonable that there should be a difference in response of the organism depending on whether nutritive or non-nutritive solutewasused to adjust the a, level ofthe medium. Scott (12) suggested that possibly only when certain minimum nutrient requirements were satisfied did limiting a, for growth become independent of nutrient supply. Fuchs and Bonde (7), as a result of their study of the nutritional requirements, stated that C. perfringens was strictly dependent on carbohydrates and similar compounds for its energy. It can be hypothesized that, in the present study, glucose acted to increase the nutrient supply of the medium to the extent that greater growth was possible. In a preliminary unreported study in the present investigation, the pH value of the cultures was recorded at various intervals during the 72-hr growth period. It was observed that at corresponding a, levels those cultures grown in FTG-G reduced the pH value of their respective media to a lower pH value than did those cultures grown in FTG-N or FTG-K. The increase in acidity of FTG-G was probably due to the production of acids from the metabolism by the organism of the added glucose. No effort was made here to ascertain whether the growth of C. perfringens would have been altered if the pH of the medium had been buffered. At lower a, levels, the inhibitory effect of low pH values on growth appeared to be diminished (Fig. 3). This was especially noticeable when comparing the highest and lowest pH values tested (7.0 and 5.5). Wodzinski and Frazier (15)(16)(17) have reported that, in their investigations with Pseudomonas fluorescens, Aerobacter aerogenes, and Lactobacillus viridescens at nonoptimal pH and temperature levels for growth, the organisms were less tolerant to low aw levels; that is, as the environment moved further away from the optimal conditions, the minimal aw necessary for growth was increased. An explanation for the difference in behavior of the organisms employed in this study and those of Wodzinski and Frazier might be that C. perfringens apparently does not have a single optimal pH value for growth. Rather, it was observed in the present study that cultures grown in FTG-1 adjusted to pH 7.0 or 6.5 gave similar growth rates and magnitudes with no consistent differences. Smith (14) studied the effect of pH on generation times with strains of C. perjringens type A and observed that there was no sharp optimal value between pH 6 0 and 7.5 for manifestation of shortest generation times. Fuchs and Bonde (7) reported an optimal pH range for growth of C. perfringens between pH 6.75 and 7.5 in a partially defined medium. The greatest differences in the response to the four pH values were observed here to be between pH 5.5 and the other three values. Perhaps then the lesser effect of pH value at reduced aw levels observed in this study might be because the majority of pH values tested were not greatly divergent from the optimal range. It should be emphasized that, in this investigation, growth was measured by turbidity readings, and the values reported for growth included both viable and nonviable cells. Lysis of cells is a possible explanation of the lower turbidity values recorded for the incubation at 46 C. The four strains employed in this investigation, on the whole, responded similarly to reduced aw and pH conditions. This homogeneity in response by strains of the same species is in agreement with the findings of Scott (11) and Christian and Scott (3), who studied strains of Staphylococcus aureus and Salmonella, respectively. However, Ohye et al. (10) suggested, on the basis of their data with C. botulinum type E, that substantial differences in the minimum a, levels of various strains might exist. The latter investigators based their conclusions on data which demonstrated a divergent growth rate response by each of four strains to lowered aw levels as the temperature of incubation was reduced. The effect of a, level on the growth of C. perfringens as demonstrated in the present study confirms the observations made by many others of a relationship between the organism and its moisture requirements. Inherent in a consideration of limiting a, levels is the whole spectrum of the microbial environment, especially the pH value and temperature of incubation. The significance of these factors lies in the fact that food, as a suspending medium, provides an environmental situation which combines various degrees of awv and pH. In general, a food may be regarded as a more complex substrate than a liquid medium in terms of its gross physical structure. The textural characteristics of the food may provide protection to the organism from adverse environmental conditions. The composition of a food may in some cases have a decided bearing on the awv level available to the microorganism. For example, as pointed out by Scott (13), foods with a high fat content have substantically reduced water contents. The work of Christian and Scott (3) indicated that Salmonella could tolerate slightly lower aw levels in a food menstruum than in liquid medium. Scott (11) reported that the lower limits of a, for growth in dried meat, dried milk, and dried soup were similar to those in liquid media. Kim (Thesis, Purdue University, 1965) found that C. perfringens manifested longer lag periods and less growth when grown in a food substrate, even when the aw levels of food and of the laboratory media were quite similar. Whether this apparent difference in response is attributable to the genera or to the varying technique employed in the experiments remains an open question. The temperature at which the food is held, the pH, and aw levels of the food all become important factors in determining the microbiological quality of a food. C. perfringens has been isolated from many food products of limited water content, including dried soup, sauce mixes, and spaghetti mixes (9). The presence of C. perfringens in these menstrua of relatively low a, levels indicates the ability of the organism to remain viable under adverse environmental conditions and suggests that the method of handling the food after rehydration is indeed important to human health.

v3-fos

2020-12-10T09:04:11.503Z

1970-11-01T00:00:00.000Z

237234174

s2

Synergistic False-Positive Coliform Reaction on M-Endo MF Medium The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium. The standard test for the detection of coliform organisms in river water has been the multipletube fermentation technique (MPN). It was not until 1957 that the Standard Methods for the Examination of Water and Wastewater (1) tentatively accepted the membrane filter technique, which, because of its ease and rapidity in providing results, is now widely used. A positive coliform test with the membrane filter technique is defined as the production of a dark colony with a metallic sheen within 24 hr incubation at 35 C on M-Endo medium (1). The sheen produced by the coliform organisms in the membrane filter technique is the result of a complex formed by the combination of sulfite and basic fuchsin with the acetaldehyde produced by the metabolic activities of the coliform (4). The accuracy of the method used in detecting coliforms has been questioned from both the false-negative and false-positive points of view. Melia (M.S. Thesis, Colorado State Univ., Fort Collins, 1965) showed that a high frequency of false-positive reactions occurred in the MPN technique with lauryl sulfate tryptose broth as the test medium. These false-positive reactions were found to be the result of a synergistic inter-action of paracolons or Proteus sp. with enterococci. Fifield and Schaufus (2) observed that false-positive reactions with the membrane filter technique utilizing the M-Endo MF medium occurred as sheen-producing colonies which are enumerated with the coliform indicator organisms. It has been suggested that the majority of the false-positive reactions of M-Endo MF medium result from the interaction of coexisting bacterial populations. These mixed bacterial cultures may produce an intermediate metabolic substance not produced by the individual organisms. This intermediate could react with the sulfite and basic fuchsin in a manner similar to that of the acetaldehyde and be responsible for the sheen characteristic of the positive coliform test. The object of this investigation was: (i) to isolate and identify populations of bacteria which produced false-positive sheen reactions on M-Endo MF medium; (ii) to determine whether the false-positive sheen was being produced by individual organisms or by two or more organisms in synergistic interaction; and (iii) to determine what metabolic intermediate(s) may be involved in such false-positive sheen production. MATERIALS AND METHODS Test organisms. This investigation was conducted with organisms isolated from water samples collected at various field stations along the upper Cache la Poudre River in Larimer County, Colo. The organisms were isolated with M-Endo MF broth by using the membrane filter technique for total coliforms. All sheen-producing colonies were further tested for the ability to ferment lactose by inoculation into lactose broth. Cultures producing acid and gas from lactose were then checked for purity and transferred to Plate Count Agar (Difco) slants for further study. A typical Escherichia coli was used as a positive control, and Serratia marcescens was used as a negative control in all biochemical studies. Media and solutions. The media used throughout this investigation were products of Difco Laboratories, Inc. In addition, a chemically defined minimal medium as described by Wendt (M.S. Thesis. Colorado State Univ., Fort Collins, 1966) was used in the biochemical studies. The medium contained (grams per liter): K2HPO4, 7; KH2PO4, 0.1; MgSO4 .7H20, 0.1; NH4H2PO4, 1; lactose, 10. The medium was adjusted to pH 6.8 to 7.2 and sterilized by membrane filtration. Growth conditions. Growth studies to determine the time to reach the late exponential growth phase were done in screw-cap tubes (16 by 125 mm) containing the chemically defined minimal medium. Incubation was at 35 C in an automatic temperaturecontrolled shaker incubator (model R26, New Brunswick Scientific Co.). Growth was determined by measuring the increase in turbidity with a Bausch & Lomb Spectronic-20 colorimeter at 420 nm. At the end of the incubation period, each culture was filtered through a 0.5-,um membrane (Millipore Corp., Bedford, Mass.), and the filtrate was collected for characterization of the intermediates. Analytical procedures. Filtrates obtained from the cultures were collected, and 2,4-dinitrophenylhydrazones were formed and extracted by the procedure developed by Smith (6) and modified by Thayer and Ogg (7). Identification of the neutral carbonyl compounds was made by the method of Thayer and Ogg (7). The paper chromatography technique of Lynn, Steele, and Staple (5) was used to identify aldehydes and ketones as their 2,4-dinitrophenylhydrazones. Descending chromatography was carried out on Whatman 3MM filter paper. Identification of hydrazones. The neutral carbonyl compounds which were subjected to paper chromatography were compared with the movement of the hydrazones of known aldehydes and ketones. The individual chromatographic zones containing the hydrazones were then eluted with ethyl acetate, and descending chromatography was repeated on the eluates to determine the purity of the zones. Individual paper strips from the chromatograms were evaluated on the Densitometer model 525 (Photovolt Corp., New York, N.Y.) by plotting distribution curves of the fractions separated on the filter-paper strip. RESULTS Colonies suspected of giving false-positive coliform reactions were obtained from M-Endo MF medium and inoculated into 0.5% lactose broth. The cultures producing gas in the lactose broth were streaked on EMB and Endo agars to determine purity. The results showed that 29% (16 of 55) of the sheen-producing colonies for study were mixed colonies. Most of the organisms isolated from the mixed colonies were of the coliform type. The mixed cultures and individual isolates of the mixtures were analyzed for their ability to reproduce a sheen on M-Endo MF medium. Individual isolates which did not produce a sheen, unless recombined to the original mixed pair, were used to study false-positive coliform reactions and to determine the metabolic intermediates responsible for the false-positive reactions. Individual isolates of the synergistic sheenproducing colonies were further studied for physiological characteristics. Results of the biochemical tests indicated that, although all isolates involved in synergistic sheen production were coliform types, there were no fecal coliforms which would grow at 44.5 C on M-FC medium. Mixed cultures showing synergistic sheen production were inoculated into the chemically defined minimal medium with lactose as the carbon source and 0.21 % sodium sulfite added as a metabolic inhibitor to prevent further metabolism of acetaldehyde. Fermentation was carried out in screw-cap and cotton-plugged tubes (16 by 125 mm) so that the increase in turbidity could be measured directly. Cultures were also grown in cotton-stoppered 125-ml Erlenmeyer flasks containing the chemically defined minimal medium. The results from the three different incubation conditions described above showed no obvious difference in the exponential growth phase. For convenience, screw-cap tubes were used throughout the fermentation studies with incubation for an 18-hr period. Hydrazones of neutral carbonyl compounds were formed and extracted from the chemically defined minimal medium in which the mixed cultures had been grown. The 2,4-dinitrophenylhydrazones of ketones and aldehydes were identified by chromatography and are listed in Table 1. A summary of the results of the chromatographic separation of the phenylhydrazones of individual isolates and mixed cultures is shown in Table 2. To determine whether the acetaldehyde produced by the mixed cultures was a result of true synergistic interaction, the following study was conducted. Each of the individual organisms making up the mixed culture was grown in the chemically defined minimal medium for 18 hr. The filtrate was then collected, and the second organism making up the mixture was allowed to grow in the filtrate until maximum growth was obtained. The 2, 4-dinitrophenylhydrazones formed in culture filtrates were subjected to descending paper chromatography as mentioned earlier. Acetaldehyde was produced only when the organisms were incubated simultaneously in the medium and not with sequential incubation of the organisms in the medium. This evidence indicates that both individual organisms making up the mixed culture had to be present throughout the fermentation process before the aldehyde was produced and, therefore, the reaction could be considered truly synergistic. DISCUSSION The observed metallic sheen on M-Endo medium was reproduced by 38% (6 of 16) of the mixed colonies when incubated in mixed culture, whereas individuals of these mixtures were unable to produce the sheen. This reaction producing the metallic sheen was probably due to a synergistic interaction of the individual organisms making up the mixtures or possibly to a situation whereby one organism supplied metabolic intermediates for the other. The remaining 62% of mixed colonies were classified into two different categories. One group of mixed cultures had an individual organism capable of reproducing a metallic sheen individually on the M-Endo medium and was shown to be a typical coliform; the other organism was a noncoliform and failed to produce a sheen. The second group of mixed cultures failed to produce a sheen either individually or in mixed culture. Lack of sheen production could have been due to an alteration in metabolism resulting from an environmental change or to catabolic repression. Another possible explanation is that a third organism in the original sheen-producing colony had been lost in the process of determining purity and this third part was necessary for the synergistic sheen production. The mixed cultures which failed to exhibit synergistic sheen production were excluded from further biochemical study. The results obtained from the paper chromatographic separations suggest that different pathways were in operation in the various mixed cultures. In mixture no. 40, glyceraldehyde, 2, 3-butanedione, acetaldehyde, and butyraldehyde were produced. Organism no. 40A produced glyceraldehyde and 2,3-butanedione, and organism no. 40B produced glyceraldehyde, 2,3butanedione, and 2-butanone. One mechanism by which acetaldehyde could have been formed was by the carboligase reaction. This reaction involves the conversion of the metabolic intermediates 2, 3-butanedione and 2-butanone to acetaldehyde (3). Since 2, 3-butanedione was isolated from all but one of the remaining mixed cultures, the mechanism mentioned above could also be responsible for the formation of acetaldehyde. In one of the mixed cultures, propionaldehyde was isolated instead of acetaldehyde. Propionaldehyde was tested with M-Endo medium to determine whether a sheen would be produced; the resultant sheen was less intense than that of acetaldehyde but could be read as a positive coliform colony. It is probable then that propionaldehyde was responsible for false-positive sheen production since no acetaldehyde was produced by this mixture. The results presented indicate that the acetaldehyde and propionaldehyde isolated from mixed cultures, but not from the individual organisms making up the mixed cultures, were responsible for false-positive sheen-producing reactions of M-Endo medium. Furthermore, from the facts that both organisms of a mixture had to grow together in the medium for the aldehyde to be produced and that sequential growth in the same medium failed to yield aldehyde, it must be concluded that acetaldehyde and propionaldehyde production which leads to false-positive coliform reaction is indeed a synergistic phenomenon.

v3-fos

2022-07-07T15:05:30.945Z

1970-01-01T00:00:00.000Z

250321578

s2

Pistia stratiotes as Effective Larvicide against Aedes aegypti World Health Organization (WHO) noted that Indonesia was the country with the highest cases of Dengue Hemorrhagic Fever in Southeast Asia. Control efforts include vector observation and monitoring, one of which is larvicides. This study aims to determine the effectiveness of Apu-apu (Pistia stratiotes) leaf extract to inhibit the growth of the third instar larvae of the Aedes aegypti. The design of this study was Completely Randomized Design (CRD). The treatment in this study was repeated 3 times using 4 treatments (negative control treatment, given extract of 10 ml, 30 ml, 50 ml). The results showed that Apu-apu leaf extract had different effects on each test concentration (10ml, 30ml and 50ml). The higher the concentration, the greater mortality of Aedes aegypti larvae. Anova test showed that Apu-apu leaf extract (Pistia stratiotes) had an effect with the F=94.667, p= 0,000 on larvae mortality. The ability of Apu-apu leaf extract to kill larvae in LC50 value was 4.0% per minute, analyzed using probit regression. P. stratiotes could be an effective larvicide against A. aegypti third instar larvae (p=0,000). Introduction Dengue Hemorrhagic Fever is commonly found in tropical and sub-tropical areas. Data from around the world shows that Asia ranks first in the number of dengue hemorrhagic fever sufferers every year. Meanwhile, from 1968 to 2009, the World Health Organization (WHO) recorded Indonesia as the country with the highest cases of Dengue Hemorrhagic Fever in Southeast Asia (WHO, 2006a;WHO, 2006b). Indonesia is a country with a wet tropical climate with high rainfall during the rainy season. The rainy season is a season that is liked by mosquitoes to breed. The disease that often becomes a problem every year in the rainy season is Dengue Hemorrhagic Fever (DHF). Data from the Surabaya City Health Office recorded the number of DHF sufferers in 2014 (816 cases), 2015 (640 cases), 2016 (938 cases), and September 2017 (302 cases). The vector of transmission of this disease is the mosquito. The dengue virus is infected by Aedes aegypti mosquito's bite (Raekiansyah & Sudiro, 2002;Shu et al, 2009;Pongsiri et al, 2012). When the virus enters the body of Aedes aegypti, the virus will infect the mid stomach of the mosquito and spread to the salivary glands within 8-12 days. During this periode Aedes aegypti can transmit the virus by biting humans. Immature Aedes aegypti can generally be found indoors and in places filled with water, such as water storage areas and others. Adult Aedes aegypti can be found around housing and fly within a radius of 400 meters. Aedes aegypti is the first agent of the tropical disease dengue fever when a person digits the Aedes aegypti mosquito infected with the dengue virus, the symptoms of dengue fever will appear within 4-7 days afterwards. Dengue Hemorrhagic Fever Virus generally causes symptoms in the form of a rash, high fever, and joint and muscle pain (Gunawan et al, 2016;Thu et al, 2004;Azami et al,2011). However, other symptoms of Dengue Hemorrhagic Fever include pain behind the eyes, swollen lymph nodes, bone pain, nausea, vomiting and headaches. Severe dengue fever can cause damage and rupture of blood vessels and can be life-threatening. To a less severe degree, the handling of dengue fever is only in the form of fluids to maintain balance in the body and anti-fever drugs. The number of patients with Dengue Hemorrhagic Fever (DHF) per 100,000 population in Central Java in the last five years was 59.2 in 2008: 57.9 in 2009: 56.8 in 201015.3 in 2011;and19.29 in 2012 (Fidayanto et al, 2013;Oliveira et al, 2010). Its spread not only in urban areas, but also spread to rural areas. Since 2007, 33 regencies/cities out of 35 regencies/cities in Central Java have been endemic areas for DHF. In 2008-2009, it has spread to all districts/cities with a fairly high number of cases. In 2010-2011, all regions experienced a decline in dengue cases. Global environmental changes or Global Environmental Change (GEC), especially Global Warming, more or less played a role in the incidence of DHF. Every season change, especially from the dry season to the rainy season, various health problems hit, including the most common is an increase in the incidence of dengue fever. Other risk factors for dengue infection include the level of host immunity, population density, vector-host interactions and viral virulence (Lam, 1994;Marjorie, 1999). The clinical manifestations of DENV infection range from asymptomatic infection or a mild flulike syndrome, also known as dengue fever (DF), to the more severe and life-threatening forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Ito et al, 2015). Drugs and vaccines to control Dengue Hemorrhagic Fever are still in the research stage. Prevention of Dengue Hemorrhagic Fever is prioritized by breaking the chain of transmission through vector control. Control efforts include vector observation and monitoring activities, namely mosquito surveys, larval surveys, egg capture surveys, insecticide spraying, 3M, 3M plus and larvicidation (Kiat et al, 2006;Sánchez et al, 2000). The only method of eradicating dengue hemorrhagic fever is vector control. Vector control is done by using chemical insecticides that can cause resistance and environmental pollution if used continuously. Therefore, controlling using natural insecticides derived from plant extracts is one of the solutions that researchers have developed so far. One of the plants that have the potential as natural insecticides is the apu-apu (Pistia stratiotes) (Che et al, 2009). Pistia stratiotes referred to as tropical duck grass or apu-apu leaf is one of the most dominant aquatic weeds in freshwater, and polluted water. This plant is raised in ponds as a shelter for other animals. The use of the apu-apu plant, especially on the leaves, is done to take advantage of the apu-apu leaves which are generally only thrown away in vain. The leaves of the apu-apu plant are generally only disposed of as waste, even though this apu-apu has biological activities that can provide various benefits to the community (Harapan et al, 2019; Sucipto et al, 2018). Present study evaluated the phytoremediation potential P. stratiotes and organic pesticides. Materials and Methods This research was carried out on 7-14 September 2021. The extraction process was conducted at the Biochemistry Laboratory of UIN Raden Fatah Palembang. The treatment with extract testing was conducted at the basic Biology Laboratory of UIN Raden Fatah Palembang. Mortality data of Aedes aegypti larvae were obtained by giving the Apu-apu leaf extract (Pistia stratiotes) 3 treatments with each different concentration. Aedes aegypti larvae were observed within 24 hours. Every 1 hour spraying based on the treatment dosage with apu-apu leaf extract is carried out. A 50ml spray bottle was used as a tool to administer the Apu-apu (Pistia stratiotes) leaf extract to Aedes aegypti larvae. After 24 hours it can be observed how many larvae experienced mortality. The data obtained were analyzed using Analysis of Variance (ANOVA) with the SPSS software application. The results of the analysis showed a significant difference, then the data were further analyzed using the Post Hoc LSD test. The further test aims to determine the location of the differences between the pairs of groups. The degree of significance used is = 0.05 (a significant difference if p 0.05). Results and Discussion The results of the treatment with the Apu-apu leaf extract (Pistia stratiotes) with each dose of 10 ml, 30 ml, and 50 ml, once every 1 hour for 24 hours can affect the mortality of Aedes aegypti larvae. Measuring average larval mortality before and after treatment are presented in Table 1 and Figure 1. This study used 4 treatments, consisting of 1 negative control treatment and 3 treatments with concentrations of 10ml, 30ml, 50ml. Each treatment was tested by spraying every 1 hour for a period of 24 hours. Based on Table 1, it can be seen that the negative control treatment remained alive for a period of 24 hours. At a concentration of 10 ml repetition I caused the death of 4 larvae from a total of 5 larvae. The second repetition was 3 animals and the third repetition was 4 animals. So the number of larval deaths was 11 from the total test, which was 15 for 24 hours. Observations at a concentration of 30 ml repetition I caused the death of 5 larvae. In the second repetition 4 dead larvae and in the third repetition 5 dead larvae. So that the total number of larvae that died during the 24-hour observation was 14 larvae from a total of 15 larvae. At the observation concentration of 50 ml repetition I as many as 5 larvae died. In the second and third repetitions, 5 larvae died. So the number of larvae that died was 15 of the total test larvae of 15. According to Chang et al, 2011;Churrotin et al, 2006;King et al, 2008, the higher the concentration level given, the faster the death rate. Giving the extract concentration to a higher level will have a high effect as well. The working power of a compound is largely determined by the amount of concentration. Each concentration has a significant effect, as evidenced by the results of the Anova analysis and tested further using Post Hoc LSD (Table 2 and Table 3). Based on the results of the Analysis of Variance (ANOVA) ( Table 2.) shows that the value is signifikan (F=94.667, p= 0,000). So it was concluded that there was a difference in the effect of apu-apu leaf extract. In Table 3. the concentration of 30ml (P2) was not significantly different from the concentration of 50ml (P3) with a significant value of 0.347. But it was significantly different with the positive control concentration (P0) and 10ml concentration (P1). Meanwhile, at other concentrations, it was significantly different. This is because the probability value exceeds the standard value of the provision, which is <0.05. So it can be said that all treatments greatly affect the positive control (P0) where the significance value is 0.00. This apu-apu leaf extract gave different effects at each test concentration. The ability of apu-apu leaf extract to kill Aedes aegypti larvae was also analyzed using probit regression analysis so that the LC50 value was known. Probit analysis was carried out to find out at what concentration the animals experienced the most mortality. LD50 is defined as a statistical sign on the administration of a substance as a single dose that can cause the death of 50% of test animals (Paloucek et al, 2007). The results of the probit analysis are presented in Table 4. In Table 4. the results of the probit analysis showed that the LC50 value was obtained at a concentration of 4.064%. Signs of larvae experiencing mortality are marked by no movement, changes in body color to transparent. The lower the LC50 value for a substance, it means that the substance has a higher activity in killing experimental animals. Andreas et al (2011), states that lower concentrations are needed to kill experimental animals in the same period of time. Apu-apu leaf extract (Pistia stratiotes) was effective at LC50. In other words, Pis. stratiotes extract can be used as an insecticide and has an effect on mortality in Aedes aegypti larvae with the highest concentration level of 50 mL with a mortality of 15. Deuis et al (2017), which states that the compounds contained in higher concentrations will have a higher effect on pest mortality as well. In addition, it is also due to the content of compounds in the apu-apu leaves (Pistia stratiotes) which are thought to cause mortality in Aedes aegypti larvae, namely flavonoids. Flavonoids are chemical compounds that have insecticidal properties. Flavonoids attack the nerves in several vital organs of insects, causing a weakening of the nerves, such as breathing and causing death. According to Zandi et al (2011), flavonoids can work as strong respiratory inhibitors and inhibit oxidation reactions. This will cause an increase in CO2 that exceeds O2, so that the test larvae will move actively to look for fresh air. Ahdiyah & Purwani (2015), stated that flavonoids interfere with energy metabolism in the mitochondria by inhibiting the electron transport system. Flavonoids have a way of working, namely by entering the larva's body through the respiratory system which will then cause withering of the nerves and damage to the respiratory system and cause the larvae to be unable to breathe and eventually die. In addition, there are also saponins which act as stomach poisons (Cania & Setyaningrum, 2013). Damage or defects in advanced stages of Aedes aegypti larvae are thought to occur due to toxic compounds that damage nervous tissue, such as alkaloids that can inhibit the process of larvae becoming pupae. Saponins found in leaves if consumed by insects can reduce the activity of digestive enzymes and food absorption (Applebaum et al, 1969). Saponins can also reduce the surface tension of the larvae skin membrane and are able to bind free sterols in the digestion of food (Panche et al, 2016;Shen et al, 2009). Sterols are precursors of the hormone ecdysone, so that the decreasing supply of sterols will interfere with the molting process in insects. Larvae can be said to be dead, it can be seen from their morphological conditions, namely if the larvae do not move anymore, the larvae settle to the bottom of the water, the larvae body is soft and the color changes from darker to pale and slightly transparent. If physiologically the death of the larvae is caused by some of the content contained in the mango peel extract. Flavonoids are chemical compounds with insecticidal properties that attack parts of the nerves in several vital organs resulting in weakening of the nerves, such as breathing to death. According to Cania & Setyaningrum (2013), saponins can act as stomach poisons and inhibit the action of the cholinesterase enzyme in larvae. Hayden et al (2015), stated that these compounds enter the larval body through the mouth as stomach poisons that can kill. In this study, it was observed that extract of P. stratiotes rendered the A. aegypti larvae inactive and motionless. Furthermore, disturbance in the normal behaviour of the larvae was observed in treatments of A. aegypti suggesting that the plant possesses larvicidal properties that may affect the either behavioural or physiology of the larvae. This possibly may be interterm to the study of (Ito et al, 2015) which reported a decrease in the feeding behavior in Anopheles and Culex after treatment with neem extract. Conclusion Apu-apu (Pistia stratiotes) leaf extract as significantly effect on mortality of Aedes aegypti larvae. The ability of Apu-apu leaf extract to kill larvae in LC50 value was 4.064% per minute.

v3-fos

2020-12-10T09:04:16.978Z

1970-08-01T00:00:00.000Z

237232559

s2

Mycotoxin from a Blue-Eye Mold of Corn High-moisture yellow dent corn became heavily molded by Penicillium martensii after storage for 6 months at 1 C. Mice ingesting corn molded by P. martensii died within a few days. The toxin was isolated and identified as penicillic acid. Large quantities of the toxin accumulated over a 3-month period on artificially inoculated corn incubated at temperatures between 1 and 15 C. At higher temperatures, the toxin disappeared within 45 days. High-moisture yellow dent corn became heavily molded by Penicillium martensii after storage for 6 months at 1 C. Mice ingesting corn molded by P. martensii died within a few days. The toxin was isolated and identified as penicillic acid. Large quantities of the toxin accumulated over a 3-month period on artificially inoculated corn incubated at temperatures between 1 and 15 C. At higher temperatures, the toxin disappeared within 45 days. Recent changes in agricultural technology have resulted in the widespread practice of harvesting high-moisture corn by picker-sheller. The corn frequently has a moisture level in excess of 20% and has sustained considerable physical damage. Such conditions are ideal for rapid molding. After harvesting, the corn must either be dried to a moisture level safe for storage or be ensiled and used for animal feed. Several commercial firms in this country and abroad have considered refrigerated storage of high-moisture corn to be an attractive alternative to drying or ensilng (6). This method of preservation allows the corn to be used for feed, or it can be channeled to the milling industry. Even under refrigeration, however, corn is known to mold, and guidelines for cold storage have been proposed (13). During storage of high-moisture yellow dent corn for 6 months at 1 C, the mold flora shifted, and Penicillium martensii Biourge predominated. This paper reports the isolation and identification of a mycotoxin from corn molded by P. martensli NRRL 3612 and the temperatures at which toxin production occurs. MATERIALS AND METHODS Source of corn from which P. martensii was isolated. This corn was a commercial single-cross yellow dent grown on the Agricultural Engineering farm, University of Illinois, Urbana. The corn was planted on 5 June and harvested by picker-sheller on 29 October 1968. The moisture level at harvest was 25% and was determined electrically and by oven drying. Microbiological exanination of corn. Fifty-gram samples of corn were shaken vigorously in 450 ml of sterile distilled water containing 25 g of sand, and serial dilutions were then made. Bacterial counts were I Presented in part at the 70th Annual Meeting of the American Society for Microbiology, Boston, Mass., 26 April-i May 1970. determined on plate count agar containing 30 1g of Acti-Dione per ml (Upjohn; cycloheximide) and mold counts on yeast extract-tryptone agar (5) containing 100,g of Achromycin per ml (Lederle; tetracycline KCI). Bacterial colonies were counted after incubation for 3 days at 32 C; molds and yeasts, after incubation for 5 days at 28 C. Animal toxicity tests. Molded corn was extracted as described below, and, after solvent removal, the residue was dissolved in propylene glycol. The extracts were tested on mice by intraperitoneal injection. Fermentation and toxin isolation. Sizable quantities of the toxin were obtained from a liquid medium. Fifteen liters of Czapek-Dox broth (9) supplemented with 0.5% yeast extract was distributed into 30 Fernbach flasks (2.8 liter), autoclaved, inoculated with an aqueous spore suspension of P. martensii, and incubated statically for 12 days at 25 C. The mycelium was removed by filtration, and the supernatant was concentrated to 0.5 liter in a vacuum evaporator. The concentrated supernatant was extracted twice by using 1 liter of chloroform for each extraction. The chloroform extracts were combined, and most of the solvent was removed by flash evaporation. After the residual oily liquid was added to 20 volumes of pentanehexane, the precipitate was recovered by filtration and then redissolved in boiling water. On cooling, white crystals precipitated. These were recovered, air-dried, and recrystallized from benzene twice, as fine needleshaped crystals (total recovery about 130 mg). Later, considerably better yields were obtained by the use of Raulin-Thom medium (4) in place of Czapek-Dox supplemented with yeast extract. Toxin identification. The molecular weight and elemental formula of the toxin were determined with a Euclid high-resolution mass spectrometer. Melting points were obtained with a Mettler FPI melting point apparatus, and excitation and emission spectra were recorded with an Aminco-Bowman spectrophotofluorometer. Ultraviolet absorption spectra were determined with a Beckman DB-G spectrophotometer. Production of toxin on corn. Fifty-gram quantities of corn were placed in 300-ml Erlenmeyer flasks with 100 ml of distilled water and autoclaved for 15 min at 121 204 C. After autoclaving, the excess water was decanted, and the flasks, stoppered with cotton plugs, were then autoclaved for 20 min. Each flask was inoculated with 1 ml of spore suspension made by suspending the spores of P. martensii from a 10-day-old slant in 50 ml sterile distilled water. The flasks were incubated at -4, 1, 5,10,15, 20, 25, 30, 32, 35, and 37 C. Isolation and assay of toxin. The toxin, penicillic acid, was assayed fluorodensitometrically by the method of Ciegler and Kurtzman (in preparation). Briefly, the method involves thin-layer chromatography of the unknown with known amounts of standard on silica gel (solvent, chloroform-ethyl acetateformic acid, 60:40:1, v/v) followed by exposure of the plate to concentrated ammonia. Penicillic acid is excited at 350 nm and fluoresces at 440 nm. The degree of fluorescence was determined with a Photovolt model 530 densitometer equipped with an automaticscanning thin-layer plate stage and a recorder equipped with an integrator. A standard curve is prepared for each analysis, Beer's law being followed between 1 and 9 ,ug of penicillic acid. The concentration of unknown is determined from the standard curve, taking into account the dilutions involved. For most assays, 50 g of molded corn was extracted with 250 ml of chloroform-methanol (90:10, v/v) in a Waring Blendor for 3 min. The first 50 ml of solvent, after recovery by filtration through anhydrous sodium sulfate, was analyzed for penicillic acid. RESULTS Yellow dent corn with a moisture level of 25 % was stored for 6 months at 1 C in sealed screwcap l-quart (ca. 1.1 liters) glass jars. Before storage, the penicillia numbered about 8,000/g, but after storage the count increased to 1,800,000/g (Table 1). Although several species of Penicillium were present before storage, only one species, P. martensii, was recovered after storage. Initially, P. martensii caused blue-eye of the kernels but eventually spread over the surface of the entire grain. Intraperitoneal injections of extracts of corn molded by P. martensii were fatal to mice in a (2) Authentic penicillic acid. matter of minutes. Mice fed the molded corn died in 3 to 5 days. The toxin was identified as penicillic acid by analyzing both authentic penicillic acid and the isolated product. High-resolution mass spectroscopy gave m/e 170.06 and an elemental formula of C8H1004 (Fig. 1). The melting point was 84.2 to 84.8 C with no depression on admixture with authentic penicillic acid. The ultraviolet absorbance in methanol showed a single peak at 221 nm with shifts to 224 nm on acidification with 0.02 N HCI and to 293 nm in 0.02 N NaOH (Fig. 2). After reaction with ammonium hydroxide, the keto form of the acid fluoresces with excitation at 350 nm and emission at 440 nm. The toxin was co-chromatographed with authentic penicillic acid in several solvent systems and both compounds, after reaction with phenylhydrazine in ammonia, gave identical excitation spectra (Fig. 3). Production of penicillic acid on corn by P. martensii was favored by low temperatures. Greatest production (12.7 mg/g) was at 5 C after 88 days, but nearly two-thirds of this amount occurred at 1 and 10 C ( Table 2). After 83 days, penicillic acid was also detected at -4 C. Considerable synthesis occurred at 15 and 20 C, but the toxin disappeared after 45 and 90 days, respectively. Only small amounts of toxin were detected at 30 and 32 C, and there was no growth or toxin production at 35 C. DISCUSSION Blue-eye is a storage disease of corn caused by several species of Penicillium. These molds grow over the embryo but under the seed coat. When sporulation occurs, a blue or blue-green color appears over the embryo because of the color of the spores. Koehler (7) reported P. notatum Westling, P. viridicatum Westling, P. palitans Westling, and P. cyclopium Westling to cause blue-eye; Semeniuk and Gilman (11) list P. expansum Link; and Semeniuk (10) added P. rugulosum Thom and P. chrysogenum Thom. Our report is the first of blue-eye being caused by P. martensii. However, the similarity of P. martensii and P. cyclopium may have resulted in it being confused with the latter. Penicillic acid was first isolated by Alsberg and Black (1) from P. puberulum Bainier. This culture, isolated from corn, produced sufficient penicillic acid on Raulin's medium to be fatal to mice and guinea pigs. Murnaghan (8) considerably expanded this early work in his studies of the pharmacology of penicillic acid. The intravenous LD50 of mice was 5 mg/20 g, and the mean lethal dose when given orally was 12 mg/ 20 g. Penicillic acid had a digitalis-like action on the heart of the frog, the rabbit auricle, the perfused heart of the cat, and a very weak action in heart-lung preparations of the dog. A dilator action on systemic blood vessels was also found and included the coronary and pulmonary vessels. In our investigation, the greatest accumulation of penicillic acid on corn inoculated with P. martensii occurred at temperatures of 10 C and below. Production at 15 and 20 C was also high, but the toxin disappeared within 45 and 90 days, respectively. Temperatures above 25 C were decidedly unfavorable for production. Several interpretations can be made of these data. Degradation of toxin at the higher temperatures may result from a nonspecific autocatalytic process or may be enzymatic; at lower temperatures, the rate of production may exceed that of degradation, or the degradation process, whether nonspecific or enzymatic, may not function. This facet of our data requires additional experimentation. Mycotoxins produced on grains at low temperatures have previously been reported and became a serious problem in the Soviet Union in the early 1930's (3). A human disease now known as alimentary toxic aleukia (ATA) became widespread as a result of the consumption of moldy grain which had overwintered in the field. Various species of Fusarium and Cladosporium were apparently responsible for ATA, with greatest toxin production occurring at -10 to 0 C. Similarly, greatest production of T-2 toxin by Fusarium tricinctum (Corda) Sacc. emend. Snyder and Hansen, which has been associated with moldy corn toxicoses, occurred at low temperatures (2). The importance of penicillic acid as a mycotoxin on high-moisture corn stored at low temperatures must be further assessed by additional field studies and animal feeding trials. Furthermore, toxin production may not be limited to P. martensii. Other fungi producing blue-eye, such as P. chrysogenum, P. palitans, and P. rugulosum, have been shown to grow at 0.5 C (12). Their toxin-producing ability at various temperatures is currently under study in our laboratory.

v3-fos

2019-06-16T13:14:00.560Z

1970-01-01T00:00:00.000Z

191154263

s2

Aplicación de recubrimientos comestibles en rodajas de plátano verde sometidas a fritura por inmersión Currently fried snacks are in great demand by Colombian consumers, however they have a high oil content, which reduces the life of the product and its sensory acceptability. An alternative to reduce the oil content, can be the use of hydrocolloids as edible coatings. Applying coatings of Guar gum, CMC and Xanthan gum at 1.2% was adequate to reduce the oil content by 43%, 31% and 23.34% respectively. Moreover, according to the implementation of the rotable central composite design for the Guar gum coating, the best operating conditions occurred at a guar gum concentration of 1.4% and a time of 90 seconds, when the banana slices undergo deep-fat frying conditions of 180 ° C and 3 minutes. Therefore, the coated plantain snack maintains a good quality with a low oil content, representing a more attractive product for consumers. hydrocolloids with thermal gelation capabilities or thickening properties have been widely studied as edible coatings. Coatings based on cellulose derivatives such as methylcellulose (MC), sodium carboxymethylcellulose (CMC) and hydroxypropylmethylcellulose (HPMC) have showed a good barrier to the absorption of oil during deep-fat frying (Albert and Mittal, 2002;Martelli et al ., 2008;Garcia et al, 2004).. Therefore, the use of hydrocolloids as edible coatings can be an alternative to meet the demands of current consumers, and thus be a promising route for obtaining Plantain snack low on calories, but investigations are still insufficient in relation to deep-fat frying while applying coatings in matrices such as plantain, since the surface of the food is important for the absorption of oil (Tavera-Quiroz et al., 2012). Therefore, the aim of this study was to determine the effect of the type of hydrocolloid, its concentration and the immersion time in solutions of hydrocolloids in terms of oil content and moisture content for plantain samples undergoing deep-fat frying at 180 ° C for 3 minutes. Raw material For the study we worked with the Dominico Harton plantain variety in the green state according to Chavez et al. (2014) (° Brix <7), it was purchased from a local market of Quito, Ecuador. Moisture was determined based on the AOAC (1997b) standard. For the analysis of soluble solids, a refractometer was used according to the AOAC (1998) standard; the acidity percentage was measured with respect to malic acid, being predominant in such fruit according to the AOAC (2005) standard. For the diameter, the middle part of the fruit was measured without exocarp with a caliper gauge (160 ± 0.05 mm). Immersion process into the film forming solutions Sodium carboxymethyl cellulose, guar gum and Xanthan were used. Aqueous solutions were prepared for each hydrocolloid (CMC, GG and GX) 0.4 to 1.2% (weight / volume) and used as Introduction Plantains and other cooking bananas, are the staple foods produced in the tropics, are an important source of carbohydrates for the people of Africa, the Caribbean, Latin America, Asia and the Pacific. Plantain production worldwide in 2013 was 37'877,804 tons, approximately 22.9% of the production comes from Latin America, standing out as major producers are Colombia, Peru and Ecuador (FAO, 2013). Much of the food market today consists of fried products of different materials, including roots, tubers, cereals, plantains, fish and chicken, these products are obtained by deep-fat frying, in the case of plantains one of the most common forms of consumption is chopped or sliced; deep-fat frying is widely used at the industrial, commercial and domestic scale for food preparation, as consumers prefer the taste, texture and appearance of fried products as well as the speed of the process compared to other cooking methods . This operation involves methods of drying and cooking through contact with hot oil, resulting in products with a high oil content (Bouchon, 2009). However, consumers currently demand products with low fat content and / or with a healthy fat mainly those fats with unsaturated fatty acids, since it is considered that the intake of lipids in excess is a factor for the presence of hypercholesterolemia and hypertension (Albert & Mittal, 2002) and in some studies it has been associated with cardiovascular disease (Iqbal et al., 2008) and obesity (Orea, 2013). Therefore, fried foods have been directed to meet consumer demand, not only in the sensory aspects but also in the nutritional aspects. The challenge for the industry is to provide healthier fried foods with less fat and pleasant sensory characteristics for these products. Much of the research to reduce the oil content and improve the quality of fried products has been focused on the process, vacuum deep-fat frying is one of the most relevant (Bravo et al., 2011), others are focused on subjecting the food matrix to pre-treatments with methodologies such as microwave and convection drying, osmotic dehydration, blanching and the use of edible coatings, in this case, the use of a plasticizer additive Propylene Glycol (50% by weight of each hydrocolloid), each solution was heated to 60 ° C and kept under constant stirring for 5 minutes, then cooled to room temperature. For the apparent viscosities of the hydrocolloid solutions a Brookfield viscometer was used with an RTD temperature sensor at a temperature of 30 ° C, using spindles # 3 for the less viscous solutions and spindles # 4 for more viscous, all measurements were made at 30 RPM. The plantains were selected, washed, peeled and sliced (3 mm thick). An immersion was conducted in an antibrowning solution of sodium metabisulfite at 30 ppm for 3 minutes. The plantain slices were coated through immersion in the hydrocolloids solutions for 15 and 30 seconds. A strainer was used to collect the coated slices and allowed them to drain for one minute, then surface drying was performed in a forced convection oven at 105 ° C for 4 minutes. Deep-fat frying process For the deep-fat frying process an electric fryer was used, with the Axis Anvil brand, model 2001.120 FFA, equipped with a temperature control system, worked at atmospheric pressure at a temperature of 180 ° C and a frying time of 3 minutes this temperature being a condition where the product absorbs more oil (Krokida et al., 2000) and the time was determined based on preliminary considerations of color and moisture. Industrial oil palm (Danolin Fri) was used in a ratio of 35 grams per liter of oil. Analysis of the fried product The moisture content was determined based on the AOAC (1997b) standard and the oil content was determined with hexane according to the AOAC (1997a) standard, the percent reduction of oil content was calculated according Martelli et al. (2008). Texture: Considering that the aim of the study was to develop a coating that reduces fat absorption, the material showing greater reduction was selected for the studies of texture. The texture, defined as breaking strength of the plantain snacks, was performed by a texture analyzing equipment TA-XT2 texture analyzer, with the brand SMS Stable Micro System. The determination was performed by 12 trials on each sample. The test involved applying a direct force over the sample, using a cylindrical probe of 1.25 cm in diameter at a descent rate of 4.0 mm / s. The snacks were kept on a base (hollow cylinder of 5.5 and 6.0 cm of internal and external diameter respectively), the sample was fractured and the peak value that represents the force was recorded (Lujan-Acosta & Moreira, 1997). Statistical analysis An analysis of variance (ANOVA) was conducted to evaluate the significant differences between the levels of each factor (hydrocolloid type, concentration and time of immersion in the film forming solutions), using the Statgraphics Centurion software. When the data showed differences between them, the comparison of measurements was performed with the Tukey test at a significance level of 0.05, the coating that showed a greater reduction of oil content was used for the realization of the response surface. Response Surface To evaluate the effect of applying the coating that showed higher values of oil reduction, a response surface methodology was used with a rotable central composite design, the effects of the independent variables were analyzed: Coating concentration (% w/v) and immersion time (min). A high level and a low level for each factor were used as shown in Table 1 and 2. Raw material The plantain selected for this research was of the Dominico hartón variety (AAB), which held values of moisture and total soluble solids of 65.0 ± 2.10 % and 3.60 ± 0.10% (° Brix) respectively, while the titratable acidity and the pulp diameter remained at 0.33 ± 0.12%, and 3.04 ± 0.15 cm respectively. These data correspond to an evaluation of 20 samples of raw materials to ensure consistency, because the characteristics of the raw material affect the quality of the final product. Table 3 shows the apparent viscosity of the various solutions, according to the Tukey's test, statistically significant differences are found for each material and concentration. In this respect it is observed that the three materials produce viscous solutions at low concentrations and this increases directly with the concentration of each material. The Guar gum provides viscous solutions at different concentrations, this may be related to their waterbinding capacity, and is of great importance since many authors directly relate viscosity with the capacity for adhesiveness in edible coatings and in the case of additives mixtures of breaded and battered; It is also important in this research because the process was carried out by immersion, which requires high viscosity solutions, because if this is too low the coating will not adhere to the tissue of the plantain and will tend to fall, a fact that favored the synergy between the plantain and the coating material mainly with the guar gum (Akdeniz et al., 2006;Garcia et al, 2009;. Altunakar et al., 2006). Moreover the Xanthan gum generates viscous solutions with 0.4%, however when the concentration raises to 1.2% it did not develop viscosity like other materials, this may be related to poor hydration of the gum particles, leading to poor dispersion, which was evident through the clumping of the particles during the resulting mixture. Varela & Fiszman (2011) in their comprehensive review, disclose that in the mixtures of chicken nuggets using (CMC, guar gum and xanthan gum) viscosity is remarkably increases with the addition of these materials, finding a relationship between the viscosity and the amount adhered to the substrate, however in this study the Xanthan gum had higher increases in viscosity than guar gum and CMC respectively; Akdeniz et al. (2006) found no significant differences in the adhesion capacity for Guar gum, xanthan and HPMC, however this was improved when combined with Guar and Xanthan gum increasing yield due to a high degree of synergy between these two gums. Immersion deep-fat frying process for coated green plantain slices In Table 4 are presented the results of deep-fat frying coated green plantain slices depending on the moisture content, oil content and reduced oil content (%). Table 4 that for both the moisture content and for the oil content, there is no statistically significant influence related to the immersion time in the coating solution (P <0.05). These results agree with those reported by Baldwin et al. (2011), as these authors state that the immersion time is not important, however, if not enough, it will not form a good coverage; Therefore for the experiments to the response surface we worked with a wider time interval to evaluate its effect. With respect to the moisture content, statistically significant differences were found for each concentration and material (although between CMC and xanthan gum there was no difference) to a level of significance of 5%. The relationship between the concentration of each coating and the moisture content was direct, a fact that is related to the ability to retain water due to the strong interaction of the resulting hydrogen bridges, between groups of molecules of Measurements with different letters represent significant differences at the 5% level of significance (P < 0.05) hydrocolloids with the water. In terms of materials, it is evident that the higher moisture content corresponds to samples coated with guar gum (> 3%) which is also related to its ability to bind and immobilize water, a product of its high viscosity. Akdeniz et al. (2006) found similar results in slices of fried carrot coated with different gums, the samples coated with HPMC, guar gum and xanthan gum showed a higher moisture content than the control samples, the higher moisture content from the samples was from the Guar and Xanthan gums; in the same way Sothornvit (2011) found that guar gum causes increased moisture retention, finding values of moisture content of 5.93% in banana snacks, with results similar to this study but significantly higher. However the moisture content is a variable to consider when edible coatings are used since, according to Lujan-Acosta & Moreira (1997) the moisture content is a critical parameter for the product to develop the typical crispness attributes of a snack and because it ensures product stability (water activity). It is observed in As for the oil content, statistically significant differences were found for each concentration and material; a significance level of 5%. A direct relationship between the concentration of each coating and the oil content is observed; the three materials also show a significant reductions in oil content, related to thermal gelation properties. Adding 1.2% of Guar gum reduces 43% of the absorbed oil content compared to the control samples, while the reduction for CMC and Xanthan gum was 31% and 23.34% respectively, this may be related to high viscosity resulting from the adhesion of the gum in the plantain, generating a good barrier against oil migration. Therefore guar gum was selected as a surface coating to assess response with broader range of time and evaluate its effect on the texture. An inverse relationship between moisture and oil content was also found, using hydrocolloids favors the reduction of oil content in fried products, but there is moisture retention. Sothornvit (2011) found similar results but with smaller reductions, solutions of 1.5% guar gum produced a reduction of 25.22% and 17.22% for Xanthan gum solutions in terms of oil content in banana snack. Akdeniz et al. (2006) used as an HPMC additive, Xanthan and Guar gum in mixtures of breaded carrot, finding the best results with guar gum, however it concluded that the mixture of Guar gum and Xanthan improves the oil barrier properties. Some studies (Singthong & Thongkaew, 2009;Garcia et al., 2002;Mai Tran et al., 2007;. Garmakhany et al, 2011) have shown that concentrations of 1 to 2% of different hydrocolloids help reduce oil absorption obtaining results similar to those of this investigation correlating the concentration with the effectiveness of the oil barrier. Table 5 shows the results of the response variables relating to the quality attributes (moisture content, oil content and texture) in deep-fat frying of coated plantain snacks covered with Guar gum. Table 6 shows the fit of the model shown in the deep-fat frying process using edible coatings, the oil content is adjusted appropriately the resulting model has a coefficients of determination (R 2 ) of 87.8%. According to the regression coefficients and figure 1a, shows the response surface of the percentage of oil content (%) versus the concentration of guar gum and immersion time in the solution, it is observed that for the interval under consideration, the concentration of Guar gum and immersion time in the solution decreases the absorption of oil, significantly, as higher as the concentration of guar gum, the lower the amount of absorbed oil, being more sensitive to the effect of the concentration of time, but according to the information in Table 6 both linear effects, quadratic and interaction of both factors were significantly influential on the oil content. Indeed, the lowest oil absorption rates are obtained with higher concentrations of the hydrocolloid solution, in this case 1.4% Guar gum and 90 a second immersion. Furthermore, the interpretation of R² (87.8%) indicates that the adjusted model explains 87.8 % of the variability of the oil content, which means a good fit. Figure 1b shows the response surface corresponding to moisture content in the plantain snack. The surface relating to the moisture content in the product corresponds to a saddle, as this increases from the center of the surface. Table 6 shows that only the linear effect of time and the quadratic effect of the concentration were significantly influential on the moisture content. surface analysis Moreover, the R² indicates that the model explains only 66.3% of the moisture content. The answer shows no significant changes in concentration, however it slightly increases the immersion time. Figure 1c shows the response surface for the analysis of texture; Table 6 shows that the linear effect of time and the quadratic effect of concentration and time are significantly influential on the texture, however the resulting model is not representative for texture as the coefficient of determination (R 2 ) is very low (36.6%). The effect of time on the texture might be related to the fact that the longer the immersion there will be a greater adhesion of the coating material, creating a tendency to increase strength, this can be explained because the film offers protection against damage which could increase mechanical strength to break or fracture (Sothornvit, 2011). In samples with weak adhesion of Guar gum, in the process of deep-fat frying there is greater cell disruption and degradation of peptic substances in the structure of plantain, resulting in a weakening of the cell walls, while in samples coated for the required time, it not only serves as a barrier to oil absorption but gives protection during the process and therefore has a stronger break force (Puncha-Arnon et al., 2008). Similar results found Tavera-Quiroz et al. (2012) on the application of coatings methylcellulose in potato snacks, concluding that such coatings with and without the addition of sorbitol does not affect the texture. Conclusions The use of edible coatings based guar gum, xanthan and CMC is feasible for fried products with lower oil content, highlighting Guar gum as more effective than other treatments in reducing oil absorption. The use of edible coatings generates moisture retention as it relates to the ability of immobilizing water which is a feature of hydrocolloids. The response surface methodology established that for the studied process low oil content (17.4%) can be achieved in the area near time = 90 seconds and Guar gum in a concentration = 1.4%. Emerging technologies based on edible coatings combined with a process of deep-fat frying, maintain the quality and addressed fried products as a healthier product, because of its lower oil content, as a new alternative for the needs and tastes of consumers.

v3-fos

2018-04-03T06:06:55.071Z

1970-09-01T00:00:00.000Z

21372022

s2

Improved Tissue and Cell Homogenizer Modification of an existing tissue homogenizer is described, which converts to a milk-like consistency even difficult to macerate tissues such as cartilage, hair, and small bones. The conventional cutting blades supplied commercially are all of the general shape seen in Fig. 1. These blades strike the tissue pieces so as to keep them out of the cutting area, where they are exposed only to turbulence. The glass or Teflon and glass homogenizers of the Ten Broeck type were inadequate for dense tissues, making it necessary to revert to sand, mortar, and pestle to macerate these specimens. The cutting head on one machine (Omni-Mixer, model 17150, Ivan S. Sorvall, Inc., Newtown, Conn.; similar to Multi-Mix, Lourdes Instrument Corp., Brooklyn, N.Y.) was modified in various ways, and the most effective design, shown in Fig. 1 and 2, was finally adopted. A stationary multihole collar, to slip over the vertical shaft, was machined from 440-C stainless steel, heat-treated, and electro-polished. The outside diameter of the collar was approximately 1 mm less than the inside of the chamber. It was fastened flush with the end of the shaft with one or two Allen screws, bearing on the vertical shaft. The rotating cutting head was machined to bear against the flat-ground bottom of the stationary collar, providing a positive, scissors-like cutting action. The center hole was threaded to fit the already threaded shaft end. Contact pressure between the rotating cutter and the collar is adjusted by loosening the collar, pressing it against the cutter, then retightening the collar on the shaft. Separate cutting heads are required: one for the microchamber (up to 5 ml), one for the 50-ml chamber, and one for both the 200-and 400-ml chambers. As seen in Fig. 2, the cutting head shaft for the microchamber required addition of an outer supporting shaft for attachment of the fixed, upper half of the cutting head. This supporting shaft is an integral part of the lower screw cover. Homogenizing action of the new cutter was tested with various tissues from the mouse: muscle, skin and hair, long bones, tails, whole limbs, and blood. HeLa cell, Staphylococcus aureus, and Eshcerichia coli suspensions were also tested. Saline diluent was used throughout. The cutter and chamber assemblies had been autoclavesterilized. Large tissue pieces were cut into smaller pieces of a few millimeters in size before homogenization. Usually 30 sec of full-speed operation was sufficient for complete homogenization. Cooling of the chamber in ice water was utilized for longer periods of cutting action. No decrease in bacterial colony counts was found after 5 min of exposure, compared to untreated controls, indicating that no bacterial cell disruption occurred. Red blood cells required 5 min for breakage of 90% of the cells, but leukocytes were completely homogenized after 1 min at maximum speed. The rupture of red blood cells was perhaps due more to turbulence than to cutting or grinding action. HeLa cell disruption required 5 min for 75% rupture, as judged by vitai staining and counting in a Levy counting chamber, Long bones from the mouse were rapidly (30 sec) converted to fragments of microscopic size, but reduction of all of these to particles under 50 jAm required 10 to 20 min of homogenization. Hair was of similar difficulty to bone, with rapid initial reduction in size, and approximately 20 min of extended treatment was required for further size reduction. A homogenizer equipped with the new cutter assembly has been completely satisfactory in service for 1 year in the large diagnostic bacteriology laboratory of this hospital, where it has been used to prepare tissues taken at autopsy for bacteriological examination. No maintenance or replacement has been necessary. It has also been used in research applications to disrupt animal tissues for enzyme extraction and for quantitative bacteriological analysis.

v3-fos

2020-12-10T09:04:10.917Z

1970-11-01T00:00:00.000Z

237231307

s2

Growth Stimulants in Plant Extracts for Leuconostoc citrovorum The growth of Leuconostoc citrovorum ML 34, an isolate associated with the malo-lactic fermentation of wine, was stimulated in part by grape, orange, cabbage, and tomato juices. The stimulatory activity of tomato serum was associated with the carbohydrate fraction. Further purification of the fraction showed that fructose was the factor responsible for initiating growth. In addition to fructose, the organism required CO2 for establishing growth. Saturated CO2 atmosphere and catalytic amounts of fructose served as substitutes for plant extracts in a complex glucose medium. The addition of plant extracts to a medium for the subsequent enhancement of growth of many lactic acid bacteria has been routinely employed in laboratories for many years. Tomato juice, an ingredient commonly added to media for culturing lactic acid bacteria, serves as an essential adjunct for the propagation of the fastidious bacteria associated with the malo-lactic fermentation of wines (6,13,17). These gram-positive, heterofermentative cocci, in addition to requiring unknown growth factors present in tomato juice, produce no slime, initiate growth at low pH (3.7), and are more tolerant to ethanol (10%) than are other leuconostocs. Based upon these and other discerning physiological properties, a new species Leuconostoc oenos has been proposed (5). L. citrovorum ML 34, a wine isolate possessing many of the taxonomic characteristics of this newly proposed species, was used to induce the malo-lactic fernentations successfully in California (8). This microorganism, likewise, is difficult to cultivate in complex media and requires unknown growth factors present in tomato and grape juices (9). Therefore, the present study was undertaken to define the conditions essential for obtaining maximum growth yields, and to determine the nature of the plant materials responsible for promoting such growth enhancement activities in L. citrovorum ML 34. 1 This contribution was approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper no. 1825. MATERIALS AND METHODS Culture. L. citrovorwn ML 34 was obtained from Ralph E. Kunkee, Department of Enology, University of California, Davis. The culture was maintained in a tryptone-glucose-yeast extract-salts medium (TGYE) supplemented with tomato juice (10%) as previously described (15). Preparation of plant extracts. Fresh tomatoes (New Yorker variety) were washed well and the juice was expressed in a paddle-finisher. The juice, pH 4.4, was centrifuged (20,000 X g) for 20 min. The tomato serum was concentrated 12-fold by vacuum distillation at 40C. The concentrate containing 897.8 g (total solids) was adjusted to pH 7.0 with granular calcium carbonate and treated with ethanol (80% saturation). After 18 hr of standing at 4 C, the alcohol-soluble fraction (411.5 g) was decanted and further treated with acetone (38% final). The alcohol-acetonesoluble fraction (334.2 g) was obtained by decantation after 3 days of standing at 4 C. Organic solvents were removed by vacuum distillation, and the various fractions were stored at -20 C. Ion-exchange chromatography. A portion of the alcohol-acetone-soluble fraction (31.8 g, pH 6.0) was passed through a column (4.5 by 42.5 cm) of the cation-exchange resin, Dowex 50 (H+). The effluent (29.2 g, pH 2.3) was passed immediately through a Dowex 3 (OH-) column of similar capacity (yield: 27.1 g, pH 6.7). A portion of this latter fraction (11.1 g) was passed through an activated charcoal (Darco, 80 mesh) column similar to the above. The clear, decolorized, aqueous effluent provided 9.6 g of material. The columns were washed with water until no sugar was detected in the effluent by anthrone reagent (12). All fractions were then adjusted to pH 5.2 with phosphoric acid (2 N) and made to a volume equiva-lent to that concentration of the original serum (29 mg/ml, dry weight). The independent fractions were sterilized by filtration and added to 5-ml quantities of double-strength TGYE broth (final volumes of 10 ml). Paper chromatography. Samples (200 mg) were spotted as compact bands on sheets (46 by 57 cm) of Whatman no. 1 high-capacity chromatography paper. Descending chromatography employing butanolacetic acid-water (5:1:4) or butanol-ethyl alcoholwater (2:1:1) solvent systems were used for irrigation. After 96 hr of development, marker strips were sprayed with silver nitrate or p-anisidine (2). The areas corresponding to the color-developed marker strips were eluted with distilled water, concentrated under vacuum, filter sterilized, and added to the TGYE assay broth. Turbidometric measurements (660 nm) served as a means of determining the biologically active areas. Other natural sources of the stimnuatory factor. Frozen orange juice concentrate, fresh grape, and cabbage juices were centrifuged, filter sterilized, and added as 10% supplements to the TGYE basal medium, pH 5.2. Destruction of the stimulatory factor. Each filtersterilized plant extract (100 ml) was fermented by Saccharomyces cerevisiae 223 for 21 days at room temperature. After completion of the yeast fermentation, as evidenced by the cessation of bubbling activity, the broths were clarified by centrifugation (20,000 X g, 20 min). The ethanol was removed by vacuum distillation at 40 C. The concentrates were reconstituted with distilled water to the original volumes and assayed as described above. Carbon dioxide atmospheres. The media (10-ml samples) were added to sterile colorimeter tubes (16 by 145 mm) and inoculated with one loop of an actively growing culture of L. citrovorum ML 34. The tubes, fitted with sterile rubber stoppers and equipped with venting ports containing glass wool, were placed in anaerobic jars for evacuation. Prior to closure of the pinch clamps, the atmospheres were displaced three times with the respective CO2 gas concentrations. All assays were run in triplicate and incubated at 32 C. The various C02-air mixtures were obtained by metering compressed CO2 and C02-free air into 20liter water-filled carboys. The volumes of water displaced by the compressed gases served to establish the ratios of final gas concentrations used. RESULTS AND DISCUSSION The addition of filter-sterilized plant extracts to a TGYE basal medium produced a 12to 30fold increase in the final optical density (OD) values ( Table 1). The cell yields indicate that the growth-enhancing properties of the juices were not unique to any particular plant extract. The rather universal occurrence of stimulatory activity suggested that the factor(s) was different from that described by Garvie and Mabbitt (6). However, there are apparent differences between grape varieties (red versus white) and the state of maturity of the tomato extracts (mature versus green). The red juice, Seibel, provided 45 % greater growth response than that obtained with the white juice, Ravat 34, and the mature tomato extract produced a twofold greater yield than its immature counterpart. It appears, therefore, that variety and state of maturity contributed significantly to the observed stimulatory effects. Since L. citrovorum ML 34 was used successfully for the induction of the malo-lactic fermentation of California wines (9), the effects of the yeast-fermented extracts were also investigated. Alcohol-free fermented extracts, when added as 10% supplements to the TGYE base, produced no significant stimulatory effects (Table 1). Results suggested that the yeast had either utilized the factor or had produced an inhibitory substance that masked its effect. As later data will show, the former appeared to be the explanation. In an attempt to identify the nature of the stimulatory factor, various fractionation steps were employed. Those fractions obtained as a result of organic solvent extraction and column chromatography were analyzed with ninhydrin and anthrone reagents, and ultraviolet absorption properties (280:260 nm ratios). It is apparent ( Table 2) that each successive fractionation step increased the cell yields and that the greatest specific activity (OD per milligram, dry weight, of tomato serum) was observed in the charcoal effluent fraction. This latter fraction, in addition to showing a threefold increase in specific activity, was ninhydrin-negative and contained less than 0.28% protein. Those factors responsible for inducing growth were best correlated to the carbohydrate-enriched fractions. Nearly 98.5 % of this latter fraction was comprised of carbohydrate, expressed as glucose, and more than 96 % of the total carbohydrate content was composed of reducing carbohydrate material. Paper chromatography of the latter fraction in neutral and acidic solvent systems, followed by color development with ninhydrin, silver nitrate, p-anisidine, and ultraviolet absorption, indicated the presence of only two distinct reducing substances. Elution ofthe bands and subsequent rechromatography indicated that glucose and fructose were the only detectable constituents present. Hydrolysis of the latter fraction with hydrochloric acid (2 N) in a boiling-water bath for 30 min failed to produce additional substances. The aqueous eluate corresponding to fructose and fructose standards (range: 0.25 to 2.0%) were compared for growth-enhancing properties in the filter-sterilized TGYE medium incubated under stationary conditions (air). The TGYE medium devoid of fructose produced no visible growth within 14 days, whereas the paper chromatographic eluate and the fructose standards produced OD values in excess of 1.80 within 9 days. It was observed, however, that 6 days of incubation were required before the onset of measurable turbidity. This extended lag period suggested that factors other than fructose, or in addition to fructose, were essential for initiating early growth. Since the original culture was isolated from a wine fermentation, and the latter fermentation under commercial conditions produces an atmosphere high in CO2 (1), the effects of CO2 concentrations upon the growth in a TGYE autoclaved medium were investigated. Increased CO2 tensions markedly decreased the lag phase (Fig. 1). The culture incubated in an air atmosphere under stationary conditions required more than 240 hr of incubation to reach an OD value of 0.10, whereas, under CO2 saturated atmosphere, the lag phase was decreased to less than 24 hr. Reductions in lag phases appear to be related to the concentration of CO2. Also, when aerobically grown cultures reached an OD of 0.030, and were then supplied with a saturated CO2 atmosphere, less than 21 hr was required to induce accelerated growth rates. The similarities in the slopes of the growth rates during the accelerated growth phase indicate that, once active growth is initiated (OD greater than 0.10), the generation times are quite similar. The above growth responses were measured in an autoclaved TGYE medium. The effects of autoclaving upon complex media and its inhibitory or enhancing properties, or both, were well documented by many investigators. It was previously reported (16) that fructose is generated by the temperatures used for sterilization of this complex medium. Thus it appears that catalytic amounts of fructose, arising as the result of autoclaving, and the elevated CO2 tensions were responsible for the observed growth responses. The actions and interactions of fructose, glucose, tomato serum, and CO2 upon growth are summarized in Fig. 2. L. citrovorwn was able to initiate only very limited growth on either fructose or glucose in the presence of air or saturated CO2. However, the additions of varying amounts of fructose (0.025 to 1.0%) or tomato serum (2.5 to 15%) to a TGYE filtered medium, incubated under a saturated CO2 atmosphere, produced marked stimulatory responses within 4 days. When grown under CO2, the addition of 0.25% fructose produced a growth response value similar to that provided by the 3% level of tomato serum grown in the absence of CO2. Those cultures grown in air produced less than 20% of the growth observed in CO2. It has been known for many years that certain heterotrophic microorganisms require CO2 for growth (14), and many of these C02-mediated reactions were extensively reviewed by Wood (19). Although high concentrations of CO2 (50% v/v) were shown to be inhibitory (A. D. King, Jr., Ph.D. thesis, Washington State Univ., 1966) and stimulatory (3) to growth and enzymatic activity, L. citrovorum responded most favorably to an atmosphere comprised solely of CO,. The CO2 served a vital function in initiating growth; however, its exact role remains to be elucidated. Since hydrogen and nitrogen atmos-pheres were no more conducive to establishing good growth than an atmosphere of air, it appears unlikely that anaerobiosis is a requirement for growth. To determine whether known growth factors would enhance early growth in an air atmosphere, compounds, added singly and as complete mixtures, were tested and found to be ineffective.

v3-fos

2018-04-03T02:52:39.892Z

1970-07-01T00:00:00.000Z

35489189

s2

Optimum Skin Blending Method for Quantifying Poultry Carcass Bacterial Optimum blending fluids and blending times for use in quantifying bacteria on poultry carcass skin by the skin "blending" method were determined. Butterfield's buffered-phosphate diluent, physiological saline solution (0.85% NaCI), peptone water (0.1% peptone), and deionized water, each at four different skin blending times of 1, 2, 3, and 4 min, were compared. The comparison was based on relative numbers of bacteria per cm2 of skin, enumerated by each combination on turkey carcasses. Peptone water and physiological saline solution each yielded significantly (P < 0.01) higher bacteria counts from turkey carcass skin samples than did Butterfield's buffered-phosphate diluent or deionized water. There were no significant differences among the four skin blending times and no significant inter- action effect between the two factors tested. The skin "blending" and dilution method for quantifying bacteria on poultry carcass skin consists of two factors, blending fluid and blending time. Either factor could affect the number of bacteria recovered and enumerated by this method. Straka and Stokes (2) demonstrated rapid and extensive destruction of bacteria in distilled water, tap water, phosphate water, and physiological saline solution. Their data indicated that water or even physiological saline was most destructive. Therefore, their use as a diluent can lead to serious errors in quantitative determination of bacteria in food. The degree of error depends on the number of sensitive bacteria present, exposure time to the fluid, and quantity of protective organic food material in the fluid. They warned that these diluent fluids can cause large errors in quantitative bacterial counts by plating. They found that 0.1% peptone in distilled water as a diluent fluid did not cause any appreciable destruction of bacteria for 1 hr. Previously, sterile water had been commonly used as a food diluent for bacteriological plate counts; also, physiological saline and phosphatebuffered distilled water have been used to a lesser extent (2). The Official Methods of Analysis of the Association of Official Analytical Chemists I Published with the approval of the Director of the Colorado Agricultural Experiment Station as Scientific Series paper number 1522. (1) specifies using Butterfield's buffered-phosphate diluent as a blending fluid for all frozen, chilled, precooked, or prepared foods. A survey of recent literature indicated that 0.1% peptone water is most frequently used as a diluent fluid for determining bacteria counts on poultry meat. Subjecting a sample of poultry carcass skin in diluent fluid to the agitation of a laboratory blendor partially disintegrates the skin, physically removes bacteria from the skin sample, and uniformly distributes the bacteria in the fluid so representative samples can be plated. Consequently, duration of this agitation could possibly affect the number of bacteria determined by plate count. The objective of this study was to compare four skin blending fluids and four skin blending times to determine the optimum combinations for use in quantifying bacteria on poultry carcass skin samples. The experiment was designed to detect whether blending fluids or blending times significantly affect bacteria counts. The null hypotheses were that no significant difference in bacteria counts occurs among the four blending fluids or among the four blending times and that no interaction occurs. These hypotheses were tested in a factorial experiment by analysis of variance. MATERIALS AND METHODS Four blending fluids, Butterfield's buffered-phosphate diluent, physiological saline solution (0.85% NaCl), peptone water (0.1% peptone in distilled water), and deionized water (A, B, C, and D, respectively, in Table 1), were compared, each at four different skin blending times of 1, 2, 3, and 4 min. The basis of comparison was the relative number of bacteria per cm2 of skin on turkey carcasses, determined by plate count for each blending fluid-time combination. Male turkey carcasses ranging in weight from 15 to 22 lb (6.8 to 10 kg) were used. For each replicate, one frozen (-29 C) turkey carcass was thawed in its plastic bag for 16 to 24 hr at room temperature (20 to 27 C). Skin samples (7.145 cm2 discs) from four parts of the carcass (breast, left and right side; and legs, left and right) were sampled for each of the four blending fluids. Thus, four skin samples were removed from each part of a single carcass (Fig. 1). Random selection of blending fluids for carcass skin samples is shown in Table 1. Sample 1 was removed consecutively from each of parts I through IV; then sample 2 was removed in the same consecutive order, as were samples 3 and 4. Each of the four blending fluids was used once for each carcass part and was randomly selected for the four skin samples in each part by use of a random number table. Skin sample 1 was removed from the same location on each leg and the same location on each side of the breast for all replicate carcasses; but the blending fluid used on skin sample 1 for each area was randomly selected, as it was for skin samples 2, 3, and 4. Each skin sample was agitated in a sterile laboratory blendor jar with 100 ml of blending fluid. The blending fluid was plated in duplicate at the 10-2 and 10-s levels in Trypticase Soy Agar plus 2% Yeast Extract after blending each skin sample for 1, 2, 3, and 4 min. After each blending time, 2.2 ml was removed from the blendor jar for plating. This volume change was adjusted by the appropriate dilution factors. Plates were incubated at 35 to 37 C for 48 hr, 25 to 28 C for 60 hr, and stored at 4 C for at least 48 hr prior to colony counting. The aerobic plate count (APC) representing each carcass with any one blending fluid-time combination was the geometric mean of APC values from the four samples, each representing one of four carcass parts. The experiment was replicated with five carcasses. The geometric means were subjected to analysis of variance and Duncan's multiple range test to determine optimum blending fluids and optimum blending times for use in quantifying bacteria on poultry carcass skin by the skin "blending" method. bBlending fluids: (A) Butterfield's buffered phosphate, (B) physiological saline (0.85% NaCl), (C) peptone water (0.1% peptone), (D) deionized water. RESULTS APC/cm2 of skin for each carcass with each blending fluid-time combination is shown in Table 2. Average APC values per cm2 of skin for each blending fluid-time combination are shown in Table 3. Peptone water and physiological saline solution yielded significantly (P < 0.01) higher APC values from turkey carcass skin than did Butteffield's buffered-phosphate diluent or deionized water. There was no significant difference in APC values between peptone water and physiological saline solution or between Butterfield's buffered-phosphate diluent and deionized water. There were no significant differences in APC values among the four skin blending times. No significant interaction occurred between the two factors tested. Results of this study are illustrated graphically in Fig. 2. With the agar-plate count technique, turkey carcass skin samples "blended" in peptone Grand avgd 790 830 1,300 1,400 aEach value represents an arithmetic mean (average) of five carcass aerobic plate counts, each of which represents a geometric mean of four skin sample counts per carcass. water or physiological saline solution yielded significantly higher carcass bacteria counts than skin samples "blended" in Butterfield's bufferedphosphate diluent or deionized water, regardless of blending time. It made no significant difference whether skin samples were "blended" for 1, 2, 3, or 4 min. In fact, in many cases (Table 2) lower counts were obtained after "blending" for 4 min, but this was not significant. DISCUSSION Prolonged "blending" could possibly rupture some bacterial cells or result in a toxic effect from overexposure to the fluid. A blending time of 1 or 2 min was sufficient for optimum results. Apparently, chains or clumps of bacteria were broken into individual cells before 1 min of blending time, or, if not, they were not further broken to any appreciable extent by blending for 2, 3, or 4 min. Based on these results and on the work of Straka and Stokes (2), it can be concluded that peptone water (0.1% peptone) is the optimum blending and diluent fluid of those tested and that the customary 2 min is an optimum blending time for use in quantifying bacteria on poultry carcass skin by the skin "blending" method. Butterfield's buffered-phosphate diluent is recommended (1) and is commonly used as a blending and diluent fluid for many types of food in microbiological analyses. With some foods, this may be a preferable blending and diluent fluid, perhaps because of its buffering capacity. However, with many materials it may not yield the maximum number of viable bacteria on plate count, as shown by this study with poultry carcass skin. If it is important to determine the maximum number of viable bacteria present in a food material by plate count, the particular food being tested should be considered as an individual case which perhaps will require a different blending and diluent fluid than some other food material.

v3-fos

2017-09-27T02:25:34.145Z

1970-09-01T00:00:00.000Z

30747635

s2

Enumeration of Byssochlamys and Other Heat-Resistant Molds' Methods for the detection of low numbers of heat-resistant molds on fruits were studied by using cultures of Byssochlamys and a number of unidentified mold isolates. Ascospore dormancy had a marked effect on viable recoveries, and the medium in which ascospores were heated influenced activation rates. Best results were obtained when fruit homogenates were heated for 60 min at 70 C in Concord grape juice, followed by culturing on acidified Potato Dextrose Agar. In recent years, there has been an increase in the spoilage of thermally processed fruit products caused by Byssochlamys and related molds (6). The problem is that their ascospores are able to survive the fill temperatures of 80 to 90 C commonly used as the process for these foods. Increasing the severity of the thermal treatment is not a solution because too often this results in a marked reduction in product quality. It appears that the best method for preventing spoilage is to control contamination. This, in turn, requires sensitive methods for the detection of viable spores on fruit and in processing lines. Numerous methods have been used in culturing for Byssochlamys. It has long been known that the ascospores exhibit a dormancy that can be broken with heat (3), and a variety of treatments, ranging from 5 min at 75 C to 35 min at 80 C, have been used (2,5,7,11). After this, the material usually has been cultured on a nutrient medium such as potato-sucrose or potato-dextrose-agar. Growth of bacterial spores that would survive the heat shock has been prevented by acidifying the agar (4) or by the inclusion of compounds such as chloramphenicol (9) or hexachlorophene (1). Little information is available regarding the effectiveness of the different activation and cultural procedures. MATERIALS AND METHODS Cultures. The named cultures of B. fulva and B. nivea were obtained from J. J. Ellis of the Northern Regional Research Laboratory, U.S. Department of Agriculture. The others were isolated in this laboratory from spoiled fruit product and from samples collected from nearby orchards and vineyards. Ascospore production. Ascospores were obtained from cultures grown as a pellicle over broth. Two media, 5% Difco Malt Extract Broth and Concord grape juice, have been used. The latter was prepared by adjusting the concentration of commercial grape concentrate to 15°Brix as measured with a hand refractometer. When only small quantities of spores were needed, a typical trial consisted of inoculating 5 ml of broth in a 16by 150-mm culture tube (10). After incubation for 28 days at 32 C, the mat and media were transferred to a sterile 50-ml chamber for blending in a Sorvall Omni-mixer homogenizer. After a visually homogeneous suspension was obtained, the material was centrifuged and washed three times in sterile distilled water. Suspensions to be used in later experiments were frozen and stored at -23 C. Activation. In most trials, 0.5 ml of washed spores was added to 4.5 ml of the activation medium. The tube then was placed in a water bath for the desired time period. The media formulas represent concentrations after dilution by the spore suspensions. To assure that different responses were not artifacts resulting from clumping or the breaking up of asci, microscopic counts, determined with a hemocytorneter, of single spores and asci were conducted routinely before and after treatment. Viable counts. Appropriate dilutions of spore suspensions were cultured on Difco Potato Dextrose Agar acidified to pH 3.5 with tartaric acid. The incubation was at 32 C for 2 to 4 days depending upon the strain. SPLIYTSTOESSER, KUSS, AND HARRISON enate at 70 C, for example, reduced the viable count by over 3 log cycles during the first 5 min, followed by a slight increase in count due to activation of dormant ascospores (10). The kinetics of activation during the initial 5 min could not, of course, be measured. Attempts to obtain pure suspensions for these studies by repeated centrifugation and by filtration (8) were not successful. Although asci could be concentrated by slow, short-time centrifugation, the method was tedious and the preparations were never completely free from conidia and hyphae. The filtration technique was not applicable because of the large numbers of conidia produced by certain strains. Fortunately, it was found that suspending homogenates in 85% ethanol for a short time destroyed the conidia and hyphae (Table 1). Heat and ethanol both reduced the viable count of NYS 1, composed mainly of conidia, by about 4 log cycles, indicating that the two treatments inactivated the same structures. With NRRL 2614, mainly asci, the counts obtained with the "ethanol only" and "washed only" (control) treatments reflected the low populations of active asci that were present. Heating these suspensions increased this population about 10-fold. It was concluded that ethanol did not activate the ascospores or affect their viability, and, therefore, the treatment was used routinely. Early studies on the effect of the heating menstruum on activation included grape juice because it represented one of the foods in which a methodology for spore detection was desired. The results of numerous trials showed an interaction between the medium and temperature. When spores in Concord grape juice were heated at 60 C, activation was completed in 60 min or less, whereas heating in water for as long as 3 hr produced no detectable increase in the viable count (Fig. 1). Spore activation also was enhanced by grape juice at 70 C, although at this temperature a significant number of the spores in water now were activated. These data indicated that grape juice accelerated the rate of spore activation. The spores from numerous strains have been activated at a variety of temperatures in grape juice. At 40 C, the lowest temperature studied, a slight increase in viable count was detected after heating for 3 hr, although 95% of the activatable spores remained dormant. At 80 C, the counts usually were lower than at 70 C, indicating that this temperature was lethal for the spores of many strains. It was concluded from this that 70 C was the optimal activation temperature for most strains. However, a lower temperature such as 60 C was more suitable for studying certain variables, because it emphasized differences in activation rates. The stimulatory effect of grape juice varied with the variety, with Concord being more active than juice from Riesling and Seibel 9549 grapes (Fig. 2). Other trials supported these data in that Concord juice could be diluted to a concentration as low as 20 Brix without reducing activation rates, whereas any dilution of Seibel 9549 juice below the original 160 Brix resulted in fewer spores being activated. Studies on the mechanism of grape juice stimulation revealed an interaction between pH and the active factor (Fig. 3). Adjusting Concord juice to pH 4 caused a marked reduction in the number of spores activated, whereas at pH 5 to 7, the viable counts were no higher than obtained in water. It is known that pH per se was not responsible for the grape juice effect, because heating spores in pH 3.5 solutions of malic and tartaric acids, the principle acids in grape juice, failed to enhance activation ( Table 2). The data ( Table 2) also show that, although glucose and malt extract broth had no effect, a 5% solution of yeast extract gave results comparable to Concord juice. It appears that the same principle was active in both menstrua since their activity responded similarly to changes in pH. Other trials have shown that the factor was still present after grape juice was dried at 100 C, autoclaved, or fermented. Work presently is underway to isolate and define the compound. Cultural conditions. At first, most-probablenumber (MPN) procedures employing broth cultures were used to enumerate ascospores on fruit samples. It was assumed that the incidence of heat-resistant spores would be low, and, therefore, the method would have the advantage that it permitted relatively large samples, 25 g or more, to be cultured. Unfortunately, the method was found to require a very long incubation period. In one trial, for example, 50-ml amounts of 5% malt broth in each of 10 milk dilution bottles were inoculated with 10 g of grape homogenate containing an average of two active asci. The bottles were incubated at 32 C, on their sides with caps loosened to facilitate aeration. The first growth, in one bottle only, was detected after 7 days, whereas 22 days was required before all of the potentially positive bottles, a total of nine, showed growth. Although the MPN recovery figures were in good agreement with the number of asci in the inoculum, the long incubation reduced the effectiveness of the method. When inoculated homogenates were cultured concurrently in broth and on agar media, the latter were found to yield maximal viable recoveries after an incubation of only 2 to 4 days at 32 C. More rapid growth because of better aeration plus the fact that colonies wvere more FIG. 4. Procedure adopted for the detection an2d eniumeration of heat-resistanit mold spores. PDA, Potato Dextrose Agar. easily seen on agar probably accounted for these results. The following agar media have been compared to determine which gave the higher viable counts when inoculated with activated ascospores: Potato Dextrose, Plate Count, 5 % yeast extract, 5% Malt Extract, Put's (9), and concentrations of Concord grape juice from 1 to 15°Brix. The results indicated that many heat-resistant molds were not particularly fastidious in that a number of strains gave comparable counts in all media. When differences were noted, acidified Potato Dextrose Agar usually provided the higher figures. For example, a study of 11 strains showed that 5 gave higher counts in this medium than in 7.5°-Brix grape juice-agar. In general, the differences were by a factor of twofold or less which indicated that the plating medium was not as important a variable as was the heat activation menstruum. Adopted methods. Figure 4 illustrates the procedures used for the detection and enumeration of low numbers of heat-resistant molds on a variety of fruit samples. To minimize the opportunity for chance contamination, a potential problem in laboratories in which Byssochiamys is routinely cultured, the sterile, screw-cap blendor jars were usually carried to the sampling site. Although the amounts varied, 50 to 100 g of fruit often was blended with 100 ml of Concord juice. A 5-min treatment usually produced a homogeneous mixture that could be readily poured into petri dishes. Heat activation was carried out in the blendor jars. The jars were enclosed in polyethylene bags before being placed in the water bath as a safeguard against leakage through the bottom bushing. A 2-hr hold assured that the contents were at the equilibrium temperature for about 1 hr. Overheating was not a problem since spores have been held for as long as 6 hr at 70 C without reducing the viable count. After heating, the entire contents of the jar were distributed into petri dishes, approximately 10 ml per dish. Equal volumes of double-strength Potato Dextrose Agar then were added to the plates. The culturing of such a large sample was required because usually the level of spore contamination was very low. The method underestimated spore populations to some extent because of material retained on the blendor walls. Most strains produced countable colonies by 48 hr at 32 C. Because a few required 396 APPL. MICROBIOL. on September 26, 2017 by guest http://aem.asm.org/ Downloaded from a longer incubation, negative plates were held for 96 hr before being discarded. The above method has been used for the examination of over 60 samples collected from orchards, vineyards, and processing lines. The procedure appears to be effective in that many contaminated samples, ranging in spore counts from under 1 to over 1,000 per 100 g of grapes, have been detected and some 40 different mold types have been isolated. Interference by other microorganisms has not been a problem. These microorganisms apparently were eliminated by heating at 70 C coupled with the low pH plating medium. LITERATURE CITED

v3-fos

2020-12-10T09:04:10.852Z

1970-09-01T00:00:00.000Z

237233397

s2

Improved Tissue and Cell Homogenizer Modification of an existing tissue homogenizer is described, which converts to a milk-like consistency even difficult to macerate tissues such as cartilage, hair, and small bones. Commercially available homogenizers failed to disrupt completely fibrous tissue or bone specimens obtained during autopsy or biopsy procedures in preparation for bacterial culture or enzyme assays. The conventional cutting blades supplied commercially are all of the general shape seen in Fig. 1. These blades strike the tissue pieces so as to keep them out of the cutting area, where they are exposed only to turbulence. The glass or Teflon and glass homogenizers of the Ten Broeck type were inadequate for dense tissues, making it necessary to revert to sand, mortar, and pestle to macerate these specimens. The cutting head on one machine (Omni-Mixer, model 17150, Ivan S. Sorvall, Inc., Newtown, Conn.; similar to Multi-Mix, Lourdes Instrument Corp., Brooklyn, N.Y.) was modified in various ways, and the most effective design, shown in Fig. 1 and 2, was finally adopted. A stationary multihole collar, to slip over the vertical shaft, was machined from 440-C stainless steel, heat-treated, and electro-polished. The outside diameter of the collar was approximately 1 mm less than the inside of the chamber. It was fastened flush with the end of the shaft with one or two Allen screws, bearing on the vertical shaft. The rotating cutting head was machined to bear against the flat-ground bottom of the stationary collar, providing a positive, scissors-like cutting action. The center hole was threaded to fit the already threaded shaft end. Contact pressure between the rotating cutter and the collar is adjusted by loosening the collar, pressing it against the cutter, then retightening the collar on the shaft. Separate cutting heads are required: one for the microchamber (up to 5 ml), one for the 50-ml chamber, and one for both the 200-and 400-ml chambers. As seen in Fig. 2, the cutting head shaft for the microchamber required addition of an outer supporting shaft for attachment of the fixed, upper half of the cutting head. This supporting shaft is an integral part of the lower screw cover. Homogenizing action of the new cutter was tested with various tissues from the mouse: muscle, skin and hair, long bones, tails, whole limbs, and blood. HeLa cell, Staphylococcus aureus, and Eshcerichia coli suspensions were also tested. Saline diluent was used throughout. The cutter and chamber assemblies had been autoclavesterilized. Large tissue pieces were cut into smaller pieces of a few millimeters in size before homogenization. Usually 30 sec of full-speed operation was sufficient for complete homogenization. Cooling of the chamber in ice water was utilized for longer periods of cutting action. No decrease in bacterial colony counts was found after 5 min of exposure, compared to untreated controls, indicating that no bacterial cell disruption occurred. Red blood cells required 5 min for breakage of 90% of the cells, but leukocytes were completely homogenized after 1 min at maximum speed. The rupture of red blood cells was perhaps due more to turbulence than to cutting or grinding action. HeLa cell disruption required 5 min for 75% rupture, as judged by vitai staining and counting in a Levy counting chamber, Long bones from the mouse were rapidly (30 sec) converted to fragments of microscopic size, but reduction of all of these to particles under 50 jAm required 10 to 20 min of homogenization. Hair was of similar difficulty to bone, with rapid initial reduction in size, and approximately 20 min of extended treatment was required for further size reduction. A homogenizer equipped with the new cutter assembly has been completely satisfactory in service for 1 year in the large diagnostic bacteriology laboratory of this hospital, where it has been used to prepare tissues taken at autopsy for bacteriological examination. No maintenance or replacement has been necessary. It has also been used in research applications to disrupt animal tissues for enzyme extraction and for quantitative bacteriological analysis.

v3-fos

2020-12-10T09:04:12.394Z

1970-05-01T00:00:00.000Z

237233067

s2

Bacteriological Examination of Commercial Precooked Eastern-Type Turkey Rolls Studies were conducted to ascertain the bacteriological condition of commercially cooked Eastern-type (foil-wrapped-oven roasted) turkey rolls during processing and storage. After 2 weeks at 5 C, numbers of aerobes on the surface of rolls, in slices, and in whole rolls reached levels of from 1 to 10 million per cm2 or per g. In stored whole rolls, coliform and enterococcus counts ranged, respectively, from about 10,000 to more than 1 million per g and from < 100 to more than 1 million per g. Postcooking processing operations in two plants did not significantly affect the total count of turkey rolls. Eight of 28 rolls obtained after handling and packaging contained coagulase-positive staphylococci. A variety of precooked poultry products are marketed for home and institutional consumption. Among the most popular of these items are precooked turkey rolls. Federal regulations (5) require that precooked turkey rolls be heated to an internal end-point temperature of 160 F (71.1 C). That such a procedure is effective in destroying salmonellae and coagulase-positive staphylococci in turkey meat has been demonstrated (3,6). The majority of ready-to-eat turkey rolls are cooked either by enclosing the raw roll in a fibrous moisture-proof casing and cooking in a hot-water bath, or by wrapping the roll in aluminum foil and oven roasting to the required endpoint temperature. The casing used in the former method is not removed and usually serves as the final package, so that additional handling of the meat is not required. Bacteriological studies relating to this type of roll have been reported (2, 3; G. A. N. da Silva, Bacteriol. Proc., p. 12, 1967). With the latter type, often referred to as the Eastern-type (4), plant employees remove the foil after cooking and insert the roll into a plastic casing which is closed at one end. The natural juices from cooked rolls are combined with spices and gelatin (according to the formulation of each plant) and the mixture is heated to a minimum of 71.1 C. A portion of this heated mixture is then added to the casing, containing the roll, after which a vacuum is drawn and the casing closed at the other end. Kinner et al. (4) showed that one of the major sources of bacteriological contamination of the Eastern-type roll is the natural juice-gelatin-spice mixture which is added before final packaging. They also showed that such mixtures should be heated to an end-point temperature of 82 C to reduce significantly numbers of coliforms and enterococci and to 93 C to reduce significantly the total bacterial count of these mixtures. The objective of this study was to evaluate the bacteriological condition of commercial Easterntype rolls immediately after processing and after various periods of refrigerated storage. Both intact packaged rolls (the form in which they would be stored by processors, distributors, or institutional users) and rolls from which slices were periodically removed during storage (to simulate a delicatessen or household practice) were evaluated. MATERIALS AND METHODS Stored roll studies. Packaged Eastern-type turkey rolls, each weighing between 5 and 7 lb (2.27 and 3.17 kg), were obtained from each of three commercial plants (A, B, and C). The rolls were from lots that had been processed 2 days previously and then held in the plant coolers. The rolls were placed in crushed ice in an insulated container and transported to the laboratory where they were placed in a laboratory refrigerator at 5 C. These rolls were examined bacteriologically as follows. (i) At intervals of 1, 4, 8, 11, 15, 18, 22, and 25 days, during storage at 5 C, two surface swabs were made on each of two rolls from each of the three plants. The same two rolls from each plant were sampled throughout the storage period, with different areas of the rolls swabbed on each sampling day. This was done by aseptically cutting, for each area, a flap in the casing of the roll of sufficient size to accommodate a sterile cardboard circular template which circumscribed an area of 12.3 cm2. After swabbing this area for 30 sec with a cotton swab moistened with 0.1% peptone solution, the flap was VOL. 19, 1970 EASTERN-TY closed and sealed with transparent tape. Serial decimal dilutions were then made and plated on plate count agar and the plates were incubated at 20 C for 72 hr. (ii) Six additional rolls, two from each of the three plants, were removed from the refrigerator at 5 C after 2, 16, and 30 days of storage. Each roll was cut approximately in half by using an electric knife with a sterile blade. One-half of each roll, with casing removed, was placed in a previously autoclaved large stainless-steel blendor jar. Sterile distilled water, equal to the weight of the half roll, was then added to the jar and the roll was blended with the water at low speed for 4.75 min. During this period, the blendor was turned off for 30 sec every 15 sec to avoid overheating the blendor motor and the sample. Approximately 1 pint (473.2 ml) of the resulting homogenate was then poured into a sterile Mason jar and the remainder was discarded. Without rinsing the jar, the second half of the roll, with an equal weight of sterile distilled water, was blended in a similar manner and a pint of the homogenate was placed in another sterile Mason jar. Serial decimal dilutions of each of these homogenates prepared in 0.1% peptone water were plated on Plate Count agar (incubated at 20 C for 72 hr) for total count, on Violet Red Bile agar for coliforms (18 to 24 hr at 35 to 37 C), on M-Enterococcus agar for enterococci (48 hr at 35 to 37 C), and on Sulfite Polymyxin-Sulfadiazine agar for Clostridium perfringens (24 and 48 hr at 37 C in anaerobicjars). Salmonella determinations were carried out on 50 g of each of the homogenates. Sufficient extra strength selenite cystine broth was added to yield a mixture equivalent to 25 g of undiluted turkey roll meat to 225 ml of "normal" selenite broth. After incubating this mixture for 18 to 24 hr at 37 C, loopfuls were streaked on Brilliant Green Sulfa, Bismuth Sulfite, and SS agars. After incubation at 37 C for 18 to 24 hr, typical Salmonella colonies were picked from these plates and transferred to Triple Sugar Iron and Lysine Iron agar slants. All cultures showing Salmonella reactions after 24 hr at 37 C were tested with Salmonella polyvalent "O" and Spicer-Edwards "H" antisera. (iii) Two sequential slices of approximately equal thickness (3 to 4 mm) were removed from each of two additional rolls from each plant after 1, 4, 8, 11, 15, 18, 22, and 25 days of storage at 5 C. After sampling, the remainder of each roll was placed in a polyethylene bag and returned to the refrigerator at 5 C until the next sampling day. Each slice was weighed and, with an equal weight of sterile distilled water, blended in a Waring blendor for 2 min at low speed. Serial decimal dilutions of this homogenate were prepared and plated on Plate Count agar, and the plates were incubated at 20 C for 72 hr. Two slices from a roll on each sampling day were identified as "outer slice" and "inner slice." Additional examinations. Employing the homogenized roll procedure described above, an additional 42 rolls obtained periodically from two plants over a 2-month period were examined (on the day after processing) for total counts, salmonellae, and C. perfringens. Coagulase-positive staphylococci in these rolls were also determined by the method of Baer (1). Fourteen of the rolls were obtained immediately after cooking, 14 were obtained immediately before the addition of the juice-spice-gelatin mixture, and 14 were "final packaged" rolls. RESULTS AND DISCUSSION Total counts of the surface of the ready-to-eat, foil-roasted rolls at various intervals during storage at 5 C are shown in Fig. 1. Counts of all rolls were relatively low after the first 4 days of laboratory refrigerator storage. After 11 days (plant and laboratory holding), five of the six rolls had at least one swab count in excess of 106/cm2, and, by the 14th day, counts of all rolls were in the range of 106 to 107/cm2. After 18 days, counts remained relatively constant at 107 to 108/cm2. An analysis of variance of the swab bacterial counts revealed no significant difference among the rolls from the three plants nor between rolls within plants. With minor exceptions, the differences on any particular sampling day between swab counts on a roll were within the range of experimental error. Only rarely were differences greater than 1 log observed. This suggests that contamination is relatively uniform over the entire surface of this type of roll. Total, coliform, and enterococcus counts of rolls stored for 2, 16, and 30 days are shown in Table 1. In all cases, counts between halves of the same rolls were in excellent agreement. No salmonellae or C. perfringens were detected in any of the rolls sampled. The rate of increase in numbers of bacteria per gram of turkey slices during storage approximated that of the swab counts of the packaged roll (Fig. 2). Total counts of the outer slice were significantly greater than those from the inner slice, although the magnitude of these differences was not very great until late in storage. Since the slices included a portion of the external surface of the roll, possibly only bacteria on the external surface were determined. The higher counts of the "outer" than of the "inner" slice might be attributed to exposure of a larger surface area which would have encouraged growth of obligate aerobes transferred from the outside of the roll to the cut surface during slicing. Growth of such aerobes in the inner portion of the roll, however, where the oxygen tension is obviously much lower, would have been restricted. In a few instances, the presence of slime on the exposed surface of cut rolls stored for 3 weeks was noted. Significant off-odors were not detected, however. Additional examinations. No significant differences were found in total counts among cooked rolls obtained at the three stages of processing. The majority of rolls obtained at the three stages had counts of less than 5000/g. Salmonellae were found in 2 of 14 rolls taken directly from the oven but not in rolls obtained at the later two stages. Coagulase-positive staphylococci were found in 8 of the 28 rolls obtained at the later two stages and in 1 roll obtained directly from the oven. C. perfringens was not detected in any of the rolls examined. Results of these examinations indicate that initial bacterial contamination of the Easterntype turkey roll is relatively low. Numbers of FIG. 2. Bacterial counts of "outer" and "inner" slices of "ready-to-eat" Eastern-type turkey rolls. Sequential pairs of slices were periodically removed from stored turkey rolls. Values are means of three slices, one from each roll, from each of three plants. bacteria may increase substantially, however, during refrigerated storage at 5 C in a matter of 2 to 3 weeks. Holding temperatures employed by processors usually range from about 34 to 40 F (1.1 to 4.4 C). At delicatessen counters, however, temperatures may be nearer to 45 to 50 F (7.2 to 10 C) so that extensive growth of bacteria as demonstrated here could occur in a relatively short period of time, with the possible development of slime. The occasional finding of salmonellae and coagulase-positive staphylococci in this type of roll emphasizes the need for continuous in-plant application of effective temperature controls both with respect to cooking of the roll itself and of spice-juice mixtures added to the rolls. Stringent adherence to plant sanitation principles and practices during handling and packaging is also necessary to minimize contamination of the cooked product with food-borne pathogens.

v3-fos

2020-12-10T09:04:17.377Z

1970-11-01T00:00:00.000Z

237234616

s2

Lyophilization of Cell-Free Marek's Disease Herpesvirus and a Herpesvirus from Turkeys Cell extracts of the JM and GA strains of Marek's disease herpesvirus and the FC 126 strain of turkey herpesvirus were lyophilized with various stabilizers. Much higher virus titers were obtained with stabilizer than without stabilizer. Titers increased even further in the case of the Marek's disease virus strains by the addition of a chelating agent, disodium ethylenediaminetetraacetate. Until now, preservation of Marek's disease herpesvirus has depended upon procedures which maintain viable cells (7). In general, the techniques which destroy cellular integrity result in loss of most of the infectivity (2); this cell association placed the Marek's disease virus with the group B herpesviruses (5). Recently, we discovered that, unlike the situation in tumor cells or whole blood cells of Marek's diseaseinfected chickens, virus replication in cells of the feather follicle epithelium is complete and large numbers of cytoplasmic, enveloped herpesvirus particles can be found there by electron microscopic examination (3, 3a). Also, by homogenization and sonic treatment of feather follicle epithelium in phosphate-buffered saline (PBS), we were able to extract cell-free virus. Adldinger (unpublished data) extracted Marek's disease virus from infected chicken kidney culture, also by sonic treatment, and learned that incorporation of a chelating agent, disodium ethylene-diaminetetraacetate (EDTA), in the suspending fluid for extraction and assay markedly increased the virus titer. Cell-free preparations of virus from either source induced Marek's disease in susceptible chickens, initiated characteristic focal lesions in chicken kidney culture, and could be stored at -65 C without loss of titer. These observations prompted us to attempt lyophilization of Marek's disease virus. Another group B herpesvirus, isolated from turkeys and identified as strain FC 126 HVT (8), was also studied since that virus is under intensive study as a potential vaccine strain for the control of Marek's disease in chickens. Two series of trials were done, one with the JM strain of Marek's disease virus (6) extracted from the skin of in-fected birds and the other with two strains of Marek's disease virus, JM and GA (4), and with strain FC 126 HVT extracted from heavily infected petri dish cultures of chicken kidney or chicken embryo fibroblast cells. MATERIALS AND METHODS Virus strains. JM strain virus for the first three trials was obtained from two batches (X-300 and X-31 1) of skin from freshly killed infected chickens by the following procedure. Strips of skin from which the feathers were clipped off at the surface were mixed 1:5 or 1:10 (w/v) with PBS (pH 6.8), minced) homogenized for 5 min (Omni-Mixer, Ivan Sorvall, Inc., Norwalk, Conn.), and sonically treated for 2 min (model W 140D Sonifer Cell Disrupter, Heat Systems-Ultrasonics, Inc., Plainview, N.Y.). The suspensions were clarified by centrifugation at 650 X g, and the supernatants were frozen at -65C. They were considered free from viable cells. For these studies, virus suspensions were thawed and then, after various treatments, refrozen or lyophilized in 1or 2-ml quantities. For trials 4 to 8, the procedure differed somewhat. Heavily infected (more than 75% cytopathic effect), 50-mm petri dish cultures were sources for JM, GA, and FC 126 viruses. Both of the Marek's disease virus strains were grown in chicken kidney culture; the turkey herpesvirus was grown in chicken kidney cells (batches 643 and 659) or in chicken embryo fibroblasts (batch 654). Supernatant fluid was discarded and replaced with 2 ml (trial 6), 2.5 ml (trials 4, 5, and 7), or 5 ml (trial 8) per culture of one of four suspending media. Cells were scraped free with a rubber policeman, and the harvests from four or five cultures were pooled (one culture per treatment in trial 8). Each suspension was sonically treated for 2 min, and 1-ml samples were frozen at -65 C or lyophilized. Cells from other cultures were harvested and frozen at -65 C with procedures designed to pre-serve whole cells and with dimethyl sulfoxide as a protectant (7). Sabilizers and diluents, In the first trial, th-following solutions were employed: (i) 20% glucose; (ii) skim milk stabilizer consisting of 8% nonfat dry miLk and 2% NZ Amine (type AS; Sheffield Chemical Co., Norwich, N.Y.) in pH 6.2 Sorensen's buffer plus 5% glucose; (iii) "SPGA," a stabilizer employed by Bovarnick et al. (1) which contains 0.218 M sucrose, 0.0038 M monopotassium phosphate, 0.0072 M dipotassium phosphate, 0.0049 M monosodium glutamate, and 1% bovine albumin powder; (iv) "SPG-NZ Amine," the same formulation as SPGA except that the bovine albumin was replaced by 1% NZ Amine (type B). The pH of the various solutions varied from about 6.2 to 7.0, a range known to have no appreciable effect on virus titer (Calnek and Adldinger, unpublished data). Additional tests with SPGA stabilizer were conducted in the second and third trials, and EDTA (0.2% final concentration) was added to some samples before freezing or lyophilization. In all trials, lyophilized samples prepared with EDTA were reconstituted with distilled water. Those prepared without EDTA were reconstituted in two ways, with and without 0.2% EDTA in the distilled water. Lyophilization. Lyophilization was carried out in a VIRTIS freeze-dryer model USM-15; (VIRTIS Co., Inc., Gardiner, N.Y.). Samples, in 5-ml serum bottles with split-rubber stoppers, were prefrozen at -65 C in a separate mechanical freezer or frozen at -60 C on the refrigerated shelves of the freeze-dryer. After drying under vacuum at 38 C for 24 hr with heat applied to the drying shelves, an additional drying period of about 15 hr was carried out with the shelf temperature at 21 C. The bottles were sealed under vacuum and stored at 4 C until virus assays were conducted. Assays were done from 1 to 10 days after the viruses had been processed. Virus assays. In all trials, each sample was inoculated onto two drained, 24-hr chicken kidney cultures immediately after thawing or reconstitution. To assure an infection rate which permitted enumeration of individual foci, 10-fold dilutions were also inoculated in some cases. The diluent was the same as the suspending medium. Cell culture methods for assay have been described (3a). Focal lesions were enumerated at 8 days (Marek's diseases virus) or at 6 days postinoculation (turkey herpesvirus). The average of the focus counts from two replicate cultures and the dilution factor were used to estimate the number of focus-forming units (FFU) per milliliter of undiluted virus. Tables 1 and 2. The studies with the JM isolate of Marek's disease virus from skin extracts (Table 1) indicated the following. (i) Virus survival after lyophilization was slightly enhanced by the use of 20% glucose as a stabilizer and markedly improved by SPGA or SPG-NZ Amine. (ii) The addition of either EDTA (0.2%) or SPGA to nonlyophilized virus suspensions increased titers more than twofold, and there appeared to be an additive effect when they were employed in combination. (iii) The highest titers were obtained from samples lyophilized with SPGA and then resuspended with water containing EDTA. Results are detailed in From the trials with cell culture-derived virus ( Table 2), the following points are significant. (i) Both strains of Marek's disease virus were extracted to a higher titer with SPGA than PBS as the suspending medium. (ii) Again, lyophilization was successful with SPGA stabilizer for Marek's disease virus, and the addition of EDTA before or after lyophilization resulted in much higher titers. (iii) The yields from extracted turkey herpesvirus-infected cultures were very much higher than those from Marek's disease virus-infected cultures although the titers of cellassociated infectivity were similar. (iv) Titers of PBS-extracted turkey herpesvirus were similar to those reported by Witter et al. (8), who employed cell culture medium for extraction. With EDTA added to PBS, the titer increase was marked (about sixfold), but EDTA added to SPGA extracts, either before or after lyophilization, had no appreciable effect. SPGA extract titers, however, were 30 to 50 times those of the PBS-EDTA extracts, and the per cent survival after lyophilization was approximately 30 to 40% versus about 5 to 6% for the respective extracts. Reassay of virus lyophilized for trials 2, 4, 6, and 7 was done after storage periods of 17 to 44 days at 4 C. Virus titers were unchanged from those in the original assays. DISCUSSION These data indicate that high yields of Marek's disease virus or turkey herpesvirus can be obtained by extraction in the presence of, or dilution in, a stabilizer and that lyophilization can be accomplished without appreciable loss (or with an increase) of titer. The potential use of lyophilization as a method for preservation of turkey herpesvirus as a vaccine virus was amply demonstrated, but the comparison of lyophilized virus and whole cell suspensions for immunizing capacity must await further study. No attempt was made in these experiments to learn the reasons for the increased titers associated with the use of both the stabilizer (SPGA) and the chelating agent (EDTA); this will be the subject of future studies. Possible explanations include dissociation of virus clumps, protection of the virion, or increased efficiency of the early events of infection. Any one or more of these might have played a role in increasing the amount of detectable infectious virus in the preparations. In addition to elucidating these factors, it will be interesting to learn if similar procedures have the same effects with other viruses and especially with other herpesviruses.

v3-fos

2020-12-10T09:04:17.644Z

1970-01-01T00:00:00.000Z

237231006

s2

Microbiological Aspects of Ethylene Oxide Sterilization The death rate kinetics of several sporeforming and nonsporeforming microorganisms, including radiation-resistant cocci, were determined by exposing them to a mixture of ethylene oxide and dichlorodifluoromethane (500 mg of ethylene oxide per liter, 30 to 50% relative humidity, and 54.4 C). Spore survivor curves obtained from tests of inoculated and exposed hygroscopic and nonhygroscopic carriers showed that the spores of Bacillus subtilis var. niger are more resistant to ethylene oxide than are spores of Clostridium sporogenes, B. stearothermophilus, and B. pumilus. The decimal reduction times (expressed as D values at 54.4 C-500 mg of ethylene oxide per liter) obtained under the test conditions for B. subtilis var. niger spores on hygroscopic and nonhygroscopic carriers exceeded the values obtained for the other organisms considered, both sporeformers and nonsporeformers. The decimal reduction times for the vegetative cells of the radiation-resistant organisms (Micrococcus radiodurans and two strains of Streptococcus faecalis) and the ATCC strain of S. faecalis demonstrated comparable resistance to ethylene oxide with the spores of C. sporogenes, B. stearothermophilus, and B. pumilus, but not those of B. subtilis var. niger. It has been found that bacterial spores can be up to 100,000 times more resistant to chemical agents than vegetative cells (5). However, Phillips (5) has also shown a comparatively narrow range of resistance to ethylene oxide between spores and vegetative cells. TIhis study developed quantitative comparative data on the death kinetics of several sporeforming and nonsporeforming bacteria after exposure to a mixture (w/w) of 12% ethylene oxide and 88% dichlorodifluoromethane. in an anaerobic incubator (National Appliance Co., Portland, Ore.) charged with illuminating gas. After inoculation, the cultures were incubated until microscopic examination revealed 90% (or better) sporulation. The spores were then harvested, washed five times, and stored in sterile distilled water under refrigeration. The M. phlei and S. faecalis cultures were incubated for 24 hr at 37 C; M. radiodurans was incubated at 30 C for 48 hr. After incubation, the cells were harvested with sterile water, washed twice, resuspended in sterile water, and then used immediately. Preparation of organisms for testing. The spore and vegetative cell suspensions were prepared for the tests as previously described (2). The strains of S. faecalis presented a problem because the cells died rapidly while being dried on the carriers. To avoid such population losses due to desiccation, plastic microcups (Bacti-Capalls, Clay-Adams, Inc., New York, N.Y.) were used as nonhygroscopic carriers. The cups were inoculated with an aqueous suspension (0.25 ml) containing 107 cells which were then transferred (without drying) to individual envelopes and immediately exposed to ethylene oxide. Exposure apparatus and procedures. The thermochemical death rate apparatus and exposure procedures previously described (2) Fig. 4b, hygroscopic carrier. inoculated carriers was the primary variable concerned. Recovery and enumeration of survivors. The recovery procedures described in reference 2 were applied to the exposed spore carriers. Exposed nonhygroscopic carriers, inoculated with cells of one of the nonsporeforming species, were transferred to 99 ml of sterile distilled water and shaken for approximately 20 min. After this, appropriate dilutions were prepared for survivor counts. The recovery procedures used with exposed hygroscopic carriers inoculated with vegetative cells were similar to those used for the hygroscopic spore carriers. Plate count agar (Difco) was used as the recovery medium for the aerobic sporeformers and nonsporeformers; TSP agar was used for C. sporogenes strains. The dilution plates of the test organisms were incubated at their optimal growth temperatures for 48 hr, and survivor counts were prepared. The dilution plates of C. sporogenes, each overlaid with TSP agar, were incubated in an anaerobic incubator charged with illuminating gas. RESUJLTS AND DISCUSSION The results of this study are shown in Fig. 1-7 and in Table 1. The thermochemical survivor curves and decimal reduction values were prepared as described in the preceding paper (2). Resistance of the sporeformers to the exposure conditions varied. The B. subtilis var. niger spores (Fig. 1) were the most resistant, particularly when dried on nonhygroscopic surfaces. Spores of the two C. sporogenes strains demonstrated similar resistance patterns (Fig. 2), as did the spores of B. stearothermophilus and B. pumilus ( Fig. 4 and 5). Among the nonsporeformers, three of the four S. faecalis strains (F6, A21, and the ATCC strain) were the most resistant to the ethylene oxide conditions (Fig. 8). The cells of S. faecalis (strain 012) and of M. phlei (Fig. 7) were the least resistant. Table 1 shows the decimal reduction times derived from the curves. Decimal reduction values, obtained by destruction methods such as steam under pressure and dry heat, have been reported by other investigators (6) as the D value at temperature of exposure (e.g., D64.4 c). Since there is only one variable in these processes, that is sufficient for D-value reporting. However, for the D value to be meaningful to ethylene oxide sterilization, temperature and concentration of gas must be considered. niger spores and were capable of surviving in liquid ethylene oxide as well as ethylene oxide mixed with a solid propellant. (We were unable to obtain a culture of Dr. Opfell's strain of S. epidermidis for testing; therefore, a comparative D value with the other microorganisms tested is not available.) Though in agreement with the work of others, our results illustrate the relative variation in resistance among microorganisms when exposed to ethylene oxide and disclose differences when organisms are dried on nonhygroscopic and hygroscopic surfaces. Such data are of value when developing specific sterilization cycles for materials of known contamination levels and when extrapolating conditions for particular sterilization applications.

v3-fos

2019-03-19T13:12:58.699Z

1970-07-01T00:00:00.000Z

237201745

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Zearalenone Production by Fusarium Species One-hundred-and-thirteen isolates of Fusarium were tested for their ability to produce zearalenone on autoclaved corn. They belonged to the following species (number of producers per number tested): F. epispheria, (0/1); F. moniliforme, (0/8); Gibberella fujikuroi, (0/3); F. nivale, (0/7); F. oxysporum, (0/15); F. roseum, (31/51); F. solani, (0/9); F. tricinctum (3/19). The isolates of individual species produced the following amounts of zearalenone per gram of corn: 3 isolates of F. roseum (0.6 to 119 μg), 3 of F. roseum “Culmorum” (1 to 210 μg), 3 of F. roseum “Equiseti” (0.6 to 2.0 μg), F. roseum “Gibbosum” (115 to 175 μg), 21 of F. roseum “Graminearum” (0.2 to 230 μg), and 3 of F. tricinctum (0.2 to 6.0 μg). All isolates of F. roseum “Graminearum” which formed the perithecial stage of G. zeae (G. roseum) produced zearalenone. Production occurred by the wild but not the appressed cultural type. Zearalenone production by F. tricinctum was confirmed by a mouse bioassay. The first report of moldy corn causing an estrogenic disturbance among swine appeared in 1928 (8). Since then, other reports have come from the United States (3,5,6,19), Australia (16), and Ireland (7). The syndrome includes vulvar hypertrophy and occasional vaginal eversion, preputial enlargement in castrated males, and prominent mammary glands in both sexes. In 1962, Stob et al. (19) demonstrated that an anabolic uterotrophic compound, crystallized and partially characterized by them, was produced by Gibberella zeae, the perfect stage of Fusarium graminearum (F. roseum "Graminearum"). Urry et al. (20) determined the compound to be an enantiomorph of 6-(10-hydroxy-6-oxo-trans-1undecenyl)-,B-resorcylic acid lactone and named it zearalenone. Partial characterization of the compound and environmental conditions suitable for its production were reported by workers at the University of Minnesota (3,10), who referred to zearalenone as F-2. Increased growth and shoot stimulation of tobacco pith callus tissue (11) are other reported effects of this compound. Since many species of Fusarium attack portions of plants that are used as food or feed, the determination of species capable of zearalenone production is of importance. F. culmorwn, F. graminearum (3), and F. moniliforme (12) whereas F. lateritum, F. tricinctum, F. nivale, F. episphaeria, F. rigidiuscula, F. roseum, and F. solani were not found to be producers (12). We report here an assessment of zearalenone production among Fusarium species and a sensitive chemical method for its determination. MATERIALS AND METHODS Sources of cultures. Identified cultures used in this investigation were received from investigators in the United States and from the American Type Culture Collection (Table 1). In addition, isolates were obtained from Indiana soil by plate dilutions on PCNB medium of Nash and Snyder (13) and from corn kernels plated on potato dextrose agar containing 100 jAg of Tergitol NPX (a nonionic detergent, Union Carbide Corp., New York, N.Y.) per ml and 30 jAg of chlortetracycline per ml. The latter two ingredients were added immediately before plate pouring Chemical screening procedure for zearalenone. Inoculum was grown in a shaken culture for 3 days at 24 C on carboxymethyl-cellulose medium (2). Inoculum (1 ml) was added to duplicate autoclaved samples (150 g each) of approximately 40% moisture corn and incubated for 3 weeks at 16 C. Selected isolates were also incubated at 16 C for 10 weeks or at 24 C for 2 weeks followed by 8 weeks at 12 C. Molded corn from each flask was extracted for 30 sec with 200 ml of anhydrous ethanol in a high-speed blendor, an additional 200 ml of ethanol was added, and a second blending was performed. The liquid portion was decanted and filtered by suction through filter paper (Whatman no. 1). Anhydrous ethanol (200 ml) was added to the remaining solid particles, and the mixture was blended and filtered as above. The extracts 31 were refiltered and reduced to a syrup in a flash evaporator. The residue was extracted with two 50-ml portions of ethyl ether, which were combined and washed with 25 ml of deionized water. The aqueous phase was discarded. The ether solution was extracted three times extracts were combined, adjusted to pH 3.5 w\ith I N HCl, and extracted three times with 25-ml portions of ethyl ether. The solvent was evaporated and the residue was dissolved in 1 ml of methanol. Silica gel G (Brinkman Instruments Inc.) thin-layer plates (250 ,m) were prepared, dried for 2 hr at 103 C, and spotted with S ,uliters of the methanol extract. The plates were developed in an unlined equilibrated tank by using a toluene-ethyl acetate-formic acid (TEF, 5:4:1) solvent system (17) and examined for zearalenone with shortwave ultraviolet light in a model C-5 Chromato-Vue chamber. Zearalenone has a RF of 0.8 in this system. Semiquantitative estimates of zearalenone in the extracts were made by comparisons with graded amounts (0.5 to 2 ,ug) of authentic zearalenone (99.3 + 0.6% pure as measured by phase solubility analysis, Tm 163.2 C; lot no. 552552, Commercial Solvents Corp.). A 0.05-,ug sample of authentic zearalenone can be detected on a thin-layer plate by this method. Zearalenone recovery efficiency. Three of four I 00-g samples of coarse-ground no. 2 yellow corn were fortified with 5 mg of authentic zearalenone. All samples were extracted by our previously described procedure and the extracts dissolved in 1 ml of methanol of spectrophotometric grade. Zearalenone extraction efficiency was judged as acceptable when 73% (average determination of 70, 74, and 75%) of the product was recovered, as measured by absorption at 236 nm. Spectrophotometric evaluation of extracts obtained by our method was ordinarily unsuitable because of interfering substances. The thin-layer procedure eliminated this difficulty and permitted assay without additional purification. Perithecial production. As an aid in identification of species, all Futsariuln isolates were checked for perithecial production on wheat culms. Two 4-cm sections of autoclaved wheat culms wvere aseptically placed on soil extract agar (9) slants, inoculated, and incubated under a 12-hr fluorescent light cycle at 23 C for 5 weeks. Mouse bioassay for zearalenone estrogenicity. Three mouse bioassays were performed to: (i) check the validity of the TLC procedures used in the screening trial, (ii) test the ability of 2 F. moniliforme isolates to produce zearalenone, and (iii) to confirm the estrogenicity of F. tricinctum extracts. Extracts from corn cultures of two F. roseum "Graminearum" cultivars used in the screening trial were checked for estrogenicity. As in the screening trial, flasks of approximately 40% moisture corn were seeded with 1 ml of inoculum and incubated for 3 weeks at 16 C. Ethanol extracts of the corn were mixed with a ground cornsupplement ration and fed to groups of 10 ovariectomized mice for 3 days. On the fourth day, the animals were killed and their uteri were weighed. To insure a greater possibility of zearalenone production, two F. moniliforme isolates were incubated for 2 weeks at 24 C and then for 8 weeks at 12 C (10). Anhydrous ethanol extracts of 400 g of molded corn were added to 50 g of ground ration, air dried, and brought to 100 g with untreated feed. The 32 APPL. MICROBIOL. ZEARALENONE PRODUCTION BY FUSARIUM test rations were fed to 10 ovariectomized mice for 3 days. The estrogenicity of three isolates of F. tricinctum was verified with corn cultures grown under the 10week incubation regime described above. Anhydrous ethanol extracts of the molded corn were spotted on TLC plates. Methanol eluates from chromatoplates were dried and mixed with 5 ml of sesame oil, and 10 ovariectomized mice were injected subcutaneously with 0.1 ml of the mixture per day for 3 days. Extraction procedures for TLC used with F. moniliforme. Three isolates of F. moniliforme tested in the screening trial were grown for 2 weeks at 24 C and then for 8 weeks at 12 C. Both the procedure of Mirocha et al. and our procedure were used to detect zearalenone. The initial extracts for the procedure of Mirocha et al. (10) consisted of: (i) blending 100 g of molded corn with 400 ml of methylene chloride for 5 min and extracting for 16 hr or (ii) extracting 100 g of molded ground corn in a Soxhlet extractor with 400 ml of methylene chloride for 12 hr. The crude extracts were taken up in petroleum ether and partitioned with acetonitrile. RESULTS AND DISCUSSION Screening of isolates for zearalenone. Initially 110 isolates, belonging to 7 species of Fusarium, were screened for zearalenone production ( Table 1). F. tricinctwn, F. roseum, and the "Culmorum," "Equiseti," "Gibbosum," and "Graminearum" cultivars of F. rosewn produced zearalenone. The number of zearalenone-producing isolates and thier range of production per gram of corn was as follows: 3 of F. tricinctum (0.2 to 6.0 ,ug), 3 of F. roseum "Culmorum" (1 to 210 jug), 3 of F. roseum "Equiseti" (0.6 to 2.0 ,ug), F. roseum "Gibbosum" (115 to 175 ,ug), and 21 of F. roseum "Graminearum" (0.2 to 230 ,ug). Zearalenone was produced by both of the duplicate samples tested, except in the case of one ofthe "Graminearum" and two of the "Equiseti" cultivars. Isolates which produced zearalenone were from different areas ofthe United States and Canada. Most of them were isolated from corn kernels, whereas others were from wheat, barley, turf grass, begonia, squash fruit, and soil. The estrogenicity of the extracts from two of the F. roseum "Graminearum" cultivars was verified with a mouse bioassay. Ethanol extracts of the two of the "Graminearum" cultivars induced uterine weights of 99.6 and 106.4 mg, as compared to 16.4 mg for the control. Freshly isolated cultures of F. rosewn, known as the wild type, generally grow rapidly with abundant aerial mycelium. Subculturing often leads to slower appressed pionnotal growth and reduced plant pathogenicity (15). Of the 52 isolates of F. rosewn screened for zearalenone, 8 grew appressed. Since none of the appressed cultures produced the toxin, it appears that zearalenone production is not associated with this cultural type. Of the 21 wild-type isolates of F. rosewn "Graminearum," 19 produced the perithecial stage of G. zeae. None of the isolates of the other species produced perithecia. Since all 21 wildtype isolates of F. rosewn "Graminearum" produced zearalenone, a close association between zearalenone production, perithecial formation, and the wild type in this taxon is indicated. Zearalenone production, estrogenicity, and morphology of F. tricinctum. Since F. tricinctum has not been reported to produce zearalenone, we attempted to verify its production by using an improved incubation protocol (10). Samples (150 g) of moist autoclaved corn were inoculated with F. tricinctwn and incubated for 2 weeks at 24 C and then for eight weeks at 12 C. Extracts of eight replicates of each of the isolates suspected to produce zearalenone (isolates FT 2, 3, and 12) and two replicates of isolates that gave no evidence of toxin production (isolates FT 10 and 16) were prepared. All 24 extracts from isolates FT 2, 3, and 12 gave fluorescent spots with the same Rp value as zearalenone. By comparison with authentic standards, the average amounts of zearalenone were 9.4, 14.4, and 13.7 ,ug/g, respectively. These levels, although low, were considerably above the average levels of 3.5, 1.1, and 0.4 ,ug/g produced in the earlier trial. Extracts from isolates FT 10 and 16 showed no zearalenone. To confirm the zearalenone production by isolates FT 2, 3, and 12, the spots on the chromatoplates corresponding to zearalenone were eluted with methanol (spectrophotometric grade) and the absorption spectra of the eluates were determined at 220 to 340 nm with a spectrophotometer (model DK-2; Beckman Instruments, Inc.). The absorption spectra closely resembled that of zearalenone with absorption peaks at 236, 274, and 314 nm. The estrogenicity of the extracts from corn infected with three isolates of F. tricinctum was tested in a mouse bioassay. Extracts were injected into mice and gave significant increases in uterine weights with progressively greater average uterine weights with increasing zearalenone levels ( Table 2). The three zearalenone-producing cultures of F. tricinctwn were isolated from soil, bluegrass, and corn kernels. The mycelium toward the center of the colony had a light-yellow tinge and the reverse was red with bands of pigmentation radiating from the center of the colony. Both macroconidia and microconidia were produced. The macroconidia had 1 to 3 septata and were unevenly tapered toward the tip. The microcondia varied from globose with a basal papilla to obclavate and were borne on bottle-shaped conidiophores. Morphologically and culturally, the zearalenone-positive and -negative isolates were indistinguishable. Further tests with F. moniliforme. Mirocha, Christensen, and Nelson reported zearalenone production by F. moniliforme, but gave no yields (12). Because of the wide occurrence of F. moniliforme in corn kernels, further examination of this material was carried out. However, no zearalenone was detected by TLC by using the extraction and purification procedures of Mirocha et al., and us. Two isolates of F. moniliforme were tested for estrogenic activity in a mouse bioassay. No significant increases in uterine weights were produced by extracts from F. moniliforme. The sensitivity of the bioassay was assumed to be at least 33 ,ug of zearalenone per g of feed since this level of authentic zearalenone, when administered for 3 days, gave a 44% increase in uterine weight. By inference, a response among the animals consuming the 100 g of feed treated with the extract from 400 g of molded corn would have resulted, if 8 Ag of zearalenone/g was present in the original molded corn. Therefore, if zearalenone was produced by the two isolates of F. moniliforme, levels would have been below 8 ,ug/g. Since zearalenone was produced by all the isolates of F. roseum "Graminearum" (G. zeae) that produced perithecia, we tested isolates of G. fujikuroi, the perfect stage of F. moniliforme. Isolates of F. moniliforme which form the sexual stage usually cause the bakanae disease on rice and produce gibberellins (18). Three isolates including ATCC 12616 and 12618 were grown on moist autoclaved corn at 24 C for 2 weeks and then for 8 weeks at 12 C. Extracts examined by TLC revealed no zearalenone.

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Non-destructive methodology in comparative physiology of buckwheat genotypes within the different origin In the presented study has been used non-destructive method for prescreening of flavonoids, anthocyanins and pigments from early stage of growth till flowering period of buckwheat genotypes of different origin. The similar increasing tendency in the changes of FLAV, ANTH and MFI indexes of Chinese genotypes compared to the tendency of Ukrainian genotypes has been observed. Genotypes of F. tataricum compared to the genotypes of F. esculentum have been shown to lower ANTH index during seedling growth. SFR index which relates to INTRODUCTION The Food and Agriculture Organization of the United Nations (FAO) supposes that the world's population in 2050 will be 34% greater than it is today. Currently, 49% of the world's population lives in urban areas, while in 2050 this will be closer to 70% (Alexandratos and Bruinsma 2012). During this time period, climate change and the development of biofuel production will present major risks to long-term food security. Population growth, urban civilization and climate change can effectively support high crop concurrence as possible sources of food, bioenergy, fiber and other industrial needs. Going forward, these problems will require innovative approaches to the genetic and agronomic components of systems of crop production and developing concurrent food market. While we are entering a period of increasingly rapid climate change, the overall objective of the project initiative is to design new strategies to maintain high yield and qualitative parameters of crop plants produced under changed environmental conditions, using novel crop plants which is promising for development in food market. Crop yield is a complex trait depending on the successful completion of different steps of vegetation (phase of reproductive organ development), which can be sensitive to environmental factors. Among innovative approaches is high interest in plant biotechnology and non-destructive methods of prescreening agriculture plants regarding content of specific secondary metabolites, regarding development of some diseases, stress reaction for the missing of nitrogen supply or to discover plant species biodiversity. In targeted plant metabolites, the main goal is to achieve a high throughput. Therefore, there is often an initial desire for a rapid pre-screening of the samples. This is especially the case when dealing with many sample numbers, where only a limited number of individuals might be expected to be different (Verhoeven et al. 2006). This is the case when searching for valuable genetic resources (e.g. those having a high content of desired compounds) within natural populations or population obtained by crosses or mutagenesis . Nowadays many scientific groups in the functional food area are looking for plants with high content of phytochemicals as an important source of active pharmaceuticals or with valuable nutritional properties (Abuajah et al. 2015;Varzakas et al. 2016). Buckwheat among these plants got a prime position as potential source of gluten free food products as well as rich source of antioxidant compounds (Saturni et al. 2010). The various types of bioactive compounds presented in different buckwheat varieties provide basic background needed for the efficient production of buckwheat foods with added value (Sytar et al. 2016). Two main buckwheat species have been commonly produced and consumed: common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (Fagopyrum tataricum Gaertn). The biodiversity of buckwheat plant species which can be used farther are wide. Therefore, its is important to develop study difference between buckwheat species of different origin. The non-invasive fluorescence-based phenomics method for determination of plant phenolics based on the strictly UV-absorbing properties, the effects of phenolic compounds on visible light-induced chlorophyll fluorescence is negligible, whereas their presences strongly suppress the chlorophyll fluorescence emission under UV excitation (Cerovic et al. 2002). This phenomenon has been successfully applied for estimation of transmittance of UV radiation by chlorophyll fluorescence (Burchard et al. 2000;Ounis et al. 2001). In the previous decades, the numerous studies examined and confirmed possibility to use the chlorophyll fluorescence signal in the estimation of phenolics and anthocyanins. In addition to self-constructed devices or standard fluorometers combined with external light sources and filters, which were used in the majority of studies, the factory-made special devices for this purpose were also introduced . In this scientific work we would like to combine the non-invasive fluorescence-based, and traditional biochemical methods (HPLC, spectrophotometry etc.), typically used for plant phenomics studies to discover growth characteristics of buckwheat species of different origin. Plant material Plants of 7 buckwheat samples from Ukraina (Fagopyrum tataricum himalaicum, Faropyrum tataricum rotundatum -red, Fagopyrum esculentum cv. Rubra -red, Fagopyrum esculentum cv. Karadag) and Chinese cultivars (Fagopyrum esculentum cv. SuQiao 1, Fagopyrum esculentum cv. YuQiao 4, Fagopyrum esculentum cv. NingQiao 1) were exposed immediately from sowing to direct sunlight in open field conditions for 52 days. The non-destructive measurements were started from early growth stage (stage of vegetation). First measurements were done at stage of two leaves (22 days after sowing (DAS)), other measurements were carried out at stage of 4-5 leaves (30 DAS), at the beginning of flowering stage when buckwheat plants had 5-6 leaves (36 DAS) and last measurements were done in flowering stage (51 DAS). The leaves for biochemical analysis were collected in the flowering phase. Cultivar Rubra with high anthocyanins content (3.87−4.41 mg/100 g DW) in the vegetative organs has been received by family selection method from chemo mutants. Cultivar Karadag is received from the Scientific Research Institute of Groat Crops in Ukraine. F. tataricum G. is a one-year plant which, among the species researched, has a better pollination of flowers and a higher grain production. F. giganteum Krot. is amphidiploid obtained after crossing F. tataricum G. with the perennial plant F. cymosum Meissn. (Krotov and Dranenko 1973). F. cymosum Meissn. is a polyploid with 32 chromosomes. The collection of buckwheat germplasm, which is maintained at the Scientific Research Institute of Groat Crops in Ukraine comprises nearly 1000 samples which are readily available for breeding research. Chlorophyll fluorescence records and analyses using fluorescence excitation ratio method The chlorophyll fluorescence analysis was done using the portable optical fluorescence sensor Multiplex-3® (Force-A, Paris, France). Multiplex-3® is a hand-operated, multi-parametric sensor based on light-emitting-diode excitation and filtered photodiode determination that is arranged to work in the field, greenhouse and laboratory conditions. The sensor of Multiplex-3® has three, redblue-green LED-matrices emitting light at 470 nm (blue), 516 nm (green) 153 and 635 nm (red). There are three integrated photodiode detectors for fluorescence recording: far-red, red and yellow (Ghozlen et al. 2010). We used values of fluorescence measured at UV (375 nm), green light (516 nm), red light (635 nm) and 735 nm (FRF). The evaluation of phenolic compounds contents in plants was done via calculation of fluorescence values detected after excitation by light of the defined wavelengths (details are below). In analogy to the spectrophotometric method for assessing leaf absorbance, the parameters were based on the Beer-Lambert's law and calculated as logarithm of the fluorescence ratio values. UV absorbing compounds (mostly flavonols) content described by flavonoid (FLAV) index (Cerovic et al. 2002;Agati et al. 2011) was estimated using the modified for-mula of Zivcak et al. (2017), as the logarithm of the ratio of the red-light induced far-red fluorescence (FRF R ) and the UV-induced far-red fluorescence (FRF UV ): Similarly, the ANTH Index that provides estimates of green-light absorbing compounds (logFER R/G ), mostly red-colored flavonoids and anthocyanins, was calculated as the logarithm of the ratio of the red-light induced fluorescence (FRF R ) and the green light-induced fluorescence (FRF G ): The correction coefficients k UV or k G was applied to measurements of fluorescence to avoid negative values . The constant values of the coefficients were used as the minimum values of the FRF UV / FRF R and FRF G /FRF R ratios found in the database that contains several thousand records from over three hundred plant species grown in diverse environments (Zivcak et al. unpublished results). The same constants have been used when processing data across all experiment and cultivars. We also calculated the modified Flavonoid Index (MFI) that provides a better estimate of total flavonoid content when plants with different colors are compared . The MFI was calculated as the logarithm of the ratio of the red-light induced fluorescence (FRF R ) and the green light-induced fluorescence (FRF G ). MFI = log[2*FRF R /(k G *FRF G + k UV *FRF UV )] The values of correction coefficients (k G , k UV ) for MFI were the same as for ANTH and FLAV. Chlorophyll content was estimated from values of fluorescence measured at 735 nm (FRF) and at 685 nm (RF) after excitation by red light (635 nm). The Simple fluorescence ratio (SFR) was calculated as: SFR = FRF R /RF R Because the diameter of the measuring area was only 50 mm, 6-7 measurements were taken on each plant in different position to account for heterogeneity in leaf color and structure. This number of measurements from the top view provides sufficient data to characterize the entire plant. Anthocyanins estimation 0.1 -0.5 g plant material was homogenized on ice with 3 ml of acidified methanol (1% HCl) and then incubated at 4 °C for 12 h with moderate shaking. The mixture was centrifuged for 10 min at 14 000 rpm at 4 °C. Absorbtion of the extracts at 530 and 657 nm wavelengths was determined spectrophotometrically. The blank was acid-ified methanol. The concentration of the anthocyanins was expressed as mg.g -1 dry weight and was calculated by formula: anthocyanins=[A530-(0.25*A657]*V/(W*1000), where A is absorbance; V is total volume of the extract (ml) and W is weight of the dry leaf tissue (g). Total phenolics estimation Total phenolic content in the buckwheat leaves extracts was determined by standard spectrophotometric method of Lachman et al. (2003) by using Folin-Ciocalteu reagent (Singleton and Rossi, 1965). 0.25 g powdered samples (freeze-dried) was extracted for 16-18 hours with 20 ml of 80% ethanol. After the time of extraction a volume of 100 µl of the plant extract was pipetted into 50 ml volumetric flask. 2.5 ml of Folin-Ciocalteau reagent was added to the extract. Then after 3 minutes (agitation) 5 ml 20% Na 2 CO 3 solution was mixed. After two hours at 25 °C the absorbance was measured on the spectrophotometer Jenway UV/Vis 6405 (Jenway, UK) at wavelength λ = 765 nm against blank. Gallic acid was used as a reference standard for plotting calibration curve. Total phenolic content was expressed as mg.kg -1 gallic acid equivalent of dry matter. Analysis of hydroxycinnamic acid derivatives Analysis of hydroxycinnamic acid derivatives has been previously developed (Mewis et al. 2010). Samples were taken after finishing the freeze-drying process where the material was ground by a flint mill (20 000 g for 2 min). A total of 20 mg ground samples from leaf suspension were extracted for 15 min using 0.75 mL 70% methanol (v/v, pH 4.0, phosphoric acid) in an ultrasonic water bath on ice. Then samples were centrifuged for 5 min at 6000 rpm. The supernatants were collected and the pellets were re-extracted twice more with 0.5 mL of 70% methanol (HPLC-Gradient grade, VWR chemicals). Coumaric acid or cinnamic acid (Sigma-Aldrich Chemie GmbH) (40 µL of 3 mM solution) was added as internal standard to the first extraction. The combined supernatants from each sample were reduced to near dryness in a centrifugation evaporator (Speed Vac., SC 110) at 25 °C. Samples were added up to 1 mL with 40% acetonitrile (HPLC Ultra Gradient Grade, Roth). The samples were filtrated using 0.22-mm filters and then analyzed with HPLC. The chromatography was performed using a DionexUltiMate 3000 HPLC System with a diode array detector (DAD-3000) with a WPS-3000 SL auto sampler, LPG-3400SD pump and a TCC-3000RS Column Compartment (Dionex Corp., Sunnyvale, CA, USA). Statistical analysis Means and standard deviations were calculated by the Microsoft Office Excel 2013. Significant differences of these data were calculated using analysis of variance ANOVA Duncan's multiple test (STATISTICA 10, Stat-Soft, Tulsa, USA). All results were expressed as mean+ standard deviations from replications n = 50. Chlorophyll fluorescence records and analyses of FLAV, ANTH, MFI and SFR indices using fluorescence excitation ratio method Chlorophyll fluorescence records and analyses using fluorescence excitation ratio method of Ukrainian and Chinese genotypes during growth periods of 51 DAS shown significant increasing of FLAV index from beginning of seedlings stage to the of flowering stage (Fig.1). The highest increase of FLAV index which is connected with flavonols content has been observed in the Chinese genotypes. The high FLAV index at the flowering stage was not depended from flavonols content on the beginning of seedlings stage (22 DAS The different tendency of ANTH index increasing from beginning of seedlings stage to the flowering stage Figure 2. Process of anthocyanins accumulation (logFER R/G -ANTH) in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 -F. tataricum rotundatum, 2 -F. tataricum himalaicum, 3 -F. esculentum cv. Rubra, has been observed for Ukrainian genotype F. esculentum cv. Rubra compared to other investigated buckwheat plant genotypes (Fig. 2). At the beginning of seedlings stage this genotype has highest ANTH index (0.50 RU) and at the flowering stage on 51 DAS (0.57 RU) too. At the flowering stage came to significant increase of ANTH index in F. esculentum cv. Rubra, on the contrary to other Ukrainian genotypes F. tataricum rotundatum and F. tataricum himalaicum, in which has ANTH index decreasing character from beginning of flowering stage. On the 51 DAS at the flowering stage in F. tataricum genotypes ANTH index was almost on the same level as at the beginning of seedlings growth after significant increasing at the 30 and 36 DAS. ANTH index for other investigated genotypes (was) ranged from 0.39 to 0.43 RU at the beginning of seedlings stage. Genotypes of F. tataricum compared to the genotypes of F. esculentum of both origin shown decreasing of ANTH index during seedling growth. All F. esculentum genotypes of both orgin has inceasing on ANTH index on 51 DAS (Fig.2). MFI, the parameter that takes into consideration the accumulation of both flavonols and anthocyanins, is a better estimate of flavonoids than FLAV . Results of statistical analyses using MFI are highly similar to those that use data of biochemical analysis . MFI index did not shown significant difference between F. tataricum and F. esculentum genotypes of both origin but highest values has been found for F. esculentum cv. Rubra during all growth stages. Based on the obtained prescreening results during 51 DAS of growing period is possible to conclude that non-destructive methodology can be used to choose genotypes with high antioxidant content. The majority of published vegetation indices for non-invasive remote sensing techniques are not sensitive to rapid changes in plant photosynthetic status brought on by common environmental stressors. The SFR index is connected with chlorophyl concentration in the leaves. In presented experiment with buckwheat genotypes of different origin the tendency of SFR index changes was different between genotypes of Ukrainian and Chinese origin (Fig.4). In the middle phase (30 DAS) of growth has been observed significant increase of SFR index almost in all experimental genotypes. On 36 DAS just two Ukrainian genotypes which characterized also high ANTH and MFI indexes has been kept tendency to increase SFR index -F. esculentum cv. Rubra and F. esculentum cv. Karadag. The Chinese genotypes shown decreasing of SFR index on 36 DAS with farther deacresing at 51 DAS of flowering stage with parallel increasing of ANTH, FLAV and MFI indexes. Total phenolics and anthocyanins estimation The biochemical analysis among experimental buckwheat genotypes of different origin has been shown that highest total phenolic contents was found in Ukrainian buckwheat Fagopyrum esculentum cv. Rubra and Chinese genotypes ( Figure 5). At the same time leaves of Ukrainian Fagopyrum esculentum cv. Rubra has been characterized by highest anthocyanins content compared to the other experimental buckwheat genotypes of different origin. This data is connected with previous research where was studied role of anthocyanins as marker for selection of buckwheat plants with high rutin content in Fagopyrum esculentum cv. Rubra (Sytar et al. 2014). Analysis of hydroxycinnamic acid derivatives HPLC prescreening buckwheat genotypes of different origin on (51 DAS) has been identified chlorogenic, p-coumaric, p-anisic, cinnamic, methoxycinnamic, ferulic and vanilic acids. It was found highest content of chlorogenic and p-coumaric acids in the Chinese genotypes F. esculentum cv. NingQiao 1 and F. esculentum cv. YuQiao 4. The highest p-anisic acid content was found for buckwheat genotypes of F. tataricum himalaicum Figure 4. Values of SFR (simple fluorescence ratio) in the leaves of investigated buckwheat plant species exposed to direct sunlight during 51 days after seedlings (numbers indicate individual cultivars of buckwheat as follow: 1 -F. tataricum rotundatum, 2 -F. tataricum himalaicum, 3 -F. esculentum cv. Rubra,. Figure 5. Content of total phenolics (5a) and anthocynanins (5b) in the buckwheat leaves determined by HPLC method (numbers indicate individual cultivars of buckwheat as follow: 1 -F. tataricum rotundatum, 2 -F. tataricum himalaicum, 3 -F. esculentum cv. Rubra, ( Table 1). It is important to admit that genotypes of F. tataricum characterized by higher p-anisic acid content compared to the experimental genotypes of F. esculentum of both origins. The nondestructive technique of infrared spectroscopy is recommended as alternative technique for routine analysis of main flavonoids like rutin, quercetin and quercitrin in aerial parts of buckwheat (Ladan et al. 2017). It can be pointed out that individual bioactive compounds compositions are suitable indicators of the physiological stage of crop plants. The phenotype of a plant is the result of a complex interaction between morphological, ontogenetical, physiological, and biochemical factors (Gratani 2014). The highest increasing of FLAV index has been observed on 51 DAS of flowering stage which is connected with flavonoids content in the Chinese genotypes. Rutin content of the grain of 22 buckwheat genotypes (F. esculentum and F. tataricum) grown in same region of origin had variation (Bai et al. 2009), so its important to use non-destructive methods for prescrening of flavonoids content for different genotypes. The high FLAV index at the flowering stage was not depended on flavonoids content on the beginning of seedlings stage (22 DAS). Other investigated genotypes for ANTH index in range from 0.39 to 0.43 RU at the beginning of seedlings stage. Genotypes of F. tataricum compared to the genotypes of F. esculentum of both origin shown decreasing of ANTH index during seedling growth. Liu et al. (2008) have shown that ethanol extracts of Tartary buckwheat sprouts had higher free radical scavenging activity and superoxide anion scavenging activity than those of common buckwheat sprouts (Liu et al. 2008). Total phenolics and rutin in tested samples were related to the antioxidant activities (Holasova et al. 2002). The SFR index is linked to the Chl concentration of leaves (Diago et al. 2016). Leaf Chl and FLAV concentration on a surface basis depends on leaf age and the amount of light radiation received during their development. Both increase with leaf expansion and light exposure until veraison, while afterwards, leaf Chl usually decreases (Louis et al. 2009) while FLAV remain unvaryingly high (Downey et al. 2003). Such tendency has been confirmed in the our experiments with buckwheat genotypes of different origin just development of SFR index changes was different between Ukrainian and Chinese genotypes origin. The antioxidant capacity can be connected with total phenolic and anthocyanin contents and variety of plant species plus maturity (Prior et al. 1998;Kim et al. 2003). Flavonoids and phenolic acids have relevant antioxidant properties (Barriada-Bernal et al. 2014). The concentration is affected by environmental conditions, age, and phenological stage (Almaraz-Abarca et al. 2013), while the qualitative phenolic profiles are more stable and vary among different groups of plants with a species-specific tendency (Emerenciano et al. 2001). It was observed that presence of p-anisic acid was typical for F. tataricum genotypes compared to the experimental genotypes of F. esculentum of both origins -Ukrainian and Chinese. p-anisic acid is one of the isomers of anisic acid which has antiseptic properties (Bhimba et . It is also used as an intermediate in the preparation of more complex organic compounds. Cinnamic acid has low toxicity and in the search for novel pharmacologically active compounds, cinnamic acid derivatives are important and promising compounds with high potential for development into drugs (Sova 2012). The high content of cinnamic acid at 52 d of flowering period found in Ukrainian F. esculentum cv. Rubra, which is characterized by high anthocyanins content. In plants, flavanone biosynthesis begins with the hydroxylation of cinnamic acid to p-coumaric acid by a membrane-bound P450 monooxygenase, cinnamate 4-hydroxylase (C4H) (Yan et al. 2005). At 52 day of flowering period the p-coumaric acid content was higher more than 2 times in Chinese genotypes compared to the Ukrainian genotypes of F. esculentum. CONCLUSION: The screening of biological active compounds of phenolic nature in the early stages of growth with non-destructive chlorophyll fluorescence techniques can be used for qualitative traits analysis of plant sprouts, especial-ly buckwheat. The high flavonoids level at the flowering stage is not dependent on flavonoids content on the begging of seedlings stage. Plant genotypes of different origin can vary in flavonoid, anthocyanins and pigments content during stages of growth but changes in their contents can be similar for representatives of the same origin. The presence of some phenolic acid can be typical for genotypes of F. tataricum compared to the genotypes of F. esculentum. To support natural plant biodiversity research it would be good to develop use of fast pre-screening methods of plants during all stages of development what can be helpful in applied food plant research.

v3-fos

2020-12-10T09:04:12.710Z

1970-03-01T00:00:00.000Z

237231718

s2

Relationship Between Rumen Ammonia Levels and the Microbial Population and Volatile Fatty Acid Proportions in Faunated and Defaunated Sheep Cheviot wethers were defaunated by using dioctyl sodium sulfosuccinate and were constantly infused with urea to provide 2.87% of the daily N intake. Defaunation resulted in higher rumen dry matter and lower rumen pH. The molar per cent propionate was higher in defaunated sheep, whereas the molar per cent butyrate and acetate was lower. Apparent nitrogen digestibility, nitrogen utilization, and nitrogen balance were higher in defaunated sheep when compared with faunated animals. Urea infusion resulted in lower apparent nitrogen digestibility, nitrogen utilization, and nitrogen balance in faunated sheep, but did not affect nitrogen metabolism in defaunated sheep. Rumen ammonia-N levels in defaunated sheep were lower than those observed for faunated animals, and urea infusion into faunated sheep increased rumen ammonia-N levels to a greater extent than did the urea infusion into defaunated animals. Significant correlations were demonstrated between rumen ammonia-N levels and C2/C3, C3/C4 and C2/C4 volatile acid ratios. From this it was concluded that, as rumen ammonia-N levels increased, there was a shift from propionate to higher proportions of butyrate and acetate. A decrease in rumen ammonia-N levels as a result of defaunation in animals fed identical rations is probably the most consistent effect recorded from studies with defaunated and faunated ruminants. The recorded values for rumen ammonia-N levels of defaunated animals as a per cent of those in control faunated animals are 47%, by Abou Akkada and el-Shazly (2), 52% by Christiansen et al. (5), 44, 50, and 39%c by Klopfenstein et al. (8) and 49 and 50% by Chalmers et al. (Proc. Nutr. Soc., p. 29A, 1968). As a consequence of this change in rumen ammonia-N levels in defaunated ruminants, any attempt to relate the effect of the presence of protozoa per se to overall animal response is confounded with a marked change in the rumen environment, i.e., greatly increased rumen ammonia-N levels. The experiment reported in this paper was designed to investigate the effect of experimentally increased rumen ammonia-N levels in defaunated and faunated sheep. MATERIALS AND METHODS Twelve rumen-fistulated, 2-year-old Cheviot wethers, averaging 30 kg, were fed ration number 3, as 1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal No. 4858. previously described (8) at 6%,7c body weight0 75 throughout the entire experiment. Due to problems encountered with sheep refusing feed, the original 4 by 4 Latin square design had to be abandoned and unequal numbers per treatment combination resulted. The four treatments imposed were (i) urea infusion into the rumen of defaunated sheep (seven observations), (ii) urea infusion into the rumen of faunated sheep (seven observations), (iii) water infusion into the rumen of defaunated sheep (six observations), and (iv) water infusion into the rumen of faunated sheep (six observations). The water and urea infusions were continuous, a volume of 460 ml being infused in 24 hr. Urea was infused at a rate to provide 0.123% of the daily ration; this represented 2.87%7o of the daily N intake. Sodium sulfide was included in the infusate to provide a N to S ratio of 15:1. Although not yet experimentally proven, this was thought to be necessary to maintain optimal N to S ratios in the rumen at times when recycling differences could otherwise prevent effective N incorporation into microbial protein. Defaunation was accomplished by using 12 ml of dioctyl sodium sulfosuccinate under the trade name Complemix (American Cyanamid Company) in a procedure only slightly modified from that used by Abou Akkada et al. (1). Volatile fatty acid concentrations were determined by using the procedure of Dehority et al. (6); all other analyses and sample collection procedures were as previously described (8). A 13-day adjustment period preceded 6 days of feed intake, urine production, and fecal output measurement. Two days later rumen samples and jugular blood samples were taken prefeeding and at 2 and 4 hr postfeeding, but the 2-hr postfeeding results are not presented for they were essentially identical to those obtained 4 hr postfeeding. Statistical analysis was by least squares and Duncan's multiple range test (7). RESULTS The mean rumen protozoal concentration for the faunated sheep was 313.2 ±i 179.4 X 103/ml, at the prefeeding sample time. Rumen dry matter was higher in defaunated sheep at all sampling times, when compared to faunated animals ( Table 1), and the difference was significant (P < 0.01) at the prefeeding sampling time. Sheep receiving the water infusion also had higher rumen dry matter percentages than did sheep which received the urea infusion. Rumen pH was slightly higher in faunated sheep and in sheep that received the urea infusion (Table 1); however, these differences were not significant. The effect of the absence of protozoa from the rumen on rumen volatile fatty acids (VFA) is outlined in Tables 2 and 3. There were no significant differences observed in total VFA concentrations at any of the sampling times between faunated and if-fannhtedarheen Thet mnlqr nrrnnrtinn of acetate was slightly higher in faun at prefeeding and 4 hr postfeeding significantly (P < 0.05) the apparent nitogen digestibility of the faunated wethers, when compared with the water-infused faunated wethers ( Table 4). The nitrogen utilization and nitrogen balance figures that were observed in this experiment were extremely high for sheep on a main- (Table 4). Defaunation resulted in a significantly (P < 0.01) higher nitrogen balance, and the infusion of urea into defaunated sheep did not affect this. However, urea infusion into faunated sheep resulted in lower nitrogen utilization and nitrogen balance, and, although not significant, these differences approached significance. When faunated sheep received the urea infusion, fecal nitrogen as a per cent of nitrogen intake was significantly (P < 0.05) higher ( Table 4). The rumen ammonia-N levels that were obtained in this study were consistent with those previously reported in the literature from defaunation studies. Defaunation of the wethers in this study resulted in significantly lower rumen ammonia-N levels at 2 (P< 0.01) and at 4-hr (P < 0.05) postfeeding (Fig. 1). The infusion of urea raised rumen ammonia-N levels in faunated sheep an average of 3.44 mg per 100 ml and in defaunated sheep an average of only 1.88 mg per 100 ml. Regression analysis, using ammonia and VFA data obtained 4 hr postfeeding from the results of Klopfenstein et al. (8) and this investigation, showed a linear relationship between rumen ammonia concentration and the C2 to C3 VFA ratio (r = 0.91; P < 0.01; Fig. 2), a curvilinear relationship between ammonia concentrations and the C2 to C4 VFA ratio (r = 0.89; P < 0.01; Fig. 3), and a curvilinear relationship between ammonia concentration and the C3 to C4 VFA ratio (r = 0.81; P < 0.05; Fig. 4). Similar relationships were demonstrated for the data taken prefeeding, but the correlation values were less. Such data indicate that, as ammonia concentrations increased, acetate and butyrate proportions increased, with butyrate increasing more than acetate. At the same time, propionate proportions decreased. DISCUSSION Higher ammonia levels in faunated animals have been consistently observed, and it has been suggested that this is due to the fact that the protozoa contribute significantly to protein degradation and deamination (8,10). Such an interpretation now appears to be entirely misleading, and, in fact, it seems that ammonia levels in the rumen of defaunated animals are lower than in faunated animals as a result of greater bacterial concentrations, thus causing greater ammonia utilization. This interpretation of the results suggests that ammonia levels are higher in faunated animals as a result of an inability to effectively utilize ammonia rather than 0 488 APPL. MICROBIOL. 1 . as a result of excessive ammonia production, a conclusion also arrived at by Smith (11). The reasons for suggesting this interpretation are given below. Firstly, greater rumen bacterial protein quantities at the expense of rumen protozoan protein should give rise to greater excretion of nitrogen in the feces, for protozoan protein is more digestible than bacterial protein (3,9). Such a relationship is shown by the results with the waterinfused animals in this experiment (27.61 versus 30.19) and by the results of Klopfenstein et al. (8). No explanation for the effect of urea infusion upon apparent nitrogen digestibility in faunated animals can be given at this time. Secondly, the quantity of urea infused in this experiment had been calculated to provide an increase of 4 mg per 100 ml of rumen fluid. This value was approached in the faunated animals (3.44 mg/100 ml), but an increase of only 1.88 mg per 100 ml was realized in defaunated animals, thus suggesting a more rapid uptake of ammonia in these animals. The slight possibility of a deficiency of urease in these animals was eliminated when it was shown that in fact urease activity (per milliliter of rumen contents) in defaunated animals was double that of faunated animals (Purser and Dehority, unpublished data). A second, but variable, effect of defaunation is a lowered dry matter digestibility as compared to faunated animals. This was not true in this experiment, but see, for example, Klopfenstein et al. (8). A large portion of any such change (3 % units) may be attributed to the decreased digestibility of bacteria, as compared with protozoa. The above value is arrived at by assuming a 20% cell yield (cell yield per 100 g of digestible substrate), equal distribution of cell matter in protozoa and bacteria in faunated animals, and digestibilities of 90 and 60% for protozoa and bacteria, respectively. A difference of 3% units would account for a large proportion of the changes recorded in the literature, but differences due to possible changes in the bacterial population composition cannot be ignored (4). The ammonia-VFA proportion relationships demonstrated in this work are potentially of very great importance to ruminant metabolism in general. For example, if a causal relationship does exist between ammonia levels and VFA proportions, the relationship is of potential value as a guide for the manipulation of rations causing milk fat depression problems. Rations markedly different in nitrogen content and energy availability were included and additional treatments in which urea infusions were the only variables were also included and all fitted the relationships. However, it remains to be seen whether the phenomenon is in fact as general as these results would suggest. One of the major aims of this work was to determine whether changes in animal response upon defaunation were the result of the presence of protozoa per se or were due to indirect effects mediated by some other factors. At this point, it seems likely that any changes in the VFA proportions as a result of defaunation are, in fact, a result of a change in rumen ammonia levels, either directly or indirectly. The explanation for this relationship cannot be given at this time, but both metabolic changes within individual organisms and population composition changes must be considered. An ammonia limitation rather than an excess would appear to be the causal factor. Two major relationships have been discussed in this paper, and a number of quite important implications follow from the acceptance of these data. Not the least is the selection of appropriate control animals to be used in defaunation work, for the results of the present experiment show that, when isonitrogenous rations are used, different rumen ammonia levels occur which apparently cause different VFA proportions. On the other hand, this secondary effect may be overcome by supplementary nitrogen to raise rumen ammonia levels, but this then means that isonitrogenous comparisons are no longer being made. It seems that careful experimentation is required to eliminate these conflicting factors.

v3-fos

2018-04-03T04:56:42.384Z

1970-09-01T00:00:00.000Z

40953658

s2

Differential Light Scattering Measurements of Heat-Treated Bacteria Effects of heat on diameter, size distribution, and refractive index of Staphylococcus epidermidis suspensions were determined accurately by computer analysis of differential light scattering data. Differential light scattering measurements have been shown (3)(4)(5) to be potentially powerful tools for studying bacterial morphology. Such measurements consist of recording the angular distribution of light scattered from a suspension of bacteria which has been illuminated by a plane parallel beam of monochromatic light. These so-called differential scattered intensities (DSI) depend upon mean size, shape, and structure of the scattering particles (3). We have applied DSI measurements to study dynamic morphological changes in cultures of Staphylococcus epidermidis during the course of heat treatment. In preparing autologous staphylococcus vaccines, many laboratories use heat as a sterilization procedure. Such a treatment supposedly does not destroy the immunogenic properties of vaccines and would be expected, therefore, to have little or no effect on cell walls. A recent study of thermally induced changes in staphylococci (1) demonstrated that optical density and light scattering properties of cells are directly influenced by heat. However, the observed optical density and light scattering changes did not correlate well with other data such as those obtained by electronic cell counter, hematocrit, and electron microscope. Results of our DSI studies will clarify the interpretation of these previously obtained nephelometric measurements. In addition, DSI measurements can detail very accurately the size and refractive index changes which occur upon heating. For the experiments described here, the culture of S. epidermidis was grown for 24 hr at 37 C on nutrient agar (Difco). Washed cells were either diluted directly into distilled water (control) or they were heated at 60 C in Heart Infusion Broth (Difco) for various periods before diluting. The heating and cooling procedures were designed to achieve rapid equilibrium. All samples were diluted to about 2 x 10 bacteria per ml for the light scattering measurements. The DSI measurements were made with a Differential 1 light scattering photometer (Science Spectrum, Inc., Santa Barbara, Calif.) with a vertically polarized He-Ne light source (632.8 nm). The instrument records the relative intensity of scattered light as a function of scattering angle, from 50 to 1500 measured with respect to the incident beam. Figure 1 presents the DSI of a washed suspension of S. epidermidis recorded immediately before heating. Figure 2 shows details of qualitative changes in light scattering in the vicinity of the two maxima after various periods of heating. In Fig. 2, we have shifted the curves with respect to scattered intensity to simplify comparisons. Since thermally induced morphological changes manifest themselves by changes in DSI, the evolution of the light scattering curves, as evidenced in Fig. 2, is a vivid indication that some morphological changes did occur. To determine quantitatively just how heating affected cell morphology, an extensive numerical analysis was performed. Data from Fig. 1 and 2 were replotted to correct for volumetric foreshortening (2,4). The corrected curves were interpreted quantitatively on the basis of Rayleigh-Gans scattering theory (3,5). By using a high-speed digital computer (CDC 6400), the scattering data were analyzed (3,5) and the analysis showed that the heat treatment (i) caused a reduction in mean cell size and (ii) resulted in an increased cytoplasmic optical density relative to cell wall. This latter change is a manifestation of the decrease in free water within the cytoplasm. In quantitative terms, the average radius derived for the control cells on the basis of the corrected curves was 432 nm (±10 nm). After 30 min of heating, this value had decreased to 403 nm (±10 nm). The refractive index ratio (5) -0.45 (40.1) for the control cells to -0.22 (±0.1) for the 30-min heat-treated cells. The average cell-wall thickness remained nearly constant at 108 nm (±20 nm) despite the heating. Finally, we deduced that the standard deviation of the cell radius increased slightly from about 106 nm (±8 nm; control) to 120 nm (±8 nm) after treatment at 60 C for 30 min. Our results show, as expected, that moderate heat treatment, although having little or no effect on cell wall, caused a measurable reduction in cell size. Some of these changes, indicated by a shift in the primary peak, occurred after only 3 min of heating. Although the data indicated an approximately linear cell shrinkage over the 30-min heating period, colony counts indicated that more than 99% of the cell population was killed after 3 min, presumably as a result of enzyme inactivation. The gross changes observed in the secondary scattering peak between 10 and 30 min of heating most probably arose, as would be expected, from protein denaturation and precipitation of internal solutes. We believe that electron micrographs taken of heated cells (1) have limited value in this type of experiment since distortion or damage may occur during preparation of specimens already weakened by heat. Furthermore, nephelometric devices which measure light scattering at a single fixed angle, such as 450 or 900, cannot be used to measure changes in cell size because such changes manifest themselves by shifts in angular location of the scattering peaks. By measuring the angular variation of scattered light, instruments such as the Differential I light scattering photometer clarify the dynamics of these structural changes. The light scattering techniques and instruments under development (5) show great promise as tools to study cell physiology and morphology under conditions where the ordinary light microscope is inadequate. Differential light scattering can bridge the gap between the light microscope and the electron microscope in the degree of resolution possible, in the elimination of the need for fixation, and in the rela-

v3-fos

2019-04-24T13:09:22.734Z

1970-05-01T00:00:00.000Z

128681002

s2

Influence of irrigation and placement of nitrogen fertilizers on the uptake of nitrogen by spring wheat. Field experiments on arable crops seldom account for more than one half of the nitrogen applied in fertilizers (Russell 1961, Cooke 1964, Kaila 1965 etc.). This may be attributed to leaching or denitrification, to volatilization or immobilization. In any case, a large part of the expensive fertilizer nitrogen is likely to be wasted. Lack of movement into the root zone of even nitrates applied as surface dressing to spring cereals was demonstrated in Finnish field experiments, and placement or working in of nitrogen fertilizers was recommended (Kaila and Hänninen 1961). Because of the usual dry period in the beginning of the summer in Finland, attention has also been paid to the possibilities of improving the efficiency of nitrogen fertilizers by irrigation (Elonen et. ai, 1967). In the present paper results are reported from a field experiment which was conducted to study the influence of sprinkler irrigation, and the placement and chemical composition of the nitrogen fertilizer on the uptake of nitrogen by spring wheat. usual dry period in the beginning of the summer in Finland, attention has also been paid to the possibilities of improving the efficiency of nitrogen fertilizers by irrigation (Elonen et. ai, 1967). In the present paper results are reported from a field experiment which was conducted to study the influence of sprinkler irrigation, and the placement and chemical composition of the nitrogen fertilizer on the uptake of nitrogen by spring wheat. Experimental In 1969 a large field trial was carried out in southern Finland, in the neighbourhood of Helsinki, in cooperation with the Finnish Research Institute ofAgricultural Engineering. Sprinkler irrigation was studied at four rates, and three kinds of nitrogen fertilizers were either placed or applied as a surface dressing. The uptake of nitrogen by spring wheat was followed by collecting samples of the aerial parts during the growing period. The soil was silty clay with an average clay content of 50 per cent. The pH of the ploughing layer was about 6 (in 0.01 M CaCl 2 ), and the content of organic C was 4-5.5 per cent. The nitrogen fertilizers studied were a Finnish ammonium nitrate-limestone, »Oulun-salpietari», urea and an American preparate of Ureaform contributed by Typpi Oy. They were applied just before sowing with a »Juko»-fertilizer drill in amounts corresponding to 120 J; 1 kg N/ha, either on the surface with the plastic fertilizer tubes outdrawn from the coulters, or at the depth of 8 cm with a spacing of 15 cm between the rows. The plots studied in the present work were irrigated with slow sprinklers in the night June 9 and June 17, at both times with 30 mm of drainage ditch water, and compared with plots without irrigation. As a basal dressing 800 kg/ha of an ammoniated Finnish PK fertilizer was placed at the depth of 7 cm with the »Juko»-fertilizer drill at right angles to the seed rows. Thus, 16 kg N, 60 kg P and 100 kg K were applied per hectare. All treatments were in four replicates. The total area of a plot was 2 X24 m 2, that of the plot harvested was 1.68 X 16 m 2. On May 8, »Ruso»-spring wheat was sown with row spacings of 12 cm at the depth of 4.5 cm. The sprouting date was May 20, that of coming to ear July 2. The yield was harvested on August 20. The growing season was very dry. The precipitation was 37 mm in May, 22 mm in June, 55 mm in July, and in August from the Ist to the 20th there was no rain. The mean temperatures did not markedly differ from those of a normal season. Plant samples were collected from each four replicate plots of the treatments studied by cutting the aerial parts from carefully measured strips at the end of the plots. The sampling area was 1.68 m 2 on June 2, 0.84 m 2 on June 16, 0.50 m 2 on June 30, and 0.42 m 2 on both July 21 and August 18. The ears were cut from the samples of July and August and analysed separately from the leaves and straw. The samples were air-dried at room temperature and ground in a Wiley mill. Total nitrogen was determined with the common Kjeldahl procedure. The results reported of the grain yields represent winnowed material. The statistical treatment of the data was performed by the new multiple range test of Duncan (1955). Results The nitrogen content of the aerial parts of wheat plants collected in June are reported in Table 1. Since the common Kjeldahl procedure was used, it is likely that nitrate, if present, was not totally included. The treatment »Without N» did not get the additional application of 120 kg/ha of nitrogen, only the 16 kg/ha in the basal dressing. On June 2, or about two weeks after sprouting, the beneficial effect of the placement of the nitrogen fertilizers is distinct. As a surface dressing, only ammonium nitrate-limestone has produced plant material with a higher nitrogen content than that without the additional nitrogen fertilization. Later, even surface-applied urea did increase the nitrogen content, but in each case, the placement significantly improved this effect. On the other hand, placement ofammonium nitrate-limestone did not increase the effect on the irrigated plots. On June 16, or about one week after the first irrigation, and on June 30, or about two weeks since the second irrigation, the nitrogen content of the irrigated plants was in most cases significantly higher than that of the unirrigated ones. This was the case even when no additional nitrogen was applied. There was in no case any significant difference in the nitrogen content of plants treated in the same way either with urea or with ammonium nitrate-limestone. Ureaform could not compete with them. The nitrogen content of the plants at later stages of development is reported in Table 2. »Straw» stands for the vegetative aerial parts. In the samples of July 21, no positive effect of irrigation on the nitrogen content can be found: the corresponding values either do not differ from each other, or the plant material from the irrigated plots is poorer in nitrogen than that from the respective unirrigated plots, as is the case with the ears from the plots surface-dressed with ammonium nitrate-limestone. In the samples collected just before harvest, or on August 18, this tendency of irrigation to lower the nitrogen content of the ears is distinct, particularly, when additional nitrogen was irrigation applied. Statistically significant difference also exists in the nitrogen content of the straw between the irrigated and unirrigated plots with placed urea. At these later stages of development, the positive effect of the placement of ammonium nitrate-limestone and urea is proved by the respective contents of nitrogen in the straw samples from the unirrigated plots. On these plots placement of urea has also significantly increased the nitrogen content of the ears. According to the nitrogen content of the samples of July and August, ammonium nitrate-limestone and urea have been equally effective. The only exception is found in the nitrogen content of the ears from the surface-dressed, unirrigated plots in July. Ureaform again, has been less effective than the two other fertilizers. On the basis of the weight of the samples and their nitrogen content, the nitrogen »yields» were calculated. These data are recorded in Table 3 expressed as N kg/ha. On June 2, there is not yet any difference between the treatments. At the later sampling dates, a distinct tendency to a marked increase in the uptake of nitrogen because of fertilizer placement is apparent. Since there was a relatively large variation in the dry matter yields of the replicates, these differences are not always statistically significant. The same holds true also with the effect of irrigation. Even in July and August it has tended to increase the amounts of nitrogen taken up by the plants, particularly on the plots treated with ammonium nitrate-limestone and urea. It may be of interest to note that in the samples of June 16, irrigation did not increase the uptake of nitrogen, if the fertilizers were placed. After the second irrigation, however, an increase also in the uptake of the placed fertilizer nitrogen is demonstrable. Both irrigation and placement tended to improve also the uptake of nitrogen from ureaform treated soil. Yet, only in the samples of June 30, more nitrogen has been accumulated on the irrigated plots from ureaform than from the soil without the additional nitrogen fertilizers. Later there is a similar tendency, but this is not statistically significant - Since the trial was harvested with a combine machine, only results of grain yield are available. Data in Table 4 show that irrigation significantly increased the dry matter yield of winnowed grains, but at the same time decreased their nitrogen content. Yet, the total amount of nitrogen harvested in grains from the plots treated with ammonium nitrate limestone or urea is distinctly increased by irrigation. Placement of these two fertilizers improved the uptake of nitrogen only from unirrigated plots. No difference may be found between the effect of ammonium nitrate limestone and urea in the dry matter yield of grain, in their nitrogen content, or in the amount of nitrogen harvested in grains. Ureaform did not give any significant response, except a slight increase in the »nitrogen yield», when the fertilizer was placed and the soil was irrigated. Means in the two corresponding columns »0» and »30 + 30» followed by a common letter do not differ atP= 0.05 Discussion The results of the present study indicate that, under these conditions, both irrigation and, apparently to a smaller extent, placement of nitrogen fertilizers distinctly improved the uptake of nitrogen by spring wheat. It may be calculated, on the basis of data in Table 4, that placement of ammonium nitrate limestone or urea did increase the amount of nitrogen in the grain yields, on the average, by 13 kg/ha without irrigation, but not significantly when irrigated. The irrigation, on the other hand, increased the nitrogen content of grain yields, averagely, by 27 kg/ha when the fertilizers were applied on the surface, and by 23 kg/ha when the fertilizers were placed. Thus, the higher dry matter yield produced by irrigation more than compensated the typical decrease in the percentage of nitrogen in the grains. responded to 120 kg N/ha. Its apparent recovery in the grain yields, calculated as the mean difference between the nitrogen yields of the respective treatments and that without additional nitrogen, is the following: without irrigation, surface dressing 31 % placement 42 % irrigated, surface dressing 54 % placement 61 % Thus, fertilizer placement with irrigation almost doubled the apparent recovery of fertilizer nitrogen in the grain yield of spring wheat. Unfortunately, straw yields were not available, but the plant samples collected on August 18, only two days before harvest, may give an approximate basis for an estimation of the apparent recovery offertilizer nitrogen in the total yield. The rather large variation due to the small sampling area, decreases, of course, the accuracy and the reliability of these estimations. The apparent recovery is, on average, the following: without irrigation, surface dressing 36 % placement 60 % irrigated surface dressing 76 % placement 96 % These percentages are fairly well in accordance with the results obtained on the basis of the grain yields, provided that there is in grains about twice as much nitrogen as in the straw yield. It is likely that the higher uptake of nitrogen from the irrigated soil is to some extent due to a more rapid mineralization of soil nitrogen. On the other hand, the larger root system of the irrigated plants (Kähäri and Elonen 1969) probably contains a larger amount of fertilizer nitrogen than does the smaller root mass in the unirrigated plots. Thus the apparent recovery of fertilizer nitrogen estimated on the basis of the aerial parts of wheat may be slightly lower than the real uptake of fertilizer nitrogen by the whole plants. This may be applicable also to the effects of the fertilizer placement. In any case, it is beyond dispute that in this field trial placement of urea or ammonium nitrate limestone, and irrigation in June twice with 30 mm water, resulted in an almost quantitative apparent recovery of the 120 kg of nitrogen applied per hectare. It is noteworthy that though irrigation decreased the nitrogen content of the dry matter in the later stages of development, it did markedly increase the nitrogen content of the wheat plants in June, in the period which in Finland is likely to be particularly important in the development of spring cereals. Analyses for other nutrients also proved the beneficial effect of irrigation on the contents of phosphorus, potassium, magnesium, and calcium in the wheat plants in the middle and at the end of June. The higher uptake of nitrogen due to the fertilizer placement, also to some extent, increased the content of potassium, calcium, and magnesium in the plants which were not irrigated. A good supply of these essential elements in the plants during the critical period apparently offered favourable conditions for a higher production. Summary The effect of irrigation and fertilizer placement on the accumulation of nitrogen in the aerial parts of spring wheat was studied in the relatively dry summer 1969, on the basis of plant samples collected at various stages of development from a field trial on silty clay soil. In addition to 16 kg/ha of nitrogen in the basal dressing, 120 kg/ha of nitrogen was applied as ammonium nitrate limestone, as urea, or as Ureaform, either on the surface, or in rows at the depth of 8 cm. 60 mm of water was applied by sprinkler irrigation, one half about three, and the other half about four weeks after sprouting. Under the conditions of this trial, spring wheat only slightly responded to Ureaform. No significant difference in the effect of urea and ammonium nitrate limestone could be detected. Placement of these two fertilizers tended to increase both the amount of dry matter and its content of nitrogen; these effects were more significant without irrigation. Though irrigation, usually, markedly increased the total amount of nitrogen in the aerial parts of the plants, this was in the later stages of development due only to higher yields of dry matter, since in July the positive effect of irrigation on the percentage of nitrogen in plant dry matter changed to a negative effect. In the grain yields the apparent recovery of nitrogen in urea or ammonium nitrate limestone was estimated to be only about 30 per cent of the 120 kg N/ha applied as surface dressing. Irrigation increased this recovery to 54 per cent, placement of the fertilizer to 42 per cent, and both placement and irrigation resulted in an apparent recovery of 61 per cent. On the basis of plant samples collected two days before harvest, the apparent recovery of nitrogen in urea or ammonium nitrate limestone by the aerial parts of wheat was estimated to be, on the average, 36 per cent from the surface-dressing, and 60 per cent when the fertilizers were placed. Irrigation increased the apparent recovery in the first case to 76 per cent and in the latter case to 96 per cent. Thus, in the field experiment the uptake of fertilizer nitrogen by spring wheat was markedly increased by irrigation and to a lesser extent by fertilizer placement. Both treatments together are recommended.

v3-fos

2020-12-10T09:04:16.706Z

1970-05-01T00:00:00.000Z

237231922

s2

Degradation of Urea Herbicides by Cell-Free Extracts of Bacillus sphaericus N′-methoxy phenylurea herbicides are degraded by induced cells of Bacillus sphaericus ATCC 12123 by liberating carbon dioxide from the ureido portion of the molecule and leaving the corresponding aniline moieties. Cell-free extracts of B. sphaericus inactivate these herbicides in the same way as reported for whole cells. A 6.6-fold purification of the crude extract was achieved by a combination of salt fractionation with ammonium sulfate and column chromatography on diethyl-aminoethyl cellulose. sphaericus inactivate these herbicides in the same way as reported for whole cells. A 6.6-fold purification of the crude extract was achieved by a combination of salt fractionation with ammonium sulfate and column chromatography on diethylaminoethyl cellulose. Substituted urea derivatives constitute a large class of compounds which show herbicidal activity. Previous reports indicate that the degradation of N'-methoxy phenylurea herbicides occurs rapidly in soils, and that soil microorganisms are mostly responsible for its breakdown (1). The isolation and identification of a soil bacterium, Bacillus sphaericus, which decomposes N'-methoxy phenylurea herbicides have been reported (5). The microorganism inactivates the herbicides by liberating carbon dioxide from the ureido portion of the molecule and leaving the halogen-substituted aniline moieties (6). This report describes the results of our study on degradation of different urea herbicides by several strains of B. sphaericus and the partial purification and properties of an enzyme from the cell-free extract of B. sphaericus. This enzyme preparation was found to be responsible for the hydrolysis of these urea compounds. MATERIALS AND METHODS Strains. A B. sphaericus strain isolated from soil (5) and ATCC strains 14577, 12300, 12123, 10208, 7055, and 7054 (provided by 0. Kandler, Botanical Institute, University of Munich) were used in this study. The bacteria were grown in 6 liters of a medium containing 0.1% yeast extract as described previously (6), but the vitamin mixture was omitted. To induce the enzyme system, bacteria were grown in the presence of 50 ,umoles of linuron (Table 2). Cells were harvested in the late logarithmic-growth phase and stored at -15 C, after being washed twice with distilled water. Herbicides. The enzymatic degradation of the substituted urea herbicides (monuron, monolinuron, linuron, metobromuron, and fluometuron) was investigated ( Table 2). The preparation of the used herbicides and the residue analysis were performed as described (6). The products of the reaction were identified by thin-layer chromatography and infrared spectroscopy. Enzymatic assays. The packed cells were ground in a mortar at 0 C with twice their weight of A1203 and then suspended in 0.1 M phosphate buffer (pH 7.0). The crude extract was centrifuged at 20,000 X g for 45 min at 4 C. All the following procedures were performed at 4 C. Enzyme activity was measured by incubating the enzyme preparation with ureido-'4C-labeled linuron (specific activity, 2,600 counts per min per jumole) in 0.1 M phosphate buffer (pH 7.0) in sealed Erlenmeyer flasks at 30 C on a shaker. "4CO2 was trapped in separate reaction vials containing 1.5 ml of 1 N NaOH. The 11CO2 was determined in a liquid scintillation counter (Beckman Instruments, Fullerton, Calif.) with a counting efficiency of about 90%. No corrections, except for background, were applied to the counting data. When the other herbicides were used as substrates, enzyme activity was assayed by determining the reaction products, namely the halogen-substituted anilines (6). The reaction was stopped after 120 min by adding 0.5 ml of 10 N H3PO4 per 10 ml of buffer to the closed system. The carbon dioxide remaining in the reaction vessels after acidification was allowed to be absorbed by the base with 30 min of additional shaking. The specific activity is defined as micromoles of herbicide degraded per minute per milligram of protein. Protein was determined by the Lowry procedure (4). Purification of the enzyme. Two milliliters of 2% streptomycin sulfate was added to 20 ml of cell-free extract of B. sphaericus (3). The precipitate collected by centrifugation was discarded. The second step of purification was achieved by salt fractionation by using ammonium sulfate from 30 to 100% saturation. The precipitates collected after centrifugation were diluted with 0.1 M phosphate buffer, pH 7.0 ( RESULTS AND DISCUSSION The N'-methoxy phenylurea herbicides could be degraded by cell-free extract from B. sphaericus only when grown in the presence of the herbicides. No measurable activity was found in uninduced bacteria. Induction, however, was possible only in the above minimal medium, but not in an enriched medium containing peptone or meat extract. B. sphaericus ATCC 12123 proved to be as active in decomposing N'-methoxy phenylurea herbicides as the wild type isolated from soil. ATCC strains 7055 and 7054 showed a considerably lower rate of degradation. However, ATCC strains 14577, 12300, and 10208 were completely inactive in decomposing substituted urea herbicides. Breakdown proceeded in the same way as described for the wild type (6). Since ATCC strain 12123 showed the highest rate of inactivation of N'-methoxy phenylureas, this microorganism was selected or further studies with cell-free systems. A crude extract decomposed the N'-methoxy phenylurea herbicides, monolinuron, linuron, and metobromuron, in the same way as reported for whole cells. Cell envelopes and a boiled extract had no activity (Fig. 1). The enzyme system responsible for hydrolyzing the herbicides was partially purified by salt fractionation and column chromatography on DEAE cellulose, yielding a 6.6-fold purification. The precipitates from 50 to 70% ammonium sulfate saturation indicated the highest enzyme activity (Table 1). Figure 2 shows the elution pattern of the enzyme from DEAE cellulose. The optimal pH level of the partially purified enzyme is shown in Fig. 3. Optimal activity ranges between pH 7.0 and 8.5. No activity was observed at or below pH 5.5. Figure 4 demonstrates that enzyme activity is linear with respect to enzyme concentrations up to 5 mg of protein. Since the activity of the enzyme system responsible for inactivating N'-methoxy phenylureas was extremely low, only a 6.6-fold purification was achieved ( Table 1). The enzyme system apparently hydrolyzed N'-methoxy phenylureas to the corresponding halogen-substituted anilines by two different processes. Enzymatic attack, resulting from a reaction with urease, would then form anilines, carbon dioxide from the ureido portion of the molecule, and a not yet defined compound derived from the N'-methoxy-N'-methylamine portion. The carbon dioxide production, however, is not related to the reaction with urease, since B. sphaericus does not possess this enzyme and none of the urea herbicides used in this investigation were degraded by soybean urease in vitro. These results may suggest that the cell-free system has amidase activity. Enzymatic attack at the amide linkage would yield a halogen-substituted phenylcarbamic acid and an unidentified metabolite. Phenylcarbamic acid is unstable and spontaneously disintegrates to the corresponding anilines and carbon dioxide (3). It is difficult to explain why the decomposition appears to be specific for N'-methoxy phenylureas. The methoxy substitution may have a weakening effect on the bond between the ureido group and the substituted nitrogen because of the electrophilic nature of the oxygen (6). There is no indication that stepwise demethylation occurs before deamination takes place (1). The highest enzymatic activity appeared in the late logarithmic-growth phase, before sporulation started (6). Therefore, a connection may exist between sporulation and appearance of the enzymatic activity in the hydrolyzation of N'-methoxy phenylureas.

v3-fos

2020-12-10T09:04:11.097Z

1970-07-01T00:00:00.000Z

237233317

s2

Recovery of Rauscher Leukemia Virus from Large Volumes of Seeded Cow's Milk and from Infected Murine Spleens Rauscher murine leukemia virus was used as an indicator agent to develop a methodology for the extraction and concentration of a theoretical leukemia virus from bovine milk and tissues. The indicator virus was seeded into cow's milk or was recovered from infected murine spleens. The tissue homogenates and the defatted milk were processed in a B-XVI rotor of a Spinco L-4 ultracentrifuge at a flow rate of 3 liters/hr. The efficiency of Rauscher virus recovery was greatest when the rotor was used without a gradient. A loss of between 0.6 and 0.7 log of total infectious virus, as determined by the spleen assay method, resulted when the seeded milk and murine spleens were processed. The procedures developed are presently being used in transmission experiments in an attempt to induce leukemia in the bovine. The finding of virus-like particles in milk from leukemic cows (7,8) and the development of methods for their recovery and partial purification have been reported (7,15,16). Similar particles have been observed in lymph node and mammary gland biopsies as well as in tissue cultures from cattle with lymphosarcoma (8,20,22,30). More recently, budding virus particles have been seen in mixed cell cultures (3,17) and in phytohemagglutinin-stimulated short-term lymphocyte suspension cultures from leukemic cattle (18). The buoyant density and the size and morphology (7,8) of the particles in milk are similar to the values reported for murine (23) and feline (25) leukemia and sarcoma viruses. Transmission experiments have demonstrated that murine leukemia is transmissable to newborn mice by inoculation of tissue extracts, filtrates, and plasma (9,12,13,19,24). Vertical transmission has also been demonstrated by foster nursing studies (4,5,13,19,20), and virus has been shown to be present in the milk of mice known to have a high incidence of leukemia (6). The transmission of feline leukemia and sarcoma in kittens by the inoculation of tissue extracts and filtrates containing virus (25) has also been confirmed. Attempts to similarly transmit leukemia in cattle by use of viable cells and tissue extracts from leukemic donors have thus far been unsuccessful (11,14,(26)(27)(28). Where responses have been reported, lymphocytosis was often the sole criterion of infection, controls were lacking, or too much time elapsed before cases occurred. Transmission studies in laboratory animals and cell culture systems with both crude and purified bovine extracts have been unsuccessful. Various methods of concentration and purification, including differential, density gradient, and zonal ultracentrifugation (1,10,29) and zonal electrophoresis, have been utilized to concentrate and purify a possible leukemia virus in the bovine system. Electron microscopic examination, which still remains the sole means of detecting virus in the bovine, has revealed the presence of viruslike particles, resembling the "C" type murine leukemia viruses, in various concentrations in all of these bovine preparations. Therefore, it was essential that a procedure be devised for the extraction of virus-like particles in sufficient quantity from bovine milk and tissues for transmission studies in newborn calves. Although the concentration of virus in miLk and tissues of affected cattle is thought to be low, large quantities of raw materials were available. To establish a procedure for the recovery of a bovine leukemia virus and to determine the effectiveness of procedures already developed, an indicator agent, murine leukemia virus (Rauscher), was added to cow's milk or was extracted from virus-containing mouse spleens. This report describes the effectiveness of the concentration procedures employed in terms of the quantitative recovery of the indicator agent as measured by its biological activity in mice (2). MATERIALS AND METHODS Milk. The milk was processed through a DeLaval model 100 cream separator at a minimum temperature of 31 C. Indicator virus. Rauscher leukemia virus recovered from the plasma of infected mice was obtained from John Moloney, National Cancer Institute, Bethesda, Md. The Rauscher virus-infected spleens were provided by Earling Jensen of the John L. Smith Memorial for Cancer Research, Maywood, N.J. Tissue extracts. Tissues were cut into sections, approximately 2 to 3 cm3, and processed immediately or sealed in plastic bags and stored in a Virtis freezer at -87 C until used. The frozen tissues in plastic bags were rapidly thawed by immersion in warm water (37 C) and immediately ground in a General commercial meat grinder (model D). The resulting finely ground tissue was suspended in 2 to 3 volumes of 0.15 M citrate buffer and blended in a Waring Blendor (model CB-5), having a 1-gal capacity (ca. 3.8 liters), for 30 sec by use of a series of 10 3-sec bursts. Centrifugation. Low-speed centrifugation was carried out in a Sorvall RC-2B centrifuge with a GSA rotor. For high-speed centrifugation, an International B-60 ultracentrifuge and an A-170 rotor were employed. Continuous-flow centrifugation was accomplished with a Beckman-Spinco model L-4 ultracentrifuge. A B-XVI rotor was used with and without a 400-ml sucrose cushion. When sucrose was used, a natural gradient was created during the centrifugation procedure. Fractions were removed by pumping from the edge with a 50% sucrose solution. Sterilization. All metallic parts that came in contact with the sample were autoclaved, with the exception of the centrifuge rotors. The rotors were assembled, with all plastic tubing connected, and sterilized in place by using 12% ethylene oxide (Oxyfume 12; Linde) at 10 lb of pressure for a minimum of 12 hr. The ethylene oxide was removed by passing dry nitrogen at 10 lb of pressure through the system, followed by the flushing of 5 liters of sterile sodium citrate buffer through the rotor. The centrifuge tubes were sterilized by either autoclave or by ultraviolet light. All equipment and glassware contaminated during the processing of the sample were disinfected by soaking in a formaldehydedetergent bath. Other. Twenty-liter sterile stainless-steel pressure vessels (Millipore Corp., Bedford, Mass.) were used to store the sample for processing through a B-XVI rotor. The vessels were immersed in an ice bath, and the sample was maintained at 5 C. Mouse spleen assay. Portions of serial dilutions of samples (0.1 ml) were inoculated intraperitoneally into 4-week-old BALB/ccr mice. After 21 days, the mice were killed, and spleen weights were recorded to the nearest milligram. Spleens of more than 180 g were considered positive. Fifteen mice were used for each dilution group. Spleen assays were performed by Gerald Spahn (Microbiological Associates, Inc.). RESULTS Because of the variation in the volume at different sampling points of milk and tissue extracts, it was necessary to compare the total amount of infectious virus present at each step in the process. Infectivity, as determined by the mouse-spleen assay method, was reported as the ED60 per milliliter of the sample. The volumes of the fluids were converted to logarithms and added to the logarithms of the infectivity titers, the result being the total amount of infectious units at each stage of the process. During the operation of the B-XVI rotor, an approximate linear gradient was created as the sucrose diffused into the covering citrate buffer. In the initial run, a flow rate of 6 liters/hr was used to process the miLk seeded with Rauscher virus. After the flow-through of the sample was completed, the rotor was maintained at operating speed (38,000 rev/min) for an additional 1 hr to sharpen the zones in the gradient. The results of this experiment are shown in Table 1. An initial reduction of virus infectivity occurred when the inoculum was mixed with the raw cow's milk. Whether this loss was due to a sampling error or to a virucidal effect of the milk was not determined. The 1.14 to 1.17 g/cm3, sucrose gradient fraction showed an additional loss of virus. After dialysis to remove the sucrose and after filtration, the original virus concentration experienced a four-log reduction of infectivity. In an attempt to increase the yield of virus, two additional Rauscher virus-seeded milk samples were processed as follows; the first observed the same procedure as described earlier with the exception of a reduced flow rate of 3 liters/hr. The second sample was processed at 3 liters/hr without a sucrose gradient, and the pellet was removed with a sterile spatula from the wall of the rotor. Results of these experiments are described in Tables 1 and 2. With the reduced flow rate, the loss of infectious units in the gradient fraction was reduced to 1.33 logs, as compared to 2.8 logs for the 6-liter/hr flow rate. When the sample was pelleted in the absence of a gradient at a flow rate of 3 liters/hr, the loss was reduced further to 0.68 log. In an attempt to develop procedures for extracting virus from tissues, 870 g of Rauscherinfected murine spleens was processed by the procedures outlined in Fig. 2. The results are shown in Table 3. The 8,700 ml of the homogenate was reduced 44-fold with a loss of 0.6 log of infectious units. The total initial infectivity was reduced from 7.84 to 7.21 logs. DISCUSSION Because of the difficulty and uncertainty involved in attempting to monitor extraction, concentration, and purification processes by electron microscopy, an indicator organism, Rauscher murine leukemia virus, was used to monitor the methodology and was seeded into the milk and recovered or was extracted from infected mouse spleens. In the initial experiment with a flow rate of 6 liters/hr, infectivity studies showed that there was a loss of 2.8 logs of the seeded Rauscher leukemia virus when the 1.14 to 1.17 g/cm3 fraction was assayed. Reduction of the flow rate to 3 liters/hr increased recovery, but there was still a loss of 1.33 logs of infectious units. Losses of such magnitude were unacceptable, especially since it was suspected that a bovine leukemia virus, if present at all in the bovine preparations, would be present in low titer. In an attempt to improve the virus recovery rate, the use of a gradient was eliminated, and the virus was pelleted directly onto the wall of the rotor. The increase in the recovery of the seeded virus by this change in methodology was demonstrated by a further reduction of the infectivity titer from 6.95 to 6.27 logs. A loss of 0.7 log of the initial seeded virus was considered acceptable, considering the volume of milk processed, the loss of fluids by mechanical operation of the system, and the limitations of the assay procedure. Therefore, it was determined that the methodology outlined in Fig. 1 would be utilized in an attempt to extract a possible bovine leukemia virus from milk obtained from cows diagnosed as positive cases of lymphosarcoma. Tissues recovered from a cow presented unusual technical problems because of the volumes of raw material available. Spleens from infected animals weighed up to 4,000 g, whereas tumors from advanced cases varied in weight from a few grams to well over 5,000 g. To process tissues of such magnitude within a relatively short period of time, the technique for the extraction of murine leukemia virus from tissue as advocated by Moloney (21) was modified and scaled up to process kilogram amounts of material (Fig. 2). In an attempt to determine the effectiveness of such large-scale tissue extraction procedures, murine spleens were processed by the outlined procedures, and the resulting resuspended pellet was titrated in mice. The loss of the indicator virus was 0.6 log which was considered to be satisfactory considering the magnitude of tissue extracted, the mechanical loss of homogenate in the system, and the limitation of the spleen assay method. A series of experiments was run to determine the effect of dialysis, filtration, and low-speed clarification of the resuspended pellet on recovery of the indicator virus. Each of these procedures resulted in considerable reduction in virus titer. Therefore, it was determined to use the crude resuspended pellet as inoculum for the transmission studies. In the event that a bovine leukemia virus might be present in milk and tissues of cattle affected with the disease, would it have properties similar to a murine leukemia virus, such as the Rauscher type used in these studies? The results of these experiments can only tell us of the recoverability of Rauscher virus in terms of the concentrations introduced and can only suggest that similar results might be obtained in recovering a bovine virus, should it have similar properties. With these limitations in mind, procedures have been developed as outlined in Fig. 1 and 2 for the extraction and concentration of a theoretical "C" type leukemia virus from cattle diagnosed as positive for lymphosarcoma. Transmission studies have been initiated utilizing these procedures in which extracts from lymph node, spleen, and milk are being used to inoculate newborn calves in an attempt to induce leukemia in these animals.

v3-fos

2020-12-10T09:04:16.975Z

1970-10-01T00:00:00.000Z

237235267

s2

Differentiation of Brucella canis from Other Brucellae by Gas Chromatography Gas chromatographic techniques allow for differentiation between a strain of Brucella canis and strains of other brucellae. A gram-negative coccobacillary bacterium isolated from placental and fetal tissues of an aborted beagle pup was characterized as a new species of Brucella, and the name Brucella canis was proposed for this previously unrecognized bacterium (1). The present study was designed to determine whether gas chromatographic techniques may be employed to distinguish between a strain of B. canis and isolates of other Brucella species. B. abortus 544, B. melitensis 16 M, B. ovis REO-1182, B. suis 1330, dog abortion agent RM 6 66 (designated herein as B. canis) obtained from the amniotic fluid of an aborted beagle fetus, and Bordetella bronchiseptica were grown at 37 C on slants containing 1.5% tryptone, 1.5% yeast extract, 0.5% glucose, 0.5% K2PHO4, and 1.5% agar (TYG medium). After 24 hr, the cells were washed from the agar with 5.0 ml of distilled water, the bacterial suspension was diluted, and 0.10 ml containing 25 X 103 to 250 X 103 cells was transferred into a screw-capped test tube containing 5.0 ml of TYG broth. After 24 hr of incubation at 37 C, replicates were examined microscopically for counts, and the cultures and samples of uninoculated medium were treated with 0.10 ml of 5 N HCI and 1.0 ml of 0.2 M HCI-KCl buffer (pH 2.0). The samples were then centrifuged at 3,000 X g, and the supernatant fluid was extracted three times with 10 ml of ether. The extracts were combined, concentrated to 0.5 ml, and dried with anhydrous Na2SO4. A 3.0-,uliter sample was injected into the gas chromatograph. The chromatographic techniques were essentially the same as described previously (2). The differentiating peaks of these strains of Brucella and Bordetella are shown in Table 1. With the electron capture detector to record the presence of microbial metabolites, the sensitivity of detection of the bacteria ranged from 42 to 490 organisms per 10 mm2 peak area. The sensi-tivity was calculated from the number of cells in the 24-hr sample which was injected into the chromatograph and the peak area of the product, the value given for sensitivity being the estimated number of bacteria to yield a peak area of 10 mm2. Each Brucella strain exhibited 10 to 15 peaks in chromatograms prepared from the spent culture media. Peaks having retention times of 25, 35, 45, 55, 60, 75, 85, 90, 390, and 940 sec were common to most of the organisms. However, as shown in Table 1, at least one compound was present in cultures of each Brucella strain which was not produced by any other organism tested. These metabolites were not found in the uninoculated medium, and the area of these ranged from 500 to 1,710 mm2. A signature for each Brucella strain was established by assigning letters to peaks in the chromatogram in order of their increasing retention times. By means of the signatures obtained, the strains examined could be characterized readily. When products elaborated by B. canis were compared with those of the other bacteria investigated, the dog pathogen was found to differ from B. abortus, B. melitensis, B. suis, B. ovis, and B. 649 Vol. 20,No. 4 bronchiseptica by eight, nine, three, eight, and four products, respectively. Some similarity in the kinds of substances excreted was evident, therefore, between B. canis and B. suis as well as B. bronchiseptica. On the other hand, most of the products formed by B. canis as well as the other brucellae were quite distinct from those synthesized by strains of Bacillus, Clostridium, and Staphylococcus. The results thus indicate that B. canis excreted metabolites similar to those generated by strains of other Brucella species. However, the brucellae investigated, although admittedly few in number, can be readily differentiated from one another by considering the presence or absence of individual compounds detectable by gas chromatography.

v3-fos

2020-12-10T09:02:49.463Z

1970-11-01T00:00:00.000Z

237232141

s2

Polysaccharide from Dry Navy Beans, Phaseolus vulgaris: Its Isolation and Stimulation of Clostridium perfringens A microbiological assay is described for determining gas produced by Clostridium perfringens (Veilon and Zuber) Holland from materials believed to be flatulent. A rationale for its use is given and analyzed. The fractionation and the results of the assay for several components of lima beans, Phaseolus lunatus L., and navy beans, P. vulgaris L., are given. In particular, a polysaccharide was isolated from dried P. vulgaris seeds. Only about one-seventh as much could be isolated from green P. lunatus seeds as from P. vulgaris seeds. The polysaccharide stimulates voluminous gas production by C. perfringens and meets all criteria of the rationale for flatulent sus pects. It has not been tested on humans; however, data from the microbiological assay correlate well with human results for green and dry lima beans and navy beans. There has been considerable interest in determining the nature of the flatulence factor of the common dry bean, Phaseolus vulgaris L.; lima bean, P. lunatus L.; and soy bean, Glycine max Merr. (3,(7)(8)(9)12). Yet a positive identification of a factor has not been made for any of these species. Progress has been slow because the ultimate assay must be performed in man, and human experiments are susceptible to uncontrollable factors such as emotions (3) and lung congestion (7). As a result, other organisms have been tried including dogs and bacteria (9,12). Richards et al. (9) first suggested a microbiological assay. Their methods suggested to us that flatulence ought to be caused by numerous foods known to be nonflatulent. The apparatus they used also proved to be very susceptible to mechanical error. We developed a more precise apparatus for measuring gas from anaerobic cultures and formulated the medium described in this report. The assay described uses beans reported to be low in flatulence as a control. The rationale of our assay, including the assumptions that we have made, is discussed. Use of the assay resulted in the fractionation of a polysaccharide from California small white beans (P. vulgaris). MATERIALS AND METHODS Bioassay. The bioassay was accomplished by measuring the volume of gas produced by Clostridium perfringens (Veilon and Zuber) Holland (ATCC 3629). The basal medium contained 0.5 ml of liquid thioglycolate medium (4), 50 mg of finely ground California small white beans, and 10 ml of 0.1 M succinate (Na+, pH 6.0). The test media contained 30 mg of the bean fraction to be tested in addition to the basal medium. Autoclaving of the media at 15 psi for 20 min provided the only cooking for the bean powder and for fractions that had not previously been cooked. To assure a vigorous inoculum, 10 ml of thioglycolate medium was inoculated and incubated overnight at 35 C. In the morning, the entire tube was used to inoculate 30 ml of thioglycolate medium in a bottle. After the bottle had been allowed to incubate for 4 hr at 35 C in vacuo, it was used to inoculate the test medium. The apparatus used for measuring gas consisted of a fermentation vessel and a gas buret attached to a manifold that was attached to a leveling bulb (Fig. 1). The fermentation vessel is a tube of about 13-ml capacity fitted with a -19/38 male joint. A glass bead was placed in the bottom to aid agitation. The gas buret consisted of an inverted 10-ml pipet fused to a 1 19/38 female joint with a side arm extending to just above the level of the medium. The lower end was sealed and openings were sawed in the side. The tip of the buret was capped with a 7-mm serum cap. The buret, manifold, and leveling bulb were filled with washed Hallikainen JY400 oil, a very light bath oil. The connections from the gas-buret side arm to the manifold and leveling bulb were made with polyvinyl chloride tubing. The manifold had a stopcock at the attachment for each of 10 gas burets and one further opening for the leveling bulb. A separatory funnel was used for the leveling bulb. The entire 716 KURTZMAN AND HALBROOK apparatus was shaken in a modified Warburg bath set at 44 C. Gas production was measured for 3 days. A blank containing the thioglycolate medium, bean powder, buffer, and inoculum (but no bean fraction) was always run. A hypodermic syringe could be used to obtain gas samples for analysis and to reset the buret to zero if more than 10 ml of gas was produced. The assay was checked for linearity and reproducibility with several substrates. As determined by the standard deviation, it reproduced very reliably ( Table 1). The assay was also linear within the limits of error expected ( ;: j : .; . 4 . . i / / * -> . } l t . : All of our dry beans were either given to us by E. L. Murphy or tested by him to insure that they were flatulent. Our green Fordhook beans were commercially mature when harvested. Fractionation. Figure 2 shows our main fractionation scheme. The scheme in Fig. 3 is better for separating the water-insoluble fractions: the starch and screenings. However, it is more laborious than the scheme (Fig. 2) for extracting the water-soluble fraction. The finely ground bean powder was prepared for the scheme in Fig. 2 by passing it through a Mikro-Samplmill with a 0.022-inch (ca. 0.05 cm) screen. The action of this machine allows only the passage of particles that are many times smaller than the screen mesh. About 55% of the dry bean powder passed through a 200-mesh screen, although a small amount would not pass a 100-mesh screen. Thus, few cells could remain intact. A batch of 300 g of bean powder was mixed with 1,500 ml of water in a Waring Blendor for 1 min. The slurry was centrifuged, and the precipitate was extracted twice in the same manner. The supernatants were combined, autoclaved, cooled, and centrifuged. The supernatant was then condensed to one-fourth its volume in a cyclone evaporator. A 10% tannic acid (J. T. Baker) solution was added to the condensed liquid at the rate of onetenth of its volume. Next, acetic acid was added until the pH fell to 4.0. This procedure removed the protein remaining after heating (Table 3), and some polysaccharide (6). An equal volume of acetone was added to the supernatant to precipitate the remaining polysaccharide. The precipitate was washed with acetone, air-dried, and labeled polysaccharide A-1. All of the other solids collected at other stages were also washed with acetone and air-dried. Later the protein tannate was suspended in saturated caffeine solution and extracted to yield a solution of free protein. The solution was precipitated with an equal volume of acetone, and the precipitate was washed with acetone. The precipitate was labeled protein A-1. RESULTS The yields and protein content of the various bean fractions are shown in Table 3. Starch represents over one-third of the total dry weight of the beans. An additional one-fifth of the dry weight of the beans was caught on the 100-mesh screen. The screenings were primarily cellulose, hemicellulose, and other insoluble materials. Heat precipitated approximately another 10% of the dry matter of the beans. This precipitate was primarily protein. The first acetone precipitate was the smallest fraction recovered from either bean by the scheme of Fig. 3. Each of the fractions was studied in corresponding pairs from green Fordhook and California small white beans. A greater gas production from a California small white bean fraction was considered to suggest the presence of the flatulence factor. C. perfringens is known to grow on starch, but we wanted to determine whether the starches of the two beans might differ in their ability to support gas production. No difference in gas production was found between the two starches (Fig. 4). The screenings showed little sign of a differential response (Fig. 4). The heat precipitates of the two beans showed a differential response, but it was the green Fordhook bean that supported the greater production of gas. The supernatant from the acetone precipitation was not assayed. It would contain oligosaccharides, peptides, and smaller compounds. The first acetone precipitates also gave differential response. The California small white fraction gave the greater gas per 30 mg. A variety of means was used to further fractionate the first acetone fraction. Ultraviolet measurements FIG. 4. Gas produced by Clostridium perfringens from bean fractions. Bars represent milliliters of gas produced by the average individual assay (30 mg of fraction) minus gas produced by basal medium. and Kjeldahl determinations suggested that the green Fordhook fraction was mostly protein, whereas the California small white fraction contains a large percentage of polysaccharide ( Table 3). The need for a simple means of separating proteins and polysaccharides suggested the scheme in Fig. 2. It proved to be very successful (Table 3; Fig. 4 and 5). The extracted residue of the scheme in Fig. 2 would be essentially a composite of the starch and screenings of the scheme in Fig. 3. The significance of gas produced from it would be obscured by the large amount of starch present. The heat precipitate and acetone supernatant were similar to the corresponding fractions of the scheme in Fig. 3; therefore, they were not of interest. The California small white and green Fordhook protein A-1 fractions contained the proteins of the corresponding first acetone precipitates. Yields were not quantitative for the protein A-1 fractions; however, the tannate yields are indicative of the protein A-1 yields. The polysaccharide A-1 fraction of California small white beans not only supported much more gas production per 30 mg than the corresponding green Fordhook fraction (Fig. 4), but it also was a much larger fraction of the bean (Table 3 and Fig. 5). These data correlate well with human flatus measurements with the same beans (7, 10). After we had shown a correlation between human flatus and gas production from the polysaccharides A-1, from green Fordhook, and California small white beans (Fig. 5), data from a third bean were desired. Dry Ventura beans are intermediate in flatulence (7,10). Figure 4 shows that polysaccharide A-1 from dry Ventura beans produced more gas in our assay. The yield of polysaccharide A-1 from dry Ventura was small ( Table 3), so that the gas produced from the polysaccharide A-1 in 1 g of dry Ventura bean was intermediate between that of green Fordhook and California small white beans (Fig. 5). Thus the results from all three beans in our assay correlate with measurements of human flatus. The hydrolyzed polysaccharide A-1 of California small white beans contained the following sugars: arabinose, galactose, xylose, and glucose. No other monosaccharides were found. DISCUSSION The use of a microbiological assay requires some assumptions. The literature contained no list of assumptions for a microbiological flatulence assay, so it was necessary to develop a list to rationalize our results. First we assumed that both a reference and a flatulent bean were necessary to the development of an assay. We desired beans that were as different as possible in flatulence but as taxonomically similar as possible. Green Fordhook (P. lunatus) was chosen as the nonflatulent control on the basis of work done on humans by Sanchez et al. (10). The California small whites (P. vulgaris) are a typical dry navy bean and were chosen as the flatulent bean because of available comparative data (7). Hydrogen is the most characteristic gas of flatus (3,9), so the assay organism should pro-718 APPL. MICROBIOL. duce hydrogen. Richards et al. (9) working with dogs and Murphy (7) working with humans found that Mexiform and its constituent iodochlorohydroxyquinoline (Vioform) inhibited intestinal gas formation, particularly hydrogen formation, but 2,4-dinitrophenol did not. Mexiform stimulates intestinal aerobic and coliform bacteria in rats according to Eisman et al. (5). These facts suggested that coliforms and intestinal aerobes should not be used. The only major hydrogen-producing inhabitants of the gut that could not be eliminated (5,7,9) were the clostridia and the Bacteroides. Only clostridia were used by Richards (1965), since he would have killed all but the sporeforming bacteria by pasteurization, and his incubation procedures were anaerobic. We found significant gas production when pasteurized human feces were used to inoculate cooked ground beans, but not when unpasteurized feces were used. We also did an experiment in which we added Vioform to our bioassay. The control produced gas, although those containing Vioform produced none. Thus we established that our assay organism was inhibited by Vioform just as flatus was inhibited. The C. perfringens culture used, therefore, met all of the criteria for an organism responsible for flatus. It seemed most likely that the basic assay as rationalized thus far would give many misleading results. As was expected when the beans were only ground, cooked, and assayed, green Fordhook beans supported more gas production than California small white beans (Fig. 4). Apparently the larger quantity of small molecules in the green Fordhook beans was responsible. An additional assumption was necessary to obviate the interference by molecules that would easily be absorbed by the gut. We made the assumption that flatulence was not due to any small molecule. Contrary evidence from bacterial experiments has been published (8, 11); however, there is also evidence from human experiments that at most oligosaccharides account for only a small fraction of the problem (3). No evidence has been published that any other small molecules produce flatus. A final assumption is that the flatulence factor is one or more chemical entities present in beans. In order for the assay to work, flatus may not be caused by a precursor that must be changed by the gut before C. perfringens can utilize it. In using a nonflatulent bean as a reference, we were making an implicit assumption that none of the nonflatulent beans not digested and absorbed by the gut is metabolized by C. perfringens. Conversely, a certain portion of the flatulent bean must not be absorbed by the gut, but must be metabolized by C. perfringens. For the success of the assay, it is necessary that the flatulent beans contain nothing that is both absorbed by the gut and metabolized by C. perfringens which is not also present in the nonflatulent bean. Even then the other foregoing assumptions must all be true, i.e., that C. perfringens is the organism or is sufficiently like the organism that produces gas in vivo, that what we are looking for has not been thrown away in our fractionating procedure, and that the flatulent factor is a chemical or chemicals present in the bean. However, the nature of our polysaccharides makes it seem reasonable that it is a flatulence factor. Another legume gum, guar gum, does not cause a body weight increase in proportion to the difference between the intake and fecal weights of rats (2). However, rats fed a neomycin supplement gained more weight and had lower fecal weights than those on a basal diet (1). It seems likely that most of the guar gum was metabolized by bacteria with the production of flatus. This is the effect that we would suggest for the bean polysaccharide.

v3-fos

2020-12-10T09:04:12.466Z

1970-05-01T00:00:00.000Z

237234716

s2

Bacteriological and Shelf-Life Characteristics of Canned, Pasteurized Crab Cake Mix The bacteriological spoilage characteristics of a canned, pasteurized crab cake mix product stored at various temperatures were investigated. A large number of bacteria, both mesophilic and psychrophilic, survived the pasteurization process. Bacillus and Micrococcus were found to predominate when the product was stored at 30 C (86 F) and 18 C (64 F), whereas Alcaligenes predominated at 2 C (36 F). The product was found to be free of Escherichia coli. Bacterial counts, trimethylamine nitrogen, volatile reducing substances, and ammonia determinations were evaluated as indices of quality for the product. Close correlation was observed between bacterial counts, volatile reducing substance values, and organoleptic tests when the product was stored at 30 C (86 F). The shelf-life of the product was approximately 6 months at 2 C (36 F), 4 days at 18 C (64 F), and 27 hr at 30 C (86 F). Canned, pasteurized crab cake mix is a relatively new product. There is little available information concerning its shelf-life and spoilage characteristics. The crab cake mix is commercially prepared by mixing crab meat with other ingredients, such as bread crumbs, eggs, mayonnaise, and other flavoring substances. The formula for the crab cake mix product is shown in Table 1. The product is packed at atmospheric pressure in 307 X 409 C enameled cans of 1 lb (454 g) net product capacity, pasteurized in a water bath at 85 to 87 C (185 to 190 F) for 110 min, and immediately cooled. The product is then kept at 0 to 5 C (32 to 41 F). A problem may arise when the consumer removes the product from cold storage in a food store. In some instances, it may take several hours before the product is returned to a refrigerator in the consumer's home, or the product might inadvertently be left unrefrigerated overnight. Bacteriological changes occur during this period, some of which are undesirable from the standpoint of product quality, and perhaps safety. This problem is of interest to consumers and the industry. Bacteriological spoilage of crab meat has been studied by Harris (15), Alford et al. (1), and Benarde (6). Pasteurization of crab meat in metal containers was first investigated by Tobin and McCleskey (27). They packed and pasteurized Present address: Office of Atom-iic Energy for Peace, Bangkhen, Bangkok, Thailand. crab meat at 15 psi for 5, 10, and 15 min. They reported that slight discoloration of the surface of the pasteurized meat was observed. Pasteurization of crab meat packed in cans in a water bath at the temperature below the boiling point of water was studied by Anzulovic and Reedy (4) and by Flynn and Tatro (14). These investigators found that pasteurization killed a large number of microorganisms including Escherichia coli, and that pasteurization prolonged the keeping quality of crab meat for a considerable time. Much research has been done by employing trimethylamine nitrogen (TMA-N) as an index of decomposition of marine fish and shellfish. Spinelli et al. (25) studied the relation of bacterial counts and increase in TMA-N with sensory evaluation in vacuum-packed king crab meat. They found that samples with TMA-N content exceeding 1.0 mg/100 g received a poor rating. There appeared to be a fair correlation between increase in bacterial counts, TMA-N content, and a decrease in sensory scores. Farber (12) claimed that the content of TMA-N was not a sensitive, reliable, or reproducible index of fish spoilage. Either the increase in TMA-N occurred during the latter stage of spoilage, or there was a variation in levels between species, or no appreciable increase took place. Volatile reducing substances (VRS) content has been found to be a useful index of freshness of fresh and canned fishery products, as indicated by Farber (11), and Farber and Ferro (13). The content of VRS was reported to correlate closely with organoleptic judgments. Burnett (8) devised a colorimetric method which used ammonia as an index of decomposition in fresh and frozen crab meat. This method is based upon the color reaction between ammonia, thymol, and bromine. The author reported that ammonia content increased uniformly and rapidly with spoilage, and that ammonia could be detected before spoilage was detected organoleptically. In crab cake mix there are ingredients of widely variable microbiological quality. The pasteurized product, therefore, is expected to have a variable spoilage pattern different from that of crab meat or fish. Generally accepted objective tests for ascertaining degree of spoilage of crab meat or fish may not be adequate for crab cake mix. The principal objectives of this investigation were: (i) to determine the shelf-life of the product at various temperatures by studying the bacteriological spoilage pattern through bacterial counts and characterization of the more prevalent bacteria in the product; (ii) to evaluate some of the existing objective tests as indices of quality and degree of spoilage of the crab cake mix; and (iii) to determine the correlation between odor and bacterial count, TMA content, VRS value, and amount of ammonia present in the product. MATERIALS AND METHODS Rate of heat penetration in canned crab cake mix was determined under commercial conditions. The method described by Alstrand and Ecklund (2) was used. Six 307 X 409 cans of 1 lb (454 g) net product capacity were used for the determination. The molded bakelite thermocouples were first placed at the geometrical centers of the test cans before filling. Filling was done in such a way that the thermocouple tips were imbedded in the product with no air space around them. The canned product was pasteurized in a steam-heated water bath, previously heated to 85 C (185 F), for 110 min. Center can temperatures of the product were read before the beginning of the pasteurization process and every 10 min throughout the heating and during the cooling operation, until the temperature returned to 60 C (140 F). A Brown po-tentiometer with automatic junction compensation was used to make temperature readings. The general plate count techniques were those outlined in Standard Methods for the Examination of Dairy Products (3). Throughout this study, BBL standard plate count (SPC) agar was used for plate counts. For estimating bacterial population in fresh crab meat and crab cake mix, SPC agar was dissolved in artificial sea water (24). Total plate counts of crab cake mix ingredients were determined. Samples of ingredients used for crab cake mix were obtained from a crab processing plant, placed aseptically in sterile bottles, and transported in ice to a laboratory where plate counts were made immediately. One gram of each of the ingredients was mixed with 9 ml of sterile water; the samples were further diluted and plated on SPC agar medium. All plates were prepared in triplicate and incubated at either 30 C (86 F), 18 C (64 F), or 2 C (36 F) to determine the microflora growing in the samples at each of three temperatures. Plates were incubated at 30 C (86 F) for 48 hr, at 18 C (64 F) for 4 days, and at 2 C (36 F) for 18 to 20 days before counting was made. The bacteriological spoilage pattern of the product was studied at 30 C (86 F) to represent the upper extremes of room temperature storage, at 18 C (64 F) to include a temperature intermediate between room and refrigerated storage temperatures, and at 2 C (36 F) to duplicate refrigerated storage temperature. The more prevalent colonies of bacteria that grew on plates incubated at the three temperatures were isolated and differentiated according to genera. Routine characterization tests were done according to Bergey's Manual of Determinative Bacteriology (7) and Guide to the Identification of the Genera of Bacteria (23). Flagella were stained by using the method of Leifson [E. Leifson, J. Bacteriol. 36:656 (Abstr.), 1938]. Oxidase activity was determined by the method of Kovacs (17). Chemical analysis of the pasteurized crab cake mix for moisture, crude fat, protein, and ash were performed in accordance with Association of Official Agricultural Chemists techniques (16). TMA-N content of pasteurized crab cake mix was determined colorimetrically as trimethylamine picrate by the method of Dyer (10). A Spectronic 20 (Bausch & Lomb) colorimeter was used. TMA-N values were read as percentage of transmittance from the TMA-N standard curve. VRS values were determined by the method of Lang et al. (18). The apparatus was slightly modified. It was found that simple test tubes (20 by 125 mm) could be used as sample vessels, since foaming was eliminated by using Antifoam A (Dow Chemical Co.). For each determination, 10 g of pasteurized crab cake mix, diluted 1:1 with distilled water, was weighed into an aeration vessel to which a few drops of the silicone antifoaming agent was added. Exactly 50 ml of 0.02 N KMnO4 in 1.0 N NaOH solution was pipetted into a reaction flask. The determination of VRS values was carried out at room temperature. Room air was drawn into the VRS apparatus at a rate of 100 liter/hr by means of a vacuum pump. The sample was aerated for 40 min. An air blank was also determined by using 10 VOL. 19,1970 ml of emulsified redistilled water, prepared by blending a few drops of the silicone antifoaming agent in 100 ml of redistilled water, in place of the sample. At the end of the aeration period, the reaction flask was removed. A 25-ml amount of 6 N H2S04 and 15 ml of 20% KI in 0.1% Na2CO3 were added. The liberated iodine was titrated with 0.025 N Na2S2O3 in 0.2% Na2CO, and 0.1% Na2B4O7. 10 HaO solution. Toward the end of the titration, several drops of 1% soluble starch in saturated NaCl solution was added as an indicator. VRS value in the sample was calculated by the equation: VRS value = titration -sample titration X normality Na2S2O3 X 1000/weight of sample in grams. The VRS value determined as described above was expressed in microequivalent GuEq) of KMnO4 per gram of sample. Ammonia content in the product was determined by the method of Burnett (8). Odor of pasteurized crab cake mix, stored at room temperature for various periods of time, was evaluated by a panel of 20 persons. The panel was untrained but was made familiar with the product. A nine-point hedonic scale was used in rating the sample. Odor evaluations were made by using four replicates. RESULTS The rate of heat penetration in crab cake mix commercially packed in 307 X 409 C enameled cans is shown in Fig. 1. Approximately 100 min was required for the center can temperature to reach 82 C (180 F), the desired temperature for pasteurization. The center can temperature was maintained at 82 C (180 F) for approximately 10 min before the cans were cooled. The pasteurization process at 85 to 88 C (185 to 190 F) for 110 m-reduced the bacterial counts in the product by a factor of approximately 20, from 6.0 X 105 to approximately 3.3 X 104 per g. Total plate counts of microorganisms in fresh crab meat and in other ingredients used for pre-APPL. MICRoBIoL. paring crab cake mix are shown in Table 2. Bread crumbs showed fairly large counts of both mesophilic and psychrophilic bacteria, whereas pepper and spice mix contained rather large numbers of only mesophilic bacteria. A large number of both mesophilic and psychrophilic bacteria were found in raw crab cake mix. Bacterial counts of the product stored at 30 C (86 F) are shown in Fig. 2. Bacterial counts from plates incubated at 30 C (86 F) and 18 C (64 F) increased with storage time, as might be expected. The increase in number of bacteria indicates typical growth curves which clearly show lag, log, and stationary phases. The product contained approximately 2 X 104 bacteria per g at zero storage time. The counts increased significantly after approximately 10 to 12 hr of storage time at 30 C. This can be explained by the fact that the rate of heat penetration in the product is slow. After 24 hr, the product showed a sign of slight spoilage, as observed by odor. Bacterial counts showed approximately 108 organisms per g. This indicates that the shelf-life of the product stored at 30 C (86 F) is in the neighborhood of 24 hr. When plates were incubated at 2 C (36 F), the number of bacteria, presumably psychrophiles, increased slightly up to approximately 12 hr of storage time at 30 C (86 F) and declined significantly thereafter. The increase in number of bacteria at the beginning indicated that the temperatures in the can of crab cake mix were suitable for psychrophilic bacteria to grow up to approximately 12 hr of storage time. The rapid decrease in the number of bacteria after 12 hr of storage is postulated to be due to the genetic behavior of the psychrophiles in the product. These psychrophiles presumably came from a cold marine environment in the Chesapeake Bay area. When exposed to high storage temperatures, they tended to die. Bacterial counts in the product stored at 18 C (64 F) for various lengths of time are shown in Fig. 3. When plates were incubated at each of the three incubation temperatures, both mesophilic and psychrophilic bacteria appeared to grow well. This is due to the fact that the storage temperature of 18 C (64 F) is within the growing ranges of both types of bacteria. However, the meso-philes seemed to grow better than the psychrophiles after 2 days of storage. After 4 days of storage at 18 C (64 F), an off odor was noted in the product. Bacterial count at this time showed approximately 7 X 107 organisms per g. Bacterial counts of the product stored at 2 C (36 F) were made at 1-month intervals for 6 months (Fig. 4). It was observed that the counts of both mesophilic and psychrophilic bacteria were fairly low, even after 6 months of storage time. A slight off odor was observed at the end of that period, but the product was still considered acceptable. The shelf-life of the product at 2 C (36 F) was considered to be 6 months. The next step in the bacteriological examination of pasteurized crab cake mix was to characterize bacteria isolated from the product stored at different temperatures. Attempts were made to characterize the more prevalent bacteria in the product stored at the three temperatures, namely, 30 C (86 F), 18 C (64 F), and 2 C (36 F). Three genera of bacteria were found to be prominent in pasteurized crab cake mix stored at the three temperatures (Table 3). They were Bacillus, Micrococcus, and Alcaligenes. Bacillus was characterized mainly according to its ability to survive the heat treatment of 65 C (149 F) for 30 min, and as catalase-positive, gram-positive, aerobic, spore-forming bacilli. Micrococcus was characterized mainly as gram-positive cocci in clumps, for producing acid from glucose oxidatively, and for having the ability to survive the heat treatment. Alcaligenes was characterized as gramnegative and oxidase-positive, for being motile with peritrichous flagella, for its inability to utilize carbohydrates, and for its ropy colonies. Attempts were made to detect E. coli in pas- Table 4. Determinations of pH in the product at each storage temperature indicate that, although pH of the samples in advanced stages of spoilage showed a lower value than that of fresh samples, the samples in between inhibited a nonuniform decrease of pH. No sig-nificant difference was found in the pH of samples in borderline stages of spoilage when compared with the pH of relatively fresh samples. Therefore, pH is not a reliable index of decomposition for pasteurized crab cake mix. Proximate composition data for pasteurized crab cake mix are presented in Table 5. The quantity of crude fat in the product is relatively high because a rather high proportion of mayonnaise is included in the formula. TMA-N content of pasteurized crab cake mix stored at 30 C (86 F) for various periods of time is shown in Fig. 5. It is evident that TMA-N content increased significantly after approximately 24 hr of storage, or when the product was approaching an advanced stage of spoilage. No significant difference was found in TMA-N content of the product stored at zero time, 6, 12, and 24 hr. These results show that TMA-N is Figure 5 also shows the VRS values of pasteurized crab cake mix stored at 30 C (86 F) for various lengths of time. It was observed that the VRS values increased progressively with an increase in storage time. The product showed VRS values of 25.85 ,uEq per g at zero storage time, which increased progressively with storage time. There was no significant difference in VRS values of samples stored for 0, 6, and 12 hr. VRS values increased significantly after 12 hr of storage at 86 F. The VRS value of the product at 24 hr of storage was not only significantly different from that at 12 hr of storage, but also significantly different from the product after 30 hr of storage. At 24 hr of storage, the product appeared to have a VRS value of 34.65 ,uEq per g and was still considered acceptable by the panel of judges. However, at 30 hr of storage, the VRS value of the product was 43.47 jAEq per g, and the product was considered unacceptable. The average odor scores for the product stored at 30 C (86 F) are also presented in Fig. 5. There was no significant difference between zero exposure and exposure to 30 C (86 F) for 6 hr. The product exposed for 12 hr at 30 C (86 F) showed no significant difference from that with zero exposure. The product exposed to 30 C for 24 hr was significantly different from any of the product exposed for shorter periods of time. This product, however, was also significantly different from the product exposed to 30 C for 30 hr which was judged unacceptable. This result indicates that the quality of pasteurized crab cake mix stored for 24 hr at room temperature was approaching the borderline stage of spoilage, although it was still considered acceptable. On the contrary, the product exposed to 30 C for 30 hr at room temperature was considered unacceptable. The borderline stage of spoilage, therefore, appeared to occur between 24 and 30 hr of storage time and was extrapolated to be approximately 27 hr at 30 C (86 F). A recovery test was employed to determine the efficiency of the colorimetric method used for detecting amount of ammonia (NH3) in the product. It was found that the determination of NH3 in the product was not reliable as a test for product quality, as the results were not reproducible. It was concluded that other substances in the product were probably extracted along with NH3 and interfered with the colorimetric determination. On the basis of the results given above, shelflife of the product was approximately 27 hr at 30 C (86 F), 4 days at 18 C (64 F), and 6 months at2C (36 F). DISCUSSION Bacterial counts have been used by many investigators to follow the deterioration of fish and shellfish. In this study, the product immediately after pasteurization had total plate counts of approximately 3.5 X 105 per g. When the product was stored at 30 C (86 F), the count increased to approximately 108 at the time of spoilage. The bacterial counts of the product stored at 30 C appeared to correlate with the degree of acceptability as indicated by odor. The bacterial counts, therefore, appeared to be a good index of spoilage of the product when stored at 30 C, as shown in Fig. 5. When the product was stored under refrigeration, the increase in bacterial counts showed no correlation with degree of spoilage. Figure 4 shows that approximately 107 bacteria per g were detected in the product held refrigerated [2 C (36 F)] for 6 months, when plates were incubated at 2 C. The count was typical for spoiled product exposed to room temperature, yet the product was still considered acceptable. These results indicate that the type of bacteria in the product is also important as an index of spoilage. The storage temperature of the product and the incubation temperature of the plates appeared to be significant when using bacterial count as an index of spoilage. A disadvantage of using plate count as an index of spoilage is the length of time involved in incubation. Bacteriological spoilage of the product is considerably retarded if the product is held refrigerated. Bacillus and Micrococcus showed optimal growth at 30 C (86 F). These bacteria are then expected to contribute very little to spoilage of the product at refrigerated storage temperatures. Alcaligenes, although showing optimal growth at 18 C (64 F), can grow well at refrigerated storage temperatures. Work done by Tobin et al. (26), Reay and Shewan (20), Campbell and Williams (9), and others indicated that the Pseudomonas-Achromobacter group of bacteria contributes significantly to the spoilage of most fishery products held at refrigerated storage temperatures. However, this group of bacteria was not found in pasteurized crab cake mix, probably as the consequence of severe heat treatment during pasteurization. This result agrees with that of Macaulay et al. (19), who reported that Alcaligenes had relatively higher heat resistance that the pseudomonads. No reports have been found in the literature indicating that Alcaligenes contributes significantly to the spoilage of fishery products. In this study, Alcaligenes was found to be abundant in pasteurized crab cake mix, but it did not appear to contribute significantly to the spoilage of the product at refrigerated storage temperatures [2 C (36 F)]. Probably for this reason, the product was considered acceptable after a 6month storage period. The TMA-N content in pasteurized crab cake mix was found not to be a sensitive index of spoilage of the product. The TMA-N content appeared to increase during the latter stages of spoilage. Early changes in quality of the product could not be detected by the TMA-N method. Determination of the VRS value of pasteurized crab cake mix can be carried out in less than 1 hr. The VRS values of the product were found to increase with length of the storage time. A close correlation was observed between the VRS values and organoleptic judgments. More significantly, quality changes in the product could be detected chemically by VRS values during the early stages of decomposition. VRS values, therefore, appear to provide a sensitive and reliable measure of the quality of pasteurized crab cake mix. The determination of ammonia in the crab cake mix product by a colorimetric method was not found reliable as an index of quality. APPL. MICROBIOL. Canned, pasteurized crab cake mix is prepared by mixing crab meat with other ingredients, some of which are heavily contaminated with microorganisms, such as the spices. The product is pasteurized in a water bath at 85 to 88 C (185 to 190 F) for 110 min. It is possible that Clostridium botulinum types A and B, which are common soil organisms, and types E and F, which have been isolated from fish and shellfish, may be present in the product and survive the pasteurization process. Moreover, the condition of the product in the can is favorable for growth of C. botulinum. As the product is a semisolid mass, anaerobic conditions could exist in it. C. botulinum type E is known to grow and produce toxin after 1 month of incubation at 38 F as reported by Schmidt et al. (21). To avoid the possibility of microbial growth and toxin production, the product would have to be stored at temperatures below 38 F. To prevent physicochemical changes caused by freezing, the storage temperature should be higher than the freezing temperature of any of the product components. Food storage temperatures in the range of about 28 to 38 F are rarely found in retail stores and homes. This presents a serious problem in marketing products like the one discussed here, and other similar meat and seafood products packed in hermetically sealed containers but not given a heat treatment that results in commercial sterilization.

v3-fos

2018-04-03T05:38:40.459Z

1970-04-01T00:00:00.000Z

43506476

s2

Chemical States of Bacterial Spores: Heat Resistance and Its Kinetics at Intermediate Water Activity Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is promi- nently nonlogarithmic for both chemical forms of the spore. Log survival varies ap-proximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, Ea, and Qlo cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed. Traditionally, bacterial spores have been considered to have two kinds of heat resistance, wet and dry. In general, wet has referred to test environments in which liquid-phase water was present, dry to measurements made in the absence of liquid water. Murrell and Scott (10) showed that in the absence of liquid water the water vapor activity (a,) or relative humidity of the test environment had a very large effect on spore heat resistance. They showed that heat resistance was maximal at environmental a, values in the 0.2 to 0.4 range. The heat resistance effects they obtained by variation of the relative humidity of the test environment were large, amounting to many-fold. Thus, those categories of spore heat resistance based on the water status of the test environment could no longer be confined to two, wet and dry, but must be increased to at least three general classes: wet, meaning in the presence of liquid water at a, values near 1; dry, meaning the total absence of water activity in the test environment; and third, resistance in the absence of liquid water but at an intermediate water vapor activity. In the last category, environmental water activity must be specified precisely because of the large variation of resistance with relative humidity. As a practical probability, most heat challenges of bacterial spores in the absence of liquid water would fall into the third category since quite rigorous precautions are required to insure the total absence of water activity (3). This inadequacy of the traditional term "dry heat" as a sufficient specification of a nonaqueous environment for spore heat resistance has also been pointed out by Angelotti et al. (5), Pflug and Schmidt (11), and others, but the term seems to persist for a description of environments with water activities from zero to near one so long as obvious liquid water is absent. Since Murrell and Scott's work (10), several recent studies of Angelotti (5), Fox and Pflug (7), Mullican and Hoffman (9), Hoffman, Gambill, and Buchanan (8), and Bruch and Smith (6) on so-called dry-heat resistance have included comments on the pertinence of these water activity effects to the results. Unfortunately, in none of these studies could the level of water activity at the lethal temperature be specified quantitatively. For ordinary aqueous heat resistance, we have shown that mature bacterial spores can exist in sensitive and resistant states (1,3,4). These different resistance states are prepared and interconverted by in vitro chemical pretreatments of the spores. The changes of aqueous heat resistance between the states can amount to about a thousand-fold. These changes or differences in heat resistance reside within the spores rather than being a response to environmental conditions during the heat resistance test since the changed resistance properties persist when the reagents used to effect the change of state are removed and the spore is transferred to a new environment for measurement of its heat resistance capacity. Thus, to the extent of three orders of magnitude, aqueous spore heat resistance for Bacillus stearothermophilus is an inducible property, and meaningful measurements of heat resistance potential are not possible without knowing the chemical state of a spore sample. Chemical events accompanying these changes of heat resistance state have been described (1,4). Later, we showed (3) that these same chemical states of the spore also have different resistances to strictly dry heat and we presented a method for dry-heat resistance measurements designed to avoid artifacts due to interference by unknown spore chemical state, water activity effects, and uncertain mechanical recovery of the dry-heat-challenged spores. Here we show that the sensitive and resistant states of spores also exist for heat resistance at intermediate environmental water activity. Characteristics of the nonlogarithmic survivor curves and their temperature dependence are given for each of the chemical states at an optimal environmental water activity. A method is presented for testing heat resistance at intermediate water activity. The method is designed to avoid the interfering factors mentioned above and also to furnish known and easily controlled environmental water activity at the test temperature. Some consequences of the existence of a maximum in the survivor versus a. relation are given for the temperature dependence of survival rates of spores encapsulated with a definite weight of water. MATERIALS AND METHODS Preparation of spore crop and the spore chemical states. Spores of B. stearothermophilus NCA 1518 were grown and cleaned as previously described (3). The preparation of the sensitive state, hydrogen form, spores, and the three preparations of the resistant state spores were the same as before (3). Method for measurement of heat resistance at controlled water activity. The general approach to water activity control was direct rather than through the use of humidity-controlling solutions. It was arranged to have a known weight of water in an otherwise evacuated sealed glass tube of known volume at a known temperature. The amounts of water required to give the desired a, at the lethal test temperature were taken from handbook tables of the properties of saturated steam. The ratio of space volume to spore weight should be chosen such that these amounts of water are large by comparison with any expected emission or uptake of water by the spores themselves. Borosilicate glass thermal death time (TDT) tubes (9 by 150 mm) were preconstricted to facilitate later flame sealing. The volume of the tubes up to the middle of the constriction (about 4 ml) was then determined by filling with water. The tubes were segregated into lots whose members had a volume variation range of less than 0.1 ml. The tubes were then filled and covered with distilled deionized water and autoclaved for 1 hr to leach out soluble alkali near the surface of the glass (2). The leaching process was repeated and the tubes were allowed to dry. Very small piles (200 Mg) of the "dry" (freeze dried) spores were weighed into the tubes. The amount (steam tables) of water required to give the desired water activity at the test temperature was then injected by a Hamilton microliter syringe as a drop on the inner wall of the tube. The area of the tube around the water drop was pressed against a piece of dry ice until the drop froze firmly. The open top of the tube was then quickly connected to a piece of gum-rubber tubing connected to, but sealed off by a spring clamp from, an oil pump vacuum. The TDT tube with the drop still frozen was then inserted up to the constriction into powdered dry ice in a Dewar flask. After about 1.5 min, the spring clamp to the preexisting oil pump vacuum was removed and vacuum was pulled for about 20 sec. The TDT tube still attached to the oil pump vacuum was then quickly sealed in a gas-oxygen flame and the seal was annealed in a smoky flame. The sealed TDT tube was then canted against the sharp edge of a slab of dry ice until needed for the lethal heating. The water drop can be moved quickly from place to place within the sealed tube by chilling a small area against dry ice. This serves as a vacuum test and was applied to each tube both before and after the lethal heating. Also, it was confirmed that no significant loss of water occurred during the above described vacuum sealing procedure. This was done by weighing, before and after drying, severed portions of previously prepared TDT tubes into which the water had been collected into one end by such spot chilling with dry ice. For the lethal heating step, the tubes were enclosed singly in flat wire cages (-1.5 by 10 by 12 cm) fabricated from 0.64-cm mesh wire (hardware wire cloth). Prior to insertion of the tubes into the cages, the chilled spot on the tube was thawed with fingers. The tubes were heated for measured times in an oil bath controlled to better than 0.01 C. After the elapsed heating time, the wire cages were quickly plunged into cool water for a few seconds. The cages were then set upright to a depth of about 2.54 cm in warm (-40 C) water for a few seconds to drive the condensed water away from the spores. The tubes were then removed from the cages, quickly wiped free of oil, and again slanted against the sharp edge of a slab of dry ice to collect all the condensed water into one spot away from the spores. If plating for survivor count was to be delayed to another day, the tubes at this point were stored in crushed dry ice. The Thomas Co., Catalog #4288, size A) and homogenized thoroughly enough to disperse them singly, as judged by direct microscopic count on oil-cleared membrane filters (12). The homogenized spore suspension was then further diluted as required with Tryptone broth for plating (Tryptone, 1%-glucose 0.5%-soluble starch, 0.1%). Incubation was for 2 days at 53 C. Direct microscopic counts were made on the Tryptone broth dilutions by the method of Snell (12) for each tube. The number of spores in the original little pile of spores was based on this direct count. This avoided needing to know and control the moisture content of the original spore sample and the problems of accurate weighing in the microgram range as well as that of correcting for possible loss in opening the evacuated tubes. After rinsing the spores out of the opened tubes, the volume of the two halves of each of the tubes was measured by filling (level meniscus) with water. With some practice in flame sealing and the preselection for volume uniformity mentioned above, the standard deviation of the measured volumes of the opened tubes within a lot was about 0.4% of the mean. However, the volume of both halves of each tube was always checked because of the obvious possibility of serious volume variation from the flame sealing operation. In actual practice, such variations did not occur. RESULTS Variation of survivors with a,. In Fig. 1 are plots for a given heat treatment of log survivors versus water activity for each chemical state of the spores. Log survivors fall off very steeply on each side of the moderately broad maximum. Fortunately, this maximum is at about the same position for each chemical state. The optimal water activity (0.28) chosen for this study was taken from the central portion of these maxima. The position of the maximum was not significantly changed when the heating time for the resistant state (preparation 3) was heated for 50 min at 150 C rather than 30 min, although the height of the maximum was reduced by about 2.5 log units. The water activity at the maximum agrees closely with that reported by Murrell and Scott (10) for their bithermal method. Survivor curve characteristics for the sensitive and resistant states. It is obvious from Fig. 2 and 3 that the log survivors versus heating time curves are indeed curves without the possibility of reasonable approximation by straight lines (log death). This curvilinearity holds for both chemical states but is most prominent in the log survivor curves for the resistant-state spores. Since the log survivor versus time plots are not linear, the D value concept is not applicable to heat inactiva- tion of spores under these conditions of intermediate water activity. For describing heat inactivation at a, = 0.28 under these conditions, an empirical expression, [(log initial count/g) -(log survivors/g)]a = ktmin + C, was chosen which gave a reasonable fit to the log survivor curves. The exponent a for VOL. 19,1970 each chemical state was chosen from the least squares slope of plots of log [(log initial count/g) -(log survivors/g)] versus log tmin. For the sensitive-state spores, a value of the exponent a of 0.58 was selected from such slopes. For the resistant state, a was taken as 0.33. The log initial count/g was 11.47 for the original unheated resistant-state preparation and 11.85 for the sensitive-state preparation. Thus, the expressions used to describe heat inactivation at a, 0.28 under these experimental conditions were (11.47 -log 5)0 33 = k'tmin + C for resistant state (1) (11.85 -log S)o -18 = k'tmin + C for sensitive state (2) where S is the number of survivors per gram of spores at heating time t in minutes, and k' and C are constants. As shown in Fig. 4, plotting (log Soriginallog S)0 33 for the resistant state and (log Soriginallog S)0 58 for the sensitive state versus time gave reasonably straight lines for several test temperatures. The least squaresdetermined slopes of these lines yielded coefficients of t in equations 1 and 2 for various temperatures. These coefficients of t were used as reaction velocity constants (k') in determining the temperature dependence of heat inactivation ( Table 1). For convenience, the Arrhenius plots for both states have been included in Fig. 5, but it should be noted that the rate expressions (equations 1 and 2) for the two states are different and so equal values of the rate constants (k') between states do not mean equal numbers of survivors will result from equal reaction times. 5, It is apparent that the temperature dependence of spore heat survival for each of the chemical states at a, 0.28 is similar to that for strictly dry heat resistance (3), even though the survivor curve shapes and the general level of heat sensitivity are quite different. The activation energies for both chemical states at both environmental humidity conditions of heat challenge (strictly dry and a, 0.28) are all about 40,000 cal. However, the log survivor versus time relations for strictly dry heat resistance are straight, that is, follow "logarithmic death," whereas these same relations for heat challenge at intermediate water activity of 0.28 are prominently curved. The general level of heat sensitivity of the chemical forms under strictly dry conditions is about like that for ordinary aqueous heat resistance. That for the same chemical forms at the intermediate water activity is much higher. DISCUSSION It is evident from Fig. 2 and 3 implications for experimental strategy which we have stressed previously (2, 3) for the a, = 0 and a, 1 conditions also hold for this intermediate humidity situation. The most important of these is that meaningful estimates of heat resistance potential cannot be made without knowing the chemical state of the spores. This knowledge of chemical state can be gained by converting deliberately a portion of the spore sample into each chemical state before measuring heat resistance at controlled water activity. As pointed out previously (2), if artifacts are to be avoided in the measurement of the aqueous heat resistance of the different spore chemical states, special care must be taken to use a testing medium inert with respect to its capacity to induce change of chemical state during the test. In fact, aqueous conditions can be arranged deliberately for the adaptation of spores to heat (4) by using a noninert suspending medium for heating. Nonaqueous heat resistance of spores, both dry and at intermediate water activity, is generally not subject to these changes of chemical state effects during the lethal heat challenge itself. However, we have pointed out how inadvertent changes of spore resistance state could occur by interaction of spores and their supporting surface in the preparatory phases of nonaqueous heat resistance measurement (3). Even in the case of aqueous heat resistance, it has been found possible to sensitize spores to heat in the presence of complex biological mixtures at their ordinary pH (2). It is not necessary to have acid conditions present during lethal heating of spores in such wet, complex biological mixtures, probably because free calcium ion is absent by virtue of the complexing action of the organic materials. Such sensitization to heat for facilitating spore killing in a practical situation should be straightforward in the case of nonaqueous heat resistance, providing the spores are or can be made physically available to the sensitizing reagent. Merely washing the sensitizing acid away before lethal heating would restore the original nonacid condition of an inert substrate on which the spores were supported. Conceivably, in some situations, the spores could be chemically sensitized before encapsulation in solid matrices. From the survivor versus a, plots of Fig. 1, some inferences may be drawn about the apparent temperature dependence of heat resistance of spores encapsulated within a given volume with a given weight (as contrasted with activity) of water which considerably exceeds the water-holding capacity of the enclosed spores, that is, where the encapsulating volume exceeds spore volume by several hundred-fold. In the absence of liquid water, specification of the water activity within an enclosed space requires knowledge of three terms: volume of space, weight of water, and temperature. Of course, if pure liquid water is known to be present at all temperatures of interest, a, is 1 by definition. For spores encapsulated in solid matrices such as plastics, crystals, tightly joined surfaces, etc., neither the volume of the encapsulating space nor the weight of water is likely to be known, leaving temperature as the only measurable variable, and water activity unknown. In Fig. 1 it may be seen that the depend- VOL. 19, 1970 569 on May 5, 2020 by guest http://aem.asm.org/ Downloaded from ence of survivors on water activity is quite sharp on either side of the maximum. This could make for an apparent unusual temperature dependence of heat resistance for spores encapsulated with a given weight of water since, when volume and weight of water (short of saturation) are fixed, a, varies inversely with temperature. If, for example, the particular tube used for the last point on Fig. 1 (Resistant State) had been heated at a temperature 100 higher, the spores would tend to be protected against the higher temperature. Instead of 0.50, the water activity at the 100 higher temperature would be only 0.39 for these encapsulating conditions. Thus, on the right side of the maximum, the lethal effect of an increase in heating temperature would tend to be compensated by the temperature-induced lowering of water activity. On the left side of the maximum in the a, versus survivor curve, the killing effect of raising the temperature would be reinforced by an a, change toward a less favorable (for survival) value. If, on the other hand, the tubes mentioned were heated at a 100 lower temperature the situation would be reversed with survival for tubes on the left side of the maximum being reinforced, whereas survival on the right side would be opposed by such a lowering of heating temperature. It is thus apparent that for such casually (with a given weight of water) encapsulated spores, it is not possible to determine temperature dependence of heat lethality in the usual sense. The temperature dependence of spore heat survival rate is commonly expressed as a z value, the temperature change required for a 10-fold change in survival rate. Such z values are considered to be a property of the spore in a given, presumably constant, environment. For example, in both aqueous and strictly dry heat environments water activity is essentially independent of temperature. However, as discussed above, when spores are encapsulated with a given weight of water, the kind of environment is also temperature dependent; water activity itself is a function of temperature. On the left side of the a, versus survivor maximum, apparent z values would be low, on the right side, high. Even in cases where the encapsulating fit around a spore is close with the resulting expected buffering of environmental water activity by the spore itself, the required lowering of equilibrium water content with increasing temperature would be expected to give some environmental disturbance. Only when volume and water content of the encapsulating space are known and manipulatable is it possible directly to determine a z value in the usual sense of its being a spore property. It appears that such considerations of environ-mental water activity temperature dependence can explain the anomalous z values reported by Angelotti et al. (5) for their paper system and possibly also for their highly torqued stainlesssteel-surface system. The cellulose moisture sorption isotherm and the geometry of the system used for drying the paper would indicate an appreciable final water content in the paper, probably at least 2%. Such a moisture content of 2%, when released by high temperature, would result in an a, of about 0.06 at 125 C, about 0.04 at 140 C. Such values lie on the steep, left side of the curves of Fig. 1 at which point lethality of heat is reinforced by temperature-induced lowering of aw, in other words, where apparent z values would be low. Doubling or halving, or more, the assumed value of 2% moisture content for the paper would not affect this qualitative argument for low z values since the a, would still be on the steep, left side of the a, versus survivor plot of Fig. 1. As we have reported (3), extremely low water activities still can elevate spore heat resistance over that at a strictly dry condition. Thus, the abnormally low z value operationally observed for the paper system appears to have been due to lack of environmental constancy during its measurement rather than being an expression of a temperature characteristic which could be interpreted as a "wet kill mechanism." Although sufficient information on environmental water activity is not available for the other unusual z value in the highly torqued stainless-steel system, its direction (high) would be expected for wetter-than-optimal conditions on the right side of the curves of Fig. 1. The cavities provided by the roughness of the no. 4 stainless finish of the washers should not have been large enough to accommodate spore size particles. One possible source for sufficient water in the environment would be water released from mechanical disintegration of most of the spores. Another recent report of an anomalously high (139 C) z value is that of Bruch and Smith (6) for spores on Teflon film. Here, the Kapton film interlayer was known to have an appreciable water-holding capacity and is a reasonable source for sufficient water to furnish a wetter-thanoptimal environment with the tendency to apparent high z values. Some inferences can be drawn on the question of efficient heating conditions for killing spores encapsulated under conditions in which environmental water activity is highly temperature dependent. For random encapsulating conditions in which the water activities of the cavities are distributed over the whole of a curve like that of Fig. 1 Murrell and Scott's comprehensive report (10) on the effect of water activity on spore heat resistance clearly established the large effect of environmental water activity on the heat resistance of several species of bacterial spores in the absence of liquid water. They showed that the optimal relative humidity for heat survival was in the range 0.2 to 0.4 a, and that in this optimum environmental a, region the great interspecies differences in heat resistance largely disappeared. There are several differences between our work here and that of Murrell and Scott (10). These differences fall in three principal categories: (i) experimental methods of water activity control, (ii) heat survivor curve shape, and (iii) knowledge and control of the chemical resistance state of the spore samples used. For the bulk of their data, Murrell and Scott used several conventional humidity-controlling solutions packaged with, but separated from, the spores by various two-container arrangements. In all these arrangements, the whole system was evacuated prior to final sealing so that equilibration of water activity would be speeded during lethal heating. They also made less extensive use of a bithermal method. Each of these variations of the methods was more than adequate to show the striking maximum in the curve relating a, and D value. However, the a, at the maximum was somewhat variable, and at particular a, values off the maximum, D value variations among the methods amounted to many-fold. It was recognized that the behavior of the controlling solutions at high temperature was somewhat uncertain and the water activity control was described as approximate. We have chosen a simpler, direct method of controlling water activity which insures that the gaseous water concentration in the spore-encapsulating environment is known unequivocally once equilibration to the lethal temperature has been achieved. The usable range is not limited to low temperatures. So long as the spore weight is kept sufficiently low that any water uptake or emission by the spores is negligible by comparison with the total water content of the whole encapsulating environment, there appears to be no reason to use humidity-controlling solutions. Both with our proposed method and that of Murrell and Scott, temperature lags will occur during the come-up and come-down periods of the lethal heating operation. Such temperature lags should be less with our method because of its smaller mass and the fact of two containers in the system of Murrell and Scott. We have made some measurements of the time required for disappearance of the water drop in our method and these times are measured in seconds. For example, in a 4-ml volume, 2.55 mg of water took 15 sec to disappear at 1480 from one spot. When the 2.55 mg of water was divided into three roughly equal spots on the wall of the evacuated tube, disappearance took about 5 sec. Water spread over the upper 1 to 2 cm of the tube was more difficult to observe precisely but disappeared very quickly. During the temperature come-up period with our method and also with arrangements 1 and 5 of Murrell and Scott (10), the spores temporarily would see a water activity lower than that intended. We did obtain increases of survival by spreading the water over the upper part of the tube as compared to letting it vaporize from a single spot, but the differences were not large. The second major difference between our work and that of Murrell and Scott lies in the radically different log survivor curve shapes. They reported a linear log survivor versus time relation and expressed survival in the usual D-value terms. Our log survivor curves, on the other hand, are, as pointed out above, definitely and prominently curvilinear and not susceptible to even rough approximation by the single D value concept. The reasons for this difference in the survival versus time relation are not definitely known. The experimental physical arrangements and means of water activity control are quite different for the two methods. Another possibility for the origin of the curve shape discrepancy may lie in the fact that Murrell and Scott determined resistance at temperatures which are, at least for the resistant form of spores like B. stearothermophilus, quite low. Under the conditions of the resultant long survival times such as curve # 2 of their Fig. 2, it would be difficult to distinguish between a curvilinear log survivor relation like our equation 1 and classic logarithmic death. Both expressions would have about the same appearance of linearity with low slope over the early portion of the log survivor drop. For example, in our Fig. 3, if only the data in the first three quarters of the time period were available, an interpretation of log death could easily be made. At such low temperatures, only in tests observing survival over several log units would the curvilinearity following the long lag period become evident. The third main category of difference between this work and that of Murrell and Scott lies in the control of the chemical state of the spore sample. Here, we converted the spores to a known heat-resistance state before measuring heat resistance. Murrell

v3-fos

2020-12-10T09:04:16.848Z

1970-01-01T00:00:00.000Z

237230886

s2

Detection of Agglutinins in Chickens Infected with JM Leukosis Virus Hemagglutinins for sheep red blood cells were detected in sera from JM virus-infected single-comb White Leghorn (susceptible S line) chickens. The agglutinins were not sedimented at 97,000 × g and were not affected by freezing and thawing, possibly indicating a soluble hemagglutinating factor. Agglutination titers were read in a relatively short period, after 4 hr of incubation at 4 C. The usefulness of this test for quick and low-cost screening of a large number of samples is indicated. Hemagglutinins for sheep red blood cells were detected in sera from JM virus-infected single-comb White Leghorn (susceptible S line) chickens. The agglutinins were not sedimented at 97,000 X g and were not affected by freezing and thawing, possibly indicating a soluble hemagglutinating factor. Agglutination titers were read in a relatively short period, after 4 hr of incubation at 4 C. The usefulness of this test for quick and low-cost screening of a large number of samples is indicated. The JM-type leukosis infections in poultry is common and widespread. The general concensus is that type n leukosis comprises the single most significant loss within and without the avian leukosis complex. Type H infections are highly contagious by direct and indirect contact (1). Chickens exposed parenterally or naturally to JM virus showed signs of paralysis within 4 to 6 weeks (4,5). Tumors of the gonads and dorsal root ganglia with ensuing mortality in many affected birds were also noted. A serological procedure for the detection of agglutinins in chickens with JM leukosis virus was investigated in this study. Both formalinized and fresh sheep red blood cells were agglutinated by fresh plasma from JM virus-infected chickens which showed signs of infection such as paralysis, tumors of the gonads, or dorsal root ganglia (or combinations of these signs). MATERALS AND METHODS Experimental animals. Single-comb White Leghorn (susceptible S line) chickens were inoculated intraperitoneally with JM-infected whole blood from birds of the same strain showing all signs of infection: paralysis and tumors of the gonads and dorsal root ganglia. Inocula. Whole blood was collected from infected paralyzed single-comb White Leghorn chickens with sterile citrated equipment. Infected tissue culture duck embryo fibroblast cells were lysed by three cycles of freezing and thawing in dry ice and 70% ethyl alcohol. Titrations. All titrations were done by using the microtiter technique of Sever et al. (3). The microtiter equipment was purchased from Cooke Engineering Co., Alexandria, Va. Phosphate-buffered saline (PBS), pH 6.4, with 1% crystalline bovine serum albumin (BSA), purchased from Sigma Chemical Co., St. Louis, Mo., was used as a diluent. A 50-pliter amount of the diluent was administered into each of the wells of disposable plastic U plates. Twofold dilutions of experimental or control chicken plasma were applied with 50-jliter dilution sticks. A 50-tliter amount of 0.125% (v/v) fresh sheep red blood cells or 0.5% (v/v) formalinized red blood cells was added to each well. Titers were read after 4 hr of incubation at 4 C. RESULTS Plasma from 42 paralyzed, JM virus-inoculated chickens was found to contain agglutinins to formalinized and fresh sheep red blood cells. Reciprocal titers as high as 256 were detected for the formalized cells and 64 for the fresh sheep red blood cells. Agglutinins were not detected in plasma from 25 control, isolated, and unexposed chickens. Centrifugation at 40,000 rev/min in a type 50 rotor of a model L Beckman preparative ultracentrifuge for 2 hr did not seem to affect the titer of the supernatant plasma, possibly indicating a soluble hemagglutinating factor. Twice freezing and thawing in dry ice and 70% ethyl alcohol did not lower the titer (Table 1). DISCUSSION The JM-type II leukosis infections in poultry are highly contagious by direct and indirect contact (1) or by the airborne route (6). Ovarian transmission of the virus to the offspring is a possibility. The signs of infection are a useful aid to diagnosis. However, earlier and quicker diagnosis is desirable. The Ouchterlony double-diffusion method is effective, but undiluted serum and 4 to 7 days are required to obtain results. Fluoresceinlabeled antibody procedures require the preparation of a fluorescein isothiocyanate-labeled antiserum for the direct method; with the sandwich technique, there is the hazard of nonspecificity of results. Hemagglutination can be used for widescale screening investigations.

v3-fos

2020-12-10T09:04:11.649Z

1970-12-01T00:00:00.000Z

237233421

s2

Effect of Trace Elements on Citric Acid Fermentation by Aspergillus niger Citric acid yields of 98.7% (sugar consumption basis) were reached in shaker flasks with mutant UV-ET-71-15 of Aspergillus niger in a resin-treated sucrose medium of the following composition (g/100 ml): sucrose, 14.0; NH4NO3, 0.20; KH2PO4, 0.10; MgSO4·7H2O, 0.025; and (mg/liter): FeSO4, 0.15 to 0.75; ZnSO4, 0.10; and CuSO4, 0.01. Yields of 75% were obtained in medium with resin-treated clarified syrup and 68% with ferrocyanide-treated blackstrap molasses. Optimal conditions included selection of appropriate pellets as inoculum at 3%, pH of 4.5, temperature at 30 C, agitation at 250 rev/min, and fermentation time of 8 days. The mutant tolerated high concentrations of trace elements. Among the problems associated with citric acid production by strains of Aspergillus niger under submerged growth conditions, those related to the control of the trace-element concentration play an important role. Recent contributors to this subject include Noguchi and Johnson (6), Millis et al. (5), and Clark (1). The optimal concentrations of such ions as Fe2+, Cu2+, Zn2, and Mn2+ vary so widely with the different strains under study that it is necessary to adjust the composition of the medium to avoid the inhibitory effects caused when these cations are present in toxic concentrations. Some of the methods we have previously used to solve this problem include (i) selection of tolerant strains, (ii) addition of substances to decrease the trace-element content of the fermentation media, (iii) treatment of the carbohydrate raw material to minimize inhibition effects, and (iv) development of potentially high-yielding mutants with a higher tolerance to trace elements. The present paper deals with the utilization of a high-yielding mutant of A. niger, previously obtained (10) by a combination of ultraviolet (UV) and ethyleneimine treatment, to study the effect of specific trace elements and phosphate on the citric acid fermentation of treated and untreated sugar sources. MATERIALS AND METHODS Raw materials. Refined commercial sugar from two types of sugar mills (producing sugars with relatively highor low-trace-element content), blackstrap molasses, and sugar cane liquor ("clarified syrup," International Society of Sugar Cane Technologists terminology) were studied. The blackstrap molasses was used either as received or after treatment with K4Fe(CN)6 by the method of Horitsu and Clark (3). The sucrose and clarified syrup, at a 14% concentration (sucrose basis), were passed through a glass column of Amberlite IR-120 prepared as follows. Appropriate amounts of the resin were washed in a glass column and were then washed successively with 2 N HCI, redistilled water, 1 N redistilled NH40H, and redistilled water. Strains. Aspergillus niger ATCC-233; the parent strain M-172, which was isolated from a Columbian soil (7); and two mutants, UV-6 and UV-ET-71-15, obtained from the latter (10) were transferred from the soil stock cultures through several successive potato dextrose agar slants to sucrose-salts agar slants. Mutant UV-ET-71-15 does not sporulate well in the usual media but does produce abundant spores in the following (g/100 ml): sucrose, 0.25; meat extract, 1.0; NaCl, 0.5; and agar, 1.5. After 4 to 6 days of incubation at 28 C, the spores were suspended in 10 ml of sterile distilled water to prepare the inoculum. Preparation of inoculum. The spores were harvested. Suspensions were prepared and standarized at a concentration of 107 to 2 X 107 and were then used to inoculate 50 ml of sterile seed media in 250ml Erlenmeyer flasks. These were incubated at 28 C on a rotary shaker at 250 rev/min for 17 to 24 hr. After that time, those flasks showing good formation of small, firm, spherical pellets were selected as inoculum for the fermentation media (3%, v/v). Seed media. Different seed media were tested and the following was finally selected (g/l00 ml): sucrose, 6; NH4NO3, 0.25; KH2PO4, 0.1; MgSO4.7H20, 0.025; agar, 0.2; and distilled water, 100 ml (pH 4.5). Fermentation media. Two previously reported media [A-1 and G (10)] and three basic media, as shown in Table 2, were first tested in a screening procedure by using the UV-6 mutant as a standard 888 for citric acid production in 250-ml shaker Erlenmeyer flasks at 250 rev/min with 50 ml of medium. When sucrose was not resin treated, either no trace elements or small quantities were added depending on the amounts initially present. All fermentation media were adjusted to pH 4.5 (except molasses at pH 6.8) and incubated at 30 C for 8 to 10 days. Estimation of growth and chemical determinations. Dry mycelial weight, pH, residual reducing sugars, citric acid production, and the presence of other acids were estimated by published methods (7,8,9). Citric acid yields were calculated as the anhydrous acid on the basis of sugar consumed. Oxalic and gluconic acids were detected by paper chromatography. The cations in the raw materials were determined by the methods of Noguchi and Johnson (6). The effect of resin treatment on the ion content of raw materials was estimated by means of absorption spectrophotometry. All cation values of the raw materials are expressed in ,ug/ml; those of culture media are in mg/liter. Table 1 gives the trace element content (tg/ml) of the raw materials used: commercial white sugar with relatively highor low-trace-element content, blackstrap molasses, and clarified syrup. Blackstrap molasses showed the highest content of trace elements. The concentration of the cations Fe2+, Zn2+, and Cu2+ tolerated by the UV-ET-71-15 mutant were 2.2, 3.8, and 0.5 mg/ liter, respectively, giving yields of citric acid from 3.0 to 5.0 g/100 ml in 10 days. It is apparent that this culture is highly tolerant of trace elements. RESULTS The UV-6 mutant was tested in the five different media given in Table 2. Untreated white sugar with a high content of trace metals was used in medium A-1; methanol and corn steep liquor were present in medium A-1; and small amounts of trace elements were in medium G. The highest yield was obtained in medium G. In the three other media, untreated white sugar with a low content of trace elements was added. These media differed in concentrations of NH4NO3, KH2PO4, and MgSO4. Results were inferior to those obtained with media A-1 and G, indicating that the addition of trace elements (either as such or present in corn steep liquor) resulted in better citric acid production. Similar results were obtained with mutant UV-ET-71-15. The trace elements of medium G were added to medium 3, and the citric acid yields of the parent strains (233 and M-172) and the two mutants (UV-6 and UV-ET-71-15) were determined. Data, as shown in Table 3, indicate the UV-ET-71-15 mutant to be superior with an average citric acid yield of 98 %. Instead of sugar, untreated molasses and mo- lasses treated with K4Fe(CN)6 were tested in medium 3 and in medium 3 with trace elements added. The yields obtained with mutants UV-6 and UV-ET-71-15 were higher in both media with the treated molasses; they were also higher when low concentrations of trace elements were added (Table 4). Resin treatment of sucrose, without added cations, also resulted in higher yields than the untreated sucrose in media 1 and 3 (Table 5); medium 3 was slightly superior. The effect of resin treatment on the traceelement content of sucrose and clarified syrup is shown in Table 6. Very small amounts of Fe2+ Cu2+, and Zn2+ remained after treatment, and Mn2+ was undetectable. The effect upon citric acid production of the addition of trace elements to media 1, 2, and 3 is shown ( Table 7). The tolerance of the UV-ET-71-15 mutant to high cation concentrations is again apparent (medium 2), and the best yields (62%) were once again obtained in medium 3. When the sucrose was resin treated and trace elements were added (Table 8), yields as high as 98 % in 8 days were reached in these modifications of either medium 1 or 3. Iron did not appear to be particularly critical for the UV-ET-71-15 mu- tant. In experiments with mutant UV-ET-71-15 and medium 3, neither oxalic nor gluconic acids Table 7. R = resin treatment; K = treatment with K4Fe(CN)6. bLow content of trace metals. c Values represent mean at 8 days in five tests. were detected by paper chromatography of the fermentation broths with the usual concentration of KH2PO4 (1.0 g/liter). However, when higher amounts of KH2PO4 (2.5 g/liter) were used, these other acids were present ( Table 9). The yields of citric acid were between 48.5 and 66.7% with untreated and resin-treated sucrose, respectively, at a KH2PO4 level of 0.5 g/liter. No cations were added in these cases. In other studies, blackstrap molasses and clarified syrup behaved in a similar manner as regards the response to prefermentation treatment. Several tests showed that the yields of citric acid in media with clarified syrup were better than molasses but not as good as white commercial grade cane sugar under the conditions of the experiments. The best yields obtained with these three substrates and the mutant UV-ET-71-15 are shown in Table 10. DISCUSSION Trace elements. The importance of trace elements in the submerged citric acid fermentation of raw commercial sugar substrates has been repeatedly pointed out (1,2,5,6,7,9). In the present investigation, great variability was found in the Fe2+, Zn2+, and Cu2+ content of the raw materials used. Reproducibility of the citric acid yields was not accomplished until the trace-element concentration of the medium was properly controlled. Although very small amounts of these trace elements were apparently necessary for good citric acid synthesis by the mutants under study, these cultures were capable of resisting unusually high cation concentrations (2.2, 3.8, and 0.5 mg of Fe", Zn2, and Cu2 per liter, respectively) in addition to those already present in the sugar substrate itself. The largest amounts of citric acid were, however, repeatedly obtained when only low concentrations of these three cations were added to the medium. Control of the trace element concentration has ensured reasonable reproducibility of the fermentation yields. Sugar treatment. The removal of excess trace elements by either ferrocyanide or resin treatment of sugar followed by the proper addition of trace elements was associated with good and reproducible citric acid yields. Our results confirm previous observations (3,4,6,7). Mutants. The development of mutants of A. niger allowed a higher average yield from all the crude substrates tested than the parent culture. Mutant UV-ET-71-15, obtained by a combined treatment of UV and ethyleneimine (10), was superior to the UV-6 mutant as well as to the parent strain. This culture has resisted higher concentrations of trace elements, thus resembling the mutants obtained by Gardner et al. (2) and Millis et al. (5), and has maintained its properties and characteristics after a storage period of 2 years in sterile soil. In the fermentation media selected, this mutant developed small pellets with a morphology similar to that described by Martin and Waters (4). Inoculum. The type and concentration of the inoculum was critical, with best results depending on the selection and number of pellets.

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2022-07-07T15:14:55.571Z

1970-01-01T00:00:00.000Z

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Screening Phytochemical and Study Insilico of Family Zingiberaceae as Anti-inflammatory Traditional medicine employs many ingredients that have been used for generations for treatment and are based on society's norms. These ingredients are referred to as jamu. The research aim was to determine the types and uses of medicinal plants, the active compound content, and the potential of traditional herbal medicine as a fever-lowering bio-computation. The traditional herbal formula was determined using a direct interview method combined with a purposive sampling technique in this study. We used the software for phytochemical screening and molecular docking. COX-2 was used to analyse proteins, and six ligands were used: Quercetin, Curcuminoid, Zingerone, Heyneanone, Zerumbone, and Sabinene. This study discovered 22 different types of medicinal plants in Bangselok village, East Java, Indonesia. The Zingiberaceae family is frequently used as an ingredient in herbal medicine. Ginger, Lempuyang, Bangle, Temu Mangga, Temu Putih, and Temu Giring contain flavonoid compounds. The docking results showed that Quercetin, Introduction Jamu is a traditional treatment in Indonesia based on the knowledge and wisdom of local communities, one of the people of Bangselok, Madura. Some plants are used as formulations of jamu making for traditional medicine (Saepudin et al., 2016). Combination of plant use on the making of Jamu based on disease type or patient complaints (Handayani, 2008). Jamu is used by the community to relieve some diseases like fever, cough, pain and to maintain body stamina. Steroids, tannins and saponins found in plants have a role in the traditional treatment success. COX-2 ιs induced by proinflammatory cytokines at the site of inflammation and enhanced COX-2-induced synthesis of prostaglandins stimulates cancer cell proliferation, promotes angiogenesis, inhibits apoptosis and increases metastatic potential. Fever is a response to an inflammatory process caused by infection with microorganisms, one of which is SARCoV -2 (Wu et al., 2020). Inflammatory signals can increase ACE 2 receptor expression in the lungs and lower the immune system, thereby increasing the risk of contracting COVID-19 (Smith et al., 2020). Virtual molecular screening analysis in several studies focused on the interaction of one active compound against COX 2 . This study uses a combination of several active compounds found in several plants that have a role in inhibiting the synthesis of inflammatory mediators. Molecular Docking provides an overview of the interaction, bonding, and affinity of ligands with substrates. In addition, it can be used to predict whether a compound has activity or not. The contribution of this study is complementary to the research data of medicinal plants, especially the Zingiberaceae family and their use and predicting new combinations of active compounds used as traditional herbal formulations as inflammatory agents. Materials and Methods Ethnobotany Observation This study was carried out in Bangselok district in Sumenep Region of the Republic of Indonesia. Data was collected through semistructured interviews with informants who knew or used plants as medicine (figure 1). This technique is commonly used in ethnobotanical studies (Pieroni et al., 2007). Interviews were conducted with selected informants, including about 10% of the total heads of family units (32 informants), to determine and explore traditional knowledge regarding medicinal plant species utilisation, usefulness, the utilised part, mode of preparation, or method of processing the plants. Respondents are traditional herbal medicine makers, age of the informants ranged from 50 to more than 65 years. The interview activities were carried out in their entirety using a questionnaire. Informant selection is based on the Snowball Sampling technique by determining the critical person. A key-person possesses strong power within society. The direction of the previous respondents determines the subsequent informants. Screening Phytochemical Medicinal Plants a. Plant Preparation Fresh plants were gathered from Bangselok, Madura. Then, plants were well cleaned and washed with water, then cut and dried using an oven 70 o C temperature. After this period, the plant's rhizome has been grinded and transformed to powder by a grinder. The powders were preserved in clean plastic containers, kept away from light, heat and moisture until use. b. Extraction Herbal Plant 1g of powdered rhizome was blended with 50 ml 70% ethanol solvents and agitation at room temperature until 1 hour. A 1 gram powdered rhizome was mixed with 50 mL 70% ethanol solvents and stirred at room temperature for 1 hour. The extracts were then collected and filtered using a 0.45-micron filter paper. The extracts were then concentrated at 40°C under decreased pressure in a rotating evaporator. The extracts were then weighted and stored at -20°C until they were used in the various assays (Zhang et al., 2018). c. Test for Flavonoid The test for flavonoid adopted is reported by Bone (2013) and Harborne (1998). Each sample (0.30 g) weighed into a beaker was extracted with 30 cm 3 of distilled water for 2 hours and filtered with Whatman filter paper number 42 (125 mm). To 10 cm 3 of the aqueous filtrate of each wood, the extract was added 5 cm 3 of 1.0 M dilute ammonia solution, followed by the addition of 5 cm 3 of concentrated tetraoxosulphate (VI) acid. The appearance of yellow colouration, which disappeared on standing, shows the presence of flavonoids. d. Test for Steroid The analytical method used is according to (Bone, 2013). Each sample (0.30 g) weighed into a beaker was mixed with 20 cm 3 of ethanol; the component was extracted for 2 hours. The ethanolic extract of each sample (5 cm 3 ) was added to 2 cm 3 acetic anhydride followed by 2 cm 3 of concentrated tetraoxosulphate (VI) acid. A violet to blue or green colour change in a sample(s) indicates the presence of steroids. e. Test for Tannins The analysis used was the method reported (Bone, 2013). Each wood powder sample (0.30 g) was weighed into a test tube and boiled for 10 minutes in a water bath containing 30 cm 3 of water. Filtration was carried out after boiling using number 42 (125 mm) Whatman filter paper. To 5 cm 3 of the filtrate were added, three drops of 0.1% ferric chloride. A brownish-green or a blue-black colouration showed a positive test. f. Test for Saponin The methodology is as reported (Bone, 2013). Distilled water (30 cm3) was added to wood powder samples (0.30 g) and boiled for 10 minutes in the water bath, and filtered using Whatman filter paper number 42 (125 mm). A mixture of distilled water (5 cm 3 ) and the filtrate (10 cm 3 ) was agitated vigorously for a stable, persistent froth. The formation of emulsion with three drops of olive oil showed a positive result. g. Prepare Protein and Ligand The proteins used in the 3D structure of COX-2 with 5F19 code are downloaded from the RCSB database (www.rcsb.org/). In contrast, the flavonoids ligands (Curcuminoid (Purwaningsih, 2014). Leaves are part of the readily available plant and are easy to mix (Hamzari, 2017). The people of Bangselok often use the rhizome part for herbal medicine because the rhizome contains many ingredients, including flavonoids, saponins, and essential oils (Muharrami et al., 2017). Gingerol can inhibit cyclooxygenase and lipoxygenase activity in arachidonic acid, causing a decrease in the number of prostaglandins and leukotrienes (Bone, 2013). Lempuyang (Zingiber zerumbet (L.) J. E. Smith) Lempuyang rhizome contains active compounds including kaempferol, quercetin, curcumin and essential oils (Zakaria et al., 2011). Lempuyang rhizome has anti-inflammatory activity, and Zingiber zerumbet extract can inhibit the action of the enzymes cyclooxygenase, lipoxygenase, myeloperoxidase and nitric oxide synthase (Jyothilakshmi, 2016). Bangle (Zingiber purpureum Roxb) Bangle rhizome has properties to reduce fever, pain and constipation. Bangle rhizome contains saponins, flavonoids, essential oils, tannins, steroids, triterpenoids, antioxidants and phenolic compounds (Chanwitheesuk et al., 2005). The phenolic compounds contained in bangle rhizomes can inhibit the activity of inhibiting inflammation by inhibiting the cyclooxygenase (COX) and lipoxygenase enzymes and inhibiting the release of histamine (Xu & Chang, 2007). Temu Mangga/Pao (Curcuma mangga Val.) Curcuma manga rhizome contains essential oils, alkaloids, flavonoids, tannins, and terpenoids ( Table 2). The phenolic compounds in mango ginger can induce glutathione-S-transferase (GST) activity, an enzyme that plays a role in the detoxification of foreign compounds in the body, and can suppress oxidative stress (Robert et al., 2012). Phytochemical Screening of Medicinal Plants used by The Community The efficacy of traditional herbal medicine depends on the processing method and the active compound content of traditional herbal medicine. The results of phytochemical screening of plants that make up traditional herbs to determine the content of flavonoids, steroids, tannins and saponins are presented in Table 3 it is shown that the reaction results in red, yellow or orange colour (Harborne, 1998). Flavonoid compounds are polar compounds because they have unsubstituted hydroxyl (-OH) groups to form hydrogen bonds. Besides that, flavonoids, which are polyphenolic compounds, can donate hydrogen atoms to free radical compounds, so the antioxidant activity of polyphenol compounds can be generated in neutralization reactions of free radicals or the termination of chain reactions that occur (Robert et al., 2012). In the extraction process, the active compounds in a plant are easily dissolved or bound by solvents according to their polarity so that a polar ethanol solution will more easily extract the flavonoids in the plant. Plant tissue Flavonoid compounds also reduce pain, antimicrobial, anti-bleeding, sedative, heart disease drugs, anti-diabetes, anti-bleeding, wound medicine, and anti-inflammatory (Ravindran & Babu, 2016). b. Stereoid The reaction of triterpenoids with Liebermann's reagent produces a red-purple colour, while steroids give a green-blue colour. Based on the ability of triterpenoid compounds and steroids to form colour by H 2 SO 4 in an anhydrous acetic acid solvent. The difference in colour produced by triterpenoids and steroids is due to different groups on the C-4 atom (Saleh & Mariana, 2011). Several steroid compounds include glucocorticoids as anti-inflammatory, allergies, fever, leukaemia and hypertension and cardenolide is a cardiac glucoside steroid used as a diuretic and heart-strengthening drug (Coutinho & Chapman, 2011 c. Tanin The test results showed that the herbal plants contained tannin compounds but the Curcuma mango Val and Curcuma zedoaria (Berg.) Roscoe did not show any tannin compounds. Tannin is a class of polyphenolic compounds commonly found in plants. Tannins can be defined as polyphenolic compounds with a molecular weight of more than 1000 g/mol and can form complex compounds with protein. Tannins have a sizeable biological role because they function as a protein depositor and metal gel. Therefore, tannins are predicted to act as biological antioxidants. (Redondo et al., 2014). d. Saponin Tanam Ginger (Zingiber officinale Roxb), Bangle (Zingiber purpureum Roxb), Temu Mangga/Pao (Curcuma mangga Val), Temu Giring (Curcuma heyneana Val. & V. Zijp) showed positive results for saponins. Saponins are generally in the form of glycosides, so they tend to be polar. The emergence of foam in the saponin test shows that saponins can become glucose and other compounds (Harborne, 1998). Saponin compounds can reduce superoxide by forming hydroperoxide intermediates, thereby preventing biomolecular damage by free radicals (Baev, 2013). Saponin compounds can provide antitussive and expectorant effects that can cure coughs. Saponins also have anti-inflammatory activity because they have been shown to inhibit the release of pro-inflammatory substances stimulated by lipopolysaccharides (Miladiyah et al., 2018). Figure 2. The NAG native ligand has a molecular weight of 221.1 g/mol, which requires an -6.7 kcal/mol energy to interact with COX 2 (domain B), while the native EDO ligand has a molecular weight of -3.4 kcal/mol (domain A). The NAG ligand native interaction occurs in the B domain (green colour), while the native EDO ligand occurs in the A domain (yellow colour) on the COX2 protein. The NAG ligand native carboxyl group forms hydrogen bonds with three amino acid residues (GLY45, CYS41, GLN461) with a 2.1-2.3 Å. Strong bonds have a length of 2.2 Å -2.5 Å, whereas most electrostatic bonds have a distance of 2.5 Å -3.2 Å, and weak electrostatic that are scattered have a distance of more than 3.2 Å (Fouzia & Salim, 2019). In general, the NAG native ligand binds to polar amino acid residues to be hydrophilic. Meanwhile, the native carboxyl group ligand EDO forms conventional hydrogen and hydrogen bonds with four amino acid residues (THR129, THR149, ASP125, ARG150) with a range of 2.2-3.0 Å. The EDO native ligand binds to polar or hydrophilic amino acid residues. Figure 2. The results of the interaction of the COX2 protein complex with NAG (a) and EDO (b) ligands. Results of Docking Complex COX 2 (Cyclooxygenase-2) with Flavonoids Compound Classes of flavonoid compounds that will interact with COX 2 protein include quercetin, curcuminoid, gingerol, sabinene, heyneanone. The results of the interaction between the flavonoid compound and COX 2 protein showed that curcuminoid, quercetin, gingerol, sabinene, native ligand NAG bind to the B domain (green colour) (figure 4a) with bond energies including -10.2 kcal/mol, -9, 7 kcal/mol, -6.2 kcal/mol, -5.9 kcal/mol, -6.8 kcal/mol ( Table 5). The heyneanone compound and the EDO native ligand bind to the COX 2 protein in the A domain (yellow) with a bond energy of -7.8 kcal/mol, -3.2 kcal/mol. Small binding affinity values predicted the best binding position (Shashank, 2013). The curcuminoid and quercetin binding sites overlap with the NAG native ligand on the amino acid residues of CYS41, GLN461, GLY45, which form hydrogen bonds and van der Waals forces ( Table 5). The similarity of the amino acid residues of the bonds formed indicates possible antiinflammatory activity (Miladiyah et al., 2018). It can be concluded that curcuminoid and quercetin can inhibit the performance of the NAG native ligand, thereby reducing prostaglandin synthesis as an inflammatory mediator. According to Fouzia & Salim (2019), natural molecules from thyme essential oil and flavonoids (Apigenin, Luteolin, Thymol, Carvacrol, Naringenine, and Chlorogenique) are highly recommended to treat inflammation by inhibition of the responsible enzyme. Heyneanone interacts with the native EDO ligand on the amino acid residues of ASP125 (Table 4) in the A domain. The carbon (C) atoms in the COX 2 alkyl group form hydrophobic bonds with curcuminoid ligands, quercetin, gingerol, heyneanone, and sabinene ( Figure 3). The bond position shows that the curcuminoid, quercetin, gingerol, heyneanone, and sabinene ligands not only bind to the nonpolar (hydrophobic) amino acid residues shown in the light brown area (positive hydrophobicity) but also bind to the polar amino acid residues (hydrophilic) which shown in the blue area (negative hydrophobicity) (Figure 3). The native NAG ligand forms hydrophobic bonds with nonpolar and polar amino acid residues, while the native EDO ligand forms hydrophobic bonds with polar (hydrophilic) amino acid residues (Figure 4b). A hydrophobic bond is a weak non-covalent bond that will occur at a distance above 3.7 Å. This distance causes the hydrophobic bond to become a fragile bond among other bonds, with a maximum distance of 3.5 Å as one factor that indicates a weak to a strong bond group. Results of Docking Complex COX 2 (Cyclooxygenase-2) with Flavonoid-Saponin Combination The COX 2 alkyl group forms hydrophobic bonds with the Curcuminoid, Quercetin, heyneanone, gingerol, sabinene, native ligand EDO and NAG ligand. The bond positions of curcuminoid, Quercetin, heyneanone, gingerol, sabinene, native EDO and NAG ligands tend to bind to non-polar (hydrophobic) amino acid residues shown in white / brown areas (positive hydrophobicity) (Figure 4). However, at the native end of the NAG, ligands bind. Polaric (hydrophilic) amino acid residues are shown in blue areas (negative hydrophobicity). Solubility plays a significant role in the therapeutic efficacy of flavonoids. The low solubility of flavonoid aglycones in water, coupled with its short residence time in the intestine and its lower absorption, does not allow humans to suffer acute toxic effects from the consumption of flavonoids, exception of a rare occurrence of allergy (Yunta, 2016). The flavonols are one such group with different compounds such as quercetin, kaempferol, myricetin, fisetin, and morin, exhibiting beneficial effects such as antiinflammatory and antioxidants, antiallergic, antiviral, as well as anticancer activity (Yunta, 2016). The saponins can inhibit the mediators of inflammation, such as histamine, serotonin and prostaglandins, along with their antioxidant property, which inhibits ROS formation and plays a significant role in inflammation (Murthy, 2006). Sabinene compounds interact in the A domain (yellow colour) with an -6.4 kcal/mol (Table 5). The binding affinity ginsenosides value of -9.6 kcal/mol is smaller than the native ligand EDO -3.3 kcal/mol, causing the native EDO ligand to move from domain A to domain B. Results of Docking Complex COX 2 (Cyclooxygenase -2 ) with Combination of Flavonoids-Steroids The results of the interaction of the combination of flavonoids (curcuminoid, quercetin, heyneanone, gingerol, sabinene) and steroids (Stigmasterol) with COX 2 show that the interaction of flavonoids (curcuminoid, quercetin, gingerol) occurs in domain B ( Figure 5) with a bond energy of -10.2 kcal/mol, -9.7 kcal/mol, -7.2 kcal/mol ( Table 7). The interaction of flavonoid groups (curcuminoid, quercetin) overlapping with the native ligand NAG on the amino acid residues of CYS41, GLN461, GLY45 in domain B by forming hydrogen bonds and van der Waals forces. The interaction of stigmasterol, heyneanone, sabinene with COX 2 occurred in domain A ( Figure 5) with bond energies of -8.5 kcal/mol, -7.8 kcal/mol, -6.3 kcal/mol ( Table 6). The difference between curcuminoid and quercetin amino acid residues with the native ligand EDO amino acid residues (PRO218, GLN454, THR212) causes the native EDO ligand to be unable to interact with curcuminoids and quercetin in domain B. The transfer of the native EDO ligand domain from domain A to domain B is caused by the difference between binding affinity and stigmasterol (Table 6). The carbon (C) atom in the COX 2 alkyl group forms hydrophobic interactions with curcuminoids, quercetin which overlaps with the NAG native ligand because it has the same amino acid residue bonds. The interaction position of non-polar (hydrophobic) amino acid residues binds to curcumonoids, quercetin, and the NAG native ligand shown in white / brown areas ( Figure 5). Sabinene, stigmasterol binds to non-polar (hydrophobic) amino acid residues which are shown in brown areas ( Figure 5), whereas native EDO ligands tend to bind to polar (hydrophilic) amino acid residues which are shown in blue areas. The similarity of interactions of amino acid residues with ligands is related to the characteristics, solubility and boiling point of the ligand complex (Them et al., 2019). Results of Docking Complex COX 2 (Cyclooxygenase -2 ) with the Combination of Flavonoids-Tannin The carbon (C) atom in the COX 2 alkyl group forms hydrophobic interactions with curcuminoids, quercetin which overlaps with the NAG native ligand because it has the same amino acid residue bonds shown in the white / brown area ( Figure 6). Sabinene and tannins bind to non-polar (hydrophobic) amino acid residues that are shown in brown areas ( Figure 6). The interaction results of the combination of flavonoids (curcuminoid, quercetin, gingerol, heyneanone, sabinene) and tannins with COX2 show that the interaction of curcuminoid, quercetin, overlap with native NAG ligands on the amino acid residues GLY45, CYS41, GLN461 in the B domain with a bond energy of -10 kcal/mol, -9.7 kcal/mol, -6.8 kcal/mol (Table 7). Tanning overlapping interactions with sabinene on the amino acid residues TRY385, TRP387 in domain A with bond energies of -9.0 kcal/mol, -6.4 kcal/mol. The interaction of heyneanone, gingerol, and native EDO ligand with COX2 occurred in domain A with bond energies of -7.8 kcal.mol, 7.2 kcal/mol, -3.4 kcal/mol (Table 7). The results of the interaction of the combination of flavonoids, steroids, tannins, saponins with COX 2 indicate that curcuminoids, quercetin, and tannins interact in the B domain (green) with a bond energy of -10.2 kcal/mol, -9.7 kcal/mol, -8, 7 kcal/mol (Table 9). they overlap between stigmasterol and ginsenosides in domains A and B with a bond energy of -9.7 kcal/mol and -9.6 kcal/mol, overlapping between the native ligand NAG and gingerol in domain A with a bond energy of -6.9 kcal/mol, 7.1 kcal/mol. These interactions among molecules of a homogeneous substance are responsible for determining their bulk properties: melting point, boiling point, viscosity, surface tension, etc (table 8, figure 7). The interactions between molecules of two different substances significantly influence the rates of chemical reactions, the effectiveness of chromatographic separations, and molecular recognition in biological processes (Murthy, 2006). The virtual molecular screening analysis carried out in this study was based on a combination of active compounds present in medicinal plants as ingredients for traditional herbal medicine. Based on the results of molecular screening analysis, it can be predicted that the combination of plant formulations is anti-inflammatory, among others, formulation of 1 ginger-temu manga, two ginger-bangle, three temu giring-bangle, temu giring-temu mango. Traditional herbal medicine is made from one or a mixture of medicinal plants. Traditional herbal medicine is Indonesian society's local wisdom, which is used to maintain stamina or relieve symptoms of diseases caused by microorganism infection or malfunctioning of the body's metabolism, such as coughing, fever, joint pain, blood circulation, anemia, etc. The active compounds found in plants as ingredients for herbal medicine have an essential role in relieving disease symptoms. The right combination of active compounds can relieve symptoms of a disease or inhibit the synthesis of specific proteins. The combination of flavonoids-tannins, flavonoids-steroids, flavonoids-saponins can inhibit prostaglandin synthesis as an inflammatory mediator through the arachidonic acid pathway. This research needs to be followed up on other proteins as inflammatory mediators, such as leukotrienes, histamine, nitric oxide, cytokines (IL-1, Tumor Necrosis Factor (TNF), interferon (INF) -C, IL-6, IL-12, and IL-18, bradykinin, serotonin or proinflammatory agents, such as interleukin-1 β (IL-1 β) and tumor necrosis factor (TNF) α. Besides inhibiting the synthesis of inflammatory mediators, herbal medicine also acts as an antioxidant. According to Them (Them et al., 2019), that the combination of alkaloids, saponins and flavonoids in the leaves of Launaea sarmentosa has a high antioxidant activity of 24.15 µg / mL compared to vitamin C of 6.45 µg / mL in the DPPH test. Based on the antioxidant activity, bioinorganic complexes (transition metal active compounds) have more potent free radical scavenging activity than the combination of active compounds or single compounds (Jabeen et al, 2017). The transition metals Fe, Mn, and Cu, are types of transition metals that naturally bond with flavonoids in the form of complex compounds (Kasprzak et al,. 2015). In order to comprehensively determine the efficacy of traditional herbal medicine, it is necessary to carry out a detailed characterization in terms of its crystal structure, bioinorganic complexes, the presence of functional groups of inactive compounds in traditional herbal medicine. Conclusion Based on the findings and discussions, it is possible to conclude that there are 22 medicinal plant inventories in Bangselok Village. In the Zingiberaceae family, phytochemical analysis revealed that the flavonoid group was dominant over steroids, tannins, and saponins. Based on the active compound content, the virtual screening results of traditional herbal formulation combinations revealed that the combination of flavonoids-steroids, flavonoids-tannins, and flavonoids-saponins is more recommended as an anti-inflammatory because it has similar interactions with amino acid residues, binding affinity values, and hydrophobicity bonds with the native NAG and EDO ligands.

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2020-12-10T09:04:10.952Z

1970-08-01T00:00:00.000Z

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Isolation of Vibrio parahaemolyticus from the Processed Meat of Chesapeake Bay Blue Crabs A method for the recovery of Vibrio parahaemolyticus from seafoods is described. By this procedure, a total of 56 biochemically positive cultures of V. parahaemolyticus were recovered from market samples of Chesapeake Bay processed blue crab (cooked, picked, packed, and refrigerated meat). All of the isolates were tested serologically, and 22 strains were serotyped according to the schema of Sakazaki as follows: K3, K5, K28, K31, K36, K37, K39, K43, and K44. These results indicate the broad distribution of these specific serotypes in a seafood harvested from the Chesapeake Bay. Japanese seafoods or marine environments. Epidemiological studies suggest that marine habitats are the most probable sources of this enteric pathogen. Further, the Japanese literature cites no evidence that V. parahaemolyticus may induce infections in man other than the typical enteropathogenesis ascribed to it. However, epidemiological data and laboratory isolations gathered by R. R. Weaver of the National Communicable Disease Center (NCDC) in the United States indicate that this organism may also be the cause of nonenteric diseases in man. Recent marine ecological studies have revealed the presence of this organism in Hong Kong, Taiwan, Singapore, Philippines, Hawaii, and Germany (4). The first isolation of V. parahaemolyticus in the U.S. was reported from the sediments and marine animals of the West Coast (2). Organisms purporting to resemble V. parahaemolyticus were isolated from the frozen sediments and waters of the Gulf and south Atlantic Coasts (8); more recently, V. parahaemolyticus was isolated from Chesapeake Bay crabs retained in commercial shedding tanks (3). The present paper describes an isolation procedure and the serotypes of V. parahaemolyticus recovered from the cooked, picked, packed, and refrigerated meat of Chesapeake Bay blue crabs offered for the retail market. MATERIALS AND METHODS Sample. Three samples of processed blue crab (Callinectes sapidus) were collected from three different firms on the Eastern shore of Maryland. The samples, which consisted of the regular white body meat, were thoroughly iced and transmitted to the laboratory for analysis. Each sample comprised 20 subsamples, each of which contained 150 g of crabmeat obtained from separate 1-lb retail cans. Thus, a total of 60 subsamples were individually examined for V. parahaemolyticus. Cultures Dilutions. Fifty gram-portions of crabmeat were added to 450-ml amounts of 3% NaCl diluent to make a 1:10 dilution. Additional 10-fold dilutions through 1:10,000 were also prepared. One-milliliter amounts of all dilutions were inoculated into parallel enrichment broths. In instances where 1-g portions of crabmeat were to be examined, 10 ml of the 1:10 dilutions were inoculated into double strength enrichment broths. Media. The two enrichment broths were glucosesalt-Teepol broth and salt-colistin broth (1), which were inoculated and incubated for 18 hr at 35 C. A loopful of each culture was streaked onto thiosulfatecitrate-bile salts-sucrose agar (1). This medium was incubated ovemight at 35 C, and any light bluishappearing colonies were subcultured onto 3% salttriple sugar-iron agar slants (1). Tubes having an alkaline slant and acid butt and being negative for gas V. PARAHAEMOLYTl( and H2S production were tested biochemically by the standard procedures of this laboratory (1). Serology. The serological identification of the isolates was accomplished by means of a slide agglutination test, employing commercially available V. parahaemolyticus K antisera (Nichimen Co., New York). The 18-hr growth from a 3% NaCl-Trypticasesoy agar slant (1) was suspended in a small volume of 3% NaCl diluent. A drop of suspension was placed on a slide, and an equal volume of a polyvalent or monovalent antiserum was added. The combination was mixed by rocking for 30 seconds to determine whether an agglutination reaction would occur. The positive reactions were recorded from 1+ to 4+, depending upon a visual estimate of the degree of agglutination. Serotypes were positively identified on the basis of 2+ or better agglutination reactions with a specific K antiserum. Those isolates that did not react with a specific K antiserum or reacted with more than one antiserum were considered nontypable. RESULTS AND DISCUSSION A total of 56 strains of V. parahaemolyticus were recovered from the 60 subsamples which were analyzed. Table 1 lists the biochemical response of these isolates compared with the response of a reference culture (S AK 5) of V. parahaemolyticus. The SAK 5 culture was characteristic of 27 known V. parahaemolyticus strains obtained from the Japanese and previously tested for biochemical reactions in our laboratory. The 56 biochemically positive isolates of V. parahaemolyticus were recovered from the three crab samples in the pattern ( Table 2). Most of them were recovered from the undiluted (1 g) and the 0.1-g portions. In the first crab sample, 6 of 20 subsamples tested were positive and gave rise to 12 cultures which were confirmed biochemically as V. parahaemolyticus, but which were not serologically typable. The remaining two crab samples, however, contained both biochemically and serologically typable cultures of V. parahaemolyticus. Thirty-four strains of the 56 isolates gave a positive response in the K polygroup antisera. Of these, five failed to react in the monovalent K antisera, seven strains reacted with more than one antiserum, and 22 strains (39.4%) responded monospecifically with fairly strong agglutination reactions (2 + to the NCDC cultures of R. R. Weaver which were originally isolated in the U. S. from human blood infections, lesions, and abscesses and the Liston-Baross isolates from Puget Sound oysters. The NCDC cultures gave the following serological results: strain A5704 agglutinated with serum K25 (4+), strain A8198 agglutinated with serum K33 (4+), and strain A6202 agglutinated with serum K17 (4+). The last strain was found by Twedt et al. (7) to agglutinate with sera K17 (3 +) as well as K3 (2 +). The Puget Sound oyster isolates OY-8 and P-OY4 both gave a K3 (4+) VOL. 20, 1970 reaction. Both of these groups of organisms were also biochemically reconfirmed as V. parahaemolyticus. Our findings indicate that 61 % of our isolates are serologically nontypable although, biochemically, these are identical with the biotype described by the Japanese (5). Nakanishi et al. (4) reported similar findings for 18%o of their isolates from Baltic Sea marine animals. There are obviously many V. parahaemolyticus serotypes from marine sources which are still unclassified. A compilation of findings outside Japan indicates that 26 serotypes (63.5 %) of the 41 specific K serotypes of Sakazaki et al. (6) have been identified thus far. These Western isolates include such categories as types recovered from marine sources only, types recovered from Western but not from Japanese marine sources, types recovered from both marine and human sources, and types recovered from human nonenteric sources. It is very likely that all of the described Japanese serotypes will be recovered from Western marine sources in the near future. Thus far, there have been no reported cases of gastroenteritis in the West in which V. para-LITERATURE CITED

v3-fos

2020-12-10T09:04:11.460Z

1970-07-01T00:00:00.000Z

237229433

s2

Action of Bacterial Growth on the Sarcoplasmic and Urea-Soluble Proteins from Muscle Comparisons of the starch-gel patterns of uninoculated aseptic control samples from rabbit and pig muscle with similar samples inoculated and incubated with Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaciens, and Kurthia zopfii were made. Results indicated that C. perfringens caused extensive alteration in the proteins or enzymes, or both, of the sarcoplasmic fraction of porcine muscle, whereas S. enteritidis and S. faecalis caused complete breakdown of only myoglobin. Neither A. liquefaciens nor K. zopfii showed any measurable amount of proteolysis in the sarcoplasmic fraction from pig muscle. Although some of the bands in the starch-gel pattern of rabbit muscle decreased in size and intensity of staining, complete proteolysis of any protein fraction was absent for all test organisms. The disc-gel patterns of the 8 m urea-soluble proteins showed that C. perfringens caused extensive proteolysis in pig muscle and a lesser extent of proteolysis in rabbit muscle. None of the other organisms utilized in this study had any measurable effect upon the urea-soluble proteins. In addition, a simple procedure for aseptic isolation of muscle samples for studying meat spoilage is outlined. Results indicate that careful sanitation and cleanliness will give suitable samples for meat spoilage investigations. Several workers (4,6) have concluded that meat spoilage microorganisms are not capable of degrading and utilizing meat and fish muscle proteins for growth. Recently, Ockerman et al. (7) demonstrated that a marked decline in the sarcoplasmic protein fraction from sterile beef muscle occurred after inoculation and incubation with Pseudomonas and Achromobacter microorganisms. Similar results with pig muscle have also been recently reported by Charpentier (2). Further support for the theory that bacteria can degrade and utilize the sarcoplasmic fraction from muscle has been obtained in our laboratory for several different species and strains of microorganisms (T. Hasegawa et al., J. Food Sci. 35: in press, 1970). However, corollary experiments involving the myofibrillar proteins from rabbit and pig muscle using a mixed culture of microorganisms from spoiled hamburger as well as pure culture experiments with several specific microbial strains and species have shown 35: in press, 1970). These studies suggest that further work is needed to ascertain the effects of various meat spoilage microorganisms on the different muscle fractions. The present investigation was undertaken to determine whether certain other species and strains of bacteria could cause proteolysis in the sarcoplasmic fraction of pig and rabbit muscle. Such studies are important in ascertaining the role different microorganisms play in meat spoilage and the mechanism whereby spoilage occurs. MATERIALS AND METHODS Sampling procedures. Seven market weight pigs [180 to 230 lb (ca. 81 to 104 kg)] produced at the Michigan State University Swine Farm were slaughtered individually over a 7-month period in a conventional manner, except that special techniques were utilized to obtain essentially aseptic muscle samples. 117 The area of the neck utilized for sticking was scrubbed thoroughly with hexachlorophene bactericidal soap. Sticking was carried out with a knife sterilized by boiling in hot water. After conventional dehairing and eviscerating, the unsplit carcass was rinsed with absolute alcohol and chilled at 1 to 3 C for 20 hr. After chilling, the alcohol rinse was repeated, and the carcass was laid on a table covered with Kraft paper in a clean room free from excessive air currents. The shoulder and ham sections were removed and the middle portion was positioned so that the dorsal midline was accessible. A cut was then made along the dorsal midline of the backfat-loin with a sterilized knife. Subsequent cuts were then made perpendicular to the midline. The backfat was stripped off, and slices about 3 cm in thickness were aseptically excised from the exposed M. longissimuis and placed in sterile containers. Both lonzgissimuls muscles were excised and handled in the same manner. The excised samples were then ground through a sterile prechilled grinder and placed in sterilized sample jars. The operator wore sterile disposable gloves during the entire sampling procedure. Rabbit carcasses were handled in essentially the same way. The carcasses were washed with absolute alcohol immediately after skinning, and the muscles were removed with a sterile knife and tweezers. The muscles were minced in a presterilized grinder and placed in sterilized beakers. Inoculation. Cultures of Salmon2ella enteritidis (13076) and Kurtl-hia zopfii (6900) were obtained from the American Type Culture Collection, Rockville, Md., whereas the Clostridium perfringens, A. liquefacienis, and Streptococclis fiaecalis cultures came from the collection of the Food Science Department at Michigan State University. Except for C. perfringens, all species were grown on all purpose plus Tween (APT) broth and, after incubation at 23 to 25 C for 48 hr, were diluted either 50-(K. zopfii) or 100-fold in buffered dilution blanks just before addition to the meat. C. perfriilgenis cultures were grown in fluid thioglycolate medium and, after 24 hr of incubation at 37 C, were diluted 100-fold in dilute peptone (0.5%) plus cysteine (0.02%) solution. The diluted cultures were added to the muscle either as it was ground (pork) or as a preground mince (rabbit). After mixing thoroughly, the inoculated samples for each treatment were divided into three or four approximately equal portions, transferred to presterilized sample jars, and covered loosely with sterilized lids. Control samples. Uninoculated control samples were handled in the same way except they were not inoculated. Thus, control samples were available for analysis at the same time as similar inoculated samples. By comparison of results of control and inoculated samples at the end of each storage period, it was possible to differentiate between the changes resulting from microbial breakdown and those resulting from incubation per se. Incubation. Samples inoculated with all microorganisms, except for C. perfrinigenis, were incubated at 10 C for 0, 8, and 20 days. The samples inoculated with C. perfrinigelns were analyzed immediately (0 days) and again after incubation at 30 C for 4 and 8 days. Bacterial counts. Plate counts were performed for all control and inoculated samples both before and after incubation. Except in the case of C. perfrin!geis, for which the inoculated and control samples were incubated at 30 C, bacterial numbers for all samples were assessed by recommended techniques (1). APT agar was used for the plating medium. The plates were inoculated aseptically and incubated for 48 hr at 23 to 25 C before counting. In a few instances in which the colonies were indistinct, the plates were held for an additional 24 hr to give 72 hr of incubation. For C. perfrin1gens, both the APT method and an anaerobic plating method were used for bacterial counts on both controls and inoculated samples. The anaerobic method utilized a sterile peptone (0.5%) plus cysteine (0.02%) solution as the diluent. Plates were poured with sulfadiazine-polymyxin sulfate (SPS) agar and incubated at 35 C for 48 hr in an anaerobic incubator. The anaerobic atmosphere was obtained by vacuumizing and nitrogen flushing of a sealed incubator cabinet (National Appliance Co., Portland, Oregon) three times. All control samples were treated identically to the inoculated samples except they were not inoculated. Extraction of the proteins. All extraction procedures were carried out at approximately 3 C. Four volumes of 0.3 M sucrose-0.01 M KCl-0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.6 were added to a weighed sample in a Waring blendor and homogenized at high speed for 1 min. The homogenate was centrifuged at 19,400 X g for 15 min. The supernatant, which consisted of the sarcoplasmic proteins, was retained for starch-gel analysis (8,9). The remaining precipitate was washed with 12 volumes of the sucrose-KCl-Tris solution and centrifuged as before. The supernatant was discarded, and 6 volumes of Weber-Edsall solution (0.6 M KCI-0.01 M K2CO3-0.04 M KHCO3) was added to the precipitate and then stored for 24 hr. Then 18 volumes of Weber-Edsall solution was added, and the suspension was mixed with the aid of a magnetic stirrer. The solution was centrifuged at 28,600 X g for 30 min. The supernatant, which contained the saltsoluble (myofibrillar) proteins, was discarded, and the residue was washed with 12 volumes of Weber-Edsall solution to more completely remove any residual salt-soluble proteins. After centrifuging as in the previous step, the supernatant was discarded. The precipitate was homogenized with 4 volumes of 8 M urea solution. Centrifugation was carried out at 28,600 X g for 30 min, and the supernatant, which contained the urea-soluble proteins, was retained for disc-gel electrophoresis. The precipitate was discarded. Protein analysis. Analysis for protein was carried out before inoculation, after inoculation, and after incubation. The procedure used was the indanetrione hydrate method described by Jacobs (5). Since the quantities of proteins in the various fractions were so small, the standard deviations were relatively large and are not reported herein. Starch-gel electrophoresis. The sarcoplasmic protein fractions were subjected to starch-gel electrophoresis by using a modification of the methods described by Scopes (8,9). The outer solution was made up of 60 mM Tris and 50 mm boric acid (pH 8.6 at 5 C), whereas the inner gel contained 12 mm Tris and 2 mM diethylenetriamine-pentaacetic acid (pH 8.25 at 5 C). Electrophoresis was carried out on the horizontal axis at 400 v (approx 36 v/cm) for 6.0 to 6.5 hr at 8 to 12 ma in a cold (3 C) room. After slicing, the gels were stained with 0.18% Buffalo Black (NBR-naphthol blue black) in methanolacetic acid-water (5: 5: 1, v/v), washed with glycerinwater-methanol-acetic acid (1:5:5:1, v/v), and photographed. Disc-gel electrophoresis. Acrylamide disc-gel electrophoresis was performed on the 8 M urea-soluble protein fraction by using minor modifications of the method of Davis (3). The gels were generally made in tubes having an inner diameter of 5 mm. The 6.5% running gel and the 5.0% spacer gel both contained 8.1 M urea. The samples were applied with a syringe underneath the buffer directly on the surface of the spacer gel. The spacer gel (7.5 mm) was added upon the acrylamide gel (50 mm). Electrophoresis was carried out at 200 v (approximately 2.5 ma/tube) in 5 mM Tris-40 mm glycine buffer for 2 hr at room temperature and at 13 to 34 ma. The gels were stained with a solution of 0.18% Buffalo Black in methanol-acetate-water (5:5:1, v/v) for 1 hr. They were then kept overnight (18 to 20 hr) in 7% acetic acid solution and destained electrically. Densitometer tracings of the gels were made with a Canalco model F Microdensitometer. Detection of enzymes. Methods for detecting certain enzymes on the starch-gel patterns were based upon the reduction of nitroblue tetrazolium by reduced nicotinamide adenine dinucleotide as described by Scopes (8,9). Exact identifications are given in another paper from our laboratory (T. Hasegawa et al., J. Food Sci. 35: in press, 1970). RESULTS AND DISCUSSION Changes in bacterial numbers and pH. The changes in log bacterial numbers and pH for both rabbit and pig muscle are shown in Table 1. The control samples were essentially free from contamination at 0 days, except in the case of the control pig muscle used in the C. perfringens trial. This indicates that it is possible to obtain aseptic samples by using careful sampling procedures without the use of aseptic isolators as utilized by Ockerman et al. (7). Obviously, the procedures described herein are simpler, easier to utilize, and require only careful attention to sanitation. A rapid increase in the numbers of C. perfringens occurred under anaerobic conditions during the first 4 days of incubation at 30 C, but from 4 to 8 days there appeared to be some decline in numbers (Table 1), indicating maximum growth had been reached and a decline in numbers may have occurred before 8 days of incubation. The exact point of maximum growth was not determined in this study, as it could have occurred either before or after 4 days of incubation. S. enteritidis (Table 1) also showed a rapid increase in numbers between 0 and 8 days of storage but little further increase between 8 and 20 days. K. zopfii (Table 1) showed continued growth throughout storage, with maximum numbers occurring at 20 days of incubation for both pig and rabbit muscle. A. liquefaciens and S. faecalis both showed little increase in numbers during incubation, but apparently reproduced slowly or remained constant since numbers were essentially the same after 20 days of storage ( Table 1). The pH values were relatively stable during storage of controls at both 10 and 30 C. Incubation with C. perfringens resulted in some increase in pH for both rabbit and porcine muscle. Rabbit muscle inoculated with A. liquefaciens and incubated for 20 days showed some increase in pH, but the pH of pig muscle did not change during storage. The pH values for samples inoculated and incubated with S. enteritidis, K. zopfii, and S. faecalis remained essentially unchanged during storage (Table 1). Sarcoplasmic proteins. The data show that incubation of control samples of rabbit muscle at both 10 and 30 C resulted in complete disappearance of myoglobin (MB), apparently due to autolysis. Similarly, at 30 C, hemoglobin (HB) disappeared from control samples during incubation for 8 days but was still present in control rabbit muscle after storage for 20 days at 10 C. This suggests that MB is more susceptible to autolysis than HB. This is not too surprising since it is well known that the molecular weight of MB is only about one-fourth of HB. However, none of the organisms studied herein caused complete disappearance of HB, MB, phosphofructokinase, aldolase (ALD), glyceraldehyde phosphate dehydrogenase (GAPDH), a-glycerophosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, phosphopyruvate hydratase, pyruvate kinase, lactate dehydrogenase (LDH), creatine kinase, or albumin during incubation of rabbit muscle. Although none of the proteins or enzymes, or both in rabbit sarcoplasm disappeared as a consequence of microbial growth, this does not prove that the organisms studied did not cause some proteolysis of the sarcoplasmic fraction. It does, however, demonstrate that complete proteolysis of individual proteins did not occur under the experimental conditions imposed during this study. The fact that some protein bands stained appreciably lighter after inoculation and incubation suggests that minor proteolysis did in fact take place during storage. In agreement with the study on rabbit muscle, MB disappeared from uninoculated control pig muscle during incubation at 30 C, but, in contrast, HB did not disappear from the control. In addition, the growth of C. perfringens at 30 C completely removed ALD, GAPDH, and LDH from the starch-gel pattern of porcine muscle, indicating considerable proteolysis. MB was still present in the uninoculated control pig muscle incubated at 10 C as was HB, indicating some resistance to autolysis. Both S. faecalis and S. enteritidis completely broke down MB, although neither of these organisms caused complete proteolysis of the other sarcoplasmic proteins or enzymes, or both, in pig muscle. Neither A. liquefaciens nor K. zopfii caused the complete breakdown of any of the sarcoplasmic proteins or enzymes, or both. Urea-soluble proteins. The disc-gel patterns for the 8 M urea-soluble proteins are shown in Fig. 1 for rabbit muscle and in Fig. 2 for porcine muscle. The disc-gel patterns for control rabbit muscle after storage for 8 days at 30 C showed 21 distinct bands (Fig. 1). After incubation with C. perfringens for 8 days at 30 C, there was considerable protein breakdown (Fig. 1). Although all 21 bands were still evident, the size and distinctness of 5 bands were definitely altered. In addition, 14 new bands were present in the pattern. This suggests that some proteolysis of the urea-soluble fraction from rabbit muscle had occurred as a result of the growth of C. perfringens. Although the disc-gel patterns were obviously different for rabbit and pig muscle, the latter also showed 21 distinct bands in control samples after incubation at 30 C for 8 days (Fig. 2). After incubation with C. perfringens for 8 days at 30 C, 6 of the bands in the disc-gel pattern had completely disappeared and 10 new bands were apparent. This demonstrates that C. perfringens caused marked protein breakdown in the 8 M urea-soluble fraction of pig muscle. Examination of disc-gel patterns of samples incubated with C. perfringens showed that proteolysis of both rabbit and pig muscle was almost as extensive after storage for 4 as for 8 days. Disc-gel patterns for samples inoculated and incubated with S. enteritidis, A. liquefaciens, K. zopfii, and S. faecalis showed no evidence of Numbers for all peaks correspond for control and inoculated samples. The absence of any number indicates that the peak disappeared from the disc-gel pattern, whereas a letter indicates the presence of a new peak. VOL. 20, 1970 121 proteolysis. This was true for both rabbit and porcine muscle.

v3-fos

2019-04-24T13:06:43.181Z

1970-01-01T00:00:00.000Z

129026815

s2

Chemical Weed Control in Sugar Beet A field experiment was carried out to evaluate the efficiency of some herbicide treatments (with and without handweeding) on sugar beet yield and yield components for two successive seasons (2010 and 2011) at Abbis farm, The treatments were Phenmedipham 6.5%+ metametron 28% + ethofumesate 6.5% (crus) with a rate of 2 kg/feddan and 2.5 kg/feddan, Phenmedipham 5.54% + desmedipham 4.34% + lenacil 2.5% + ethofumesate 6.93 (betanal maxxpro), acetochlor (harness), Phenmedipham 7.5% + desmedipham 1.5% + ethofumesate 11.5% (betasana trio), handweeding twice and Unweeded check, all previous herbicide treatments were repeated twice either with or without handweeding. The dominant weed in both seasons was Beta vulgaris . The results in the first season showed that the best herbicide treatments which gave maximum weed reduction were handweeding twice, Acetochlor + handweeding, (Phenmedipham 6.5% + metametron 28% + ethofumesate 6.5%) 2.5 kg/fed + handweeding , (Phenmedipham 6.5% + metametron 28% + ethofumesate 6.5%) 2 kg/fed + handweeding, (Phenmedipham 7.5% + desmedipham 1.5% + ethofumesate 11.5%) handweeding and (Phenmedipham 5.54% + desmedipham 4.34%+ lenacil 2.5%+ ethofumesate 6.93%) respectively) with no significant difference between them. and diameter of the were not the tested herbicides, also the percentage of were all treatments, but in the first season, the highest sugar yield per was found in the case of five INTRODUCTION Sugar beet is an important crop of arable rotations throughout the major growing regions of Egypt. It provides a valuable break crop returning organic matter to the soil and preventing the buildup of disease. The root of the beet has a sugar content of around 17%. Harvesters cut off the top leaves of the sugar beet which are used as animal feed for cattle and sheep or are ploughed back into the land as a natural fertilizer. The root is then cleaned to remove any soil attached to it before it is transported. Roots awaiting delivery to the factory are stored in protected storage to maintain the highest possible quality and sugar. Weeds are known to cause crop yield losses, hamper harvest, reduce quality of the harvest product, and perhaps harbour insects and diseases that may harm the crop. Yield losses are of the greatest concern and have been predicted using early season assessments of the weed population such as weed seedling density, relative time of emergence, weed pressure, and relative leaf area (Schweizer and May, 1993;Dieleman and Mortensen, 1998). Sugar beet is a poor competitor with weeds in arable fields because it is slow growing early in the season and has a low canopy in its first year of a biennial life cycle (May, 2003). Sugar beet is not competitive with emerging weeds until it has at least 8 true leaves. The total potential losses from weeds would be between 50 and 100% of the potential crop yield (May, 2001). Weeds that emerge 8 weeks after sowing, and particularly after the sugar beet plants have eight or more leaves, are less likely to affect yield (Scott et al., 1979). The most competitive are annual weeds, mostly broadleaved species that emerge with, or shortly after, the crop, grow taller than the crop and produce dense shade. These weeds often grow to a height two to three times that of sugar beet by midsummer, sugar beet herbicides seldom have a wide enough weed control spectrum or sufficient residual activity to control all weeds, and tank mixes and sequences of different herbicides are commonly used in order to provide a broad spectrum of weed control (May and Wilson, 2006). Weeds damages in developing countries, by applying different methods of control, resulted in 10% decrease in performance of sugar beet. Without any control of weeds, damages fluctuate between 10 to 100% according to environmental conditions and genus of cultivated plant, economically (Kropff and Vanlour, 1993). Due to that chemical methods are among the most important ways of management in cultivating farms, using appropriate paths to improve the performance of herbicides must be considered. Handweeding 10-20 weeks after planting sugar beet can keep the field clean until harvest time (Dawson, 1977). The aim of this study is to find out the best treatment to control the weeds specially broad leaf weeds in sugar beet under Egyptian conditions. MATERIALS AND METHODS A field experiment was carried out in Abbis farm (Faculty of Agriculture farm), Alexandria, Egypt for two successive seasons 2010 and 2011) to estimate the effect of some herbicidal treatments on both broad and narrow leaf weeds. This experiment was designed as a randomized complete block design with four replicates, tables (1 and 2) shows the herbicides application time and rate. All tested herbicides were applied with a CP3 knapsack sprayer with red fan type nozzle. Handweeding twice and unweeded check were included in both season. All cultural practices like fertilization and irrigation were applied as usual in sugar beet production. Herbicides were evaluated after 45 days from application by collecting all weeds grown in 1m 2 randomly, weeds were sorted and weighted. Percentage of weed reduction of each weed species as well as total of all weeds were calculated, also the effect of tested herbicides on yield, yield components (root length and diameter) were measured. Betasana trio 0.9 L + 0.9L 0.9 L (two leaves stage) then 0.9 L (after 8 days from first treatment) 5 Handweeding 6 Unweeded check Percentage of sugar (Brix) was also measured by a hand refractometer (Atago N1, Brix 0~32 %). Statistical analysis of data were carried out by assistat software version beta (Silva and Azevedo, 2009). Tables (3) and (4) shows that the dominant weed in the first season was Beta vulgaris with a a percentage of infection (51.6%) followed by Medicago polymorpha (33.4%), Phalaris minor (8.8%), Marva parviflora L. RESULTS AND DISCUSSION (3.2%) and Vicia monantha Retz. (3.1%). In the second season the dominant weed was also Beta vulgaris with a percentage of infection (52%) followed by Medicago polymorpha L. (32.4%), Phalaris minor (9.6%), Marva parviflora L.( 3.3%) and Vicia monantha Retz. (2.7%). On the other hand, tables (3) and (4) also showed that the highest weed control in the first season was found in the case of handweeding twice, harness + handweeding, crus 2.5 kg/fed + handweeding, crus 2 kg/fed + handweeding, betasana trio + handweeding and betanal maxxpro + handweeding with no significant differences between them with a percentage of weed control (98.7, 97.7, 96.7, 96.2, 95.8 and 93.7), respectively, which indicated the role of handweeding in sugar beet weed control, the least percentage of control was observed in the case of betanal maxxpro (42.8% control). This is due to the ability of these treatments together with the handweeding to control the dominant weeds (Beta vulgaris and Medicago polymorpha). Concerning with narrow leaf weeds (Phlaris minor) in the first season, the best control was found in the case of harness+handweeding (97.9%), followed by handweeding twice (96.9%), the least control was in the case of betasana trio (46.1%). In the second season, handweeding twice gave the best %control (96.5%) followed by crus 2kg/fed and crus 2.5kg/fed as they gave 90.7 and 90.6 % control, respectively. The least control was shown by betanal maxxpro (57.5%). In the second season (table 6), the highest yield was found in the case of crus 2.5 kg/fed + handweeding and handweeding twice (34.23 and 33.18 ton/fed , respectively) with no significant differences between them, also there were no significant differences between harness + handweeding, betasana trio + handweeding, crus 2 kg/fed + handweeding and betanal maxxpro + handweeding ( 28.56, 27.51, 26.59 and 26 ton/fed, respectively), the least yield per feddan was found in the case of unweeded check (10.63 ton/fed), harness and crus 2 kg/fed gave a low yield 19.91 and 17.64 ton/fed, respectively with no significant differences between them. These results agreed with Mohammad et al (2011), Who found that herbicides such as Metamitron and PDA (Phemedipham + Desmedipham + Autophpmisete) and mixtures of Clopyralid and desmedipham and mixture of Desmedipham and trisulfuron methyl increased the performance of root yield up to 73.66, 70.73, 67.23 and 60.33 ton/hactar. Conecrning with yield components, the highest length of root in the first season was found in the case of betanal maxxpro (31.56cm) with no significant difference between all treatments except in the case of harness betanal maxxpro + handweeding and betasana trio with and without handweeding. In the second season, there was no significant difference between all treatments which indicated that the herbicide treatments don't affect the sugar beet root length. Table (5) also showed that there was a very slight difference in root width of sugar beet in the first season as the highest width was found in the case of crus 2 kg/fed + handweeding (39.88cm) with no significant difference between all treatments except in the case of harness and handweeding twice (30.58 and 30.63 respectively) which was the least. Similarly in the second season (table 6) crus 2 kg/fed + handweeding recorded the highest width (38.3cm) with a significant difference with unweeded check, harness + handweeding, harness, betasana trio + handweeding and betasana trio (31.50, 31.35, 30.90, 30.03 and 28.83cm, respectively). The data in table (5) showed that the percentage of sugar (brix) of the crop was not affected in the first season extremely by the treatments, the highest results was noticed in the case of betanal maxxpro + handweeding (16.83%) with no significant difference between the rest of treatments except in the case of harness which was the least (12.50%), there were no significant differences between either betanal maxxpro + handweeding and the rest of treatments or harness and the rest of treatments which was common between them. In the second season, there were no significant differences between all treatments except in the case of harness least result (13.35%) which was illustrated in table (6). Betanal maxxpro+ handweeding, betasana trio and handweeding were in between harness and the rest of treatments with no significant difference. These results indicates that the percentage of sugar was not affected by all tested herbicides. This study agreed with Tevor et al (2006), who studied a post combination of desmedipham plus phenmedipham at 0.045 + 0.045 kg ai/ha (desphen) or desmedipham plus phenmedipham plus ethofumesate 3 (1:1: 1 ratio) (desphenetho) at 0.09 kg ai/ha plus triflusulfuron at 0.004 kg ai/ha plus clopyralid at 0.026 kg ai/ha plus 1.5% methylated seed oil received registration in 1998 and 2000 in North Dakota and Michigan, respectively and found that herbicide rates are reduced by 80%, compared to standard-split applications. Sugar beet populations and recoverable white sucrose per hectare did not differ among post herbicide treatments. Abdallahi and Ghadiri (2004) found that maximum reduction in weed biomass was observed with desmedipham plus phenmedipham plus ethofumesate at 0.23 + 0.23 + 0.23 kg ai/ha and desmedipham plus phenmedipham plus propaquizafop at 0.46 + 0.46 + 0.1 kg ai/ha. Efficacy of grass herbicides was reduced when they were combined with pyrazon. Highest crop injury in both years was observed with desmedipham plus phenmedipham plus ethofumesate at 0.23 + 0.23 + 0.23 kg/ ha. Highest and lowest root yields in both years were produced in weed-free and weedy check plots, respectively. All herbicide treatments produced lower sugar beet yields than the hand-weeded check. Of the herbicide treatments evaluated, the highest sugar beet yields were with desmedipham plus phenmedipham plus propaquizafop at 0.46 + 0.46 + 0.1 kg/ha in 2001 and with desmedipham plus phenmedipham plus ethofumesate at 0.23 + 0.23 + 0.23 kg/ha in 2000. Sucrose content and other sugar beet characteristics were not affected by the herbicide treatments Also, it was found that planting pattern had proper effect on weeds biomass that best results were obtained in twin row planting 60 cm. Also, mechanical control at 4 leaves stage of sugar beet had the best effect on weeds density and biomass. metamitron plus combination of phenmedipham + desmedipham + ethofumesat had also the best effect on weeds density and biomass. (Zargar and Rostami, 2011). The results in table (5) indicated that sugar yield per feddan in the first season increased in five treatments (crus 2.5 kg/fed + handweeding, betanal maxxpro + handweeding, Handweeding twice, crus 2 kg/fed + handweeding and crus 2.5kg/fed) as they gave 5.4, 4.98, 4.73, 4.64 and 4.35 ton/fed, respectively with percentage from unweeded check 251.87, 232.02, 220.56, 216.33 and 202.64 %, respectively, also the results in this table showed that there was no significant difference between betasana trio+ handweeding and harness + handweeding as they both gave 4.12 ton/fed. The least sugar yield was found in the case of unweeded check (2.15 ton/fed), harness (2.6 ton/fed) with percentage from unweeded check (121.36%) and crus 2 kg/fed (3.09 ton/fed) with percentage from unweeded check (144.1%) with no significant differences between them. The unweeded check gave the least sugar yield per feddan (1.7 ton/fed), also harness and crus 2 kg/fed gave lower sugar yield than the rest of treatments (2.64 and 2.84 ton/fed, respectively) with percentage from unweeded check 155.15 and 166.79%, respectively. The previous results in both seasons indicated the important role of herbicide treatments with the aid of handweeding in increasing the sugar beet yield as well as sugar yield in sugar beet crop.

v3-fos

2018-04-03T03:46:45.293Z

1970-07-01T00:00:00.000Z

36489415

s2

Endocarpic microorganisms of two types of windrow-dried peanut fruit (Arachis hypogaea L.). The endocarpic microorganisms of peanut fruit dried in either a random windrow (plants left as they fell from the digger) or an inverted windrow (plants inverted to expose fruit to sunlight) were different from that of freshly dug fruit. Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium were the dominant fungi found associated with shells (pericarp) of freshly dug fruit. The dominant fungi of shells of windrowed fruit included Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria. Seeds of freshly dug fruit were dominated by Penicillium and Aspergillus. The only dominant species in seed of windrowed fruit was Penicillium. Microorganisms were isolated from shells and seed of freshly dug fruit at a frequency of 79% and 52%, respectively. The percentage of infestation was reduced by drying in the field. This was particularly true of the inverted windrow. The proportion of shells and seed infested with a microorganism was reduced 13% and 36%, respectively, after field drying for 5 to 7 days in random and inverted windrows. Microorganisms were isolated much more frequently from shell pieces (73%) than from seed (36%). Since the first discovery in 1960 (14) that a fungus commonly associated with peanut fruit (Arachis hypogaea L.) could produce a metabolite toxic to some animals, numerous reports on the microorganisms associated with mature fruit (5-7, 10, 12), overmature fruit (3), and damaged fruit (1,16) have appeared in the literature. As fruits mature in the soil, they become more susceptible to invasion by members of the microbial community of the surrounding soil (13). Thus, there is established an endocarpic (11) [or endogeocarpic (7)] microbial community in the fruit (5,18). Porter and Garren (18) reported that, from freshly dug fruit, microorganisms were isolated from 90% of the shells and over 63% of the seed. Other reports (2,3) indicate that fungal invasion may continue after fruits are removed from the soil. Garren (6) showed that isolation procedures, including the use of different media, temperature, and soil additives, influenced the microbial population associated with peanut fruit. In the now widespread mechanical harvesting of peanuts, plants are frequently lifted from the soil with fruits intact and windrowed in the field until the moisture content is reduced to 20 to 30%. The fruits are then combined, and drying is completed with forced air. During the period of windrow drying, changes in the microbial community associated with peanut fruit have been noted. Jackson (9,11) found that the "fungal communities from windrowed peanuts were distinctly different from communities which developed in the soil." Dickens (4) found that fielddrying was accelerated considerably in the inverted windrow and was less favorable for fungal growth than random windrows. The objectives of this study were to characterize the dominant endocarpic microorganisms of mature peanut fruit (i) at the time of digging, and (ii) after partial drying in two types of field windrows. MATERIAIS AND METHODS Peanut plants of the cultivar Virginia Bunch 46-2 were grown in a Norfolk fine, sandy loam soil at Holland, Va., in 1966Va., in , 1967Va., in , 1968Va., in , and 1969. Agronomic practices approved for Virginia-type peanuts were used. Planting dates were between 10 and 16 May. Soil fungicides and nematocides were not used. Plants were harvested during the time commercial peanuts were being harvested. Plants were mechani- and 2, 9, 21 October 1969. Plants were exposed for 5 to 7 days in a random windrow or in an inverted windrow (Fig. 1). In the random windrow, plants were left as they fell from the digger with most fruit covered with foliage and in contact with the soil. In the inverted windrow, plants were turned to expose most of the fruit to direct sunlight. After 5 to 7 days in each windrow, mature handpicked fruit was shelled, and pieces of shell (ca. 1 cm2) and seed with intact testa were surface-disinfested for 3 min in 0.5% NaOCl and plated (four per plate) on rose bengal-streptomycin-agar (15). Therefore, fungi growing onto this medium from surface-disinfested shells and seed should not have come from surface propagules, but from propagules produced by a thallus well-established therein. After incubation for 7 days at 25 C, most of the thalli that grew on to the medium from shells and seed could be identified. Approximately 1,200 shell pieces and 1,200 seed were plated in each of the 4 years of the study. At each reading, the percentage of shell pieces and seed from which at least one microorganism grew was determined. This was recorded as the proportion of shells and seed infested with some microorganism. We could identify most of the thalli of the fungi that grew from shells and seed. Thus, at each reading, we determined the isolation frequency of the dominant fungi in these shells and seed. RESULTS The percentages of shells and seed of freshly dug and windrowed peanut fruit that were infested with some microorganism during the 4 years of the study are given in Table 1. From this data, the following may be deduced. An Drying in a random windrow did not significantly reduce the number of microorganisms associated with shells. An average of 52.1 % (range 45.8 to 66.6) of freshly dug seed examined during the 4-year period yielded microorganisms. The proportion of shells and seed infested with a microorganism was reduced by an average of 31.5% by drying in a random windrow and by 60.4%o in an inverted windrow. An average of 42.2% fewer seed yielded microorganisms when dried in an inverted windrow than in a random windrow. In the whole of this study, a larger proportion of shells (73%) than seed (36%) was infested with some microorganism. The dominant shell and seed fungi (classified as a dominant if the isolation frequency from shell or seed was 6% or over) isolated during 1966, 1967, 1968, and 1969 are given in Table 2. The dominant fungi associated with shells of freshly dug peanuts included Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium. Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria dominated the shells of windrowed fruit. The dominant fungi of seed of freshly dug fruit included Penicillium and Aspergillus while that of seed from windrowed fruit was dominated only by Penicillium. More than one-fifth of the shells of freshly dug fruit yielded Chaetomium spp., mainly C. globosum Kunze ex Fr. ( Table 2). The isolation frequency of this fungus dropped slightly in the inverted windrow (15.2%) but was not changed in the random windrow (20.5%). The isolation frequency of Chaetomium from seed was much lower than that obtained from shells. In fact, only 3.9% of the seed from freshly dug fruit, 3.7% of the seed from random windrows, and 2.1 % from the inverted windrows gave rise to this fungus. Penicillium was isolated at a frequency of 17.5% from shells of freshly dug fruit (Table 2). Each windrow-type greatly reduced the isolation frequency of this fungus. Penicillium was the predominant genus in the seed of freshly dug and windrowed fruit, and approximately 28% of all seed plated from freshly dug fruit gave rise to this fungus. Its isolation frequency dropped considerably after drying in the windrow. More shells and seed yielding at least one thallus of Penicillium spp. were taken from random windrows than from inverted windrows. Many different species of this fungus were observed; however, over onehalf were P. funiculosum Thom. and P. janthinellium Biourge. A higher proportion of freshly dug fruit was infested with Trichoderma, mainly T. viride Pers. ex Fr. and other species including Trichodermalike isolates that were probably Gliocladium spp., than fruit from either windrow type ( Table 2). The type of windrow had little effect on the isolation frequency of Trichoderma. In all instances, the isolation frequencies of this fungus were higher in shells than in seed. The isolation frequency of Rhizoctonia was greater in windrowed fruit than in freshly dug fruit ( Table 2). The average isolation frequency from windrowed fruit was 6.7% compared to 3.5 % from freshly dug fruit. More shells than seed were infested with Rhizoctonia. More fruit yielding at least one thallus of Fusarium spp. were taken from windrows than were taken from freshly dug lots ( This fungus was obtained more readily from fruits taken from random windrows than from fruit taken from inverted windrows. A higher proportion of freshly dug and randomwindrowed fruit were infested with Rhizopus, mainly R. stolonifer (Ehr. ex Fr.) Vuill. and R. arrhizus A. Fischer than fruit in the inverted windrow (Table 2). This fungus was found more frequently in shells of random-windrowed fruit (5.5 %) than in shells of freshly dug fruit (4.2%). However, twice as many seed with at least one thallus of Rhizopus spp. were taken from freshly dug lots than from random windrows. The isolation frequency of Sclerotium from shells was much higher in fruits that were windrowed (8.5 %) than from freshly dug fruit (2.7 %; Table 2). More seed than shells yielded at least one thallus of Aspergillus spp. (Table 2). These were mainly A. flavus and A. niger v. Tiegh. Aspergillus spp. were isolated more readily from freshly dug fruit than from windrowed fruit. More fruit in the random than in the inverted windrow yielded at least one thallus of Aspergillus spp. Some A. flavus was isolated each year from shells and seed of freshly dug and windrowed fruit. The isolation frequencies of this species from seed of freshly dug, random, and inverted fruit were 5.2%, 3.9%, and 2.6 respectively. Shells with at least one thallus of Alternaria spp. were more numerous in windrowed fruit (8.3 %) than in freshly dug fruit (0.6%) ( Table 2). Seed were rarely infested with this fungus. In the 4 years of this study, the data (Table 1) show that without exception more shells and seed were infested in 1968 than in any other year. This may be attributed in part to the environmental conditions that prevailed during these years ( Table 3). The rainfall in October 1968, was about five times that of 1966, about four times that of 1967, and about two times that of 1969. VOL. 20Y 1970 The mean temperature for October was slightly higher during 1968 than during the other years. Also, the growing season of 1968 was prolonged because of the lateness of the first killing frost. The relation of rainfall on the windrow to changes in microbial infestation of peanut shells and seed is shown in Table 4. The differences in microbial infestation of freshly dug fruit and that of samples of the same fruit after 5 to 7 days in the windrow seemed almost inversely proportional to the amount of rain falling on the windrow. If no rain fell on the fruit while they were in the windrow, the proportion of fruit components infested with at least one microorganism decreased greatly and rapidly. For example, no rain fell on those plants dug and windrowed on 5 October 1966 and 25 October 1968, and fewer fruit parts yielding at least one microorganism were found in samples taken from these windrows than in samples taken from the windrows immediately after digging. On the other hand, much rain fell on plants dug on 2 November 1966 and 18 Octo- ber 1968, and these were the only two instances in which the proportion of fruit parts yielding at least one microorganism in samples taken from windrows was greater than that of the samples taken from the windrows immediately after digging. Frequency of shell infestation was affected more by rain than was the frequency of seed infestation. The moisture content of freshly dug fruit (determined on wet weight basis after drying for 4 hr at 130 C) during 1967 and 1968 averaged approximately 52% (Table 5). After field-drying for 5 to 7 days, fruit moisture content was reduced to 32% in the random windrow and to 22.7%, in the inverted windrow. DISCUSSION The degree of microbial infestation of shells and seed from windrowed peanut fruit was less than that of freshly dug fruit. Also, fewer shells and seed from fruit dried in inverted windrows were infested with microorganisms than were shells and seed from fruit dried in random windrows. These changes in the microbial community are to be expected, because the environment surrounding the windrowed fruit is different from that of fruit in the soil. At the time of removal from the soil, the fruit moisture averaged over 50%. However, once plants were placed in the windrow, the moisture content dropped rapidly. The drying rate of fruit was much more pronounced in the inverted windrow than in the random windrow. The average moisture content of seed dried for 6 days in the random windrow and inverted windrow in 1967 and 1968 was 31.9%zo and 22.6%, respectively. Others have reported similar findings (4,9). Fruits on plants in a windrow, especially an inverted windrow, undergo rapid dehydration which no doubt retards growth and development of the endocarpic fungi and which may account, in part, for the decrease in prevalence of the microbial populations. Another, and perhaps even greater, difference between fruit in the soil and fruit on plants in a windrow is relative humidity. The humidity level in the soil is high and remains fairly constant unless the soil moisture is depleted (19). Thus, the subterranean peanut fruits are usually surrounded by an atmosphere of very high humidity, except when surface soil is very dry. On the other hand, fruits on plants in a windrow are exposed to a wide range of relative humidities. Usually the above ground humidity is low during the day except during periods of inclement weather, and the relative humidity at night is high and often exceeds 90%. Most of the fungi found associated with peanut fruit have been called molds. Studies on molds (17) show them to be hydrotolerant, with optimum relative humidities of at least 90%. These high-optimum relative humidities for peanut fruit fungi may account in part for the reduction in the number of microorganisms present in the windrowed fruit. For example, the proportion of seed taken from windrows which yielded at least one thallus of Penicillium spp. was only 22 to 32% of that of freshly dug fruit. These factors, plus others undescribed, may act either separately or in combination to reduce the population of some of microorganisms associated with drying peanut fruit. A reduction in the isolation frequency of a fungus during windrowing may result from dehydration of the fruit and exposure to variable relative humidities. A similar reduction in the inverted windrow may be the result of more rapid dehydration and variable humidities, coupled with the effects of solar radiation. On the other hand, the isolation frequencies of some of the principal microorganisms of fruit increased while they were drying in the windrow. The isolation frequency of Fusarium and Alter-naria increased 4-fold and 16-fold, respectively, when fruits were subjected to windrow drying. Others (11) have also shown that the isolation frequency of Fusarium from peanut fruit increased when plants were windrowed. The increase in the isolation frequency of these two genera in windrowed fruit is of significance, especially since Garren et al. (8) recently reported that these fungi were capable of producing mycotoxins. McDonald and Harkness (16) showed that the isolation frequencies of most of the microorganisms associated with undug peanut fruit increased during periods of rainy weather. In our study, the isolation frequencies of most microorganisms tended to decrease more slowly in shells and seed when fruit drying in the windrows was exposed to appreciable rain than when not so exposed (Table 4). This was probably due to such factors as rehydration of the fruit, increased relative humidities, and lowered light intensities. The possibility of mycotoxin contamination of the fruit also increases under these conditions. Thus, growers must exercise extreme care in thehandling of such fruit. The isolation frequency of the well-known toxicogenic fungus A. flavus (14) was relatively low in freshly dug fruit each year of this study.

v3-fos

2020-12-10T09:04:12.883Z

1970-02-01T00:00:00.000Z

237231500

s2

Determination of Untreated Whole-Milk Effects on In Vitro Antibacterial Activity The effect of fresh whole milk without pasteurization or other pretreatment on in vitro antibacterial activity of selected compounds was determined in broth dilution. The milk was collected by hand directly from dairy goats, or by syringe or cannula from bovine quarters showing low bacterial counts. Antibacterial activity was determined in 50% (v/v) milk-broth medium against sensitive mastitis-etiologic strains of Streptococcus agalactiae and Staphylococcus aureus. The indicator salt 2,3,5-triphenyltetrazolium chloride was incorporated in the milk broth medium to determine inoculum growth. Contaminant interference was circumvented through early as well as late readings and comparisons with uninoculated control tubes, with and without the test compounds. Application of the method with more than 75 compounds, including nitrofurans, antibiotics, and other chemicals uncovered marked degrees of milk interference. The method warrants routine use among preliminary screens to relate in vitro with in vivo observations of antimicrobial activity. Similar procedures may be used with serum, skim milk, or mastitis-milk media for separating effects due to protein, lipid, or other elements in product evaluation. The effect of fresh whole milk without pasteurization or other pretreatment on in vitro antibacterial activity of selected compounds was determined in broth dilution. The milk was collected by hand directly from dairy goats, or by syringe or cannula from bovine quarters showing low bacterial counts. Antibacterial activity was determined in 50% (v/v) milk-broth medium against sensitive mastitis-etiologic strains of Streptococcus agalactiae and Staphylococcus aureus. The indicator saJt 2,3, 5-triphenyltetrazolium chloride was incorporated in the milk broth medium to determine inoculum growth. Contaminant interference was circumvented through early as well as late readings and comparisons with uninoculated control tubes, with and without the test compounds. Application of the method with more than 75 compounds, including nitrofurans, antibiotics, and other chemicals uncovered marked degrees of milk interference. The method warrants routine use among preliminary screens to relate in vitro with in vivo observations of antimicrobial activity. Similar procedures may be used with serum, skim milk, or mastitis-milk media for separating effects due to protein, lipid, or other elements in product evaluation. Few studies have been conducted to demonstrate the effect of milk on the antibacterial activity of compounds. The effect of milk on antibiotics has been reported by Marshall, Price, and their co-workers (8,10,11), but these have involved agar diffusion media and pretreated (autoclaved, skim, methoxylated, or pasteurized) milk fractions. These and other investigators (3, 5-7, 9, 12) indicated potential effects of raw milk components on microbial inhibitors, e.g., protein, lipid, pH, and divalent-ion interference with diffusion and activity. Preservation of physical, chemical, and biological properties of the milk, therefore, would seem necessary for the accurate evaluation of the interaction of antimicrobial agents and milk components. In developing sensitive microbiological assays to select products for therapy, easily applied in vitro methods are sought to simulate the in vivo condition. In seeking compounds active against etiological agents of mastitis, it is important to determine the effect of milk on chemicals showing initial low minimal inhibitory concentration (MIC) values. The in vitro vessel closely resembles the glandular environment with minimal absorption and maximal medium effects being present. A direct contact method excluding diffusion and using untreated raw milk is desirable. This report describes a procedure suitable for this purpose. MATERIALS AND METHODS Antibacterials. More than 75 compounds have been screened thus far with this method, including nitrofurans and other synthetic compounds prepared by The Norwich Pharmacal Co., Chemistry Division, as well as commercial preparations of chlortetracycline hydrochloride (National Formulary reference standard), erythromycin (Abbott Laboratories, North Chicago, Ill.), sodium penicillin G (USP reference standard), and streptomycin sulfate (USP reference standard). Stock solutions of the synthetic compounds containing 2,000 to 20,000 pliters/ml were prepared immediately before use in sterile water or dimethyl formamide (DMF) as required by their solubility. The antibiotics were prepared at similar concentrations by the procedures outlined by Grove and Randall (4) for routine potency assays. Milk effect on activity. Standard twofold tube dilution procedures were employed for duplicate or triplicate tests of the compounds' in vitro activity against Streptococcus agalactiae V.StB-1 and Staphylococcus aureus V.Mi-1 mastitis isolates. Working solutions of the antibacterials described above were subsequently diluted 1:100 in tryptone soy broth (TSB) or the same medium containing 50% fresh whole milk (MB) for serial dilution, and thus the ratio of MIC values with and without milk was obtained. Concentrations of the antibacterials were chosen for test- hydrochloride ing which were similar to and higher than those found effective against the same strains using TSB alone. Concentrations of 25 to 50 ,ug/ml of 2,3, 5-triphenyltetrazolium chloride (TTC) were added to the media containing milk to determine presence or absence of growth, and identical concentrations were included in the plain broth medium as an additional control for compound potentiation (see below). Contaminant interference from the normal milk flora was circumvented by taking early (14 to 17 hr) as well as later (20 to 24 hr, 40 to 48 hr, or sometimes 5 day) turbidity readings after incubation at 37 C. Growth responses at each dilution in the 50% MB medium were compared further to inoculated control tubes, with and without test compound, and to similar preparations using Bacto-Skim Milk (Difco), heat-treated milk (autoclaved 13 min at 121 C), or sterile calf serum. MIC value changes, indicative of interference with compound activity, were expressed as quotients, e.g., MIC in milk broth/MIC in tryptone soy broth, the higher the ratio the greater the interference of the medium additive. RESULTS AND DISCUSSION In addition to the antibiotics listed earlier, several antibacterials prepared in our laboratories were used for selected comparisons or gave results of special interest referred to below. The chemical name of each compound is given in Table 1, and specific details of their synthesis and activity may be found in reports by Burch (1, 2), Gever (G. Gever, U.S. Patent 2,802,002, 1957), Snyder and Benjamin (13,14), and Thomsen (15). Fresh milk effects on antibacterial activity in TSB were sought for subsequent comparison with those of serum or other fluid supplements, and 50% additive concentrations were chosen as optimal for the studies. Preliminary experiments after multiplication of each test strain in 0, 50, or 100% fresh cow's or goat's milk-broth demonstrated similar maximal growth in broth and milkbroth media. Further experiments indicated no significant difference in MIC activity ratios with either caprine or bovine milk supplements, and goat's milk was selected for the study because it generally had minimal or no contaminant interference. The results of subsequent experiments with compound 4 ( Table 2) showed little or no difference in ratios taken from the early or late observations (up to 48 hr) when either fresh milk or serum supplements were added to the media. Readings made at 20 to 24 hr were employed routinely as shown in Table 3, except when contamination interference required use of the 14to 17-hr data. Good replicate agreement was also obtained unless this contamination was excep- Ag/ml of TTC were used routinely, therefore, and no instances of potentiation were observed from data compared with earlier primary-screen results or with a broth series of compound dilutions, without the TIC indicator. The antibiotic test findings in Table 3 are in general agreement with values reported by Price and his co-workers (10,11). Chlortetracycline activity against S. agalactiae was niinimally affected by the untreated milk, as compared to an 8to 16-fold increase in the MIC value for S. aureus. Erythromycin, penicillin, and streptomycin showed similar differences in effect on the two test organisms with corresponding reductions of 2-as compared with 32-fold, 2as compared with 4-fold, and 2-to 8-fold as compared with 8to 16-fold, respectively. Effects on nitrofuran derivatives activity also varied, and milk or serum interference was not predictable from reported solubility data. Of special interest were the differential effects of milk and serum on the in vitro activity of quinazoline compounds no. 5-7. This suggests specific lipid, rather than protein, inhibition of the heterocycic derivatives, and studies are indicated to relate the finding with in vivo data. Considered with other studies designed to correlate in vitro and in vivo antimicrobial activities, this method lends itself to routine use among preliminary screens for potential products, especially mastitis evaluation. In addition to comparisons with serum, further milk modifications may be studied, and effects due to pH change, microor macromolecules, leukocytes, or other elements, may be determined for better understanding of compound action.

v3-fos

2020-12-10T09:04:12.488Z

1970-02-01T00:00:00.000Z

237234243

s2

Growth from Spores of Clostridium perfringens in the Presence of Sodium Nitrite The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations. Although sodium nitrite plays an important role in the preservation of canned cured meats, the actual mechanism by which it prevents spoilage is obscure. It is believed that the stability of these meat products is due to the ability of the curing salts, sodium nitrite and sodium chloride, to block some stage in the outgrowth or germination, or both, of the indigenous bacterial spores which have been heat-injured by the brief thermal processing that these canned meats undergo. The mild thermal processing presumably renders the spores more sensitive to the curing salts; however, the transitional stages of spore germination or outgrowth, or both, at which sensitization occurs remainunclear. Sodium nitrite, when heated in a laboratory medium, forms a bacterial inhibitor which has been found to be highly effective in preventing the growth of several clostridial species (11,12). However, recent evidence indicates that such an inhibitory product may not be active in cured meat products (9). It is known that commercially acceptable concentrations of sodium nitrite may interfere with the growth of bacterial cells (2). Another postulated role of sodium nitrite in the preservative system of cured meats may be its ability to induce spore germination, thus making the spores susceptible to subsequent heat processing (4). Indeed, The present study was initiated to determine whether the inhibition of microbial growth from spores by commercially acceptable levels of nitrite (0.02% maximum residual nitrite) occurs at both the level of germination and outgrowth. MATERIALS AND METHODS Preparation of spore supenions. Spores of C. perfringens strain FDI, obtained from the Food and Drug Administration, and C. perfringens NCTC 8798 (Hobbs type 9) were used. Strain FD1 produces heatsensitive spores that require no prior heat shock for germination, and strain NCTC 8798 produces heatresistant spores that require heat shock for optimal germination (Labbe and Duncan, unpublished data). The organisms initially were grown in 15 ml of Fluid Thioglycoilate Medium (BBL); successive 10% inocula were made at 16-hr intervals until a 1,500-ml culture was obtained. The latter culture was inoculated into 15 liters of D-S sporulation medium (6). All incubations were at 37 C. When the number of free spores reached a maximum (about 15 hr for FDI and 20 hr for NCTC 8798), the culture was harvested by continuous-flow centrifugation. The spores were resuspended in distilled deionized water and were subjected to ultrasonic treatment to remove spores still in their sporangia. Subsequently, the spores were washed several times with cold deionized water until a suspension of clean spores was obtained. During 353 cleaning and storage in deionized water, about 3 to 4% of the spores became phase dark. A stock spore suspension adjusted to an optical density (OD) of about 325 Klett units was prepared, and the remainder of the spores was lyophilized. Assessment of initiation of germination and outgrowth. Initiation of germination was measured as a decrease in OD at 660 nm by using a Bausch & Lomb Spectronic-20 colorimeter; initiation was confirmed by determining loss of spore-phase brightness by using a Zeiss phase-contrast microscope. Some of the data are presented as per cent germination with the decrease in OD of the positive control taken as 100% germination. NA Spore outgrowth was determined by the increase in OD that occurred subsequent to an initial decrease in OD due to initiation of germination. Outgrowth was confirmed by phase-contrast microscopy. In studying the inhibitory effect of sodium nitrite upon germination and outgrowth, a complex medium was used which contained a final concentration of 2.5% Brain Heart Infusion (Difco) plus 0.27% added yeast extract. The pH was adjusted to give a final pH of 7.0 or 6.0. In this medium, outgrowth of non-heatinjured spores began after 15 to 20 min. Reagents for the nitrite-induced germination experiments were prepared as concentrated stock solutions in deionized water and diluted as necessary. A final reaction mixture volume of 6 ml was used. Fresh stock solutions of sodium nitrite were prepared before each experiment. Strain FD1 spores initiated germination and grew without prior heat shock; strain NCTC 8798 spores were heated at 70 C for 15 min to initiate germination. In all germination and outgrowth studies, the reaction mixtures, contained in screw-cap tubes (16 by 125 mm), were preheated to the appropriate temperature in a thermostatically controlled water bath; spores were then added to a final concentration of about 3 X 107/ml. Preparation of heat-injured spores. Heat-injured spores were prepared by heating 5 ml of a stock spore suspension containing about 2 X 108 spores/ml in a screw-cap tube (16 by 125 mm); the spores were heated either at 90 C for 5 min in the case of FD1 or at 100 C for 18 min in the case of strain NCTC 8798. These times and temperatures resulted in an approximately 0.33 D value (time in minutes for inactivation of 90% of the spores) for spores of both strains. Viable counts of heat-injured spores were made by means of pour plates by using TSN agar (10). Incubation was at 37 C for 24 hr (strain FD1) or 48 hr (strain NCTC 8798) under a gas mixture of 90% nitrogen and 10% carbon dioxide. RESULTS Four mechanisms for possible inhibition of microbial growth from spores by sodium nitrite were investigated. These included (i) prevention of germination of heat-injured spores in a complex medium, (ii) prevention of germination of heatinjured spores in a complex medium by an inhibitory product formed by prior combination of sodium nitrite with a component in the medium incubated at 45 Cfor 15 min in a germination medium consisting of 2.5% Brain Heart Infusion and 0.27% yeast extract. Sodium nitrite was added to the medium in the concentrations indicated either before or after autoclaving for 15 min at 121 C. Broken lines, heatinjured spores; solid lines, unheated spores; A, sodium nitrite added before autoclaving; *, sodium nitrite added after autoclaving. during autoclaving, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Inhibition of germination. The effect of adding sodium nitrite to a complex medium before or after autoclaving (15 min at 121 C) on the germination of heat-injured or non-heat-injured FD1 spores is shown in Fig. 1. At pH 7 and at sodium nitrite concentrations less than about 0.3%, heatinjured spores were more susceptible to inhibition of germination by nitrite regardless of whether nitrite was added before or after autoclaving. Addition of sodium nitrite before autoclaving the medium did not reduce the per cent germination of heat-injured spores, as compared to the germination obtained when nitrite was added after autoclaving. However, germination of non-heatinjured spores was reduced when nitrite was added before autoclaving. At pH 6, the results were nearly the reverse of those obtained at pH 7.0. At ably due primarily to species differences. Heat-injured FD1 spores were much more sensitive to nitrite than non-heat-injured spores, with a concentration of 0.02% preventing outgrowth at pH 6 and 0.1% preventing outgrowth (7), on the other hand, noted inhibiheat-injured Clostridum perfringens strain FDI spores. tion of several Bacillus species in yeast-glucose-Incubation conditions were the same as those described agar by 0.075 to 0.25% nitrite. The various efin Fig. 2, except for the concentrations of sodium fects of sodium nitrite on germination are probnitrite used. FIG. 4. Effect of sodium nitrite on the outgrowth of non-heat-injured Clostridium perfringens strain NCTC 8798 spores. Spores were heat-shocked at 70 C for 15 min and incubated under the same conditions as those described in Fig. 2, except for the concentrations of sodium nitrite used. at pH 7, since the OD at concentrations of 0.01% was greater after 150 min than that of the control. Similar experiments were conducted with NCTC 8798, a heat-resistant strain. This strain was more sensitive to nitrite than was FD1; concentrations of 0.02 and 0.3 % were required for inhibition of outgrowth of non-heat-injured spores at pH 6 and 7, respectively (Fig. 4). As with strain FD1, heat-injured spores were more susceptible to inhibition of outgrowth by sodium nitrite both at pH 6 (0.01%) and pH 7 (0.04%; Fig. 5). Although commercial heat treatments employed in the processing of canned cured meats may destroy heat-sensitive spores, it is unlikely that all spores of resistant strains are so affected. That the latter may be more sensitive to inhibition of outgrowth by sodium nitrite is therefore of added importance. Sodium nitrite-induced Germination. There have been several reports on induction of spore germination by sodium nitrite ( low that to which spores are subjected during the thermal processing of canned cured meats. We have confirmed that nitrite induction of germination does occur with C. perfringens and have investigated the possibility that this may be a factor in preservation ofcanned cured meats. If commercially acceptable concentrations of nitrite induced germination of the spores in a meat product during processing, the spores would become heatlabile and would therefore be inactivated by the thermal process. The effect of nitrite concentration on the induction of germination of strain FD1 spores is shown in Fig. 6. There is a limiting concentration (about 1.5%) above which the level of initiation of germination will not increase under the stated conditions. The effects of temperature and pH are shown in Table 1. As the temperature increased, the per cent initiation of germination increased at pH 6. Complete germination occurred in 90 min at low temperatures (25 C) and at pH 6 only if the nitrite concentration was sufficiently high (6%). Essentially no germination occurred at pH 8. Nitrous acid appears to be the effective agent, since the extent of initiation of germination was greatest at pH 6. No germination occurred after 2 hr at pH 6 when the spores were incubated at temperatures of 25, 37, 45, 60, 75, or 90 C with as much as 0.02% sodium nitrite, the maximum 96 a Germination mixtures contained 1% NaNO2 and 33 mm phosphate buffer. Germination was determined after 1 hr of incubation. allowable concentration in canned cured meats. Slight decreases in OD in the control tubes were observed at 75 and 90 C after 24 hr. Microscopic examination of these controls revealed spores with a phase-dark periphery but a refractile core. It is unlikely that most of these spores remained viable since this strain, FD1, is itself heat-sensitive with a D9o equal to about 15 mi. It may be concluded that, although sodium nitrite may induce bacterial spore germination, concentrations that are commercially acceptable will not. DISCUSSION The method by which sodium nitrite may act to prevent microbial growth of heat-injured spores in canned cured meats was investigated by using C. perfringens. Four possible mechanisms were tested: (i) prevention of germination of heatinjured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, (iv) induction of germination (which would render the spore susceptible to subsequent heat treatments). When sodium nitrite is heated in a complex medium, it combines with some component in the medium to yield an unknown but extremely potent inhibitory agent; e.g., autoclaving 3.5 ,ug/ ml (0.00035%) in a medium for 20 min at 109 C produces enough inhibitor to inhibit 8 X 106 vegetative cells in 50% of the trials (12). This product has been shown to be effective against vegetative cells of several Clostridium species (11). However, the present results indicate that, even when using concentrations of sodium nitrite up to 10 times greater than commercially acceptable levels, the inhibitory agent, if indeed produced, is not effective in preventing initiation of germination of most of the intact or heat-injured C. perfringens spores. For instance, at pH 6, about 0.5% nitrite was required to reduce the per cent germination of heat-injured strain FD1 spores to 50% of the control, whether sodium nitrite was added before or after autoclaving. It has been found (9) that the inhibitory agent formed by heating nitrite in a complex medium may have little effect in the preservation of canned cured meats. A meat suspension heated with 150 ,ug (0.015%) of sodium nitrite per ml was inhibitory to C. botulinum; however, the inhibition was thought to be due to residual inorganic nitrite since dialysis removed the inhibitory activity. Furthermore, the inhibitory factor produced by heating nitrite in a complex medium could be inactivated by nonfat meat solids. The present results show that sodium nitrite at greater than commercially acceptable concentrations does not prevent initiation of germination of the majority of non-heat-injured or heat-injured C. perfringens spores, regardless of whether it is added before or after autoclaving the complex germination medium. However, a small number of the spore population may actually have been blocked from initiating germination. These blocked spores would not have been detected by the technique used for assessment of spore germination, since extinction measurements are of little value in estimating germination above about 90% (8). It would be difficult to distinguish a low number of nitrite-blocked spores from the "superdormant" spores (8) that may be present in a normal spore population. The fact that a small number of spores may be found by postprocessing analysis of canned cured meats (13) indicates that either inhibition of the initiation of germination of the heat-injured spores has occurred or that the spores represent a superdormant fraction. Indeed, superdormancy may have been enhanced by heat injury. Although sodium nitrite alone did not prevent germination of the majority of the spore population, it may have inhibited germination of small numbers of spores. This inhibition should be greater in combination with the curing salt sodium chloride. Sodium nitrite and sodium chloride are not solely responsible for the preservation of canned cured meats. Heat plays an important role, although it is not sufficient to destroy all bacterial spores. The thermal treatment is thought to injure the spores, making them more sensitive to curing salts in the outgrowth medium (3,14). This phenomenon was observed in the present studies with heat-damaged C. perfringens spores. At pH 6.0, concentrations of 0.02 and 0.01% sodium nitrite inhibited outgrowth of heat-damaged spores of a heat-sensitive and a heat-resistant strain, respectively. Unheated spores required 0.04 and 0.02%, respectively. It should be pointed out that the incubation temperature was 45 C for 3 hr. It is conceivable that, with additional incubation, inhibition might have been overcome. A temperature of 45 C was used since it is near the optimum for growth of C. perfringens. Roberts and Ingram (14) pointed out that the injured spore may be affected by the temperature prevailing while it is struggling against the inhibitory salts. Undoubtedly, inhibition of outgrowth of germinated spores by sodium nitrite is an important aspect of canned meat preservation, since commercially acceptable concentrations were effective. Nitrite-induced germination has been observed with several organisms. We confirmed its occurrence with C. perfringens. High temperature, low pH, and high sodium nitrite concentrations yielded optimal germination. Undissociated nitrous acid presumably was the effective agent. Duncan and Foster (4) postulated that the phenomenon of nitrite-induced germination may be involved in the stability of the preservative system. Once germinated by sodium nitrite, the spores would be destroyed by the heat treatment rendered the particular product. This seems unlikely, since, in the present experiments, germination was not obtained after 1 hr at 90 C with as much as 0.02% sodium nitrite, the maximum concentration allowable in commercial use. Whether spores of other organisms would be similarly affected is not known. An important factor in the safety and stability of canned cured meats which must be mentioned is that of cell numbers (13). In the present experiments, concentrations of about 107 spores per ml were used; this is much greater than the actual spore load in meats. Concentrations of sodium nitrite lower than those used here may have been equally effective against germination and outgrowth had we employed a smaller number of spores. In these experiments no attempt was made to investigate the additive effect that a combination of sodium chloride plus sodium nitrite might have in inhibiting growth from spores. Conceivably, the combined inhibitory effect would be greater than that reported for nitrite alone.

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Epidemiological parameters to delineate weather-disease interactions and host plant resistance against leaf blight in small cardamom (Elettaria cardamomum Maton) Small cardamom, the versatile spice of Zingiberaceae is cultivated in diverse agro-climatic regions of India especially in South India. Leaf blight incited by Colletotrichum gloeosporioides sensu lato is one among the major challenges encountered across different cardamom growing tracts. In the present investigation, epidemiology of cardamom leaf blight was studied and an attempt was made to decipher the resistant nature of Malabar, Mysore and Vazhukka genotypes based on epidemiological parameters. The average percent disease index recorded in Malabar, Mysore and Vazhukka varied from 23.41 to 27.72, 18.79 to 20.34 and 18.74 to 20.38, respectively. The disease exhibited a positive correlation with respect to Tmax and Tmin in all the genotypes, however, significant correlation was observed only in Malabar and Mysore with respect to Tmax. Whereas, rainfall and rainy days had negative correlation with leaf blight in all genotypes however found to be non-significant. The average infection rate was maximum (0.000429) in Malabar, whereas it was 0.000124 and 0.000186 in Mysore and Vazhukka, respectively. The area under disease progress curve registered the highest for Malabar (8814.15) and lowest in Vazhukka (6531.02) while, Mysore type recorded 6612.96 indicating that, Vazhukka and Mysore types might posses horizontal resistance and Malabar with vertical resistance. In the light of above results, plant protection measures could be scheduled based on the take-off level and genotypes with horizontal resistance could be promising candidates in resistance breeding programmes. Introduction Small cardamom (Elettaria cardamomum Maton) is a commercial Zingiberaceous spice originated and evolved in the biodiversity rich forest ecosystem of Western Ghats, India. Besides its centre of origin, cardamom is widely cultivated in Sri Lanka, Guatemala, Papua New Guinea and Tanzania (Ravindran 2002). The diverse species has morphologically evolved into Malabar, Mysore and Vazhukka genotypes distinguished primarily based on plant stature, leaf, panicle and capsule characters. A myriad of biotic and abiotic factors acts as major 23 impediments to cardamom cultivation consequently leading to decline in gross cropped area and productivity. Among the foliar diseases, leaf blight incited by Colletotrichum spp. is noticed in all cardamom growing areas, inflicting damage invariably to all the genotypes. Divergent species belonging to the hemi-biotrophic ascomycetous pathogen, Colletotrichum incites leaf blight (Chethana et al. 2016). Though the disease is prevalent during the entire cropping season, it generally exacerbates and assumes epiphytotic proportions during post-monsoon period, probably favoured by an escalation in atmospheric temperature (Thomas & Bhai 2002). The anamorphic phase (conidia) plays a pivotal role in the epidemiology of leaf blight facilitating dissemination and pathogenesis evidently favoured by conducive environmental factors. Delineating vulnerable crop stages with respect to weather factors and disease pattern helps to formulate and implement plant protection strategies economically and efficiently. Correlating the disease progression with weather variables adequately supports decision making process besides providing an indirect indication on pathogen dynamics. The apparent infection rate (r) in conjunction with area under disease progress curve (AUDPC) values could be effectively employed as epidemiological parameters to define vertical or horizontal nature of disease resistance ( Van der Plank 1963;Nagarajan 1983). Adopting plant protection measures based on apparent infection rate helps targeting the initial inocula thereby checking disease spread. Definitive information on epidemiology of cardamom leaf blight and the role of r and AUDPC parameters in disease progression as well as attempts towards deciphering the nature of resistance based on epidemiological parameters is scanty in cardamom-Colletotrichum host-pathosystem. Hence, the present investigation was formulated to delineate the relationship between disease progression vis-à-vis weather variables and an attempt was made to interpret the resistant nature of Malabar, Mysore and Vazhukka genotypes based on epidemiological parameters. Recording leaf blight incidence in field gene bank accessions The study was carried out at ICAR-Indian Institute of Spices Research Regional Station, Appangala, Madikeri, Karnataka, India (12 o 26'N Latitude, 75 o 45'E Longitude, 920 meters above MSL) during 2015-16. The experimental materials comprised of 119 field gene bank accessions and three released varieties (Appangala 1, IISR Avinash and IISR Vijetha) representing Malabar (41 accessions), Mysore (29 accessions) and Vazhukka (52 accessions) genotypes. The accessions used in the study were collected during exploration and collection programmes undertaken at diverse agro-climatic regions of major cardamom growing tracts and maintained in the in situ Field Gene Bank of the experimental location. The accessions/varieties (4 to 5 years old) were established under natural vegetation of shade trees during 2011 and each accession had five clumps each. The area under the experiment was maintained under uniform shade level and recommended package of practices were adopted to raise the crop with timely adoption of recommended plant protection chemicals to manage insect pests (thrips and panicle/shoot borer) and diseases (rhizome and root rot) other than leaf blight. Since, foliar diseases like leaf blotch, Phytophthora leaf blight and rust were either totally absent or less prevalent, foliar sprays with any of the recommended chemicals were not taken up. The incidence of leaf blight invariably relied on natural inoculum was visually recorded monthly employing 1-6 disease rating scale based on severity of foliar symptoms and percent disease index (PDI) was calculated. For scoring leaf blight incidence, peripheral portion of the clump comprising of senile tillers were avoided and the scoring was based on the manifestation of foliar symptoms on the inner tillers (minimum 8-12 tillers) of the clump. The PDI of all the accessions representing each genotype was calculated and averaged to depict the monthly average PDI for each genotype. Data on different weather variables such as maximum (T max ) and minimum (T min ) atmospheric temperature, rainfall and number of rainy days were also recorded daily and the monthly average values were computed. In order to study the relationship between various weather parameters and disease incidence by correlation studies, monthly averages of these weather factors was used. Computing r and AUDPC values The apparent infection rate (r) is a parameter used to analyze the momentum of epidemic development. The apparent infection rate was calculated for each genotype based on the formula suggested by Van der Plank (1963). where, r is the apparent infection rate, x 1 is the disease index at initial time (t 1 ), x 2 is the disease index at subsequent observations (t 2 ). The apparent infection rates were further employed to identify the highest and lowest infection periods with respect to each genotypes. The AUDPC value was computed using the formula suggested by Wilcoxson et al. (1975). where, y i is the disease incidence at i th day of evaluation (initial observation), y i + 1 is the observation after the successive defined period, t i+1 -t i is the period between two observations and n is the number of successive evaluations. Results and discussion The percent disease index (PDI) observed in different cardamom genotypes along with weather variables recorded during October, 2015 to September, 2016 are presented in Table 1. Among the genotypes, Malabar comprised of 41 accessions and Mysore and Vazhukka comprised of 29 and 52 accessions, respectively. The period of observation spanned reproductive as well as vegetative phases of developmental stage and post-monsoon as well as monsoon periods as far as the weather pattern is concerned. Leaf blight incidence in cardamom genotypes and variation in weather variables In Malabar genotype, the maximum PDI (27.72%) was recorded during May, 2016 whereas the minimum (23.41%) was observed during October, 2015. In Mysore type, the PDI was in the range of 18.79% to 20.34% which was the minimum during July, 2016 and the disease registered as peak during April, 2016. Biju et al. In Vazhukka, the variation in PDI was 18.74% to 20.38% of which the minimum was noticed during December, 2015 while; the maximum was recorded during September, 2016. The maximum atmospheric temperature (T max ) varied from 24.3 o C to 33.7 o C in which September, 2015 had the minimum and April, 2016 recorded the maximum. Whereas, the minimum atmospheric temperature (T min ) was in the range of 13.1 o C to 18.7 o C during January and May, 2016 respectively. The average monthly rainfall ranged between 0-653.4 mm. During the period of observation, the rainfall was maximum during June, 2016 and minimum during February-March 2016. The rainy days were highest during August (29 days) and no precipitation was recorded during January to March 2016 (Table 1). Crop architecture and microclimate appears to be the deciding factors which favour incidence and proliferation of various diseases in the spice ecosystem. Nevertheless, the dynamic weather variables, often manifested in the form of erratic rainfall and temperature fluctuations over a period of time have unforeseen effects which may be even pose a threat to the production system in which these spice crops are grown either as principal or component crops. Murugan et al. (2017) observed that, climate change as evidenced through rise in ambient temperature and erratic rainfall immensely contributed to disease outbreaks in cardamom, demanding nonjudicious application of fungicides. Patterns in disease progression and correlation with weather variables The disease exhibited a positive correlation with respect to T max and T min in all the genotypes, however, significant correlation was observed only in Malabar and Mysore with respect to T max . Whereas, rainfall and rainy days had negative correlation with the disease in all genotypes however found to be non-significant (Table 2). In Malabar, the disease progression followed a high-low-high pattern. The disease exhibited increasing trends during November, December, January, March, April, May, August and September, whereas, decreasing trends were observed during October, February and June-July. In Mysore genotype, leaf blight incidence registered peaks during November, March-April and August-September. While, the lowest incidence was noticed during October, December-February and May-July. In Vazhukka, decreasing trends in the PDI was observed during October-November, June-July and increasing pattern was noticed during November, January-March, May and August-September. In general, increasing as well as decreasing trends in disease progression were observed in all the genotypes during different months with a uniform increase during November, August and September whereas, a uniform decreasing trend was noticed during October, June and July. Though the impact of weather on diseases incited by Colletotrichum are well described by earlier workers, deciphering disease progression and host plant resistance through epidemiological parameters are not attempted in cardamom-Colletotrichum hostpathosystem. Alterations in temperature and moisture regimes influence population dynamics and pathogenicity of infectious agents and also might impact host physiology (Coakley et al. 1999;Chakraborty & Datta 2003;Mina & Sinha 2008). Physiological changes in host plants might result in increased resistance, leading to evolution of pathogens to prevail over host-plant resistance (Caffarra et al. 2012). Foliar diseases incited by Colletotrichum species are generally favoured by temperature in the range of 25 to 35 o C, atmospheric humidity of 80-90% and rainfall. Singh et al. (2009) reported that, anthracnose of guava incited by C. gloeosporioides attained a peak during September which had a negative correlation with maximum and minimum temperatures. The disease also exhibited a positive correlation with precipitation defined in terms of rainfall and number of rainy days. The minimum and maximum temperature regime for development of guava anthracnose was found to be 10 and 35 o C, respectively with an optimum of 30 o C (Tandon & Singh 1969). Bainik et al. (1998) reported the temperature range 28-34.2 o C favours development of mango anthracnose. Though relative humidity had positive correlation with disease development, rainfall and number of rainy days had non-significant correlation (Ann et al. 1994). The sporulation in Colletotrichum is favoured by temperatures in the range of 20-25 o C, while temperatures above 30 o C have an inhibitory effect (Kendrick & Walker 1948;Slade et al. 1987;Mello et al. 2004). Cowger & Mundt (2002) illustrated that, interaction of components conferring genotypic resistance with environmental factors is required to modify the effects of host diversity with respect to disease progression. In cardamom system, it is speculated that Colletotrichum might have remained as cryptic dormant endophyte during anti-epidemic phase and subsequently activated during postmonsoon period particularly favoured by temperature. C. gloeosporioides colonized the internal foliar niche as endophyte in sweet orange which served as inoculum reservoirs Categorization of accessions based on resistance towards leaf blight The reaction exhibited by the genotypes and their categorization based on resistance/ susceptibility levels towards leaf blight are presented in Table 3 Trends in r and AUDPC values The rate of disease spread as expressed in terms of r-value exhibited an erratic pattern irrespective of the genotypes. However, in general, the occurrence of anti-epidemics (where the r value attained a negative phase) was found to be uniform in all the genotypes during June-July. The average r-value was the highest (0.000429) for Malabar and least in Mysore (0.000124), whereas in Vazhukka it was 0.000186 (Table 4). The take-off level (an increase in r-value after attaining the anti-epidemic phase) was generally observed during July-August which coincided with an average T max of 24. Non-judicious application of synthetic molecules might lead to several far-reaching consequences as it contributes immensely to the evolution of novel races of pathogens with fungicide resistance and pesticide residues in the produce. Besides deciphering the speed at which the disease spreads in a population, the r-value could be effectively employed to identify the stages at which the plant protection measures can be adopted, primarily targeting the initial inoculum (X o ). In the present study it is observed that, in general, the r-value exhibited the trend of anti-epidemic phase during May-June and a take-off phase during July-August. Hence, undertaking plant protection measures with recommended fungicides before the commencement of monsoon (May-June) and during July-September targeting the low inoculum levels would have significant adverse effect on the disease progression. From the perspectives of epidemiology and host plant resistance it is concluded that, an increase in temperature during post-monsoon and precipitation have significant implications on disease progression. Introduction India is the second largest producer of onion next to China. Maharashtra state is the leading producing state in India. Onion is cultivated in three distinct seasons namely Kharif, late Kharif and Rabi. The soil moisture affect the quality of bulb and yield which is greatly influenced by the irrigation system. Onion is a shallow rooted crop needs light but frequent irrigation either by flood, sprinkler, or drip. The productivity of onion in India is 17.33 ton ha -1 which is low compared to world average. Managing the amount of applied irrigation water is critical to achieve optimum yield and quality. Most of the onion grown in India is under surface irrigation, which is relatively inexpensive, but inefficient in the amount of water use. Irrigation through drip is a new technique to increase agricultural production and to enhancing the efficiency of water use (Kusçu et al. 2009;Shock 2013;Enciso et al. 2015). Drip irrigation lends itself to automation, more so than either surface or sprinkler irrigation. Keeping this in mind, an experiment was planned on onion to study the feasibility of onion cultivation under drip irrigation. system fertigation was done with drip tank in 7 splits with 7 days intervals per day 2-3 hr. First irrigation was operated immediately after transplanting and light irrigation was done three days after transplanting for better and uniform initial establishment of crop. Materials and methods Recommended crop production and protection practices were followed as and when required to get good healthy crop. Following critical precautions were taken while conducting the experiment viz. irrigation interval followed uniformly; the operating pressure of drip system was 1.0 -1.5 kg cm -2 . In both the systems, irrigation was stopped at 15-20 days before crop harvesting. The bulbs were harvested at full maturity stage. After proper curing and neck cutting, the observations on yield and yield contributing characters and marketable bulb yield, percent of A (>6.5 cm), B (4.5-6.5 cm) and C (< 4.5 cm) grade bulbs on weight basis separately recorded and quantity of water applied was also measured. Results and discussion The results revealed that all growth and yield parameters of onion are significantly influenced by irrigation methods. et al. 2012). The results further revealed that drip irrigation provided lower bolting (0.56%) and doubles (2.28%) as compared with surface system and it was observed that during Kharif season bolting did not record in any treatments because the average minimum temperature is above 17ºC is not favourable to initiate bolting, however doubles were recorded (Table 2). Nashik comes under shadow of a southwest monsoon; hence the erratic summer monsoon experienced by this region sets in last part of June and extends till the 2 nd week of October. The mean average rainfall varied from 548.0 mm to 862.0 mm during 2013 to 2015. Rainfall was coincided with bulb initiation and bulb development phases, due to heavy rain fall during Kharif season poor bulb development was observed. This is the reason and other climatic reasons during Kharif season 36.32% yield decreased as compared with Rabi, however by adopting drip irrigation system in Kharif season considerable yield was increased over surface irrigation method because drip irrigation raised bed system up to some extent remove excess water and avoid water logging due to slow and steady runoff water as compared with surface flat bed system where crop was affected. Over all during Kharif season in drip irrigation, the gross yield and marketable yield increased 14.27% and 18.30%, respectively. It is evidenced from the results, properly designed and managed drip irrigation raised bed has many advantages over surface irrigation including: elimination of surface runoff, high uniformity of water distribution, high water usage efficiency, flexibility in fertilization, prevention of weed growth and plant disease during rainy season. The results further revealed that highest 'A' grade (>6.0 cm) bulb (63.07%), 'B' grade (4.0-6.0 cm) bulb (24.82%) and lower 'C' grade (<4.0 cm) bulb (12.10%) were recorded in drip irrigation system during Rabi season and in Kharif also higher 'A' grade bulb (28.97%), 'B' grade bulb (36.28%) and lower 'C' grade bulb (34.76%) recorded in drip irrigation system (Table 3). Drip irrigation ensures optimum growth, better bulbing and early maturity of crops by assuring optimum soil moisture, water, air and nutrients throughout the crop growing period resulting uniform bulb obtained is directly correlated to the highest bulb size and productivity, whereas in surface irrigation yield decreased due to deep percolation and water is lost beyond the active absorption zone of the root system as an onion The benefits of drip irrigation system over surface irrigation are illustrated in Table 4, that applied water in drip system is very lower in for production of 5.62 kg and 3.49 kg of onion in drip and surface irrigation, respectively. Whereas in Kharif season 1000 litre of water by drip produces 4.18 kg, while in surface it is 2.67 kg, therefore drip irrigation system well suited for shallow rooted onion. The Cost: Benefits ratio also high in drip (1:2.69) while in surface irrigation it is 1: 1.68. The Cost: Benefits ratio in drip suggests that despite higher initial cost of the drip system, the drip irrigation is more profitable than the surface irrigation. Based on the obtained results of Rabi and Kharif seasons of the effect of drip irrigation on yield, yield components and morphological characteristics of onion, as well as water use efficiency and water saving it concluded that drip irrigation is highly significant effect on all studied parameters. To achieve a high production potential of onion, adopting drip irrigation should be maintained during the both Rabi and Kharif seasons. , but intensive farming that need high input of mineral fertilizers is considered as serious damage that may affect soil health, sustained production and balanced environment (Anwar et al. 2007). Taking into consideration of the deleterious effect of continuous applying of mineral fertilizer on the soil structure, organic farming could consider as a suitable replacement of inorganic fertilizer for improving microbial population, and soil organic matter (Shahram & Ordookhani 2011). Reza et al. (2015) claimed that substituting chemical fertilizers by organic manures and biofertilizers, could consider as a good farming system improve the ecosystem and soil health as a step for achieving sustainability in agriculture. Besides this, for medicinal and aromatic plants (MAPs), the real value is given to the quality rather than quantity, so that organic farming is considered as a suitable approach that enhances the performance of these crops. However, complete replacement of inorganic fertilizers by the organic manures is not advisable owing to their very low nutrient concentration and in turn requirement in huge quantities which may not be possible due to scarcity of such materials. In this endeavor, a blend of organic and inorganic fertilizers is important for improving the yield, maintaining soil health and keeping favorable ecological conditions on long-term basis. Amran (2013) and El-khyat (2013) revealed that application of organic manure along with half dose of chemical fertilizer had a positive impact on the oil yield of Pelargonium graveolens and Rosmarinus officinalis. The quality of the essential oil in basil is determined by the percentage of its volatile molecules which is affected by soil and climate conditions. Combined application of organic manure and inorganic fertilizer is considering the best tool that can be used to improve the yield and quality of these plants. Despite the importance of the basil crop, information on different aspects of growth, development, influence of organic manure and inorganic fertilizers on herbage and oil yield is very meager. In this context, this study was aimed to find out the "Influence of FYM, inorganic fertilizer and bio-fertilizers on herbage, oil yield, essential oil content and oil quality of sweet basil. Material and methods Field experiments were conducted in the experimental field at ICAR-Indian Institute of Horticultural Research (ICAR-IIHR), Bangalore during the kharif season of 2015 and 2016. The experimental station is located at an altitude of 890 m above mean sea level and 13 o 58" N latitude and 77 0 29" E longitudes. The nine treatments of experiment contain T 1 (FYM (10 t ha -1 ) + 100% recommended N through FYM), T 2 (FYM (10 t ha -1 ) + 100% recommended N through FYM + bio-fertilizer), T 3 (FYM (10 t ha -1 ) + 75% recommended N through FYM), T 4 (FYM (10 t ha -1 ) + 75% recommended N through FYM + biofertilizer), T 5 (FYM (10 t ha -1 ) + 50% recommended N through FYM), T 6 (FYM (10 t ha -1 ) + 50% recommended N through FYM + biofertilizer), T 7 (recommended FYM (10 t ha -1 only), T 8 (recommended NPK (160:80:80 kg ha -1 ) only, and T 9 (recommended FYM 10 t ha -1 ) + recommended NPK (160:80:80 kg ha -1 ). Treatments were replicated thrice in a randomized complete blocks design. Physical and chemical properties of the initial experimental soil are presented in (Table 1). The nutrients were supplied in the form of straight fertilizers like urea (160 kg N ha -1 ), single super phosphate (80 kg P 2 O 5 ha -1 ) and muriate of potash (80 kg K 2 O ha -1 ). Fifty per cent of nitrogen and full dose of phosphate and potash were applied as basal dose and the remaining fifty per cent of N was applied after 45 days of transplanting in T 8 and T 9 treatments. For biofertilizers, Arka Microbial Consortium (AMC) developed by ICAR-IIHR was used in the experiment and it contains N fixing, P and Zn solubilizing and plant growth promoting microbes in a single carrier. After 15 days of transplanting, recommended dose of AMC @5 kg ha -1 was applied at 2 cm deep to individual plants and immediately covered with soil. Similar method of application was also followed for ratoon crop after harvest of main crop in T 2, T 4, and T 6 treatments. Quantities of added fertilizers are given in (Table 2). 40 Each experimental plot size was 4.8 m long and 4 m wide with spacing of 40 cm between the plants and 60 cm between the rows. There was a space of 0.5 meter between the plots and 0.5 meter between replications. Basil variety Cim-Saumya (CIMAP) was sown in two nursery beds of 6.0 m in length with 1 m in width and 10 cm height. Forty days old (40) healthy and uniformly rooted seedlings of sweet basil were transplanted to the field. Weeding was done manually and drip irrigation was given daily for half an hour in the early stages and subsequently irrigation was given depending on the soil moisture condition. Fresh weight from each plot was converted to per hectare and it was expressed in tones (t). In order to determine the essential oil content (%), a sample of 100 g of basil fresh herb from the each plot were collected and mixed with 500 ml distilled water and then were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus (Darzi et al. 2012). The quality of basil oil samples was analyzed by gas Al-mansour et al. FYM=Farm Yard Manure; Rec.=Recommended and *BF=Bio-fertilizer chromatography (Varian 3800 series) using VH-5 column for GC and VH-5 MS column for GCMS 30 m x 0.2 mm with 0.2 mm film thickness, oven temperature programmed at 60 0 C for 5 min then 210 0 C hold for 1 min then 240 0 C hold it for 1 min and helium gas as a carrier at 1 ml min -1 . Injector and detector temperature were 270 0 C and 240 0 C, respectively. Methyl chavicol and Linalool constituents of the oil were identified based on their retention time by comparing with the peak retentions times of those authentic standards obtained from Sigma, Aldrich, Bengaluru and run under identical conditions, then it were estimated in respect to total components and expressed as percentage. The data recorded from the experiment were analyzed using SAS 9.3 version of the statistical package (SAS Institute Inc 2011). Analysis of variance (ANOVA) was performed using SAS PROC ANOVA procedure. Means were separated using Fisher 's protected least significant difference (LSD) test at a probability level of p<0.01. Fresh herbage yield Fresh herbage yield of basil differed significantly due to application of different levels of N through FYM along with and without biofertilizers and inorganic fertilizer in the main crop and ratoon during two years of the experiment. It is evident from the Table 3 that the application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) i.e., T 9 recorded significantly the highest herbage yield in the main crop (39.96 t ha -1 ) and ratoon (19.37 t ha -1 ). The lowest fresh herbage yield per hectare was obtained in T 7 applied with FYM (10 t ha -1 ) alone (22.92 t ha -1 ) and in ratoon (10.76 t ha -1 ). Similar trend was also reflected in total herbage yield of basil. Application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) i.e., T 9 recorded significantly the highest total herbage yield (59.3 t ha -1 ) while, the lowest value (33.7 t ha -1 ) was recorded in T 7 applied with FYM alone. Nutrients through chemical fertilizer is expected to be more available that reflect on its uptake by plants leading to enhance the growth and yield. On the other hand, combined application of organic manure along with inorganic fertilizer regulated the supply of nutrients which in turn increased the yield (Merestala 1996). Similar findings were also reported by Mohamad et al. (2014) in Ocimum basilicum. Essential oil content Essential oil content plays a key factor in selecting the "adequate" combination of fertilizers in sweet basil cultivation. Application of different levels of FYM, bio-fertilizers and inorganic fertilizer on essential oil content (%) in the main crop and ratoon showed a significant difference (Table 3). Application of recommended NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) recorded maximum essential oil content ( 0.48 and 0.45 %) whereas the lowest essential oil content was recorded with recommended dose of FYM alone in T 7 (0.31 and 0.17%) in the main crop and ratoon, respectively. The content of soil nutrient enhanced with application of organic manure, that had positive effect on the growth parameters, herbage and oil yield (Khalid et al. 2006). Oil yield Oil production is the most important parameter in basil farming. The results of different levels of N through FYM, bio-fertilizers and inorganic fertilizer on oil yield of main crop and ratoon recorded during the two years of the field experiment showed a significant increase in oil production which can be attributed to the increase of nitrogen doses either through organic or inorganic form (Table 3). In the present study, oil yield per hectare increased with the increase of FYM doses, but with application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) recorded maximum oil yield in the main crop (199.7 kg ha -1 ) and in ratoon (107.58 kg ha -1 ). The lowest oil yield per hectare was obtained in T 7 applied with FYM (10 t ha -1 ) alone in the main crop (73.8 kg ha -1 ) and in ratoon (26.94 kg ha -1 ). Highest total oil yield was observed in T 9 applied with NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) and the lowest total oil yield was recorded in T 7 applied with FYM alone. Integrated nutrient management improve the chemical, physical and biological soil proprieties that reflect positively on plant growth and oil yield (Patra et al. 2000). These results are similar to the observation of Zeinab (2005), Dadkh (2012) and El-naggar et al. (2015). Oil quality The most important volatile molecules identified in basil essential oil are methyl chavicol as phenolic compound and linalool as monoterpene (Mondello et al. 2002) andSajjadi (2006). The quality standard of the essential oil in basil is determined by polyphenols concentration (Toor 2006). Comparing the integration peaks, there was a significant difference in methyl chavicol percentage within the treatments. The results in (Table 4) showed that T 9 applied with NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) recorded the highest percentage of methyl chavicol in the main crop of 2015 (52.3%) while, in the main crop of 2016, application of FYM (10 t ha -1 ) +100% recommended N through FYM + bio fertilizers i.e., T 2 recorded maximum methyl chavicol percentage (63.78%). In ratoon, application of FYM (10 t ha -1 ) + 100% recommended N through FYM + bio fertilizers recorded the highest methyl chavicol percentage (59.39 and 59.67%) during first and second year, respectively. The lowest percentage of methyl chavicol was recorded with T 7 (40.05 and 46.2%) in main crop and in ratoon (40.22 and 41.49%) of 2015 and 2016, respectively. Linalool content increased with dosage of nutrients application as shown in ( The outcome of the present investigation revealed that the maximum fresh herbage yield, essential oil content, oil yield and its best quality was obtained with application of recommended FYM (10 t ha -1 ) + recommended NPK (160:80:80 kg ha -1 ) for both main as well as in ratoon basil crop. Addition of biofertilizers also increased the content of principle ingredient in basil essential oil (methyl chavicol and linalool). Hence, the incorporation of full dose of recommended FYM along with 50% of recommended N through inorganic fertilizer as basal and the remaining fifty per cent as top dressing at 45 days after transplanting may be recommended for basil crop to realize higher herbage and oil yield and better oil quality in sweet basil. These districts contribute significantly to the plantation and spice economy of the country and houses main plantation crops like tea, coffee, rubber, and spice crops such as cardamom, black pepper and ginger. As these crops are mostly grown as rainfed, analysis of rainfall and identification of trend is essential for designing proper crop management plans in the light of current climatic change. Materials and methods Four exclusive WG districts viz., Kodagu from Karnataka, The Nilgiris from Tamil Nadu, Wayanad and Idukki from Kerala were selected for the study. These are high altitude districts have dense forest cover and enjoy higher rainfall. The altitude of Wayanad district is 700 to 1,200 masl lies in the latitude between 11°27' and 15°58' N and the longitude 75°47' and 70°2 7' E. Idukki's mean altitude is 1,200 m but many peaks rise above 2000masl and the district lies between 9°15' and 10°2' N and 76°37' to 77°25' E. The Nilgiris altitude varies between 300 m and 2,789 masl situated between 11°08' to 11°37' N and 76°27' to 77°4' E longitude. Kodagu has the lowest elevation range between 900 to 1,750 masl located between 11°56' to 12°56' N latitude and 75°22' to 76°11' E longitude. The rainfall data of these districts for hundred years (1901 to 2000) were purchased from IMD, Pune and used for rainfall analysis. A non-parametric Mann-Kendall test was used for the detection of rainfall trends (Mann, 1945;Kendall, 1975 According to this test, the null hypothesis H0 assumes that there is no trend (the data is independent and randomly ordered) and this is tested against the alternative hypothesis H1, which assumes that there is a trend at P d"0.05. Results and discussion Major plantation and spice crops like tea, coffee; black pepper and cardamom are produced in higher elevation of the WG districts where their relative spread and yield are high compared to lower elevation areas or plains. These crops used to grow under rainfed condition as the rainfall receipt was high and distribution also was good. The yield of these crops now often fluctuates mainly due to higher inter-annual variability in rainfall. Results of study conducted to find out the rainfall trend of potential plantation and spice producing WG districts viz., Idukki and Wayanad in Kerala, The Nilgiris in Tamil Nadu and Kodagu (Coorg) in Karnataka are presented and discussed here. The Nilgiris Mean annual rainfall was 1839.7mm (Table 1) Rainfall analysis of western ghats 48 (20%) like tea, coffee and the remaining areas are used for vegetables. They also stressed that many places experience sever water scarcity and moisture stress during summer months due to frequent dry spells even during monsoon periods that adversely affects the productivity of plantations and vegetable crops in this region. They also strongly advocated strengthening water harvesting structures to capture excess rainfall received between 20 th and 45 th weeks and utilize for farming. Kodagu Mean annual rainfall of this district was 2715.7mm (Table 2) with 17.0% C.V. Monthly rainfall pattern signaled that receipt was less during winter and summer months and July is considered the rainiest month (876.6mm) with a coefficient of variation of 34.8%. The rainfall amounts from southwest and northeast monsoons as well as summer rainfall were 2143.1mm, 310.5mm, 252.4 mm that respectively accounts 78.9%, 11.4%, 9.3% of annual rainfall. The rainfall receipt between May and October was more than 100 mm per month. Sufficient soil moisture would be available for crop production during these months. Mann-Kendall Statistics (S) and Sen's slope analysis have indicated that there was a positive trend in annual rainfall. Although few months showed negative trend in monthly rainfall, the 'p' values proved that as such there was no significant trend in monthly rainfall except for August where 'p' value was less than 0.05%. Raju et al. (2013) have classified the Kodagu climate as per-humid to humid type. Increasing rainfall trend was also reported by Mallappa et al. (2015) for Kodagu district. The district is popular for cardamom and coffee + black pepper cropping system and contributes substantially for national production. Ankegowda et al. (2010) have identified the length of growing period for this region between 21 st and 43 rd week (21 st June to 28 th November) with more dependable rainfall with lesser C.V. and suggested crop production operations to be carried out during this period for various crops. Idukki Idukki's mean annual rainfall was 2979.4mm (Table 3) with very high percentage of C.V. of (25.8%). Highest rainfall among months was received by July (673.7 mm) with 45.7% C.V. The monthly rainfall between May and October was above 100 mm and contribution to annual rainfall was maximum ( (2014) have noticed a decreasing monthly and annual rainfall trend in Kerala which they attribute to anthropogenic green house gas (GHG) emissions due to increased fossil fuel use, land-use change due to urbanisation and deforestation, proliferation in transportation associated atmospheric pollutants. Murugan et al. (2000;2009;2011) have reported the changes in rainfall trends of this region and discussed possible negative impacts on crops and plants as well as ecosystem hydrology because cardamom, coffee, tea and native forest plants are highly sensitive to precipitation changes. -2004), and only 28.2 percent of the years only fell under early or late monsoon years. Further, it was also noticed that if the monsoon was early that season would receive deficit rainfall, and no trend was seen when the monsoon was late. They also reported that there was decline of 6.8% in annual rainfall over the period which was more evident in monsoon rainfall since the last sixty years. Similar negative trend for the district was reported by Sushant et al. (2015). In another study by Kumar & Srinath (2011) showed that weakening of the early phase of the southwest monsoon precipitation; increasing polarisation of daily rainfall and more frequent heavy rainfall days that hastened maturation of a variety of crops. Gaetaniello et al. (2014) have observed that the variability in rainfall was more influential than the air temperature fluctuation in this district. During the study period (1951 to 2008), the monsoon rainfall decreased, while maximum and minimum daily temperature increased and farmers perceived Rainfall analysis of exclusive WG districts indicated that there was a temporal and spatial variation in rainfall receipt among these districts and high mean annual rainfall was received in Wayanad (3381.0 mm) followed by Idukki (2979.4 mm), Kodagu (2715.7 mm) and Nilgiris (1839.7). Decreasing trend in mean annual rainfall was noticed in Idukki, Wayanad and The Nilgiris, whereas in Kodagu the annual rainfall was stable (Fig. 1). The trend was significant for The Nilgiris and Wayanad. Similar trend was also observed for southwest monsoon rainfall. The decline in annual rainfall and the southwest monsoon was noticed for The Nilgiris and Wayanad. July was the rainiest month in all these districts. The negative trend was significant for January, May, June, July and August for the The Nilgiris, whereas in Kodagu no significant trend for mean monthly rainfall was observed except for August. At Idukki, significant negative changes were noticed for January, March, October and December months rainfall; whereas in Wayanad, January, March, April and July months rainfall showed significant negative trend. These negative trends in these important plantation and spices producing districts of the WG would affect the agricultural economy and hydrological systems. Rainfall analysis of western ghats The availability of quality planting material is one of the major issues that black pepper growers face in India. The conventional propagation methods have several limitations due to low success rate, poor rooting, spread of soil borne pathogens and poor survival rate of transplanted rooted cuttings (Rini et al. 2016). In order to overcome these problems, a technological intervention is needed to boost both the production and supply of quality planting materials. In the context of promoting organic cultivation, the use of biofertilizers for quality seedlings/cuttings production secures much importance because they are eco-friendly, low cost, capable of improving crop yields and quality sustainably. Biofertilizers improve growth rate of plants and soil health as they act as plant strengthners, phytostimulators, plant health improvers, and have the potential to fix nitrogen (Babalola 2014). The fertility of the soil is also restored by biofertilizers so that plants were better protected from getting any diseases (Amna 2010). Fish emulsions are used as a source of nitrogen during the early or vegetative stage of development to boost plant growth and also this has been documented to promote seedling growth in many crops T 4 0.2% Humic acid @100 ml plant -1 T 5 0.5% Fish extract @100 ml plant -1 T 6 PSB @5 g plant -1 + Azospirillum @5 g plant -1 + 0.2% Humic acid @100 ml plant -1 + 0.5% Fish extract @100 ml plant -1 T 7 PGPR Mix I @5 g plant -1 + 0.2% Humic acid @100 ml plant -1 + 0.5% Fish extract @100 ml plant -1 T 8 Untreated control Local Karimunda cuttings were planted in polythene bags size of 20 × 15 cm filled with potting mixture composed of garden soil, sand, and farm yard manure in 1:1:1 proportion. The potting mixture had an initial nutrient status of organic carbon (1.98%), N (0.704 mg/100 g) available P (4.01 mg/100 g) and K (43 mg/100 g) with a pH of 5.3. The treatments were superimposed and evaluated in vivo under greenhouse condition. Commercial formulations of phosphorus solubilizers (5x10 7 cfu), Azospirillum (5x10 7 cfu) and PGPR Mix I (Consortium of Azospirillum lipoferum, Azotobacter chroococcum, Bacillus megaterium and Bacillus sporothermodurans each with 5 × 10 7 cfu) were obtained from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The humic acid containing 12% potassium humate was used in this study. Fish extract was prepared as per the standard procedure. Equal quantity of fish and jaggery was taken, sliced, mixed and kept in closed container with periodical stirring up to 30 days, after that the solution was strained through a muslin cloth and stored. The experiment was conducted using Completely Randomized Design (CRD) with four replications. Five plants were kept in each replication.In addition to this fiveplants/ treatment were also maintained for destructive sampling to study the root characters. Talc based formulation of PSB, Azospirillum, PGPR Mix I, humic acid and fish extract individually and in combination were applied to the root zone of one month old black pepper cuttings grown in polythene bags. The treatments were given twice at fortnightly interval (when the plants were 30 and 45 days old). Observations on the height, number of leaves, leaf area and root characters were recorded 60 days after second treatment application. The experimental soil (potting media) was analyzed for physicochemical properties like pH, organic carbon and available N, P and K content. The leaf chlorophyll content was estimated as per the protocol of Sadasivam & Manickam (1992). The data collected from the experimentwere processed statistically with appropriate statistical tool for the interpretation of the results. There was no significant difference with respect to morphological characters before treatment application. Butgood improvement invegetative characterswas noted after the application of biofertilizers and organic supplements.Black pepper rooted cuttings responded well to combined inoculation of biofertilizers and organic supplements compared to individual inoculation and control ( Table 1). Application of PSB along with Azospirillum, humic acid and fish extract (T 6 ) resulted increased plant height (98.66 cm), numbers of leaves (12.16), number of roots (13.50), root length (28 cm) and highest leaf area (63.18 cm 2 ) 60 days after second treatment application compared to all the other treatments. Similar increaseof growth was recorded in black pepper for combined inoculation of biofertilizers such as Azospirillum, Phosphobacteria and VAM (Kandiannan et al. 2000;Bopaiah & Khader 1989). In addition to this, fish emulsions have been reported to increase the nitrogen accessibility (Weinert et al. 2014). According to Chen & Aviad (1990) the application of humic substances increase root length and produce more secondary roots. As a result, the plants were capable of absorbing more available nutrients from the soil which in turn resulted better establishment and subsequent growth and development. Thus, the combined inoculation of PSB and Azospirillum along with humic acid and fish extractperformed best through improving the morphological characters of the cuttings compared to their individual application. Total chlorophyll content was maximum in plants treated with PSB along with Azospirillum, humic acid and fish extract (0.64 mg/100 g) followed by the combined application of PGPR Mix I along with Azospirillum, humic acid and fish extract (0.60 mg/100 g). The least quantity of chlorophyll was observed in control plants (0.42 mg/100 g). This can be attributed to the increased uptake of nutrients leading to enhanced chlorophyll content. Pereira et al. (2015) reported that maize plants when inoculated with Azospirillum under different dosages of nitrogen enhanced the chlorophyll content. Our best treatment also includes Azospirillum as one component. This contributes to the increased chlorophyll content of the treated plants. Nutrient status of the potting mixture 60 days after second treatment application (Table 2) showed that, readily available nutrient content of potting mixture was improved through the application of biofertilizers and organic supplements compared to control. Among the treatments, PSB along with Azospirillum humic acid and fish extracttreated soil showed higher levels of organic carbon (3.17%), available phosphorus (17.83 mg/100 g) and potassium (84.66 mg/100 g) whereas, available nitrogen was highest for combined application of PGPR Mix I along with Azospirillum, humic acid and fish extract (1.05 mg/100 g). Increased availability of nutrients in the potting mixture was attributed through combined application of biofertilizers and organics. Bio inoculants have had influence in increasing the organic carbon content of turmeric (Sumathi et al. 2011). Biofertilizers like Azospirillum and PSB were highly beneficial to plants through augmentation of nitrogen and phosphorus content in soil, thus making these two essential nutrients available to the plant and also produce phytohormones like auxins (Singh et al. 2011;Rocheli et al. 2015). In our experiment also treatments consisting of PSB along with Azospirillum, humic acid and fish extract (T 6 ) resulted in high available organic carbon, P & K compared to control which is responsible for increased growth parameters observed. The increase in P availability could be attributed to the application of PSB which produces organic acids that act as a chelating agent and thereby, releases P into the soil solution and making it more available resulted in improved root growth. These findings are in agreement with the findings of Naik & Hari babu (2007) and Sharma et al. (2009) in guava. It is evident that, the combined application of PSB (5 g) along with Azospirillum (5 g), 0.2% humic acid (100 mL plant -1 ) and 0.5% fish extract (100 mL plant -1 ) followed by PGPR Mix I (5 g) along with 0.2% humic acid (100 mL plant -1 ) and 0.5% fish extract (100 mL plant -1 ) produced healthy black pepper rooted cuttings with good morphological characters than those of sole application of any of these biofertilizers and organics. Therefore, this technology could be effectively advocated to produce black pepper root cuttings with lusty growth in nurseries. 60 The demand for healthy planting material of high yielding black pepper varieties is on the increase and the country needs large quantity of quality planting material to meet the increasing demand. The present study was aimed to establish a model nursery to produce disease free healthy planting material of improved varieties of black pepper by creating awareness among farmers with good agricultural practices and by adopting nonchemical bio-intensive management strategies. et al. 2016) in different combinations were used as growth promoters as well as bioagents for incorporating into the nursery mixture. The experiment was designed in a two factor CRD with four improved varieties of IISR and five treatments. The five treatments were T1-T. harzianum + P. chlamydosporia, T2-Streptomyces strains 2+9, T3-Streptomyces strains 5+9, T4-Metalaxyl-Mz+ Carbsosulfan and T5-control without any amendments. The individual treatments were incorporated with the solarized nursery mixture separately and filled in polythene bags (15 cm × 10 cm) @250 g bag -1 . T. harzianum and P. chlamydosporia were made in liquid form with water and added @100 mL (cfu 10 9 mL -1 ) each to 100 kg potting mixture. Streptomyces spp. grown as broth culture in Nutrient broth and 100 mL (cfu 10 10 mL -1 ) mixed with 1 kg vermicompost and grown for 5 days (cfu10 8 mL -1 ) ) and applied @1 kg 100 kg -1 potting mixture. The treatment imposed poly bags were arranged inside the nursery and planted with single node virus free (indexed) rooted cuttings as mentioned above and were allowed to grow by serpentine method. When the number of rooted nodes in the serpentine reached around 10, the rooted middle cuttings were cut and separated leaving three plants at the tip and nucleus plant at the end and were kept for establishment to a 3-4 leaf stage in the same nursery. Five plants each were taken from each treatment and observed for biometric growth parameters. The biometric observations were recorded on height of the plant, fresh and dry weight of the plant, number of roots, root length and root biomass. The soil was analysed for the presence of targeted pathogens like Phytophthora capsici and nematodes (Radopholus similis and Meloidogyne incognita), pH and dehydrogenase activity (DHA). The data were analyzed by using PROC ANOVA procedure of SAS 9.3. Least square means statements were used for mean separation. After nine months of growth by serpentine method, the variety IISR Malabar Excel and IISR Thevam produced the maximum number of plants in T1 (T. harzianum + P. chlamydosporia (59 nos. and 51 nos. respectively) followed by T2 (Act 2+9) in case of Malabar Excel (45nos) and T3 (Act 5+9) (45 nos) in case of Thevam, from a single node cutting. An average of 6-7 plants/month/cutting was produced from these varieties with the treatment T1, while it was only 4 plants/month/ cutting in control. In case of IISR Girimunda, the performance was almost the same with all the three bioagent combinations when compared to Metalaxyl-Mz + Carbsosulfan and control. However, IISR Shakti showed comparatively lesser performance with bioagent combinations. The results of the study clearly showed the response of varieties to bioagents. In all cases, the number of plants produced with Metalaxyl-Mz + Carbsosulfan (T4) was comparatively lesser when compared to control ( Table 1). The root system was also healthy in all treatments except for control where the root was not profusely grown. No disease of any kind was observed in any of the plants. Though there is no difference between varieties in fresh weight of the plant, the dry weight ( Fig. 1) is significantly superior in treatment with Streptomyces strains (Act 2+9) and is at par with T. harzianum and P. chlamydosporia. Not much difference was observed in the number of leaves between treatments but, there is difference in the height of the plants (Fig. 2) where bioconsortia showed increased height when compared to Metalaxyl-Mz. + Carbsosulfan and control. Difference was observed in number of roots, root length, and root fresh and dry biomass (Tables 2 to 5). Since the nursery mixture was solarised and irrigation was limited to once in two days, there was no incidence of soil borne infections caused by Phytophthora capsici, Sclerotium rolfsii or nematodes (the common diseases otherwise observed in nurseries), in any of the treatments including control ( Table 6). The pH of the soil in untreated plants ranged from 4.55-5.66. It is interesting to note that the pH of the soil is raised to neutral in treatments with T. harzianum + P. chlamydosporia where it ranged from 6.86-7.63. In the case of Streptomyces combinations the pH ranged from 4.85 to 6.85 (Table 6) and the dehydrogenase activity which reflects the total oxidative activity of soil microflora, (Liang et al. 2014) was unaffected by the incorporation of bioagents like Trichoderma, Pochonia or Streptomyces sp. (Table 6). So without the addition of external nutrients, the micro flora enriched solarized mixture supported the growth of plants as well as prevented the incidence of infection caused by nematodes, Phytophthora or any other soil borne pathogens of black pepper. This may be due to the increased microbial activity either through the production of IAA or other growth promoting traits including siderophore production which is observed in the case is an important tree spice, yielding two spices, namely, the nutmeg (dried seed) and the mace (dried aril surrounding the seed). Nutmeg is hitherto considered to be predominantly dioecious in nature (Flach 1966). However, of late this concept is undergoing a paradigm shift, as monoecious trees are being reported often across the country (Krishnamoorthy et al. 1996;Krishnamoorthy et al. 2012;Rema et al. 2014). Though some literature is available on the reproductive biology of dioecious nutmeg (Armstrong & Drummond 1986), there is no report available for monoecious nutmeg trees. Here we attempt to study the floral diversity of monoecious nutmeg. The study on sex differentiation and variability in monoecious nutmeg was carried out at ICAR-Indian Institute of Spices Research (IISR), Kozhikode during June to September 2015. Total of 53 trees of 18 year old were selected randomly from the germplasm maintained at ICAR IISR and were studied for sex of tree from December 2014 onwards. The sex of flowers were recorded in all the trees at monthly interval. Out of the 53 trees of the selected population 6 trees are found to be monoecious. These 6 monoecious trees are used in the present study. Within the 6 monoecious trees observations on inter and intra floral variability in monoecious for anther length, number of anthers, filament length, ovary length and flower opening type were recorded. Total of 100 flowers / month were collected from each tree and observations were recorded for variability during peak flowering season June -September, 2015. Pollen viability test was done using Acetocarmine staining method. Pollen viability estimated using glyceroacetocarmine (Marks 1954). The pollen viability percentage determined as the ratio of the number of viable pollen to the total pollen number. ANOVA for the floral attributes were done using SAS 9.3 software. Based on the observations made on the phenological measurements inter and intra flower variability in monoecious nutmeg flowers are prominent. Three types of flowers are recorded in monoecious trees namely, pistillate, staminate and hermaphrodite flowers. The structure of hermaphrodite in nutmeg is reported for the first time. The flowers are borne on the leaf axil, flowering habit of the three types of flowers are seen in cymes as well as solitary in the same tree. All the types of flowers were light creamy yellow colour in monoecious tree, with thick gamosepalous perianth which bursts as trilobed or bilobed (less frequent) lobes during anthesis as reported earlier (Armstrong & Drummond 1986). However, addition to this tetralobed and pentalobed are observed rarely in monoecious trees (Fig. 1). Tetralobed lobes are reported in M. fatua, this type of lobe opening helps in more access for insects (pollinators) to enter the flower (Sharma & Armstrong 2013). The androecium of staminate flowers consisted of adnate 7-13 anthers, with average of 9.30 anthers and 4.09 and 3.18 mm of anther length and filament length respectively, androecium with 8-10 anthers were reported earlier by Armstrong & Drummond (1986) in staminate flowers. The gynoecium consists of single ovary with a mean length of 5.28 mm with bifid stigma in the pistillate flowers (Tables 1 & 2). Intra flower variability is evident in case of hermaphrodite flowers. In hermaphrodite flower the androecium ranges with 1-4 anthers, in the form of fused filament or free filament or both; some anthers are fused with the gynoecium (Fig. 2); the length of the anther varies from 3- 3). Analysis of variance of trees for flower types and floral attributes like flower type, number of anthers, length of anthers, length of filaments and length of ovary are found to be highly significant (Table 1). High coefficient of variation is recorded for hermaphrodite (183.84%) and pistillate (171.71%) flowers ( Table 2). The pollen viability of the hermaphrodite flowers are found less as compared to the staminate flowers. The percentage of pollen viability is 79.74 and 94.77 in hermaphrodite and male flower respectively (Table 3). In the population studied, the average segregation of staminate, pistillate and hermaphrodite flower in the collected samples is 87.72, 19.22 and 5.77, respectively with approximate ratio of 17: 4:1. The occurrence of hermaphrodite flower in monoecious tree ranged from 0-9%. In 90% of the flowers studied the stamen length is found shorter than the stigma thereby excluding the chance of selfing. Moreover the pollen viability is also found less in these flowers. Variability in flowering phenology among individuals has direct impact on their fitness (Mauricio et al. 2013). However, the influence of environment factors on sex expression and floral variability need to be studied in detail in nutmeg. Cinnamon (Cinnamomum verum L.) or dalchini is one of the ancient tree spices grown in India. Though both bark as well as leaves are known to possess aromatic components mainly cinnamaldehyde and eugenol, the bark of this species is valued as a spice. It has been used in the form of dried bark, bark powder, oil and oleoresins. Cultivation of this spice in India is mainly confined to States of Andaman and Nicobar Islands, Kerala, Karnataka, North Eastern India and parts of Tamil Nadu and Maharashtra. Combined estimate of cinnamon and tejpat suggests that in India, it's being grown on 2,770 ha area with about 5,050 t production (Indian Horticulture Database 2014). However, the production is not enough to meet the domestic demand and hence, cinnamon is being imported from other countries of the world (Indian Spices 2016). Increasing the productivity through development and adoption of improved technologies is a key factor in reducing the dependence on import. Soil and climatic conditions of the Andaman and Nicobar Islands are well suited for the cultivation of cinnamon (Parthasarathy et al. 2009) and presently it is cultivated in about 150 ha yielding 40 t annually. Quality of cinnamon is assumed to be the finest in the islands compared with other parts of the country (Singh & Sankaran 2012). Large availability of interspaces in the coconut and arecanut plantations in the Andaman and Nicobar Islands could be successfully utilized for its cultivation (Waman et al. 2016). However, existing plantations in the islands are of seedling origin. One can easily notice variations in cinnamon seedlings for leaf size, shape and colour of new flush, apart from the distinct chemotypes (Krishnamoorthy et al. 1988). Ergo, considerable variability is noticed in the yields and quality of the final produce and hence, vegetative propagation is of interest (Rema et al. 1997). The present report concerned an effort to identify the most appropriate time for carrying out air layering under island condition. The present investigation was carried out in the Division of Horticulture and Forestry of ICAR-Central Island Agricultural Research Institute, Port Blair, Andaman and Nicobar Islands during 2015 and 2016. The islands exhibit typical tropical climate with average temperature of 18-31°C and annual rainfall of 3,100 mm distributed over May to December. Further, the relative humidity ranges between 60-90% in a year, while the average lies near to 70-80%. For layering, healthy shoots of 25-30 cm length and 1.0-1.5 cm thickness were selected. Leaves and small branches near the ringing area on selected shoots were removed and two circular cuts were given to the shoots for removing a ring of bark of about an inch width. Commercial formulation of rooting hormone (Lipsa, Kolkata) was used for root induction and ringed portion was covered with soil: farmyard manure (1:1) before wrapping with polythene (20 cm × 20 cm). Experiment consisted of six treatments i.e. T 1 : layering on July 3, T 2 : layering on July 23, T 3 : layering on August 12, T 4 : layering on September 1, T 5 : layering on September 21 and T 6 : layering on October 11. Experiment was laid out in completely randomized design with 20 layers in each treatment. Various parameters were recorded at the time of separation (90 days after layering) and data was subjected to analysis of variance using Web Agri Statistical Package 2.0 (WASP 2.0, ICAR-RC for Goa, Ela, India). Air layering was performed for six times during rainy season under island condition. Generally, rooting process is facilitated by the rains (Ranaware et al. 1995) and hence, the dry periods in the islands were avoided during the experimentation. Pooled analysis of two years data revealed that percentage root induction varied between 35.0% and 87.5% amongst the treatments studied (Table 1). Maximum rooting percentage was obtained in layers done on 3 rd July (87.5%) followed by those done on 23 rd July, while it was the lowest in layering performed on 21 st September. Though rooting response varied considerably during different seasons, the mean number of primary roots per layer did not vary significantly (Table 1). Length of longest root varied significantly amongst the studied treatments (Table 1). Maximum length of primary root was observed in propagules obtained from cinnamon layered on 1 st September (8.5 cm), which remained on par with layers of 23 rd July. Similar to rooting percentage, length of root was found to be the lowest (3.6 cm) in layers of 21 st September batch. Thickness of root ranged from 2.04 to 2.74 mm; however, the differences were non-significant amongst the treatments studied. Waman & Bohra Similar to present communication, significant variations for rooting response and growth parameters have been reported from different agro-ecological regions of the country viz., West Bengal (Banerjee et al. 1982), Karnataka (Hegde et al. 1989) and Maharashtra (Ranaware et al. 1995). These reports have suggested the positive effects of rainy period on layering success; however, the raining season and pattern vary significantly in different regions and hence, location specific studies are required. As no reports are available for island conditions, present study was conducted. Cinnamon propagation technique Fennel (Foeniculum vulguare mill.) is a highly cross pollinated and very important seed spice crop exhibiting 82.2% to 91.4% natural out crossing (Ramanujam et al. 1964). The hybridization of diverse genotypes followed by selection in segregating crop will be helpful in identifying heterozygous and heterogenous progeny. Insects plays an important role in maintaining heterogeneity in fennel crop. The existing varieties were developed using mass selection. Fennel seeds are having medicinal importance as carminative, cardiotonic, stimulant, vermicide and lactagogue (Lal 2014 environments and over the years for high yield and other yield attributing and quality traits. Phenotypically stable genotypes are of great importance because environmental conditions vary from season to season. Wider adaptation to a particular environment and consistent performance of recommended genotypes is one of the main objectives in breeding programme. A differential response of fennel genotypes when grown under different environments in the Rabi season has been reported by few scientists (Lal 2014;Drazic et al. 2014). Hence information on availability of stable high yielding varieties of Fennel is lacking. Keeping this in view, the present investigation was undertaken to determine the genotype x environment (year) interaction on stability parameters and to identify the stable and responsive genotypes for yield and yield contributing characters of fennel for Chhattisgarh. The inter-and intrarow spacing was kept at 45 cm and 20 cm, respectively. The recommended package of cultural practices was followed to raise a good crop. In each plot, five competitive plants were identified randomly for recording data on days to 50% flowering, plant height (cm), primary branches plant -1 , umbels plant -1 , umblets umbel -1 and seed yield plot -1 (kg). The data recorded during three years were subjected to stability analysis according to the model proposed by Eberhart & Russel (1966) and three stability parameters mean (m), regression coefficient (bi) and the deviation from linearity (S 2 di) were estimated. (Table 2). For days to flower, umbelets /umbel and umbels plant -1 FNL 69 had recorded wider stability (above average mean, bi = 1 and S 2 di = 0). The genotype FNL 70 was stable for plant height, primary branches plant -1 , umbelets umbel -1 and umbels plant -1 but had low seed yield, indicating its adaptation to stress environments (Lal 2008) while genotype FNL 71 recorded stability for plant height and umbels plant -1 . For umbels umbel -1 FNL 72 had high stability across the environments of Raigarh. The genotypes FNL 69, FNL 70 and FNL 71 were among the top entries which had their mean umbels plant -1 greater than the average of all the genotypes with regression coefficient (bi =1) and non-significant deviation for regression (S 2 di =0). This indicated their high stability over the different environments of years (Lal 2008). The genotype FNL 68 and RF 101 had above average mean for umbels plant -1 , S 2 di = 0 but the value of bi #1, indicating their adaptation to high input conditions. For umbels plant -1 genotype RF 205 and FNL 67 had bi = 1 and S 2 di = 0, but their mean was low, indicating their adaptation to stress environments (Table 3). The genotypic differences were found to be highly significant for all the traits in each environment (year). The mean genotypic values from different year were subjected to pooled analysis. The mean sum of squares (MSS) due to genotypes (G) and environments (E) were significant for all the traits except for primary branches plant -1 when tested against MSS due to genotype x environments. It revealed the non significant differential response of the varieties to the changing environments. The results were in close conformity to the findings of Lal (2008), Verma & Solanki (2015). The MSS due to G × E when tested against pooled error, were found highly significant for all the characters. Thus stability analysis was carried out for all the traits. The variance due to G × E were divided in to G × E (Linear) and due to pooled deviation (Non-linear). The G × E (Linear) mean squares were found significant for all the traits except primary branches plant -1 indicating the presence of predictable components where as significance of pooled deviation for seed yield plot -1 , days to 50% flowering, plant height (cm), primary branches plant -1 and umbels plant -1 showed the presence of non-predictable components. These observations indicated that some reliable predictions about G × E interaction as well as its unpredictable components can be made for these traits. Hence, both components contributed significantly in determining the stability of genotypes (Lal 2008;Verma & It was observed that national check (RF 205) showed wider stability for seed yield, days to flower, primary branches plant -1 , umbels plant -1 and umbelets umbel -1 while local fennel had wider stability for seed yield (kg ha -1 ), plant height, umbels plant -1 and umbelets umbel -1 . While none of the test entries showed superior stable performance over the national and local checks during all the three years. This appears to be evidence for much greater genotype × environment interaction for the entries to be evaluated than for the pure lines. This is somewhat unexpected in view of the greater homeostasis in unfavorable environments usually found in heterozygous genotypes. This needs further study. (125), highest yield plant -1 (1052.5g), yield ha -1 (337.63q) and premier fruit quality score (9.11) with maximum net return (Rs.326407.28) and benefit: cost ratio (3.41) was also reported in same treatment. Comparatively minimum time (15 hours) required for one hectare irrigation was also reported in drip irrigation on raise bed with plastic mulch. This led to lower population of white fly plant -1 (4.53), minimum weed infestation (1.53 weed m -2 ), leaf curl (5.50%) and fruit rot (5.0%) incidence than other treatment combinations. The minimum growth, yield and profitability were reported in check basin method of irrigation without mulch (T 1 treatment). to mulch includes early production, more yield and reduced insect and disease problems (Pattanaik et al. 2003). Linear Low Density Poly Ethylene (LLDPE) plastic films have been proved as better mulch because of their puncture resistance quality, thinness and lower cost (Panda 2004). Numerous experiments have reported the benefits of LLDPE mulch in several crops, but research is limited on response of chilli production in western Rajasthan by this method. Keeping this in background, the present study was undertaken to study the effect of different irrigation methods and mulching on chilli crop and compare the result with the conventional method of growing the crop under surface irrigation without mulch. Keywords A field experiment was conducted at Agricultural Research Station, Mandor, Jodhpur (Rajasthan), India during kharif seasons of year 2016-17. The soil of experimental plot was of sandy loam texture with average pH range 8.5, having organic carbon 0.55%, available N 180 kg ha -1 , P 27.5 kg ha -1 and K 250.0 kg ha -1 during experimentation. The experiment was conducted in a completely randomized design having nine treatments comprising by different irrigation methods and mulching viz., T 1 = Check basin method, T 2 = furrow irrigation method, T 3 = Raise bed with trench method, T 4 = Flat bed with drip irrigation, T 5 = Flat bed + plastic mulch + drip irrigation, T 6 = Raise bed with drip irrigation, T 7 = Raise bed + organic mulch + drip irrigation, T 8 = Raise bed + plastic mulch + drip irrigation, T 9 = Sprinkler irrigation method. In well prepared field, transplanting of Chilli seedlings variety RCh 1 of 35-40 days old were planted in pair row method with a spacing of 45cm x 45 cm/90 cm (33,333 plant ha -1 ) during last week of June. In check basin and sprinkler system of irrigation the bed size is 2 x 2 meter and in all other methods is 1 × 4 meters. The cultural practices of the crop were followed as per the recommendations. The organic material and LLDPE silver colour film of 100-micron thickness was used for mulching around the plant. The lateral lines of 12 mm diameter LLDPE pipes were laid along with crop rows. The laterals were provided with inlet drippers of 8 litre hr -1 discharge capacity. All the observations were taken from five randomly selected plant of each replication throughout the investigation period at appropriate time by adopting standard method for growth, development, fruiting behavior and yield. Seedling survival per cent (after transplanting in main field at 15 DAT and 30 DAT) was recorded by following formula; Survival percent = [Total survival transplanted plants / Total transplanted plants] × 100 Plant height (at 45 DAT and at harvesting) was measured from soil surface upto the highest shoot tip by straightening all branches. Stem girth was measured 1 cm from the base of the stem using vernier calliper. Observation of number of branches, days taken to first flower initiation, duration of fruiting period and number of fruit plant -1 was recorded by standard counting method. Number of roots, root length was measured by destructive method of uprooting the plants and taking measurement by standard method. Length of fresh fruits measured by scale and fruit diameter using vernier calliper and expressed in centimeter. Fruit set per cent was recorded by following formula; Fruit set per cent = [Total number of fruit set plant -1 / Total number of flowers plant -1 ] × 100 Fruit weight was determined by weighing method at the time of harvesting and expressed in gram fruit -1 . The total fruit yield plant -1 and hectare -1 was calculated by weighing total marketable fruits and has been expressed in gram and quiantal respectively. Further, the net return was calculated by subtracting cost of each treatment from gross return. The gross return was calculated from yield multiplied by average market rate during the period of investigation. The benefit cost ratio was calculated by dividing net return to total cost of cultivation. Benefit-Cost ratio and net profit were carried out to determine the economic feasibility of the crop using surface and drip irrigation as suggested by Tiwari et al. (1998a). The seasonal system cost of drip irrigation system included depreciation, prevailing bank interest rate, and repair and maintenance cost of the system. The fixed cost of drip irrigation system was determined to be Rs 112,000 ha -1 . The useful life of drip system was considered to be 10 years. The system cost was evaluated by distributing the fixed cost of system over life period of drip irrigation set. For calculating depreciation, the life of the drip irrigation set and 10% junk value was considered. The interest was calculated on the average of investment of the drip irrigation set taking into consideration the value of the set in the first and last year @10% per annum. Cost of repairs and maintenance of set is @2% of initial cost of the drip irrigation set per year. The cost of cultivation includes expenses incurred in land preparation, interculture operation, fertilizer, crop protection measures, irrigation water and harvesting with labour charges. Therefore, total seasonal cost was worked as: depreciation, interest, repairs and maintenance cost of set + cost of cultivation + cost of mulch. The income from produce was calculated using prevailing average market price of capsicum @ Rs 1250 q -1 . Disease incidence (leaf curl and fruit rot) and quality of fruits was measured by visual inspection (Five member team of crop experts and plant pathologist). White fly population plant -1 and weed infestation meter -2 was calculate by simple counting method. The time required for irrigation was calculated as per actual required time of irrigation of specified area by different methods of irrigation. To test the significance of variance of data obtained from crop growth, yield and economics of variance technique for completely randomized design was done by standard procedure prescribed by Panse & Sukhatme (1985). Significance of difference among the treatments effect was tested by 'F' test and critical difference (CD) was calculated, wherever the results were significant. The results revealed that, the irrigation methods and mulching are significantly influenced growth attributes at all the growth stages ( Table 1). The maximum seedling survival per cent at 15 DAT (95.10%) and 30 DAT (91.70%) was recorded in T 8 treatment, which was significantly superior to other treatment but at par with T 6, and T 7 treatments. The maximum survival per cent of seedling in T 8 treatment might be due to more favourable moisture condition for seedling transplanting and reestablishment of roots than others. The height of plant under treatment T8 (47.10 cm at 45 DAT) and treatment T6 (62.60 cm at harvest) was found highest among all other treatments and is 67.19% and 13.40% higher than the T 1 treatment. About to number of branch plant -1 , maximum value was recorded in treatment T8 (14.93) followed by treatment T7 (12.53) and the lowest value was in treatment T1 (7.17). Maximum stem girth at harvest (2.36 cm) and highest number of roots plant -1 (138.50) were observed in T 8 treatment whereas longest root system (10.50 cm) was observed in T 3 treatment. The minimum stem girth (1.68 cm) and the number of roots plant -1 (53.57) were observed in T 1 treatment whereas shortest root system (7.97 cm) was observed in T 9 treatment. The higher available moisture status in soil favourably influences the uptake of nutrients which maintains the cell turgidity, cell elongation, photosynthesis and respiration at optimum level, leading to favourable growth and development of plant in terms of plant height, number of branches plant -1 , stem girth and number of root plant -1 in the present study. The highest increase in vegetative growth in drip irrigation with mulching might be due to the availability of soil moisture as well as favourable temperature at optimum level for plant growth development (Pattanaik et al. 2003;Paul et. al. 2013). The lowest value of vegetative growth in T1 might be because of unfavourable moisture regime (moisture stress or excess moisture) in the soil through surface irrigation and competition of weeds for nutrients (Pattanaik et al. 2003;Agrawal & Agrawal 2005). The increased growth attributes might have supplied water and nutrients in adequate proportion, which resulted in triggering the production of plant growth hormone, viz., indole acetic acid (IAA) and higher number of leaves and roots throughout the cropping period (Sankar et al. 2008). The drip irrigation in combination with mulch significantly increased the yield of chilli as compared to drip irrigation without mulch (Table 2) and surface irrigation methods. The minimum days (42.38) required for first flower initiation was reported in T 9 treatment whereas the maximum days (51.39) was required in T 5 treatment. Among various treatments, highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm), duration of fruiting (71.38 days), fresh weight of fruit -1 (8.42 g), maximum number of fruits plant -1 (125), highest yield plant -1 (1052.5 g) and yield ha -1 (337.63 q) was recorded under T 8 treatment, whereas lowest yield (153.45 q ha -1 ) was recorded under T 1 treatment. This might be due to water stress during the critical growth period and fruit development stage coupled with aeration problem in first few days immediately after irrigation. Another reason to get low yield by surface irrigation without mulch might be due to less availability of nutrients for crop growth due to leaching and high weed competition between the crops (Pattanaik et al. 2003). In drip irrigation system on raise bed with plastic mulch the water is applied at a low rate for a longer period at frequent intervals near the plant root zone through lower pressure delivery system, which increases the availability of nutrients near the root zone with a reduction in leaching losses and minimum weed competition. More nutrient availability, especially near the root zone might have increased the translocation of photosynthetes to storage organ of chilli resulting in an increased weight of fruits. This result corroborated the findings of Singh (2007), Sankar et al. (2008), Paul et al. (2013) and Kumar et al. (2016). Irrigation methods and mulching also significantly influenced the gross return, net return and benefit cost ratio in chill (Table 3). Maximum net profit of Rs. 326407.28 ha -1 with B: C ratio of 3.41 was recorded in T 8 treatment followed by Rs 296192.61 ha -1 with B: C ratio of 3.11 in T 5 treatment and lowest net profit of Rs 119007.80 ha -1 with a B: C ratio of 1.63 in T 1 treatment (Table 3). It is observed that, the drip irrigation with mulched treatments T 5 , T 7 and T 8 gave better net return with higher B: C ratio ha -1 than their corresponding treatments without mulching in conventional irrigation method. The highest net return (US$ 7098 ha -1 ), incremental net return (US$ 1556 ha -1 ), and incremental benefit-cost ratio (7.03) were found for 50% water application with straw mulch (Biswas et al. 2015). The results are in conformity with the findings of Singh (2007), Sankar et al. (2008) and Kumar et al. (2016). Apart from reducing water consumption, drip irrigation with mulching also helps in reducing cost of cultivation and improving productivity of crops as compared to the same crops cultivated under flood method of irrigation (Paul et al. 2013). Irrigation time significantly pretentious by different irrigation methods. The minimum time required in irrigation (14.50 hours) in T 5 treatment which closely followed by T 8 treatment. Drip irrigation method with or without mulching required less irrigation time than without mulching in conventional irrigation method. There was significant effect of LLDPE mulch over drip irrigation system alone. Drip irrigation with LLDPE mulching (T 5 & T 8 treatment) saving irrigation time (21.40 hour ha -1 and 21.35 hour ha -1 ) upto 60 per cent by reducing water losses and increased irrigation efficiency. The increase in water saving per cent in trench method (T 2 ), drip irrigation system alone (T 4 ), drip irrigation system with LLDPE mulch (T 5 & T 8 ), drip irrigation with organic mulch (T 7 ) and sprinkler system (T 9 ) over conventional surface irrigation by check basin method (T 1 ) was 38.8%, 46.6%, 60.0%, 54.4% and 41.9% respectively. The highest water use efficiency of 592 kg ha -1 mm -1 was obtained with 50 per cent water application under polyethylene mulch (Biswas et al. 2015). Drip irrigation with mulching helps to achieve yield gains of upto 100 per cent, water savings of upto 40-80 per cent, and associated fertilizer, pesticide, and labour savings over conventional irrigation systems in capsicum crop (Paul et al. 2013). Similar trend has been reported in water use efficiency for okra crop by Tiwari et al. (1998a) and for tomato crop by Singh (2007). Occurrence of chilli leaf curling and fruit rot was detected throughout the investigation period. The best performance, with a marked reduction in leaf curling and fruit rot and improve fruit quality was observed in drip irrigation with LLDPE mulching (Table 3). The minimum incidence of fruit rot (5.0%), leaf curing (5.5%) and highest quality score of fruit (9.11) was observed in T 8 treatment which was closely followed by T 7 and T 5 treatments where as maximum incidence of fruit rot (20.02%) and leaf curling (15.84%) was reported in check basin method of irrigation (T 1 treatment) whereas minimum quality score (6.50) was observed in T 9 treatment. Presence of white fly and weed was observed throughout the investigation period. The minimum population of white flies (4.53 plant -1 ) and weed infestation (1.53 weed m -2 ) was observed in T 8 treatment whereas highest incidence of white flies (17.43 plant -1 ) and weed infestation (30.03 weed m -2 ) was observed in T 1 treatment. This is due to the fact that in drip irrigation with mulching significantly reduced additional moisture level in field environment which in turn increase quality of fruits and reduce disease infestation, white fly population as well as it also trim down weed seed germination, growth and development. The increase in quality of fruits was due to the effective utilization of applied nutrients, water and significantly reduced weed growth; disease incidence and increased rate of photosynthesis, sink capacity and accumulated more amounts of dry matter and finally increased quality of fruits and yield. Conventional surface irrigation methods without mulching provide favorable environmental condition for increase insect population and development of disease as well as germinate and develop high density weed plants. The beneficial effect of drip irrigation and black LLDPE mulch in capsicum, tomato and okra was also reported earlier by Horo et al. (2003); Singh (2007) The combination of raised bed + drip irrigation system with LLDPE mulching is observed to be economical and cost effective as compared with conventional surface irrigation without mulching. Thus, the use of drip irrigation system either alone or in combination with mulching, could increase the chilli yield quality of fruits and profitability. It also reduces white fly population, disease incidence (root rot and leaf curling) and minimise with crop weed competition. Drip irrigation with mulching increase water use efficiency by significant reduction in irrigation time ha -1 . It is concluded that the drip irrigation method with LLDPE mulching is suitable for chilli production in arid and semi arid condition of western Rajasthan. Swelling factor (cc g -1 ) 11.4 9.1 Patel et al. GI-4 recorded 89.67% less shattering than that of check variety, which revealed that Gujarat Isabgol 4 was non shattering in habit as compared to Gujarat Isabgol 3 which is prone to high seed shattering. The post dipped seed weight of GI 4, was 0.115 kg threshed seeds, which was 96% higher than that of the post dipped seed yield of GI-3 (0.037 kg threshed seeds) (Table 4). Hence, Gujarat Isabgol 4 recommended for cultivation.

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Enzymatic Removal of Diacetyl from Beer Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at — 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors. Diacetyl has long been considered a serious off-flavor component in beer (18). It has been the subject of several recent reports (3,5,6) and also has been reported as a growing problem in frozen orange juice (2,8,9,10). Marketing demands for beer have shifted in favor of a mild-flavored product, making the diacetyl problem even more acute. Without the masking effect of stronger flavor components, diacetyl becomes increasingly noticeable and objectionable (13). Changes in the composition of wort, acceleration of production, and introduction of continuous fermentation have created conditions favorable to formation of diacetyl off-flavor. Brewers have been only partially successful in controlling diacetyl levels in beer, usually at the expense of production efficiency. Extended lagering of the beer assures more complete diacetyl removal, but the longer holding periods and storage requirements make the process burdensome. The addition of fresh whole yeast cells to fermented beer (Krausening) is another means of reducing the diacetyl content, but yeast autolysis 1 Technical Paper no. 2825 from the Oregon Agricultural Experiment Station. may produce additional off-flavors in the finished product. Addition of valine to discourage diacetyl production by feedback inhibition also has been advocated (6,13). Research carried out in the Department of Microbiology at Oregon State University has revealed the ability of several bacterial species to destroy diacetyl in milk cultures (4,15,16). By using the organism Aerobacter aerogenes 8724, Seitz et al. (16) demonstrated a diacetyl-destroying enzyme identified as diacetyl reductase. In a preliminary report, Bavisotto et al. (1) presented findings suggesting the possible commercial use of diacetyl reductase to control diacetyl off-flavor in beer. Subsequently, Tolls et al. (17) studied methods of using the enzyme in eliminating diacetyl from beer and pointed out limitations of the enzyme in flavor control. The present report further substantiates possible industrial use of the enzyme by providing a means to stabilize the enzyme in beer and to eliminate the use of substrate quantities of the expensive cofactor reduced nicotinamide adenine dinucleotide (NADH). MATERIALS AND METHODS Enzyme preparation. The organisms used as sources of enzyme were A. aerogenes 8724 and Streptococcus diacetilactis 18-16. Both are maintained in the culture collection of the Department of Microbiology, Oregon State University; the A. aerogenies strain was originally obtained from the American Type Culture Collection, where the S. diacetilactis strain also has been deposited (ATCC no. 15346). Both organisms were maintained and grown by using citrate broth described by Sandine et al. (14). Cells were grown in 8-liter quantities by using aerated carboys (nonaerated in the case of S. diacetilactis) or an aerated and pH-regulated New Brunswick microfermentor (capacity of 12 liters). Cells were harvested after 36 to 48 hr of growth with a Sorvall RC-2 refrigerated centrifuge operated at 5,000 X g for 15 min. The cells were then washed three times and resuspended in 0.1 M potassium phosphate buffer at pH 7.0. Cell suspensions (50-ml quantities with the consistency of light cream) were sonically treated for 20 min in a Raytheon 10-kc sonic oscillator, and the cell debris was removed by centrifugation at 15,500 X g for 1.5 hr. The resulting supernatant fluid was dialyzed through three 2-liter distilled water changes over a 30-min period and then lyophilized or stored frozen as the crude, unpurified extract. Protein determinations for each extract were done by the method of Lowry et al. (7). Crude extracts of diacetyl reductase were partially purified by using a column (2.5 by 45 cm) packed with G-200 Sephadex gel in 0.1 M potassium phosphate buffer at pH 7.0. Samples (4.0 ml) were added to the column, and 50-drop portions were collected every 5 min; 25 samples were assayed for enzyme activity. In other attempts at enzyme purification, protein precipitation by ammonium sulfate was carried out. The purified samples were further analyzed for activity maintenance after lyophilization and extended storage at room temperature, 5 C, and -20 C. Activity was also analyzed as a function of decreasing pH (7.0 to 3.0) and in a 5% aqueous ethyl alcohol. Enzyme assays. The Gilford model 2000 continuous-recording spectrophotometer was used to measure enzyme activity recorded as the decrease in absorbancy of NADH at 340 nm. The total system (standard assay conditions) contained 2.7 ml of potassium phosphate buffer at pH 7.2, 0.1 ml of diacetyl reductase (5 mg/ ml), 0.2 ml of NADH at a concentration of 2 mg/ml, and 0.1 ml of diacetyl at a concentration of 860 ,g/ml. The resulting absorbancy decrease plots were converted to units of enzyme activity by two methods. The first used a definition of one unit of enzyme as the amount of enzyme which under standard assay conditions caused a 50% reduction of the absorbancy when 1 /T, the reciprocal of time in seconds, equalled (1). The other means of determining enzyme activity was to record the change in absorbancy with time (chart speed, 2 inches/min), measured on the linear portion of the curve. Specific activity in each case was determined by calculating the units of enzyme present per milligram of enzyme extract tested. Diacetyl determinations. Diacetyl was assayed by using the colorimetric method of Owades and Jakovac (11) as modified by Pack et al. (12). Use of diacetyl reductase in beer. The enzyme system used for studies on diacetyl removal from beer consisted of gelatin (Swifts' Superclear GIO) to protect the enzyme, yeast cells (Fleischmann's dry yeast, Standard Brands, Inc., New York) to regenerate NADH, and the crude lyophilized enzyme. The mixture was prepared by warming 10 g of gelatin in 50 ml of distilled water to 40 C, at which temperature it was held until the gelatin was dissolved. After cooling to just above room temperature, the pH of the gel was adjusted to 7.0 i 0.1 with 1 M NaOH before addition of the yeast and enzyme. One gram of yeast suspension and 0.1 g of dissolved, lyophilized enzyme preparation were then added, mixed, and spread as a thin film on sheets of polyethylene. After 24 hr at room temperature (25 C), the gel dried to a translucent, flexible film that could then be peeled from the polyethylene, cut into pieces approximately 1 cm square, and stored at 5 C. Beer samples were spiked to 0.5 ppm of diacetyl by the addition of diluted, pure diacetyl (K & K Laboratories, Jamaica, N.Y.). The gel-yeast-enzyme was added to the beer at the rate of 360 mg per 120 ml of beer; each 120 ml of beer sample contained 324.4 mg of gelatin, 32.4 mg of yeast, and 3.2 mg of enzyme. The beer samples then were incubated for periods of time ranging from 12 to 120 hr at 25 C and at 5 to 7 C. Final diacetyl concentrations after incubation were determined by using the method of Owades and Jakovac (11). Each component of the gel-yeastenzyme system was prepared separately and in combination with one of the other components, and analyzed in beer to determine the role of each in the diacetyl removal system. The standards set for the satisfactory performance of the enzyme system in beer required that diacetyl be reduced from 0.5 to 0.1 ppm in a minimal amount of time or that it be reduced to 0.2 ppm or less within 48 hr. All tested combinations were incubated in beer at 5 to 7 C for 48, 72, and 96 hr and analyzed in duplicate during separate trial periods (four to six tubes per trial). Procedures used to measure leakage of yeast and enzyme from the diacetyl removal system into beer included plate counts on potato dextrose agar and reuse experiments to show that elements of the gel-yeastenzyme system remained intact and active through repeated uses in fresh-beer samples. Use of diacetyl reductase in orange juice and distillers' products. The same conditions used to measure the ability of the enzyme system to remove diacetyl from beer were employed to test the effectiveness of diacetyl reductase in removing the compound from orange juice samples or distilled liquors. The orange juice was obtained commercially as the frozen concentrate; the liquor samples were supplied by Hiram Walker and Sons, Peoria, Ill. RESULTS Enzyme assays. Table 1 shows specific activity values of crude and partially purified enzyme fractions prepared from A. aerogenes. The apparent protective influence of protein and the inactivating effect of lyophilization may be noted. Also, less than two-and fourfold purification was obtained by ammonium sulfate and Sephadex chromatog-raphy, respectively; freezing and thawing had little affect on enzyme activity. The influence of storage temperature and time on the activity of diacetyl reductase fractions appear in Table 2. These results indicate that crude, lyophilized, and ammonium sulfate-precipitated enzyme fractions need to be stored at -20 C to maintain activity for extended periods. The activity of diacetyl reductase as a function of decreasing pH and in 5 % aqueous ethanol is shown in Fig. 1. The lability of the enzyme to increasing concentrations of hydrogen ion is apparent; 5% ethanol also was harmful to the enzyme. At pH 7.0 and 6.0, only about 5 min was required for the complete reduction of the diacetyl (29 ppm). The protective effect of gelatin against this adverse influence of pH is illustrated in Table 3; even at pH 4.1, complete removal of diacetyl (29 ppm) was affected by enzyme mixed in 1.5% gelatin in less than 1 hr. It also may be seen that 2.57% gelatin was too high a concentration to allow the reaction to proceed. The gelatin also protected against inactivation by 5%X, alcohol. Use of diacetyl reductase in beer. Table 4 shows the effect of the individual components (gelatin, enzyme, or yeast) on the level of diacetyl remaining in beer (pH 4.1) after incubation at 5 C for 2, 4, and 9 days. Gelatin or enzyme alone were inactive, but yeast alone was active. Yeast and enzyme were no more active than yeast alone, suggesting the enzyme was not functional under these conditions, again because of the pH effect. Gelatin and enzyme also were inactive because there was no NADH for the enzyme. The complete system was the most active, reducing all the diacetyl in 4 days even at this low temperature. The effect of varying the gelatin, yeast, and enzyme concentrations on the rate of diacetyl reduction in beer by using the protected enzyme system may be seen in Fig. 2-4, respectively. It is clear that the yeast cells are the most important variable, though the gelatin and enzyme concentrations also are important. When these data were submitted to computer regression analysis, the equation and correlation coefficient shown in Table 5 resulted. From the equation, the optimal concentration of the three components was determined. Use of diacetyl reductase in orange juice and distillers' products. Diacetyl analyses on distillers' samples along with proof data are given in Table 6. Successful reduction of diacetyl reductase was only measureable with the low-proof samples, since no color reaction occurred when diacetyl analyses were made on the three high-proof samples. a Results are expressed as in Table 1; -, indicates not determined. b Frozen extracts were placed at the temperature indicated and held for the period shown before assay. c Lyophilized enzyme was stored at 22, 5, and -20 C and assayed after the periods indicated. d Ammonium sulfate. In orange juice, the protected enzyme system functioned, but complete removal of diacetyl was not achieved (Fig. 5). DISCUSSION The results presented in Tables 1 and 2 on the stability of crude diacetyl reductase preparations from A. aerogenes emphasize that partially puri- fied enzyme becomes much more labile to freezing and lyophilization. The crude extract, however, is quite stable to these treatments but, unfortunately maintains activity for extended periods only when stored at -20 C. This imposes a storage inconveinence regarding commercial use of the enzyme. Whether this limitation will apply to the gelatin protected enzyme system will require further study. However, preliminary results have revealed that gel-yeast-enzyme mixtures stored at 25, 5, or -20 C lose no activity up to 4 months of storage. Although initial experiments were complicated by flakes of gelatin-containing enzyme which made absorbancy readings somewhat inaccurate, the data (Table 3) suggested that gelatin would be a suitable protective agent for the enzyme in adverse pH and alcohol environments. Although this was true (Fig. 2), some method of regenerating cofactor for the enzyme was needed to avoid the necessity of adding substrate quantities of NADH. When it was noted that yeast cells alone and, more especially, when incorporated into gelatin were active in reducing diacetyl in beer (Table 4), it became apparent that yeasts were providing a constant source of reduced cofactor to allow the enzyme to function. This may occur through the coupling of the enzyme to alcohol or lactic dehydrogenase (1). Figure 3 emphasizes dramatically the dependency of diacetyl reductase on the presence of yeast cells when exogenous NADH is not provided. In fact, even in the absence of enzyme (Fig. 4), the yeast cells function quite well in reducing diacetyl since they contain diacetyl reductase (17) in addition to the NADHregenerating system. These findings suggest the possibility of using yeast cells immobilized in gelatin in the absence of added diacetyl reductase to control diacetyl offflavors in fermented berverages. This could be done by using the strains of yeasts being used in the actual fermentation to minimize the possibility of contaminating beer with a foreign yeast. In this regard, the gelatin flasks prepared in this study, which contained enzyme and yeast cells, were tested for leakage of cells into beer. In three experiments in which 120 ml of beer was incubated at 5 C for 96 hr with 360 mg of the enzyme system, an average of 3 cells per ml were recovered; when an equivalent amount of yeast was similarly plated, 35,000 cells per ml were recovered. Thus, 99.991% of the yeasts are retained in the gelatin, even though the flakes were prepared by cutting the film. Preparation of the mixture in droplet form would probably prevent any leakage, and further work on this is in progress. Where it might be desirable to use yeast cells exclusively for diacetyl control, studies need to be made on conditions necessary in preparation of the cells to minimize cost and maximize the activity of the cells for diacetyl reductase and NADH regeneration. VOL. 19, 1970 In the brewing industry, a variety of chillproofing (proteolytic) enzymes are currently used to enhance the clarity of the finished product. Usually, less than a pound of these agents is used per barrel of beer, and it was of interest to com pare this customary rate of enzyme addition with that found necessary in the present study for the diacetyl reductase system. From the regression analysis (Table 5), the optimal concentration of the enzyme component was about 68 mg per 120 ml of beer; this is equivalent to 0.15 lb per barrel. Where the total weight of the enzyme system is concerned (490 mg/120 ml), this is equivalent to about 1.1 lb per barrel of beer. Although this may seem like a sizable quantity, the gelatin-yeastenzyme system is recoverable from the beer after the diacetyl is reduced and does not actually represent an addition to the beer that might affect quality after processing. Furthermore, it was determined in this study that the gelatin-protected system was reusable after recovery from treated beer. In a typical experiment in which the initial use reduced the diacetyl in beer from 0.5 ppm to none in 96 hr, the first reuse left 0.23 ppm with complete removal by 192 hr. Even a third reuse of the recovered flakes lowered the diacetyl to 0.07 ppm after 192 hr. Attempts to contain the gelatin flakes in gauze bags suspended in the beer to facilitate easy removal resulted in some inhibition of diacetyl removal; reduction of diacetyl from 0.5 to 0.23 ppm occurred in 96 hr, whereas control beer with the flasks evenly distributed throughout contained no diacetyl after this period. Thus, it would appear that the protected enzyme system should be free to mix with the beer for maximal diacetyl removal efficiency. It is clear from the results of this study (Table 6 and Fig. 5) that further research is necessary to establish the applicability of diacetyl reductase to the removal of diacetyl from orange juice and distillers' products. The preliminary data would appear to justify further studies in this regard. The suitability of the protected enzyme system, however, to control this flavor defect in beer seems well established. Results on the application of the system on a commercial scale will be reported in the near future.

v3-fos

2020-12-10T09:04:11.505Z

1970-07-01T00:00:00.000Z

237234180

s2

Survival of Virus in Chilled, Frozen, and Processed Oysters Samples of whole and shucked Pacific and Olympia oysters, contaminated with 104-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 104 PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area. Research by European and East coast workers has shown that viruses can survive in the sea long enough to be taken up by shellfish (2,3,13). Similar findings have been reported recently for West coast species (8). The major site of virus accumulation in these species has proven to be the digestive tract (11,12,15). However, the extent of the hazard presented by the survival of virus, through the processes of freezing or cooking, has not been fully examined. Therefore, a study was made of virus survival in chilled and frozen oysters and in oysters processed by stewing, frying, steaming, and baking. This latter study was considered of particular value because the Pacific oyster, the principal oyster grown on the West coast, is normally cooked prior to being eaten. MATERIALS AND METHODS Oyster samples. The Pacific oysters (Crassostrea gigas) and Olympia oysters (Ostrea lurida) used in these experiments were obtained from a Shelton, Wash., oyster grower in lots of 48 to 50 oysters. The shells were cleaned of external debris and superficially disinfected by dipping in a 1%-to hypochlorite solution; they were then rinsed with tap water and dried. Oysters to be used in experiments were kept in 5-gal (ca. 19 liters) stainless-steel aquaria to which was added 3,500 ml of filtered seawater of 28%0 salinity. Water temperature was maintained at 13 C by cold-air circulation in a constant temperature bath. Aeration and water circulation were provided by means of air hoses placed in the aquaria. Virus. Attenuated poliovirus Lsc-2ab was used in all experiments. This virus is representative of enteric viruses and has been used commonly in studies dealing with the uptake of virus by shellfish. The strain was obtained from the Department of Preventive Medicine, University of Washington. The seed virus contained 3.5 X 108 virus plaque-forming units (PFU)/ ml. Stock virus was propagated on primary African green monkey (MK) kidney cell cultures. The stock virus pool, diluted with Hanks balanced salt solution (BSS) at 30 C and adjusted by plaque formation determinations to contain approximately 108 PFU/ml, was kept at -20 C until used. Tissue culture. Cell suspensions of primary MK kidney tissue (BBL) were used throughout experiments. Monoayer cultures were prepared in 3-oz prescription bottles (ca. 90 ml). Hanks BSS, to which were added 0.2% Casamino Acids and 10% calf serum, was used for cell growth, and Earle's BSS containing 2% calf serum was used for cell maintenance. One hundred units of penicillin G per milliliter, 100 jig of streptomycin sulfate per ml, and 2.5 ,ig of Fungizone per ml were incorporated in all media to prevent the growth of bacteria, molds, and yeasts. Plaque assay technique. The assay method used was essentially that of Hsuing and Melnick (9). The medium was decanted from monolayer bottles, and 0.5 ml of appropriately diluted sample was added to the bottles. All samples and all experiments were run in duplicate. Immediately after inoculation, the bottles were rotated five times to insure even distribution of the inoculum over the cell sheet. All inoculated bottles were incubated at 36 C for 1 hr and 15 min to allow for virus adsorption. After incubation, cell sheets were washed once with 4 ml of warm (30 C) Hanks BSS and then overlaid with 10 ml of growth medium containing 1.5% purified agar (Difco) and 0.0017% neutral red (BBL). This procedure was conducted in semidarkness to prevent photoinactivation of virus and cell sheets by the neutral red. After the agar solidified, the bottles were rotated flat side up and then covered and returned to the 36 C incubator. Bottles were observed for the appearance of plaques on the third to fifth days. Each plaque was marked with a Magic Marker pen, and the total number observed was written on the side of the bottle. The total count on the last day of observation was considered the final count. The sum of the counts of two bottles multiplied by the dilution factor represented the total virus content in each milliliter of inoculum. Survival of virus in chilled Olympia oysters. To determine the survival rate of virus in Olympia oysters held at storage temperatures, the following study was conducted. Forty-eight 4-year-old Olympia oysters were exposed to attenuated poliovirus I for 48 hr, dipped in a 1% hypochlorite solution to disinfect the shell surfaces, washed in distilled water, and sealed in polymylar pouches, eight per pouch. Samples were placed in a refrigerator set at 5 C and assayed for virus content at 0, 5, 10, 15, 20, 25, and 30 days. To determine the rate of virus inactivation in the various anatomical regions of the oyster, the shellfish were carefully dissected as aseptically as possible. The gills, mantle, and palps were first dissected out, then the digestive area, and the remaining body. Ten per cent (w/v) homogenates of each in nutrient broth were prepared, and the samples were clarified by low-speed centrifugation (1,200 X g for 20 min at 10 C) in a Sorvall RC 2-B refrigerated centrifuge. Serial decimal dilutions in nutrient broth were prepared, and all samples were assayed. Survival of virus in frozen oysters. To determine the ability of virus to withstand inactivation in frozen whole oysters, a long-term study was conducted. Fifty shucked Pacific oysters were inoculated directly in the gut region with an inoculum containing 104 virus PFU/ml. The inoculum consisted of poliovirus Lsc-2ab in nutrient broth. The oysters were sealed in polymylar pouches, five to a pouch, and quick frozen at 36 C in a blast freezer for 24 hr. Suspensions of poliovirus (3 ml) in nutrient broth were also quick frozen for 24 hr to serve as controls. Samples were removed from the freezer and stored frozen at -17.5 C. Samples were assayed for virus content at 0, 2, 4, 6, 8, 10, and 12 weeks, by using identical assay procedures (see above). Survival of virus in stewed, fried, baked, and steamed oysters. (i) For stewed oyster studies, 20 mediumsized shucked Pacific oysters were inoculated with 0.5 ml of an attenuated poliovirus suspension in nutrient broth containing 104 virus PFU/ml. Oyster stew, in milk, was prepared by standard cookbook methods. The inoculated oysters were placed in boiling milk in a 2.5-liter stainless steel kettle. Normal recipe procedure requires the oysters to stew for 5 min; however, cooking time was extended to 8 min. Samples of milk and oysters were removed at 0, 2, 4, and 8 min. A 5-ml amount of milk and five oysters constituted a sample. The milk was aseptically pipetted into 20-ml sterile test tubes, and the oyster samples were sealed in polymylar pouches. All samples were cooled to room temperature (23 C). Samples not assayed immediately were held at -20 C until tested. Controls consisted of inoculated oysters which were not stewed but were sealed in polymylar pouches and held at -20 C until assayed. (ii) For fried oyster studies, 20 medium-sized shucked Pacific oysters were inoculated with a poliovirus suspension in the identical manner described in the preceding experiment. The oysters were prepared for frying by standard cookbook recipes. Oysters were coated in a batter of egg, bread crumbs, and seasoning and fried in Wesson Oil at a temperature of 177 C. Frying time was extended from the 5 to 8 min called for in the recipe to 10 min. Samples of five oysters each were removed for assay at 3-, 6-, and 10-min intervals, sealed, in polymylar pouches, allowed to cool, and tested. The zero-hour and control samples consisted of inoculated but unprocessed oysters. (iii) For the baked oyster study, 20 medium-sized Pacific oysters were inoculated with poliovirus, as described in preceding experiment, and then prepared by standard cookbook methods. The oysters were rolled in a batter of egg and bread crumbs, plus seasoning, and then baked for 20 min in an oven at 121.5 C. Samples were removed for assay at 5, 15, and 20 min. Oysters used as zero-hour and control samples were inoculated and rolled in batter but not baked. (iv) For the steamed oyster study, 25 medium-sized Pacific oysters were placed in three 5-gal (ca. 19 liters) aquaria containing 3,500 ml of filtered seawater (28% salinity) at 13 C. To each aquarium was added sufficient poliovirus Lsc-2ab to yield a count of 105 virus PFU/ml of seawater. The oysters were contaminated for 48 hr and then removed from the aquaria. Shell surfaces were disinfected by dipping in a 1% hypochlorite solution and then rinsed in distilled water and dried. Oysters used in experiments were placed in metal pans and held under flowing steam for 30 min in an autoclave. Samples were removed at 0, 5, 15, 20, and 30 min, by using metal tongs. They were placed in polymylar pouches and allowed to cool to 25 C before being assayed. The control samples were contaminated oysters not subjected to steaming. Temperature determinations. Temperature determinations were made in all processing studies to correlate the rate of virus inactivation with the rise in temperature inside processed shellfish. All processing procedures were repeated in duplicate with noncontaminated oysters. Four oysters were used in each experiment. A thermocouple wire was inserted into the gut area of each shellfish, and the temperature change was recorded on a Brown Recording Potentiometer. The shellfish were processed as described above. Thus, temperature increase inside the shellfish could be determined for the duration of each processing procedure. To insert thermocouples into oysters uscd in steaming experiments, small holes (4 mm) were drilled into the bills of the shells and thermocouple wires were inserted. The thermocouple wires were taped down with plastic tape to prevent them from slipping out of the shells. RESULTS Survival of virus in chilled olympia oysters. Poliovirus in Olympia oysters proved to be very stable under storage conditions (Table 1). After 5 days of storage at 5 C, virus numbers in the oysters were reduced by 10%, with 75% of the total virus still viable in the digestive area. After 15 days of storage, the total virus count was reduced by 60%, with 36% of the total virus still surviving in the digestive area. Because of decomposition, it was impossible to dissect the oysters after more than 15 days of storage. A second study was made over a 30-day period in which virus survival in the whole oyster was measured. Results are summarized in Fig. 1. After 10 days of storage, there was less than a log reduction in the total virus present and 46% of the virus were still viable. After 30 days of storage, the oysters were badly decomposed. At this time, the total virus count was reduced by 2 logs, but 13% of the virus still remained viable even under these conditions. Survival of virus in frozen oysters. Poliovirus was found to be very stable in whole Pacific (Fig. 2). After 4 weeks of storage, the total virus count was reduced by little more than 0.5 log. By 12 weeks of storage, the original virus titer in the oysters was reduced by one log. Approximately 91% of the virus was viable after 2 weeks of storage, 40% was still viable at 6 weeks, and 10% still survived after 12 weeks of storage (Table 2). Stewed oyster studies. The inactivation of poliovirus in stewed oysters was relatively rapid. The stewing process caused the oysters to split open after 2 min of cooking time, thereby releasing some of the virus into the hot milk. This fact probably accounted for some of the virus inactivation. However, even after 8 min of stewing, 10% of the virus still survived in the oyster, and 7% was recovered from the milk ( Table 3). Fried oyster studies. The initial inactivation of poliovirus in the oyster was slow, with 61 % of the 14t 0 . virus still viable after 3 min of processing. The virus were rapidly inactivated as processing continued (Table 4). When the exper.iment was terminated, after 10 min of frying, the virus count had been reduced by approximately 2.5 logs. This represents a 13% survival of poliovirus. Baked oyster studies. The rate of virus inactivation was initally rapid but declined as processing proceeded (Table 5). Virus titer was reduced by approximately 2.5 logs after 20 min of baking, at which time the experiment was terminated. Virus titer was rapidly reduced during the first 5 min of baking, with only 24% of the virus still surviving at this time. However, at the conclusion of the study, 13 % of the virus still survived in the baked oysters. Steamed oyster studies. The inactivation of poliovirus in steamed oysters appeared to progress in four distinct stages, two of rapid decline and two of longer, more gradual decline. The total virus count was decreased by 1 log after 15 min of steaming and by 2 logs at the end of the experiment. At this time, although the oysters had been steamed for 30 min, approximately 7% of the virus was still viable (Fig. 3). Heat penetration studies. The results of heat penetration determinations for the above studies are presented in Tables 2 through 6. Temperature rise was rapid inside oysters processed by stewing and frying, but less so in baked and steamed oysters. Routinely, exposure to a temperature of (1). In these studies, 6.5 min of heating was required for stewed oysters to reach this temperature, 5.5 min for fried oysters, 16 min for baked oysters, and 20 min for steamed oysters. Under experimental conditions, only in the case of steaming did the actual internal temperature of the processed oysters attain that of input (processing temperature). This was as expected and is due in part to the processing times used and in part to the kinetics of heat penetration. DISCUSSION Survival of virus in chilled Olympia oysters. It has been reported that enteroviruses would persist for a considerable length of time in inoculated, chilled, commercial food products. Thus, Lynt (14) observed that type 1 poliovirus and types Bi and B6 coxsackievirus would survive for at least 1 month in representative samples of commercially prepared foods stored at 10 C. The results of our studies are in agreement with the observation of Lynt and show that virus can survive for a considerable length of time in chilled oysters. After 15 days of storage at 5 C, the total poliovirus remaining in the Olympia oysters was reduced by only 60%. Significantly, three-fourths of the remaining virus was recovered from the digestive tract. After 30 days of storage, although the oysters were badly decomposed, 13%8o of the poliovirus was still viable, indicating that the breakdown products of oyster decomposition have little effect on the virus. These findings are significant since the Olympia oyster, a small cocktail oyster, is normally not held either in grocery or household refrigeration for more than 5 days. Obviously, refrigeration for this length of time will not be sufficient to inactivate even low numbers of virus. The reasons for the prolonged survival of virus in shellfish are not known. The tendency of virus to aggregate and their incorporation by ionic bonding into shellfish mucous may be means by which virus are able to survive in chilled oysters. However, further research is needed to clarify this matter. Survival of virus in frozen oysters. Lynt (14) reported that poliovirus type 1 and coxackievirus types Bi and B6 would survive for 5 months in inoculated frozen foods held at -20 C, findings subsequently confirmed by Heidelbaugh and Giron (6). Results of the present studies with Pacific oysters show that virus also survive for considerable lengths of time in frozen oysters. In our studies, the titer of poliovirus in frozen Pacific oysters was reduced by less than 10% after 4 weeks of storage at -17.5 C, and by only one log after 12 weeks of storage at this temperature. The de facto survival of virus in frozen oysters is of public health significance. Since the advent of modern freezing techniques, there is considerable interstate shipment of frozen shellfish. If the shellfish are harvested from an area contaminated with virus, or if the animals become secondarily contaminated during handling, freezing will not inactivate these pathogens. Hence, these shellfish can serve as vectors for the dissemination of virus diseases. Survival of virus in stewed, fried, baked, and steamed oysters. Viruses are known to be inactivated by heat, which causes coagulation and breakdown of the virus protein coat. However, the medium in which viruses are held has been shown to influence virus sensitivity to thermal inactivation (4,19,20). Results of processing studies show that virus in oysters survived the inactivating effects of heat. Survival of the virus ranged from 7 to 13%o in different home cooking procedures. Virus resistance to inactivation was influenced by the method of processing used. Thus, virus survival was greater in fried and baked oysters (13 %) than in steamed and stewed shellfish (770). It has been proven that most of the virus in contaminated oysters is found in the digestive tract (11,15). The heat penetration studies have shown that, at the processing times normally used to cook oysters, the internal temperature of the shellfish is not sufficient to inactivate all of the virus which may be present. These findings are of great public health significance, since it is often assumed that cooked oysters are ipso facto safe foods. We believe that the possible accumulation by, and survival of, virus in marine food products is an area in which more research should be conducted.

v3-fos

2020-12-10T09:04:10.771Z

1970-09-01T00:00:00.000Z

237230732

s2

Differential Light Scattering Measurements of Heat-Treated Bacteria Effects of heat on diameter, size distribution, and refractive index of Staphylococcus epidermidis suspensions were determined accurately by computer analysis of differential light scattering data. Differential light scattering measurements have been shown (3)(4)(5) to be potentially powerful tools for studying bacterial morphology. Such measurements consist of recording the angular distribution of light scattered from a suspension of bacteria which has been illuminated by a plane parallel beam of monochromatic light. These so-called differential scattered intensities (DSI) depend upon mean size, shape, and structure of the scattering particles (3). We have applied DSI measurements to study dynamic morphological changes in cultures of Staphylococcus epidermidis during the course of heat treatment. In preparing autologous staphylococcus vaccines, many laboratories use heat as a sterilization procedure. Such a treatment supposedly does not destroy the immunogenic properties of vaccines and would be expected, therefore, to have little or no effect on cell walls. A recent study of thermally induced changes in staphylococci (1) demonstrated that optical density and light scattering properties of cells are directly influenced by heat. However, the observed optical density and light scattering changes did not correlate well with other data such as those obtained by electronic cell counter, hematocrit, and electron microscope. Results of our DSI studies will clarify the interpretation of these previously obtained nephelometric measurements. In addition, DSI measurements can detail very accurately the size and refractive index changes which occur upon heating. For the experiments described here, the culture of S. epidermidis was grown for 24 hr at 37 C on nutrient agar (Difco). Washed cells were either diluted directly into distilled water (control) or they were heated at 60 C in Heart Infusion Broth (Difco) for various periods before diluting. The heating and cooling procedures were designed to achieve rapid equilibrium. All samples were diluted to about 2 x 10 bacteria per ml for the light scattering measurements. The DSI measurements were made with a Differential 1 light scattering photometer (Science Spectrum, Inc., Santa Barbara, Calif.) with a vertically polarized He-Ne light source (632.8 nm). The instrument records the relative intensity of scattered light as a function of scattering angle, from 50 to 1500 measured with respect to the incident beam. Figure 1 presents the DSI of a washed suspension of S. epidermidis recorded immediately before heating. Figure 2 shows details of qualitative changes in light scattering in the vicinity of the two maxima after various periods of heating. In Fig. 2, we have shifted the curves with respect to scattered intensity to simplify comparisons. Since thermally induced morphological changes manifest themselves by changes in DSI, the evolution of the light scattering curves, as evidenced in Fig. 2, is a vivid indication that some morphological changes did occur. To determine quantitatively just how heating affected cell morphology, an extensive numerical analysis was performed. Data from Fig. 1 and 2 were replotted to correct for volumetric foreshortening (2,4). The corrected curves were interpreted quantitatively on the basis of Rayleigh-Gans scattering theory (3,5). By using a high-speed digital computer (CDC 6400), the scattering data were analyzed (3,5) and the analysis showed that the heat treatment (i) caused a reduction in mean cell size and (ii) resulted in an increased cytoplasmic optical density relative to cell wall. This latter change is a manifestation of the decrease in free water within the cytoplasm. In quantitative terms, the average radius derived for the control cells on the basis of the corrected curves was 432 nm (±10 nm). After 30 min of heating, this value had decreased to 403 nm (±10 nm). The refractive index ratio (5) -0.45 (40.1) for the control cells to -0.22 (±0.1) for the 30-min heat-treated cells. The average cell-wall thickness remained nearly constant at 108 nm (±20 nm) despite the heating. Finally, we deduced that the standard deviation of the cell radius increased slightly from about 106 nm (±8 nm; control) to 120 nm (±8 nm) after treatment at 60 C for 30 min. Our results show, as expected, that moderate heat treatment, although having little or no effect on cell wall, caused a measurable reduction in cell size. Some of these changes, indicated by a shift in the primary peak, occurred after only 3 min of heating. Although the data indicated an approximately linear cell shrinkage over the 30-min heating period, colony counts indicated that more than 99% of the cell population was killed after 3 min, presumably as a result of enzyme inactivation. The gross changes observed in the secondary scattering peak between 10 and 30 min of heating most probably arose, as would be expected, from protein denaturation and precipitation of internal solutes. We believe that electron micrographs taken of heated cells (1) have limited value in this type of experiment since distortion or damage may occur during preparation of specimens already weakened by heat. Furthermore, nephelometric devices which measure light scattering at a single fixed angle, such as 450 or 900, cannot be used to measure changes in cell size because such changes manifest themselves by shifts in angular location of the scattering peaks. By measuring the angular variation of scattered light, instruments such as the Differential I light scattering photometer clarify the dynamics of these structural changes. The light scattering techniques and instruments under development (5) show great promise as tools to study cell physiology and morphology under conditions where the ordinary light microscope is inadequate. Differential light scattering can bridge the gap between the light microscope and the electron microscope in the degree of resolution possible, in the elimination of the need for fixation, and in the rela- NOTES tive speed at which these measurements can be obtained.

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2017-09-27T02:25:05.056Z

1971-10-01T00:00:00.000Z

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Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide Aqueous suspensions of Bacillus subtilis NCTC 8236 spores, surviving gamma irradiation from a cesium-137 source, exhibited an enhanced rate of inactivation compared to nonirradiated spores when heated with 0.04% phenylmercuric nitrate. The enhanced rate of inactivation, observable from survival curves, was noted when spores were irradiated with 150,000 rad under air in either the presence or absence of the bactericide. The magnitude of the enhanced inactivation rate increased as the irradiation dose under air increased from 150,000 to 300,000 rad. The inactivation rates of spores surviving irradiation with 150,000 rad under either oxic or anoxic conditions did not exhibit a simple quantitative relationship. The enhancement effect was observed when the severity of the heat treatment was increased by either reducing the pH from 8 to 6 or raising the temperature from 70 to 90 C. Phenylmercuric nitrate is a commonly used mercurial bactericide. Mitchell (5) noted the suitability of 0.004% phenylmercuric nitrate for inclusion in ophthalmic vehicles to facilitate sterilization by heating at 100 C. In the British Pharmacopoeia 1968 (The Pharmaceutical Press, London) sterilization process of "heating with a bactericide," 0.002% phenylmercuric nitrate is added to thermo-labile injections which are then heated at 98 to 100 C for 30 min to effect sterility. It has been reported by us (1,3) that prior treatment of Bacillus subtilis spores with low doses of gamma irradiation enhances their rate of inactivation, compared to nonirradiated spores, by various heat treatments with phenolic bactericides. Accordingly, it was decided to attempt to establish a similar enhancement effect in the inactivation of the spores when phenylmercuric nitrate was used as bactericide. Such studies would indicate the usefulness of a sterilization process based on combined minimal dose of gamma irradiation and thermal treatment with a bactericide. MATERUILS AND METHODS The preparation and storage of the stock spore suspension of B. subtilis NCITC 8236, the viable count procedure, the dosimetry of the cesium-137 gamma irradiation facility, and the utilization of the heat I Present address: Justus Liebig-Universitat, Giessen, Germany. source have been described (1)(2)(3). The count of viable spores in the stock suspension used was ca. 2 X 109 organisms per ml and did not diminish significantly (P = 0.05) in viability during storage at 5 C throughout the 9 months of usage in the experiments described. Spores of equivalent total count, obtained from the stock suspension, were used for determining comparable survival curves. The method of construction and statistical analysis of divergence between survival curves has been described (3). Medium. Preliminary experiments indicated the suitability of nutrient agar (Oxoid) containing 1% dextrose, sterilized by autoclaving at 10 psi for 15 min, to which was added aseptically at 55 C 0.3% sodium thioglycolate previously sterilized by filtration. The sodium thioglycolate was prepared and assayed just prior to incorporation into the medium as described by Steel (6). Chemicals. Glass-distilled water was used to prepare the medium and chemical solutions. Laboratory grade phenylmercuric nitrate and thioglycolic acid and analar grade dextrose, sodium phosphate, sodium acid phosphate, and sodium hydroxide reagents were obtained from The British Drug Houses, Ltd. Phenylmercuric nitrate carryover. Serial dilutions of disinfection mixtures containing phenylmercuric nitrate were made in sterile water. The appropriate serial dilutions used for the enumeration of surviving spores were adjusted to contain 0.00004% phenylmercuric nitrate, as preliminary experiments showed that this procedure minimized the carryover effect of the bactericide on viable counts. treatment at 80 C. There was no observable alteration in the pH of the suspensions after treatment, but the phenylmercuric nitrate was noted to have undergone less than 5 % radiolytic decomposition, when estimated by the 2 method recommended by Eldridge and Sweet 4 (4). This alteration in the effective concentration of the bactericide could affect the magnitude of the divergence between the survival curves discussed. The inactivation experiments were repeated with the exception that the 0.04%r phenylmercuric nitrate, phosphate buffer (pH 7), was added to the nonirradiated and irradiated spores just prior to the commencement of the heat treatment. The survival curves (Fig. 1) indicated that the spores surviving irradiation were also more readily inactivated than nonirradiated spores by heating with the bactericide in the absence of its radiolytic products. RESULTS It was considered desirable to cause inactivation of spores through several log cycles of surviving fraction for the realistic evaluation of the sterilization procedure. Preliminary experiments indicated that a suitable concentration of phenylmercuric nitrate for such a study was 0.04%/ when proposed experimental conditions were varied. The divergence of survival curves determined for replicate experiments was insignificant, whereas the divergence of all the comparable survival curves reported in this communication was significant, unless otherwise indicated. Effect of presence or absence of phenylmercuric nitrate during irradiation on spore inactivation. Volumes of spore suspension in 0.04% phenylmercuric nitrate, phosphate buffer (pH 7), were either nonirradiated or irradiated with 150,000 rad under air. Figure 1 shows that spores surviving irradiation were more readily inactivated than nonirradiated spores by subsequent heat inactivation of spores, both nonirradiated and irradiated with 150,000 rad under air, increased when heated at 80 C in the presence of 0.04% phenylmercuric nitrate. Spores surviving irradiation were more readily inactivated than nonirradiated spores at each pH. 2 Effect of temperature on spore inactivation. Figure 5 shows survival curves for aqueous suspensions of spores, either nonirradiated or irradiated with 150,000 rad under air, before heating at either 70 or 90 C in the presence of 0.04% phenylmercuric nitrate, phosphate buffer (pH 7). The severity of the bactericidal treatment was increased by raising the temperature, and it was observed that spores surviving irradiation showed an enhanced rate of inactivation compared to nonirradiated spores at both tempera- tures. DISCUSSION When aqueous suspensions of spores were irradiated, damage induced in the spores must be attributable to both direct and indirect action of radiation. It is probable that one or several (2), and nitrogen (3), 0.04% phenylmercuric nitrate in phosphate buffer (pH 7) being added to each spore suspension just before the heat treatment. air, or irradiated with 300,000 rad under air, and to each suspension was added 0.04% phenylmercuric nitrate, phosphate buffer (pH 7) just before heating at 80 C. Figure 2 shows that the enhanced inactivation rate of spores surviving irradiation, compared to nonirradiated spores, after heating with the bactericide increased in magnitude as the dose of prior gamma irradiation applied to the spores was increased. Effect of gas present during irradiation on spore inactivation. Samples of aqueous spore suspension were sealed under air, oxygen, or nitrogen and then irradiated with 150,000 rad. One volume of each irradiated spore suspension was mixed with two volumes of 0.06% phenylmercuric nitrate, phosphate buffer (pH 7) just prior to heating at 80 C. The survival curves obtained are shown in Fig. 3 and do not indicate a simple quantitative relationship between the inactivation rates of spores surviving irradiation under the various gases. Effect of pH on spore inactivation. From the survival curves shown in Fig. 4, it was apparent that as the pH decreased from 8 to 6 the rate of (2,4), with 0.04% phenylmercuric nitrate in pH 8 phosphate buffer (1, 2) and 0.04% phenylmercuric nitrate in phosphate buffer (pH 6) (3, 4) addedjust before the heat treatment. a simple quantitative relationship. The enhance--~-2 ment effect was also noted when the severity of the moist heat treatment with the bactericide was increased by either raising the temperature from 70 to 90 C or reducing the pH from 8 to 6. These results are qualitatively similar to those reported previously (3) when using 0.2%0 chlorocresol as a bactericide. Such findings indicate that the enhancement effect, induced by prior irradiation of the spores, may be exhibited in other systems by using heat treatment with different bactericides. The enhanced bactericidal efficiency of a combined process, based on minimal gamma irradiation and heating with a bactericide treatment, may be of use for the sterilization of substances which undergo significant degradation with conventional sterilization techniques. The 3 combined process should facilitate the ready inactivation of those organisms in the mixed flora of the substances which are particularly sensitive to inactivation by any of the individual . 4 bactericidal agents comprising the process. Findings reported in this and previous com- radiochemical reactions in the spore initiated a sequence of events, which resulted in either spore inactivation or a predisposition of surviving spores to inactivation during storage and heat treatment with the bactericide. The enhanced rate of inactivation of spores surviving irradiation, compared to nonirradiated spores, caused by heating with phenylmercuric nitrate, phosphate buffer (pH 7), was observable in the presence or absence of the radiolytic products of the bactericide. The magnitude of the enhancement effect was observed to increase as the dose of prior gamma irradiation applied to the spores was increased. However, the enhanced inactivation rate, caused by moist heat treatment at 80 C with 0.04% phenylmercuric nitrate of the spores surviving irradiation under either oxic or anoxic conditions, did not exhibit

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2019-04-22T13:13:08.394Z

1970-01-01T00:00:00.000Z

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Effect of irrigation methods and mulching on growth and yield parameters of chilli (Capsicum annum L.) in arid condition The research work was carried out to study the impact of various irrigation methods and mulching on plant growth, production and profitability of chilli cv. R.Ch. 1 at Agricultural Research Station, Mandor, Jodhpur during July, 2016 to February, 2017. The results of surface irrigation were compared with drip irrigation system under no mulch and in conjunction with plastic mulch. The results revealed that the crop was irrigated by drip irrigation on raise bed with 100 micron Linear Low Density Poly Ethylene plastic mulch (T8 treatment) exhibited significantly higher seedling survival at 15 and 30 days after transplanting (95.16% and 91.70%), highest plant height (47.10 cm at 45 DAT and 54.60 cm at harvest), highest number of branches (14.93) plant-1, maximum stem girth (2.32 cm) number of roots plant-1 (138.5), highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm) and fresh weight of fruit-1 (8.42g) was observed. The maximum number of fruits plant-1(125), highest yield plant-1 (1052.5g), yield ha-1 (337.63q) and premier fruit quality score (9.11) with maximum net return (Rs.326407.28) and benefit: cost ratio (3.41) was also reported in same treatment. Comparatively minimum time (15 hours) required for one hectare irrigation was also reported in drip irrigation on raise bed with plastic mulch. This led to lower population of white fly plant-1 (4.53), minimum weed infestation (1.53 weed m-2), leaf curl (5.50%) and fruit rot (5.0%) incidence than other treatment combinations. The minimum growth, yield and profitability were reported in check basin method of irrigation without mulch (T1 treatment). 82 to mulch includes early production, more yield and reduced insect and disease problems (Pattanaik et al. 2003). Linear Low Density Poly Ethylene (LLDPE) plastic films have been proved as better mulch because of their puncture resistance quality, thinness and lower cost (Panda 2004). Numerous experiments have reported the benefits of LLDPE mulch in several crops, but research is limited on response of chilli production in western Rajasthan by this method. Keeping this in background, the present study was undertaken to study the effect of different irrigation methods and mulching on chilli crop and compare the result with the conventional method of growing the crop under surface irrigation without mulch. A field experiment was conducted at Agricultural Research Station, Mandor, Jodhpur (Rajasthan), India during kharif seasons of year 2016-17. The soil of experimental plot was of sandy loam texture with average pH range 8.5, having organic carbon 0.55%, available N 180 kg ha -1 , P 27.5 kg ha -1 and K 250.0 kg ha -1 during experimentation. The experiment was conducted in a completely randomized design having nine treatments comprising by different irrigation methods and mulching viz., T 1 = Check basin method, T 2 = furrow irrigation method, T 3 = Raise bed with trench method, T 4 = Flat bed with drip irrigation, T 5 = Flat bed + plastic mulch + drip irrigation, T 6 = Raise bed with drip irrigation, T 7 = Raise bed + organic mulch + drip irrigation, T 8 = Raise bed + plastic mulch + drip irrigation, T 9 = Sprinkler irrigation method. In well prepared field, transplanting of Chilli seedlings variety RCh 1 of 35-40 days old were planted in pair row method with a spacing of 45cm x 45 cm/90 cm (33,333 plant ha -1 ) during last week of June. In check basin and sprinkler system of irrigation the bed size is 2 x 2 meter and in all other methods is 1 × 4 meters. The cultural practices of the crop were followed as per the recommendations. The organic material and LLDPE silver colour film of 100-micron thickness was used for mulching around the plant. The lateral lines of 12 mm diameter LLDPE pipes were laid along with crop rows. The laterals were provided with inlet drippers of 8 litre hr -1 discharge capacity. All the observations were taken from five randomly selected plant of each replication throughout the investigation period at appropriate time by adopting standard method for growth, development, fruiting behavior and yield. Seedling survival per cent (after transplanting in main field at 15 DAT and 30 DAT) was recorded by following formula; Survival percent = [Total survival transplanted plants / Total transplanted plants] × 100 Plant height (at 45 DAT and at harvesting) was measured from soil surface upto the highest shoot tip by straightening all branches. Stem girth was measured 1 cm from the base of the stem using vernier calliper. Observation of number of branches, days taken to first flower initiation, duration of fruiting period and number of fruit plant -1 was recorded by standard counting method. Number of roots, root length was measured by destructive method of uprooting the plants and taking measurement by standard method. Length of fresh fruits measured by scale and fruit diameter using vernier calliper and expressed in centimeter. Fruit set per cent was recorded by following formula; Fruit set per cent = [Total number of fruit set plant -1 / Total number of flowers plant -1 ] × 100 Fruit weight was determined by weighing method at the time of harvesting and expressed in gram fruit -1 . The total fruit yield plant -1 and hectare -1 was calculated by weighing total marketable fruits and has been expressed in gram and quiantal respectively. Further, the net return was calculated by subtracting cost of each treatment from gross return. The gross return was calculated from yield multiplied by average market rate during the period of investigation. The benefit cost ratio was calculated by dividing net return to total cost of cultivation. Benefit-Cost ratio and net profit were carried out to determine the economic feasibility of the crop using surface and drip irrigation as suggested by Tiwari et al. (1998a). 83 The seasonal system cost of drip irrigation system included depreciation, prevailing bank interest rate, and repair and maintenance cost of the system. The fixed cost of drip irrigation system was determined to be Rs 112,000 ha -1 . The useful life of drip system was considered to be 10 years. The system cost was evaluated by distributing the fixed cost of system over life period of drip irrigation set. For calculating depreciation, the life of the drip irrigation set and 10% junk value was considered. The interest was calculated on the average of investment of the drip irrigation set taking into consideration the value of the set in the first and last year @10% per annum. Cost of repairs and maintenance of set is @2% of initial cost of the drip irrigation set per year. The cost of cultivation includes expenses incurred in land preparation, interculture operation, fertilizer, crop protection measures, irrigation water and harvesting with labour charges. Therefore, total seasonal cost was worked as: depreciation, interest, repairs and maintenance cost of set + cost of cultivation + cost of mulch. The income from produce was calculated using prevailing average market price of capsicum @ Rs 1250 q -1 . Disease incidence (leaf curl and fruit rot) and quality of fruits was measured by visual inspection (Five member team of crop experts and plant pathologist). White fly population plant -1 and weed infestation meter -2 was calculate by simple counting method. The time required for irrigation was calculated as per actual required time of irrigation of specified area by different methods of irrigation. To test the significance of variance of data obtained from crop growth, yield and economics of variance technique for completely randomized design was done by standard procedure prescribed by Panse & Sukhatme (1985). Significance of difference among the treatments effect was tested by 'F' test and critical difference (CD) was calculated, wherever the results were significant. The results revealed that, the irrigation methods and mulching are significantly influenced growth attributes at all the growth stages ( Table 1). The maximum seedling survival per cent at 15 DAT (95.10%) and 30 DAT (91.70%) was recorded in T 8 treatment, which was significantly superior to other treatment but at par with T 6, and T 7 treatments. The maximum survival per cent of seedling in T 8 treatment might be due to more favourable moisture condition for seedling transplanting and reestablishment of roots than others. The height of plant under treatment T8 (47.10 cm at 45 DAT) and treatment T6 (62.60 cm at harvest) was found highest among all other treatments and is 67.19% and 13.40% higher than the T 1 treatment. About to number of branch plant -1 , maximum value was recorded in treatment T8 (14.93) followed by treatment T7 (12.53) and the lowest value was in treatment T1 (7.17). Maximum stem girth at harvest (2.36 cm) and highest number of roots plant -1 (138.50) were observed in T 8 treatment whereas longest root system (10.50 cm) was observed in T 3 treatment. The minimum stem girth (1.68 cm) and the number of roots plant -1 (53.57) were observed in T 1 treatment whereas shortest root system (7.97 cm) was observed in T 9 treatment. The higher available moisture status in soil favourably influences the uptake of nutrients which maintains the cell turgidity, cell elongation, photosynthesis and respiration at optimum level, leading to favourable growth and development of plant in terms of plant height, number of branches plant -1 , stem girth and number of root plant -1 in the present study. The highest increase in vegetative growth in drip irrigation with mulching might be due to the availability of soil moisture as well as favourable temperature at optimum level for plant growth development (Pattanaik et al. 2003;Paul et. al. 2013). The lowest value of vegetative growth in T1 might be because of unfavourable moisture regime (moisture stress or excess moisture) in the soil through surface irrigation and competition of weeds for nutrients (Pattanaik et al. 2003;Agrawal & Agrawal 2005). The increased growth attributes might have supplied water and nutrients in adequate proportion, which resulted in triggering the production of plant growth hormone, viz., indole acetic acid (IAA) and higher number of leaves and roots throughout the cropping period (Sankar et al. 2008). The drip irrigation in combination with mulch significantly increased the yield of chilli as compared to drip irrigation without mulch (Table 2) and surface irrigation methods. The minimum days (42.38) required for first flower initiation was reported in T 9 treatment whereas the maximum days (51.39) was required in T 5 treatment. Among various treatments, highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm), duration of fruiting (71.38 days), fresh weight of fruit -1 (8.42 g), maximum number of fruits plant -1 (125), highest yield plant -1 (1052.5 g) and yield ha -1 (337.63 q) was recorded under T 8 treatment, whereas lowest yield (153.45 q ha -1 ) was recorded under T 1 treatment. This might be due to water stress during the critical growth period and fruit development stage coupled with aeration problem in first few days immediately after irrigation. Another reason to get low yield by surface irrigation without mulch might be due to less availability of nutrients for crop growth due to leaching and high weed competition between the crops (Pattanaik et al. 2003). In drip irrigation system on raise bed with plastic mulch the water is applied at a low rate for a longer period at frequent intervals near the plant root zone through lower pressure delivery system, which increases the availability of nutrients near the root zone with a reduction in leaching losses and minimum weed competition. More nutrient availability, especially near the root zone might have increased the translocation of photosynthetes to storage organ of chilli resulting in an increased weight of fruits. This result corroborated the findings of Singh (2007), Sankar et al. (2008, Paul et al. (2013) and Kumar et al. (2016). Irrigation methods and mulching also significantly influenced the gross return, net return and benefit cost ratio in chill (Table 3). Maximum net profit of Rs. 326407.28 ha -1 with B: C ratio of 3.41 was recorded in T 8 treatment followed by Rs 296192.61 ha -1 with B: C ratio of 3.11 in T 5 treatment and lowest net profit of Rs 119007.80 ha -1 with a B: C ratio of 1.63 in T 1 treatment (Table 3). It is observed that, the drip irrigation with mulched treatments T 5 , T 7 and T 8 gave better net return with higher B: C ratio ha -1 than their corresponding treatments without mulching in conventional irrigation method. The highest net return (US$ 7098 ha -1 ), incremental net return (US$ 1556 ha -1 ), and incremental benefit-cost ratio (7.03) were found for 50% water application with straw mulch (Biswas et al. 2015). The results are in conformity with the findings of Singh (2007), Sankar et al. (2008) and Kumar et al. (2016). Apart from reducing water consumption, drip irrigation with mulching also helps in reducing cost of cultivation and improving productivity of crops as compared to the same crops cultivated under flood method of irrigation (Paul et al. 2013). Irrigation time significantly pretentious by different irrigation methods. The minimum time required in irrigation (14.50 hours) in T 5 treatment which closely followed by T 8 treatment. Drip irrigation method with or without mulching required less irrigation time than without mulching in conventional irrigation method. There was significant effect of LLDPE mulch over drip irrigation system alone. Drip irrigation with LLDPE mulching (T 5 & T 8 treatment) saving irrigation time (21.40 hour ha -1 and 21.35 hour ha -1 ) upto 60 per cent by reducing water losses and increased irrigation efficiency. The increase in water saving per cent in trench method (T 2 ), drip irrigation system alone (T 4 ), drip irrigation system with LLDPE mulch (T 5 & T 8 ), drip irrigation with organic mulch (T 7 ) and sprinkler system (T 9 ) over conventional surface irrigation by check basin method (T 1 ) was 38.8%, 46.6%, 60.0%, 54.4% and 41.9% respectively. The highest water use efficiency of 592 kg ha -1 mm -1 was obtained with 50 per cent water application under polyethylene mulch (Biswas et al. 2015). Drip irrigation with mulching helps to achieve yield gains of upto 100 per cent, water savings of upto 40-80 per cent, and associated fertilizer, pesticide, and labour savings over conventional irrigation systems in capsicum crop (Paul et al. 2013). Similar trend has been reported in water use efficiency for okra crop by Tiwari et al. (1998a) and for tomato crop by Singh (2007). Occurrence of chilli leaf curling and fruit rot was detected throughout the investigation period. The best performance, with a marked reduction in leaf curling and fruit rot and improve fruit quality was observed in drip irrigation with LLDPE mulching (Table 3). The minimum incidence of fruit rot (5.0%), leaf curing (5.5%) and highest quality score of fruit (9.11) was observed in T 8 treatment which was closely followed by T 7 and T 5 treatments where as maximum incidence of fruit rot (20.02%) and leaf curling (15.84%) was reported in check basin method of irrigation (T 1 treatment) whereas minimum quality score (6.50) was observed in T 9 treatment. Presence of white fly and weed was observed throughout the investigation period. The minimum population of white flies (4.53 plant -1 ) and weed infestation (1.53 weed m -2 ) was observed in T 8 treatment whereas highest incidence of white flies (17.43 plant -1 ) and weed infestation (30.03 weed m -2 ) was observed in T 1 treatment. This is due to the fact that in drip irrigation with mulching significantly reduced additional moisture level in field environment which in turn increase quality of fruits and reduce disease infestation, white fly population as well as it also trim down weed seed germination, growth and development. The increase in quality of fruits was due to the effective utilization of applied nutrients, water and significantly reduced weed growth; disease incidence and increased rate of photosynthesis, sink capacity and accumulated more amounts of dry matter and finally increased quality of fruits and yield. Conventional surface irrigation methods without mulching provide favorable environmental condition for increase insect population and development of disease as well as germinate and develop high density weed plants. The beneficial effect of drip irrigation and black LLDPE mulch in capsicum, tomato and okra was also reported earlier by Horo et al. (2003); Singh (2007);Vankar & Shinde (2007), Bhardwaj & Sarolia (2012), Paul et al. (2013). The combination of raised bed + drip irrigation system with LLDPE mulching is observed to be economical and cost effective as compared with conventional surface irrigation without mulching. Thus, the use of drip irrigation system either alone or in combination with mulching, could increase the chilli yield quality of fruits and profitability. It also reduces white fly population, disease incidence (root rot and leaf curling) and minimise with crop weed competition. Drip irrigation with mulching increase water use efficiency by significant reduction in irrigation time ha -1 . It is concluded that the drip irrigation method with LLDPE mulching is suitable for chilli production in arid and semi arid condition of western Rajasthan. Isabgol (Plantago ovata Forsk) is a short duration, more remunerative and medicinally important crop of arid and semiarid regions. In India, it is largely grown in Gujarat, Rajasthan, Madhya Pradesh and Haryana. The area under Isabgol in India during 2014-15 is 1.09 lakh hectares. The production recorded 72 thousand MT with productivity of 660 kg ha -1 . In Gujarat, area mostly falls in Banaskantha, Kachchh and Patan districts with acreage of nine thousand hectare with production and productivity 5000 MT and 556 kg ha -1 , respectively during 2015-16 (Anonymous 2016a). During the last decade, area and production of isabgol has decreased to the tune of 343% and 281%, respectively, mainly due to problems of seed shattering. At the time of maturity, unseasonal rain or heavy dew leads to failure of the crop which is the fact for reduction of area under Isabgol (Anonymous 2016b). The objective of the study was to evolve non-shattering isabgol cultivars. Isabgol growing area of Kachchh in Gujarat was surveyed during 2009-10 and subsequently genotypes were evaluated. First three years (2010-11 to 2012-13) 13 genotypes 07,10,13,15,16,20,21,22,23,24,25 The stability analysis of variance and stability parameters viz., linear regression coefficient (bi) and deviation from regression (S 2 di) of genotype means over environment were computed as suggested by Eberhart & Russell (1966). 89 plants in each replication were selected at maturity stage. Entire spike were dipped in water and then observed for seed shattering from selected plants. The percentage of seed shattering calculated by using a following formula. Gujarat Isabgol 4 recorded high mean with regression coefficient (bi) near unity and deviation from regression (S 2 di) around zero for seed yield, indicating GI-4 has average responsiveness and are highly stable over environments ( Table 2). The new culture has compact spike and did not separate easily even after dipping in water. Only 7.25% seeds shattered after dipping in water. Swelling factor (cc g -1 ) 11.4 9.1 Patel et al. GI-4 recorded 89.67% less shattering than that of check variety, which revealed that Gujarat Isabgol 4 was non shattering in habit as compared to Gujarat Isabgol 3 which is prone to high seed shattering. The post dipped seed weight of GI 4, was 0.115 kg threshed seeds, which was 96% higher than that of the post dipped seed yield of GI-3 (0.037 kg threshed seeds) (Table 4). Hence, Gujarat Isabgol 4 recommended for cultivation.

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2020-08-27T09:04:47.275Z

2020-01-01T00:00:00.000Z

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Phytochemical Screening and Antidiabetic Activity Test of Extracts and Fractions of Lactuca Indica (L.) In Streptozotocin- Induced Diabetic Mice Design: The design of this study was experimental in which the extraction and fraction of Lactuca indica leaves were tested for the value of decreasing blood glucose levels of mice after induction of streptozotocin. Antidiabetic activity tests were divided into 12 groups. Group I (baseline) group II (negative control) were given CMC 0.5%, Group III (positive control) were given metformin 65 mg/kg BW, while Groups IV to XII were given Lactuca indica leaf extract and fractions at their respective doses -each 100, 150 and 200 mg/kg BW Interventions: The variable that was intervened in this study was the concentration of extract used Main outcome measure: The main measurement results in this study were to determine extracts and fractions that we're able to reduce blood glucose levels in mice. Results: The antidiabetic effect of Lactuca indica leaves shows that EELL, EAFLL and NHFLL have antidiabetic effects, this is supported by the chemical content contained in the extracts, namely flavonoids, tannins, saponins, glycosides and triterpenoids/steroids. The most effective activity to reduce blood glucose levels in streptozotocin-induced mice is EAFLL 100 mg/kg BW Conclusion: ethyl acetate fraction of Lactuca indica leaves has an effective antidiabetic activity at a dose of 200 mg/kg BW has given antidiabetic activity in mice induced by STZ, on the 9th day BGL mice have dropped below 120 mg/dl. INTRODUCTION iabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the pancreatic b-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The basis of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues 1 . The management of diabetes is a global problem until now and successful treatment is not yet discovered. The modern oral hypoglycemic gents produce undesirable and side effects. Therefore, it needs to find an alternative drug to avoid adverse event from conventional treatment i.e. herbal medicine from plant 2 . One of the plants that have the potential to be developed is the Lactuca indica leaf. Lactuca indica (Compositae) is edible wild lettuce widely distributed in Asia and has been popularly used as folk medicine. The whole plant of Lactuca indica, prepared by using boiling water or an ethanolic solution, has been taken orally or topically administered in various medications for its anti-inflammatory and antibacterial activities and to treat intestinal disorders. However, limited scientifically proven information is available on the bioactivities, pharmacological functions, and specific clinical efficacies of this plant 3 . Lactuca indica is used in medicine and has pharmacological effects on human health, has antiinflammatory and antibacterial properties, can also reduce intestinal disorders 3 . Besides being used as a diuretic, Lactuca indica shows a direct secondary effect on epithelial cells that can protect against Escherichia coli infection in urinary tract infections 4 . Lactuca indica leaf extract has potential as an anti-hepatitis virus 4 . Lactuca indica is an edible wild vegetable, used as an antiinflammatory, antibacterial, free radical antidote (DPPH IC¬50) 12.2 ± 0.02 in hot water extract, DPPH IC50 6.1 ± 0.01 (µg / ml) in the ethyl acetate fraction and DPPH IC50 19.4 ± 0.02 in the water fraction) and leukemia treatment 3 . Based on the description above, the ethanol extract and nhexane and ethyl acetate of Lactuca indica leaves were tested as antidiabetic in mice (Mus musculus L.) induced by streptozotocin (STZ) to find the most effective fraction to reduce BGL (blood glucose level), as comparison used metformin. Plant and Chemical Material Lactuca indica sample were collected from North Tapanuli, North Sumatera, Indonesia. The part of the plant being used is the leaves. Lactuca indica leaf has been determined by the Herbarium Bogoriense Indonesian Institute of Science, it is known that the species is Lactuca indica (L.) with the Family Compositae. Extraction and fraction The dried Lactuca indica leaves (1000 g) were crushed using a blender. After that, using the maceration method the leaf powder is soaked with 80% ethanol. The solvent was evaporated at low pressure and the temperature not exceed 60 0 C using a rotary evaporator, then dried using a freeze dryer. Next, the crude extract is taken (90 g) for liquid-liquid extraction to obtain nonpolar (using nhexane) and semi-polar (using ethyl acetate) 5,6 . Mice were housed in polycarbonate cages in a room with 12 hrs day-night circle. They were fed on a standard pellet diet and water ad libitum. The study was approved by Animal Research Ethics Committees (AREC) of University of Sumatera Utara, and then experiments were conducted according to the ethical norms and AREC guidelines. Induction of diabetes Experimental diabetes was induced by a single intraperitoneal injection of 55 mg/kg of STZ, freshly dissolved in NaCl, 0.9%. After 3 days of STZ injection, mice with fasting glucose above 200 mg/dl were considered as diabetic and included in the study. Experimental design The animals used for the experimental design of the antidiabetic test were divided into twelve groups consisting of four animals for each group. the twelve groups consisting of 1 basic group, 2 control groups and 9 test groups consisting of a dose of 100 mg / Kg BW, 150 mg / Kg BW, 200 mg / Kg BW, summarized as follows: The treatment begins after the animal tested positive for diabetes, this is the 1st day of treatment, every two days measurements of blood glucose levels are taken. Testing is carried out for 2 weeks, namely on days 3, 5, 7, 9, 11, 13 and 15. Statistical analysis All the data were expressed as mean±standard deviation. The significant difference of data between different groups was compared by ANOVA followed by Duncan's test. Phytochemical screening The results of Lactuca indica leaf fractionation screening showed different chemical compounds in different extracts in Table 2. Extraction and Fractionation The extraction process by the way of maceration of 1000 g Lactuca indica leaves simplicial powder produced 140.8 g of Lactuca indica leaves ethanol extract. Then the 90 g ethanol extract was fractionated using a separating funnel to obtain 12,7 g n-hexane fraction, 61,32 g ethyl acetate fraction and 15,55 g fraction remaining. Anti-Diabetic test Blood glucose levels are measured enzymatically with a glucometer. Gluco test strips are inserted into the glucometer so that the glucometer will turn on automatically. blood glucose level testing using healthy mice that have been acclimated fast for 18 hours then weighed and their blood glucose levels measured. The treatment begins after the animal tested positive for diabetes (day 1) Every day given test material, BW is weighed every two days intervals, then measurements of blood glucose levels for two weeks are on days 1, 3, 5, 7, 9, 11, 13 and 15. The results of observations on average BGL (Table 3) and (Figure 1). The results obtained that EELL, EAFLL and NHFLL have antidiabetic effects, this is supported by the chemical content contained in the extracts, namely flavonoids, tannins, saponins, glycosides and triterpenoids/steroids. The hypoglycemic mechanism is thought to be caused by flavonoids which can inhibit glucose reabsorption from the kidneys and can increase blood glucose solubility so that it is easily excreted in urine [10][11][12] . So it is suspected that the flavonoid group in the leaves of Lactuca indica can reduce blood glucose levels. CONCLUSIONS Lactuca indica leaves can provide effects especially EAFLL and NHFLL both provide effective antidiabetic activity. EAFLL with a dose of 100 mg/kg BW has given antidiabetic activity in mice induced by STZ, on the 9th day BGL mice have dropped below 120 mg/dl

v3-fos

2018-04-03T00:28:18.901Z

1970-05-01T00:00:00.000Z

11792575

s2

Bacteriological and shelf-life characteristics of canned, pasteurized crab cake mix. The bacteriological spoilage characteristics of a canned, pasteurized crab cake mix product stored at various temperatures were investigated. A large number of bacteria, both mesophilic and psychrophilic, survived the pasteurization process. Bacillus and Micrococcus were found to predominate when the product was stored at 30 C (86 F) and 18 C (64 F), whereas Alcaligenes predominated at 2 C (36 F). The product was found to be free of Escherichia coli. Bacterial counts, trimethylamine nitrogen, volatile reducing substances, and ammonia determinations were evaluated as indices of quality for the product. Close correlation was observed between bacterial counts, volatile reducing substance values, and organoleptic tests when the product was stored at 30 C (86 F). The shelf-life of the product was approximately 6 months at 2 C (36 F), 4 days at 18 C (64 F), and 27 hr at 30 C (86 F). Canned, pasteurized crab cake mix is a relatively new product. There is little available information concerning its shelf-life and spoilage characteristics. The crab cake mix is commercially prepared by mixing crab meat with other ingredients, such as bread crumbs, eggs, mayonnaise, and other flavoring substances. The formula for the crab cake mix product is shown in Table 1. The product is packed at atmospheric pressure in 307 X 409 C enameled cans of 1 lb (454 g) net product capacity, pasteurized in a water bath at 85 to 87 C (185 to 190 F) for 110 min, and immediately cooled. The product is then kept at 0 to 5 C (32 to 41 F). A problem may arise when the consumer removes the product from cold storage in a food store. In some instances, it may take several hours before the product is returned to a refrigerator in the consumer's home, or the product might inadvertently be left unrefrigerated overnight. Bacteriological changes occur during this period, some of which are undesirable from the standpoint of product quality, and perhaps safety. This problem is of interest to consumers and the industry. Bacteriological spoilage of crab meat has been studied by Harris (15), Alford et al. (1), and Benarde (6). Pasteurization of crab meat in metal containers was first investigated by Tobin and McCleskey (27). They packed and pasteurized Present address: Office of Atom-iic Energy for Peace, Bangkhen, Bangkok, Thailand. crab meat at 15 psi for 5, 10, and 15 min. They reported that slight discoloration of the surface of the pasteurized meat was observed. Pasteurization of crab meat packed in cans in a water bath at the temperature below the boiling point of water was studied by Anzulovic and Reedy (4) and by Flynn and Tatro (14). These investigators found that pasteurization killed a large number of microorganisms including Escherichia coli, and that pasteurization prolonged the keeping quality of crab meat for a considerable time. Much research has been done by employing trimethylamine nitrogen (TMA-N) as an index of decomposition of marine fish and shellfish. Spinelli et al. (25) studied the relation of bacterial counts and increase in TMA-N with sensory evaluation in vacuum-packed king crab meat. They found that samples with TMA-N content exceeding 1.0 mg/100 g received a poor rating. There appeared to be a fair correlation between increase in bacterial counts, TMA-N content, and a decrease in sensory scores. Farber (12) claimed that the content of TMA-N was not a sensitive, reliable, or reproducible index of fish spoilage. Either the increase in TMA-N occurred during the latter stage of spoilage, or there was a variation in levels between species, or no appreciable increase took place. Volatile reducing substances (VRS) content has been found to be a useful index of freshness of fresh and canned fishery products, as indicated by Farber (11), and Farber and Ferro (13). The content of VRS was reported to correlate closely with organoleptic judgments. Burnett (8) devised a colorimetric method which used ammonia as an index of decomposition in fresh and frozen crab meat. This method is based upon the color reaction between ammonia, thymol, and bromine. The author reported that ammonia content increased uniformly and rapidly with spoilage, and that ammonia could be detected before spoilage was detected organoleptically. In crab cake mix there are ingredients of widely variable microbiological quality. The pasteurized product, therefore, is expected to have a variable spoilage pattern different from that of crab meat or fish. Generally accepted objective tests for ascertaining degree of spoilage of crab meat or fish may not be adequate for crab cake mix. The principal objectives of this investigation were: (i) to determine the shelf-life of the product at various temperatures by studying the bacteriological spoilage pattern through bacterial counts and characterization of the more prevalent bacteria in the product; (ii) to evaluate some of the existing objective tests as indices of quality and degree of spoilage of the crab cake mix; and (iii) to determine the correlation between odor and bacterial count, TMA content, VRS value, and amount of ammonia present in the product. MATERIALS AND METHODS Rate of heat penetration in canned crab cake mix was determined under commercial conditions. The method described by Alstrand and Ecklund (2) was used. Six 307 X 409 cans of 1 lb (454 g) net product capacity were used for the determination. The molded bakelite thermocouples were first placed at the geometrical centers of the test cans before filling. Filling was done in such a way that the thermocouple tips were imbedded in the product with no air space around them. The canned product was pasteurized in a steam-heated water bath, previously heated to 85 C (185 F), for 110 min. Center can temperatures of the product were read before the beginning of the pasteurization process and every 10 min throughout the heating and during the cooling operation, until the temperature returned to 60 C (140 F). A Brown po-tentiometer with automatic junction compensation was used to make temperature readings. The general plate count techniques were those outlined in Standard Methods for the Examination of Dairy Products (3). Throughout this study, BBL standard plate count (SPC) agar was used for plate counts. For estimating bacterial population in fresh crab meat and crab cake mix, SPC agar was dissolved in artificial sea water (24). Total plate counts of crab cake mix ingredients were determined. Samples of ingredients used for crab cake mix were obtained from a crab processing plant, placed aseptically in sterile bottles, and transported in ice to a laboratory where plate counts were made immediately. One gram of each of the ingredients was mixed with 9 ml of sterile water; the samples were further diluted and plated on SPC agar medium. All plates were prepared in triplicate and incubated at either 30 C (86 F), 18 C (64 F), or 2 C (36 F) to determine the microflora growing in the samples at each of three temperatures. Plates were incubated at 30 C (86 F) for 48 hr, at 18 C (64 F) for 4 days, and at 2 C (36 F) for 18 to 20 days before counting was made. The bacteriological spoilage pattern of the product was studied at 30 C (86 F) to represent the upper extremes of room temperature storage, at 18 C (64 F) to include a temperature intermediate between room and refrigerated storage temperatures, and at 2 C (36 F) to duplicate refrigerated storage temperature. The more prevalent colonies of bacteria that grew on plates incubated at the three temperatures were isolated and differentiated according to genera. Routine characterization tests were done according to Bergey's Manual of Determinative Bacteriology (7) and Guide to the Identification of the Genera of Bacteria (23). Flagella were stained by using the method of Leifson [E. Leifson, J. Bacteriol. 36:656 (Abstr.), 1938]. Oxidase activity was determined by the method of Kovacs (17). Chemical analysis of the pasteurized crab cake mix for moisture, crude fat, protein, and ash were performed in accordance with Association of Official Agricultural Chemists techniques (16). TMA-N content of pasteurized crab cake mix was determined colorimetrically as trimethylamine picrate by the method of Dyer (10). A Spectronic 20 (Bausch & Lomb) colorimeter was used. TMA-N values were read as percentage of transmittance from the TMA-N standard curve. VRS values were determined by the method of Lang et al. (18). The apparatus was slightly modified. It was found that simple test tubes (20 by 125 mm) could be used as sample vessels, since foaming was eliminated by using Antifoam A (Dow Chemical Co.). For each determination, 10 g of pasteurized crab cake mix, diluted 1:1 with distilled water, was weighed into an aeration vessel to which a few drops of the silicone antifoaming agent was added. Exactly 50 ml of 0.02 N KMnO4 in 1.0 N NaOH solution was pipetted into a reaction flask. The determination of VRS values was carried out at room temperature. Room air was drawn into the VRS apparatus at a rate of 100 liter/hr by means of a vacuum pump. The sample was aerated for 40 min. An air blank was also determined by using 10 VOL. 19,1970 ml of emulsified redistilled water, prepared by blending a few drops of the silicone antifoaming agent in 100 ml of redistilled water, in place of the sample. At the end of the aeration period, the reaction flask was removed. A 25-ml amount of 6 N H2S04 and 15 ml of 20% KI in 0.1% Na2CO3 were added. The liberated iodine was titrated with 0.025 N Na2S2O3 in 0.2% Na2CO, and 0.1% Na2B4O7. 10 HaO solution. Toward the end of the titration, several drops of 1% soluble starch in saturated NaCl solution was added as an indicator. VRS value in the sample was calculated by the equation: VRS value = titration -sample titration X normality Na2S2O3 X 1000/weight of sample in grams. The VRS value determined as described above was expressed in microequivalent GuEq) of KMnO4 per gram of sample. Ammonia content in the product was determined by the method of Burnett (8). Odor of pasteurized crab cake mix, stored at room temperature for various periods of time, was evaluated by a panel of 20 persons. The panel was untrained but was made familiar with the product. A nine-point hedonic scale was used in rating the sample. Odor evaluations were made by using four replicates. RESULTS The rate of heat penetration in crab cake mix commercially packed in 307 X 409 C enameled cans is shown in Fig. 1. Approximately 100 min was required for the center can temperature to reach 82 C (180 F), the desired temperature for pasteurization. The center can temperature was maintained at 82 C (180 F) for approximately 10 min before the cans were cooled. The pasteurization process at 85 to 88 C (185 to 190 F) for 110 m-reduced the bacterial counts in the product by a factor of approximately 20, from 6.0 X 105 to approximately 3.3 X 104 per g. Total plate counts of microorganisms in fresh crab meat and in other ingredients used for pre-APPL. MICRoBIoL. paring crab cake mix are shown in Table 2. Bread crumbs showed fairly large counts of both mesophilic and psychrophilic bacteria, whereas pepper and spice mix contained rather large numbers of only mesophilic bacteria. A large number of both mesophilic and psychrophilic bacteria were found in raw crab cake mix. Bacterial counts of the product stored at 30 C (86 F) are shown in Fig. 2. Bacterial counts from plates incubated at 30 C (86 F) and 18 C (64 F) increased with storage time, as might be expected. The increase in number of bacteria indicates typical growth curves which clearly show lag, log, and stationary phases. The product contained approximately 2 X 104 bacteria per g at zero storage time. The counts increased significantly after approximately 10 to 12 hr of storage time at 30 C. This can be explained by the fact that the rate of heat penetration in the product is slow. After 24 hr, the product showed a sign of slight spoilage, as observed by odor. Bacterial counts showed approximately 108 organisms per g. This indicates that the shelf-life of the product stored at 30 C (86 F) is in the neighborhood of 24 hr. When plates were incubated at 2 C (36 F), the number of bacteria, presumably psychrophiles, increased slightly up to approximately 12 hr of storage time at 30 C (86 F) and declined significantly thereafter. The increase in number of bacteria at the beginning indicated that the temperatures in the can of crab cake mix were suitable for psychrophilic bacteria to grow up to approximately 12 hr of storage time. The rapid decrease in the number of bacteria after 12 hr of storage is postulated to be due to the genetic behavior of the psychrophiles in the product. These psychrophiles presumably came from a cold marine environment in the Chesapeake Bay area. When exposed to high storage temperatures, they tended to die. Bacterial counts in the product stored at 18 C (64 F) for various lengths of time are shown in Fig. 3. When plates were incubated at each of the three incubation temperatures, both mesophilic and psychrophilic bacteria appeared to grow well. This is due to the fact that the storage temperature of 18 C (64 F) is within the growing ranges of both types of bacteria. However, the meso-philes seemed to grow better than the psychrophiles after 2 days of storage. After 4 days of storage at 18 C (64 F), an off odor was noted in the product. Bacterial count at this time showed approximately 7 X 107 organisms per g. Bacterial counts of the product stored at 2 C (36 F) were made at 1-month intervals for 6 months (Fig. 4). It was observed that the counts of both mesophilic and psychrophilic bacteria were fairly low, even after 6 months of storage time. A slight off odor was observed at the end of that period, but the product was still considered acceptable. The shelf-life of the product at 2 C (36 F) was considered to be 6 months. The next step in the bacteriological examination of pasteurized crab cake mix was to characterize bacteria isolated from the product stored at different temperatures. Attempts were made to characterize the more prevalent bacteria in the product stored at the three temperatures, namely, 30 C (86 F), 18 C (64 F), and 2 C (36 F). Three genera of bacteria were found to be prominent in pasteurized crab cake mix stored at the three temperatures (Table 3). They were Bacillus, Micrococcus, and Alcaligenes. Bacillus was characterized mainly according to its ability to survive the heat treatment of 65 C (149 F) for 30 min, and as catalase-positive, gram-positive, aerobic, spore-forming bacilli. Micrococcus was characterized mainly as gram-positive cocci in clumps, for producing acid from glucose oxidatively, and for having the ability to survive the heat treatment. Alcaligenes was characterized as gramnegative and oxidase-positive, for being motile with peritrichous flagella, for its inability to utilize carbohydrates, and for its ropy colonies. Attempts were made to detect E. coli in pas- Table 4. Determinations of pH in the product at each storage temperature indicate that, although pH of the samples in advanced stages of spoilage showed a lower value than that of fresh samples, the samples in between inhibited a nonuniform decrease of pH. No sig-nificant difference was found in the pH of samples in borderline stages of spoilage when compared with the pH of relatively fresh samples. Therefore, pH is not a reliable index of decomposition for pasteurized crab cake mix. Proximate composition data for pasteurized crab cake mix are presented in Table 5. The quantity of crude fat in the product is relatively high because a rather high proportion of mayonnaise is included in the formula. TMA-N content of pasteurized crab cake mix stored at 30 C (86 F) for various periods of time is shown in Fig. 5. It is evident that TMA-N content increased significantly after approximately 24 hr of storage, or when the product was approaching an advanced stage of spoilage. No significant difference was found in TMA-N content of the product stored at zero time, 6, 12, and 24 hr. These results show that TMA-N is Figure 5 also shows the VRS values of pasteurized crab cake mix stored at 30 C (86 F) for various lengths of time. It was observed that the VRS values increased progressively with an increase in storage time. The product showed VRS values of 25.85 ,uEq per g at zero storage time, which increased progressively with storage time. There was no significant difference in VRS values of samples stored for 0, 6, and 12 hr. VRS values increased significantly after 12 hr of storage at 86 F. The VRS value of the product at 24 hr of storage was not only significantly different from that at 12 hr of storage, but also significantly different from the product after 30 hr of storage. At 24 hr of storage, the product appeared to have a VRS value of 34.65 ,uEq per g and was still considered acceptable by the panel of judges. However, at 30 hr of storage, the VRS value of the product was 43.47 jAEq per g, and the product was considered unacceptable. The average odor scores for the product stored at 30 C (86 F) are also presented in Fig. 5. There was no significant difference between zero exposure and exposure to 30 C (86 F) for 6 hr. The product exposed for 12 hr at 30 C (86 F) showed no significant difference from that with zero exposure. The product exposed to 30 C for 24 hr was significantly different from any of the product exposed for shorter periods of time. This product, however, was also significantly different from the product exposed to 30 C for 30 hr which was judged unacceptable. This result indicates that the quality of pasteurized crab cake mix stored for 24 hr at room temperature was approaching the borderline stage of spoilage, although it was still considered acceptable. On the contrary, the product exposed to 30 C for 30 hr at room temperature was considered unacceptable. The borderline stage of spoilage, therefore, appeared to occur between 24 and 30 hr of storage time and was extrapolated to be approximately 27 hr at 30 C (86 F). A recovery test was employed to determine the efficiency of the colorimetric method used for detecting amount of ammonia (NH3) in the product. It was found that the determination of NH3 in the product was not reliable as a test for product quality, as the results were not reproducible. It was concluded that other substances in the product were probably extracted along with NH3 and interfered with the colorimetric determination. On the basis of the results given above, shelflife of the product was approximately 27 hr at 30 C (86 F), 4 days at 18 C (64 F), and 6 months at2C (36 F). DISCUSSION Bacterial counts have been used by many investigators to follow the deterioration of fish and shellfish. In this study, the product immediately after pasteurization had total plate counts of approximately 3.5 X 105 per g. When the product was stored at 30 C (86 F), the count increased to approximately 108 at the time of spoilage. The bacterial counts of the product stored at 30 C appeared to correlate with the degree of acceptability as indicated by odor. The bacterial counts, therefore, appeared to be a good index of spoilage of the product when stored at 30 C, as shown in Fig. 5. When the product was stored under refrigeration, the increase in bacterial counts showed no correlation with degree of spoilage. Figure 4 shows that approximately 107 bacteria per g were detected in the product held refrigerated [2 C (36 F)] for 6 months, when plates were incubated at 2 C. The count was typical for spoiled product exposed to room temperature, yet the product was still considered acceptable. These results indicate that the type of bacteria in the product is also important as an index of spoilage. The storage temperature of the product and the incubation temperature of the plates appeared to be significant when using bacterial count as an index of spoilage. A disadvantage of using plate count as an index of spoilage is the length of time involved in incubation. Bacteriological spoilage of the product is considerably retarded if the product is held refrigerated. Bacillus and Micrococcus showed optimal growth at 30 C (86 F). These bacteria are then expected to contribute very little to spoilage of the product at refrigerated storage temperatures. Alcaligenes, although showing optimal growth at 18 C (64 F), can grow well at refrigerated storage temperatures. Work done by Tobin et al. (26), Reay and Shewan (20), Campbell and Williams (9), and others indicated that the Pseudomonas-Achromobacter group of bacteria contributes significantly to the spoilage of most fishery products held at refrigerated storage temperatures. However, this group of bacteria was not found in pasteurized crab cake mix, probably as the consequence of severe heat treatment during pasteurization. This result agrees with that of Macaulay et al. (19), who reported that Alcaligenes had relatively higher heat resistance that the pseudomonads. No reports have been found in the literature indicating that Alcaligenes contributes significantly to the spoilage of fishery products. In this study, Alcaligenes was found to be abundant in pasteurized crab cake mix, but it did not appear to contribute significantly to the spoilage of the product at refrigerated storage temperatures [2 C (36 F)]. Probably for this reason, the product was considered acceptable after a 6month storage period. The TMA-N content in pasteurized crab cake mix was found not to be a sensitive index of spoilage of the product. The TMA-N content appeared to increase during the latter stages of spoilage. Early changes in quality of the product could not be detected by the TMA-N method. Determination of the VRS value of pasteurized crab cake mix can be carried out in less than 1 hr. The VRS values of the product were found to increase with length of the storage time. A close correlation was observed between the VRS values and organoleptic judgments. More significantly, quality changes in the product could be detected chemically by VRS values during the early stages of decomposition. VRS values, therefore, appear to provide a sensitive and reliable measure of the quality of pasteurized crab cake mix. The determination of ammonia in the crab cake mix product by a colorimetric method was not found reliable as an index of quality. APPL. MICROBIOL. Canned, pasteurized crab cake mix is prepared by mixing crab meat with other ingredients, some of which are heavily contaminated with microorganisms, such as the spices. The product is pasteurized in a water bath at 85 to 88 C (185 to 190 F) for 110 min. It is possible that Clostridium botulinum types A and B, which are common soil organisms, and types E and F, which have been isolated from fish and shellfish, may be present in the product and survive the pasteurization process. Moreover, the condition of the product in the can is favorable for growth of C. botulinum. As the product is a semisolid mass, anaerobic conditions could exist in it. C. botulinum type E is known to grow and produce toxin after 1 month of incubation at 38 F as reported by Schmidt et al. (21). To avoid the possibility of microbial growth and toxin production, the product would have to be stored at temperatures below 38 F. To prevent physicochemical changes caused by freezing, the storage temperature should be higher than the freezing temperature of any of the product components. Food storage temperatures in the range of about 28 to 38 F are rarely found in retail stores and homes. This presents a serious problem in marketing products like the one discussed here, and other similar meat and seafood products packed in hermetically sealed containers but not given a heat treatment that results in commercial sterilization.

v3-fos

2020-07-09T09:06:17.862Z

1970-01-01T00:00:00.000Z

228283937

s2

ANTIOXIDANT ACTIVITY IN BRASSICA NAPUS L. PLANTS EXPRESSING LOX-DEPENDENT BAR GENE © SAKHNO L.O., LYSTVAN K.V. Production of reactive oxygen species, including free radicals, is an integral part of plant metabolism. Antioxidant activity of plant tissues characterizes an ability to overcome excessive amounts of compounds such as superoxide and hydroxyl radicals, singlet oxygen, and hydrogen peroxide. It is provided by various antioxidant systems. There are enzymes (superoxide dismutase (SOD), peroxidase, catalase) and non-enzyme (proline, glutathione, ascorbic acid, fatty acids, polyphenols (tocopherols, anthocyanins, flavonoids, aromatic oxyacids)) antioxidants [1]. An increase in plant antioxidant activity influences positively on both the ability to grow faster as well as resist stresses of different origins [2–3]. Antioxidant activity has been intensively studying in crop species [4–6] and medicinal plants [7–8]. Previously we produced transgenic canola plants using our own Agrobacterium tumefaciensmediated protocol and pICH 3744 vector which was kindly provided by Icon Genetics (Halle, Germany) [9]. The feature of this genetic construction was a location of the coding sequence for BAR gene without its own promoter near the right border of TDNA and between two lox A and M sites which were the elements of Cre/lox recombination system of P1 phage. The BAR gene from Streptomyces hygroscopicus confers resistance to Bayer’s herbicides with phosphinothricin (РРТ, glufosinate) as active ingredient. These herbicides inhibit glutamine synthase [10] that plays a key role in assimilation of nitrogen and regulation of its metabolism in plants. SOD activity, as a measure of antioxidant activity, has been determined in seeds of commercial herbicide-resistant varieties of canola and maize and compared to activity of the untransformed progenitors [11]. We studied SOD activity in leaf tissue of transgenic canola plants expressing the lox-dependent BAR gene [12] in non-stress conditions. This parameter, however, has not yet been explored under stress (herbicide application). In addition, data concerning total radical scavenging activity in herbicide-resistant plants are absent. Investigation of these characteristics assist in determining if there are unintended biochemical changes in transgenic canola plants in comparison with untransformed canola plants. Production of reactive oxygen species, including free radicals, is an integral part of plant metabolism. Antioxidant activity of plant tissues characterizes an ability to overcome excessive amounts of compounds such as superoxide and hydroxyl radicals, singlet oxygen, and hydrogen peroxide. It is provided by various antioxidant systems. There are enzymes (superoxide dismutase (SOD), peroxidase, catalase) and non-enzyme (proline, glutathione, ascorbic acid, fatty acids, polyphenols (tocopherols, anthocyanins, flavonoids, aromatic oxyacids)) antioxidants [1]. An increase in plant antioxidant activity influences positively on both the ability to grow faster as well as resist stresses of different origins [2][3]. Antioxidant activity has been intensively studying in crop species [4][5][6] and medicinal plants [7][8]. Previously we produced transgenic canola plants using our own Agrobacterium tumefaciensmediated protocol and pICH 3744 vector which was kindly provided by Icon Genetics (Halle, Germany) [9]. The feature of this genetic construction was a location of the coding sequence for BAR gene without its own promoter near the right border of T-DNA and between two lox A and M sites which were the elements of Cre/lox recombination system of P1 phage. The BAR gene from Streptomyces hygroscopicus confers resistance to Bayer's herbicides with phosphinothricin (РРТ, glufosinate) as active ingredient. These herbicides inhibit glutamine synthase [10] that plays a key role in assimilation of nitrogen and regulation of its metabolism in plants. SOD activity, as a measure of antioxidant activity, has been determined in seeds of commercial herbicide-resistant varieties of canola and maize and compared to activity of the untransformed progenitors [11]. We studied SOD activity in leaf tissue of transgenic canola plants expressing the lox-dependent BAR gene [12] in non-stress conditions. This parameter, however, has not yet been explored under stress (herbicide application). In addition, data concerning total radical scavenging activity in herbicide-resistant plants are absent. Investigation of these characteristics assist in determining if there are unintended biochemical changes in transgenic canola plants in comparison with untransformed canola plants. Plant material and growth conditions. Spring canola plants cv Kalinovskii were used as the controls for transgenic ones (lines T 3 5/44/1 and T 3 5/44/2). Analyzed transformants were homozygous plants from the third generation. They were produced after self-pollination of primary lines and chosen as ones demonstrating the highest resistance to Basta herbicide treatment under greenhouse conditions among seven tested lines from the T 1 generation. Plants were grown on agar-solidified MS medium without hormones [13] in Magenta TM Boxes under in vitro conditions (+23 С, 4000 -5000 lux, 14 (light) /10 (dark) h). The upper buds were also cultured on MS media supplemented with phosphinothricin (10 mg/L). Sterile solution of herbicide was added to the media after autoclaving. After four weeks of growth the fresh weight (FW), total soluble protein content, total radical scavenging activity, and SOD activity were measured. Fresh leaves (100 mg) was ground with 1 mL of 50 mM Tris-HCl buffer (pH 8.0) in a mixer mill Retsch MM 400 (Germany) with a vibration frequency of 25 Hz for 3 min and then centrifuged at 13000 g (4°С) for 15 min. The supernatant was used for the analyses. Determination of total soluble protein (TSP) content. TSP was determined using Bradford's method [14] with bovine serum albumin (Fermentas, Lithuania) as a standard. The optical density of the reaction mixture was detected at 595 nm by BioPhotomether Eppendorf, v.1.35 (Germany). SOD activity assay. SOD activity was measured using a method of nitroblue tetrazolium photo-chemical oxidation [15] with slight modifications [12]. The absorbance by the reaction mixture in illuminated probes was read at 550 nm by BioPhotomether Eppendorf (Germany) versus the ones of dark probes. Statistical analysis. The experiments were repeated three times independently, and each data point was the mean of three replicates. The mean, standard deviation, confidence interval and Student's t-test for statistical significance were calculated using Microsoft ® Office Excel 2003 (Microsoft Corporation) standard functions. Results and discussion We have analyzed some growth and biochemical parameters of transgenic canola plants expressing the lox-dependent BAR gene. Despite the introduction of the BAR gene without its own promoter, transformed canola shoots were regenerated and selected on media containing phosphinothricin [9]. It had been supposed that expression of the target gene occurred due to its special location in the vector, namely near the right border of T-DNA and between two lox sites [18]. Transgenic T 3 5/44/1 and T 3 5/44/2 lines and untransformed control plants produced fresh weight without differences when they were cultivated on media without phosphinothricin (Fig. 1 A). The upper buds of control plants had no ability to photosynthesis, bleached, formed no roots, and died under herbicide pressure but transgenic lines formed roots and new green leaves. Transgenic plants produced similar fresh weight on media both with phosphinothricin and without this herbicide (Fig. 1 A). Similar results were obtained for N. tabacum [19][20], tomato (Lycopersicon esculentum) and potato (Solanum tuberosum) [19] that expressed the BAR gene introduced via other vectors. Fresh shoot weight exhibited no significant difference in transformed Lotus corniculatus shoots expressing BAR gene when they were grown in vitro before and after Basta application [21]. Leaves of 28-day-old plants were used for biochemical analyses. TSP content was similar in plants tested under normal growth conditions (Fig. 1B). No significant changes were detected in transgenic plants under growth on the media with PPT as well as after cultivation without it. SOD activity exhibited no significant differences in leaves of control (25.2±1.5 U mg -1 protein) and transgenic canola plants (24.8±1.2 and 26.0±0.9 U mg -1 protein for T 3 5/44/1 and T 3 5/44/2, respectively) in growth on MS medium. Furthermore, the changes in SOD activity were not detected in transgenic plants grown with PPT or in its absence ( Fig. 2A). When SOD activity was tested in commercial canola seeds, an decrease in SOD activity by 25 % was shown for plants of MS1/RF1 event and no significant changes were detected for ones of MS8/RF3 event [11]. Both events have the BAR gene in their nuclear genomes. Most transgenic plants including ones expressing heterologous SOD genes possess SOD activity similar to initial plants in non-stress conditions [22]. Sometimes it was lower as in the case of FeSOD overproducing tobacco plants which showed approximately one-half of the endogenous FeSOD activity that was found in nontransgenic plants [23]. Increase in SOD activity due to heterologous gene expression may reach up to 5-fold but the transgenic canola plants failed to survive past the first generation [24]. There are two groups of transgenic canola plants obtained in our experiments characterized by elevated SOD activity under normal growth conditions [12]. They expressed heterologous human interferon alpha 2b gene (HuInf-α2b) and CYP11A1 gene for cytochrome P450 SCC from bovine adrenal cortex mitochondria. Improved tolerance to osmotic and heat stresses for these plants in comparison with untransformed ones has been demonstrated [25][26]. The results of the DPPH assay revealed no significant differences between the control and the BAR gene canola plants cultivated without herbicide addition nor transgenic lines grown on media with or without PPT (Fig. 2 B). Antioxidant activity of canola leaves measured as total radical scavenging activity is higher than most other crops [27]. B. napus plants possessing antioxidant activity up to 4-fold higher than in original controls have been obtained as a result of introduction РАР1 (Production of Anthocyanin Pigment 1) gene from Arabidopsis thaliana into the canola nuclear genome [28]. These transgenic plants exhibited purple leaves. Differences in their coloration depended on changes in anthocyanin synthesis induced by expression of the heterologous РАР1 gene. The BAR gene was used as the selective marker in the vector bearing the РАР1 gene. It had no influence on antioxidant activity in the obtained transgenic canola plants. In our experiments expression of lox-dependent BAR gene in canola plants produced no significant changes in total radical scavenging activity in comparison with untransformed plants under normal growth conditions. In addition, simi-lar results from the DPPH assay were detected in transgenic plants grown on media with or without phosphinothricin. Conclusions We have found no significant differences in antioxidant activity from leaf extracts of untransformed canola plants under in vitro growth conditions in comparison with ones of phosphinothricinresistant plants of the T 3 generation. This lack of effect was found for total radical scavenging activity measured by DPPH-assay and SOD activity evaluated photometrically using a nitroblue tetrazolium assay. No significant changes in parameters investigated were observed in transgenic plants cultivated on media with herbicide addition compared to ones grown on media without herbicide. Fresh weight and total soluble protein content were similar in transgenic and untransformed canola plants under growth without phosphinothricin. Thus, loxdependent BAR gene introduction and expression resulted in no significant differences in leaf antioxidant activity in transgenic canola plants comparing to untransformed controls. Authors appreciate Dr. Valeriia B. Belokurova (Institute of Cell Biology and Genetic Engineering National Academy of Sciences of Ukraine, Kyiv) for improving English in the manuscript.

v3-fos

2020-12-10T09:04:11.454Z

1970-12-01T00:00:00.000Z

237232519

s2

Amino Acid-Induced Inhibition and Stimulation of Saccharomyces carlsbergensis The response pattern of Saccharomyces carlsbergensis (ATCC 9080) to pantothenic acid in Atkin's medium was changed dramatically by adding small amounts of casein hydrolysate (0.032 to 0.32 mg/ml) to the assay medium. Under static, mildly anaerobic conditions, growth at low pantothenic acid levels was reduced by 54 to 69%, whereas at saturating or near saturating pantothenate concentrations marked stimulation of growth (up to 41%) was observed. Under aerobic conditions, inhibition but not stimulation of growth occurred. It is recommended that Atkin's medium for the assay of pantothenic acid with S. carlsbergensis (ATCC 9080) be modified to include 0.6% acid-hydrolyzed casein (Vitamin Free Casamino Acids, Difco) to prevent erroneous growth responses, which may result if significant amounts of amino acids are present in natural materials being assayed for this vitamin. During recent studies on pantothenic acid transport, it was deemed necessary to assay cellular extracts of Lactobacillus plantarum for free pantothenate. The assay method of Atkin et al. (1) with Saccharomyces carlsbergenesis (ATCC 9080) was employed since this organism responds only to free pantothenic acid (2)(3)(4)6). It was considered very likely that the extracts to be assayed would contain significant quantities of various amino acids since the lactobacilli had been grown in a medium high in amino acid content. Previously, Hertz found that the maximum growth of S. cerevisiae was markedly stimulated by amino acids in Snell's biotin assay medium (5,7). Therefore, the effects of various concentrations of amino acids (added as Vitamin Free Casamino Acids, Difco) on the growth response of S. carlsbergensis in Atkin's pantothenic acid assay medium were studied. The results in this paper demonstrate that small amounts of casein hydrolysate alter the growth response of the organism such that erroneous pantothenic acid values could easily be obtained. Medium. The medium of Atkin et al. (1) was the basic medium used in all of these studies. Preparation of inoculum. Inoculum cells were grown overnight at 32 C in Atkin's medium containing 10-1 jug of pantothenic acid per tube (6 ml). The cells were harvested by centrifugation, washed three times in about 5 ml of sterile distilled water, and resuspended to 0.6 to 0.8 mg (dry weight) per ml. Assay procedure. Most assays and experiments were carried out in Pyrex tubes (18 by 150 mm). Pantothenic acid levels of 0, 2, 4, 6, 8, 10, 20, 30, 50, and 100 X 10-2 jg per tube were used in each assay except where indicated. Water was added to each tube to bring the volume to 3.0 ml. Then, to each tube was added 3.0 ml of nonsterile, double-strength Atkin's medium supplemented with various amounts of Casamino Acids (Difco) such that Casamino Acid concentrations of 0, 0.005, 0.05, 0.1, 0.5, 0.6, 0.8, 1.0, and 2.0% resulted after dilution with the pantothenate solutions. The tubes were covered with stainless-steel caps (Bellco Glass, Inc., Vineland, N.J.), shaken to mix the contents, heated for 10 min in flowing steam, cooled, inoculated with one drop per tube of the inoculum suspension described above, shaken to mix the inoculum, and incubated in a water bath at 32 C for 20 to 24 hr at which time near maximum growth was obtained. The tubes were then cooled in ice water to stop growth, and the turbidities were determined in a Klett-Summerson photoelectric colorimeter fitted with a red (no. 66) filter and adjusted to zero on a medium blank. In some experiments, when the cells were grown under highly aerobic conditions, the assay vessels were 25-ml Erlenmeyer flasks. During these experiments, the flasks containing 10 ml of medium were shaken at 180 rev/min in a controlled environment incubatorshaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 32 C. Temperature variation was 0.5 C. Chemicals. Calcium pantothenate was obtained from Sigma Chemical Co., St. Louis, Mo.; casein hydrolysate (Vitamin Free Casamino Acids) was from Difco Laboratories, Detroit, Mich.; and other chemicals required to make the different media were from Mallinckrodt Chemical Works, St. Louis, Mo., Sigma, or Difco. Nitrogen was determined by nesslerization as described in Manometric Techniques (8). RESULTS Effect of Casamino Acids on growth of S. carlsbergensis at various pantothenic acid levels. From Fig. 1A it can be seen that the presence of Casamino Acids mediated two distinct effects on the response of S. carlsbergensis (ATCC 9080) to pantothenic acid. At low pantothenate concentrations, growth was dramatically depressed (54% with 0.005 % and 60% with 0.05% at 6 X 10-2, and 63% with 0.05% at 10-1); however, at higher concentrations, those adequate to satisfy most or all of the organism's pantothenic acid requirement, marked stimulation of growth occurred (26 and 41% with 0.1 % at 2 X 10-1 and 5 x 10-1, respectively). The data represented in Fig. lB show that increasing the content of Casamino Acids from 0.1 to 0.6% modified the growth depression somewhat but did not significantly affect growth stimulation. Further increases up to 1% exhibited little additional effect on growth (Fig. 1C). Since pantothenic acid is somewhat heat-labile, the possibility existed that casein hydrolysate mediated some pantothenic acid destruction during the heating process and that this effect might be more evident at low concentrations. Accordingly, pantothenic acid concentrations of 10 , 5 x 10-s, and 10-1 ,g were heated in flowing steam for 20 min in 5 ml of Atkin's medium and in Atkin's medium supplemented with 0.1 and 0.5% Casamino Acids. The media were cooled and assayed, without further heating, for pantothenic acid by using the L. plantarum (ATCC 8014) assay method (2). Results of this assay demonstrated that there was no destruction of pantothenic acid during heating in Atkin's medium with or without supplementation with Casamino Acids. Thus, the decreased growth of S. carisbergensis in the presence of casein hydrolysate was due to some effect on the metabolism of the yeast and not on pantothenic acid itself. Influence of aeration on the Casamino Acids effect on the growth of S. carisbergensis. Since S. carisbergensis grows better under highly aerobic conditions and since most pantothenic assay pro- cedures with this organism call for aerobic conditions, the effect of aeration on the Casamino Acids-induced growth effects were investigated (Table 1). Marked depression of growth still occurred at low concentrations of Casamino Acids (0.05%), but the stimulatory effect was eliminated under these conditions. Also, at 0.5% Casamino Acids, marked stimulation of growth occurred at pantothenate concentrations (6 X 10 and 10-1) which had exhibited inhibition under both aerobic Ten-ml amounts of Casamino Acids-supplemented Atkin's medium containing the indicated pantothenic acid were shaken in 25-ml Erlenmeyer flasks at 180 rev/min for 20 hr at 32 C. Inoculum was 0.15 mg of cells (dry weight) per flask. b Klett readings. ( Table 1; 0 and 0.05% Casamino Acids) and static conditions ( Table 2; 0, 0.05, and 0.5% Casamino Acids, respectively). Comparison of turbidity readings and nitrogen content. While reading turbidities during the course of these experiments, it was noticed that yeast cells growing at low pantothenic acid concentrations, especially in the presence of Casamino Acids, formed large particulate masses consisting of many cells aggregated or otherwise joined together. Therefore, it was reasoned that since the cells were clumped together and not evenly dispersed in the medium, these turbidity readings might be considerably lower than they should be, based on the amount of growth actually present as measured by cellular nitrogen or cell mass. Accordingly, cells were grown under a variety of nutritional conditions both aerobically and in stationary tubes. Growth was measured turbidimetrically and by dry weight and nitrogen content of washed cell suspension. Comparison of the results obtained from one such experiment (Table 2) shows that the same pattern of inhibition and stimulation was evident from either nitrogen content or turbidity values. Although only nitrogen versus turbidity under static conditions is reported in Table 2, dry weight determinations followed the same pattern, and both dry weight and nitrogen content of aerobically grown cells reflected the turbidity observations reported in Table 1. Thus, the phenomena observed by turbidimetric determinations were true reflections of the growth of this yeast under the experimental conditions applied. DISCUSSION The results presented in this paper suggest the need for modification in the medium used to assay pantothenic acid with S. carlsbergensis (ATCC 9080). As little as 0.3 mg of Vitamin Free Casamino Acids (Difco, 0.005%) per tube (6 ml) markedly affects the growth response of this yeast to pantothenate, and 3 mg (0.05%) per tube results in a dramatic bimodal inhibition and stimulation of growth (Fig. 1A). In fact, when one considers that Casamino Acids is 38% salt, the actual content of amino acid mixture per tube is reduced to 0.186 and 1.86 mg, respectively. Thus, it is quite probable that many natural materials contain quantities of amino acids sufficient to affect the pantothenic acid response of the organism and lead to erroneous results. Incorporation of 0.6% vitamin free acid-hydrolyzed casein eliminates the probability of error from amino acids that may be present in natural material, since further increases in concentration of the amino acid mixture up to 1% have no significant effect on the response of the assay organism at any assayable pantothenic acid level. Preliminary results indicate that microgram quantities of lysine and methionine are the primary amino acids responsible for growth inhibition, that methionine also stimulates growth at higher pantothenate levels, and that apsartic and glutamic acids primarily cause the marked stimulation of growth at high or low pantothenate levels. The individual amino acid effects, their relationships to pantothenic acid metabolism, and their modes of inhibition and stimulation presently are being investigated as are the morphological changes induced by pantothenic acid deficiency. ACKNOWLEDGMENTS I thank Ralph Germinario for assistance rendered during the early phases of this study.

v3-fos

2018-04-03T01:12:58.840Z

1970-10-01T00:00:00.000Z

28530720

s2

Differentiation of Brucella canis from Other Brucellae by Gas Chromatography Gas chromatographic techniques allow for differentiation between a strain of Brucella canis and strains of other brucellae. A gram-negative coccobacillary bacterium iso- lated from placental and fetal tissues of an aborted beagle pup was characterized as a new species of Brucella, and the name Brucella canis was proposed for this previously unrecognized bacterium (1). The present study was designed to determine whether gas chromatographic techniques may be employed to distinguish between a strain of B. canis and isolates of other Brucella 1330, dog abortion RM 66 (designated herein as B. canis) obtained from the amniotic fluid of an aborted beagle fetus, and Bordetella bronchiseptica were grown at 37 C on slants containing 1.5% tryptone, 1.5% yeast extract, 0.5% glucose, 0.5% K2PHO4, and 1.5% agar (TYG medium). After 24 hr, the cells were washed from the agar with 5.0 ml of distilled water, the bacterial suspension was diluted, and 0.10 ml containing 25 X 103 to 250 X 103 cells was transferred into a screw-capped test tube containing 5.0 ml of TYG broth. After 24 hr of incubation at 37 C, replicates were examined microscopically for counts, and the cultures and samples of uninoculated medium were treated with 0.10 ml of 5 N HCI and 1.0 ml of 0.2 M HCI-KCl The samples were then centrifuged A gram-negative coccobacillary bacterium isolated from placental and fetal tissues of an aborted beagle pup was characterized as a new species of Brucella, and the name Brucella canis was proposed for this previously unrecognized bacterium (1). The present study was designed to determine whether gas chromatographic techniques may be employed to distinguish between a strain of B. canis and isolates of other Brucella species. B. abortus 544, B. melitensis 16 M, B. ovis REO-1182, B. suis 1330, dog abortion agent RM 6 66 (designated herein as B. canis) obtained from the amniotic fluid of an aborted beagle fetus, and Bordetella bronchiseptica were grown at 37 C on slants containing 1.5% tryptone, 1.5% yeast extract, 0.5% glucose, 0.5% K2PHO4, and 1.5% agar (TYG medium). After 24 hr, the cells were washed from the agar with 5.0 ml of distilled water, the bacterial suspension was diluted, and 0.10 ml containing 25 X 103 to 250 X 103 cells was transferred into a screw-capped test tube containing 5.0 ml of TYG broth. After 24 hr of incubation at 37 C, replicates were examined microscopically for counts, and the cultures and samples of uninoculated medium were treated with 0.10 ml of 5 N HCI and 1.0 ml of 0.2 M HCI-KCl buffer (pH 2.0). The samples were then centrifuged at 3,000 X g, and the supernatant fluid was extracted three times with 10 ml of ether. The extracts were combined, concentrated to 0.5 ml, and dried with anhydrous Na2SO4. A 3.0-,uliter sample was injected into the gas chromatograph. The chromatographic techniques were essentially the same as described previously (2). The differentiating peaks of these strains of Brucella and Bordetella are shown in Table 1. With the electron capture detector to record the presence of microbial metabolites, the sensitivity of detection of the bacteria ranged from 42 to 490 organisms per 10 mm2 peak area. The sensi-tivity was calculated from the number of cells in the 24-hr sample which was injected into the chromatograph and the peak area of the product, the value given for sensitivity being the estimated number of bacteria to yield a peak area of 10 mm2. Each Brucella strain exhibited 10 to 15 peaks in chromatograms prepared from the spent culture media. Peaks having retention times of 25, 35, 45, 55, 60, 75, 85, 90, 390, and 940 sec were common to most of the organisms. However, as shown in Table 1, at least one compound was present in cultures of each Brucella strain which was not produced by any other organism tested. These metabolites were not found in the uninoculated medium, and the area of these ranged from 500 to 1,710 mm2. A signature for each Brucella strain was established by assigning letters to peaks in the chromatogram in order of their increasing retention times. By means of the signatures obtained, the strains examined could be characterized readily. When products elaborated by B. canis were compared with those of the other bacteria investigated, the dog pathogen was found to differ from APPL. MICROBIOL. bronchiseptica by eight, nine, three, eight, and four products, respectively. Some similarity in the kinds of substances excreted was evident, therefore, between B. canis and B. suis as well as B. bronchiseptica. On the other hand, most of the products formed by B. canis as well as the other brucellae were quite distinct from those synthesized by strains of Bacillus, Clostridium, and Staphylococcus. The results thus indicate that B. canis excreted metabolites similar to those generated by strains of other Brucella species. However, the brucellae investigated, although admittedly few in number, can be readily differentiated from one another by considering the presence or absence of individual compounds detectable by gas chromatography.

v3-fos

2018-04-03T01:31:22.283Z

1970-04-01T00:00:00.000Z

29909559

s2

Genetic and phenotypic relationships between ovulation rate and body weight in the mouse The genetic and phenotypic regressions and correlations between ovulation rate and body weight were examined in a random bred strain (Q) of laboratory mice during the course of three experiments. These experiments were (1) a sib analysis; (2) selection for natural and induced primiparous ovulation rate; and (3) replicated selection for 6-week weight. The following results were obtained: INTRODUCTION The complex relationships between body weight, litter size and fitness in the mouse have been the subject of many discussions. Both body weight and litter size can be readily changed by selection, reported first by Goodale (1938) and Falconer (1960 a) respectively, and additive genetic variation in these two traits has invariably been found in outbred populations of mice. In addition to demonstrating the presence of genetic variation, the results of selection experiments 172 R. B. LAND have also indicated that the two traits are not under independent genetic control, but have led to conflicting conclusions about the nature of the relationship between them. On the one hand, selection for body weight has always led to changes in litter size in the same direction as that of selection, but on the other, when the change in body weight was examined following selection for litter size (Falconer, 1960 a), it was found that body weight had increased following selection for both high and low litter size. However, when litter size is considered in terms of its components-ovulation rate and embryonic mortality-a much clearer picture emerges. Whenever body weight has been changed by selection, either directly or as a correlated response, ovulation rate has been found to change in the same direction. This relationship was first reported many years ago (MacArthur, 1944) but the magnitude of the correlation has not been estimated. The first object of the work described here was to estimate the magnitude of the genetic correlation between ovulation rate and body weight in an outbred strain of mice. This was done in two ways, by estimating the components of variance and covariance in a half-sib analysis, and by examining the changes in body weight following two-way selection for ovulation rate and vice versa. Ovulation rate can not only be considered as a component of litter size, but may also be considered in terms of its own components-the activity of circulating follicle-stimulating hormone (FSH), and the sensitivity of the ovary to this hormone. When the correlated changes in ovulation rate have been analysed in terms of these components, it has been found that when the change has followed selection for body weight, most of the change in ovulation rate has been due to changes in FSH activity (Fowler & Edwards, 1960;Edwards, 1962), whereas changes in ovulation rate following selection for litter size were mainly due to ovarian sensitivity (McLaren, 1962)-even though these changes were also accompanied by changes in body weight. The second object of the present study was to compare the genetic correlation between ovulation rate and body weight with that between ovarian sensitivity and body weight. Ovarian sensitivity was measured as the number of eggs shed in response to exogenous gonadotrophins (the induced ovulation rate); and the genetic correlation between body weight and ovarian sensitivity was estimated by recording the changes in body weight following two way selection for induced ovulation rate. The phenotypic correlation between body weight and ovulation rate was also examined in the mice used for the half-sib analysis and at different stages during the selection programmes. MATERIALS AND METHODS This investigation was made possible by the use of mice from experiments which were primarily designed for other purposes. These were: (1) a study of growth (Monteiro & Falconer, 1966); (2) a study of ovulation rate (Land & Falconer, 1969); and (3) a further study of growth (Falconer, unpublished). The first incorporated a sib analysis, the second two way selection for both natural and induced ovulation rate, and the third replicated two way selection for body weight. These experiments are described in more detail below. The stock of mice used for all experiments was the genetically heterogeneous Q-strain, the background of which is described by Land & Falconer (1969). Experiment 1. Sib analysis Seventy-one males were each mated to three females, providing groups of full and half-sib offspring. Females from each group were obtained and their natural ovulation rate scored when they were between 6 and 8 weeks of age (Land & Falconer, 1969). All females were weighed at 6 weeks of age, and on the day that their ovulation rate was scored. The components of variance of ovulation rate and body weight and the covariance between them were calculated, and the results are given in Table 1. Experiment 2. Selection for ovulation rate Four selected lines were established, and maintained together with an unselected control line (Land & Falconer, 1969). Each line was maintained by eight pair matings with minimal inbreeding. Natural ovulation rate was scored as the 1144 * A = additive genetic; Ew = environmental within full-sib families; Ec = environmental common to full-sibs; D = dominance. Variance and covariance due to epistatic interactions is assumed to be negligible. number of eggs shed at natural oestrus. Induced ovulation rate was scored as the number of eggs shed in response to treatment with 4 i.u. pregnant mares' serum (PMS) at 17.00 h on the day after weaning, followed by 3 i.u. human chorionic gonadotrophin (HCG) at 12.00 h 2 days later. The hormones used to induce ovulation were 'Gestyl' (Organon Ltd.) PMS and 'Pregnyl' (Organon Ltd.) HCG. The ovulation rate was scored after the females had had their first litter of young weaned from them. The ovulation rate is therefore that of primiparous females, and in this respect differed from the ovulation rate scored in the sib analysis which was that of virgin, or nulliparous, females. The mean ovulation rate and body weight of each line was estimated each generation as the mean of family means. The body weight of each female was recorded at 3 and 6 weeks of age, and at the time of scoring; these are presented graphically in Fig. 1. It can be seen that there is very little difference between the mean 3-week weights of any of the lines. Differences can, however, be seen between the mean weights of the 5 lines at 6 weeks of age and at the time of scoring, the differences at 6 weeks being in the same direction but smaller than those at the time of scoring. The divergence in body weight at 6 weeks of age and at the time of scoring between the high and low natural and the high and low induced lines are illustrated in Fig. 2, together with the divergence in ovulation rate. The correlated response of 6-week and scoring weight after twelve generations of selection were estimated from the regression of the response on the selection differential in eggs to be 2-79 and 5-39 g respectively for the natural lines, and 2-90 and 5-20 g respectively for the induced lines. The results of this experiment which are relevent to the calculation of the genetic and phenotypic relationships between body weight and ovulation rate are summarized in Table 2. The divergence in body weight (• •) at 6 weeks of age and at the time of scoring between the lines selected for high and low natural ovulation rate and high and low induced ovulation rate; plotted against the cumulative selection differential in eggs. The regressions of the divergence in body weight on cumulative selection differential ( ) and the actual divergences in eggs ( --) are also given. Experiment 3. Selection for body weight A replicated body weight selection experiment was started by Professor D. S. Falconer in 1963. Selection was applied to body weight at 6 weeks of age, the heaviest individuals were selected in the large lines, the lightest in the small lines. Each pair of lines, together with their control was replicated six times, making 18 lines in all. Selection was carried out within families and each line was maintained by eight matings in each generation, with minimal inbreeding. This experiment is still in progress, and the parameters of body weight quoted in this paper are the results of intermediate calculations generously provided by Professor Falconer. 1, data given in the present paper; 2, Land & Falconer (1969). The ovulation rates of females discarded from all 18 lines were scored after five generations of selection. The animals from the selection lines were of course selected from the total number of available animals on the basis of their phenotypic body weight. The mean ovulation rate for each line Avas therefore corrected to that which it would have been if all individuals had been scored. This was done as follows: the regression of ovulation rate on body weight was first calculated for each line; analysis of covariance indicated that the regression coefficients did not differ significantly within the control or small lines, but that significant differences were present between the six large lines (P<0-05). The individual regression coefficients were therefore used to correct the ovulation rates of the large lines, the mean for all lines being the mean of corrected means. In the control and small lines the best estimate of the regression coefficient within each set of lines was used to correct the mean of uncorrected means. Variation between replicates has been ignored for the purpose of the genetic analyses, and the direct and correlated responses were assessed in terms of the divergence between the means of the high and low lines. Each line was given equal weighting. The mean ovulation rate, 6-week weight before and after selection and the regression of body weight on 6-week weight in each of the lines are given https://doi.org/10.1017/S0016672300001506 Published online by Cambridge University Press in Table 3, together with the corrected ovulation rates. The divergence of body weight was 6-0 g and the corrected divergence of ovulation rate 2-9 eggs. In addition to the results given above, the square root of the within family heritability of 6-week weight and the within family additive genetic standard deviation of 6-week weight were also needed. These were calculated to be 0-602 and 1-163 g respectively. The genetic correlations and regressions presented in the next section were calculated from the results of these experiments by the methods described by Falconer (19606). 13-3 120 * The differences between individual regressions were statistically significant (P<0-05). RESULTS (1) The genetic correlation between body weight and ovulation rate All individuals were killed at the time of scoring, and consequently although the body weight of an individual could be recorded several times during its life, the ovulation rate of that individual could only be recorded once. It is therefore possible to examine the relationship between ovulation rate and body weight at various times up to and including the time of scoring. The genetic correlation between the natural ovulation rate of nulliparous females at 8 weeks of age and their body weight at the time of scoring could only be calculated from the sib analysis (Expt 1), and was found to be 0-33 + 0-58. The correlation between the same ovulation rate and body weight at 6 weeks of age can, however, be calculated from both Expt 1 and the body weight selection experiment (Expt 3). These experiments give estimates of 0-45 + 0-59 and 0-87 respectively. Unfortunately it is not possible to calculate the standard error of the realized genetic correlation, and hence there is no logical way of pooling these estimates. In addition to the results obtained directly from the ovulation rate selection experiment (Expt 2) summarized in Table 2, the genetic correlation between primiparous ovulation rate and body weight at the time of scoring is also dependent upon the heritability of body weight at the time of scoring, which has not R. B. LAND been estimated. Monteiro & Falconer (1966), however, observed that there is little variation in the heritability of body weight between 6 and 8 weeks of age, and consequently the heritability of primiparous scoring weight has been assumed to be the same as that of 6-week weight. The genetic correlations between natural primiparous ovulation rate and body weight at the time of scoring and at 6 weeks of age were calculated and found to be 0-60 and 0-42 respectively. These estimates suggest that the genetic correlation between body weight and ovulation rate may decline either with parity, or with age at the time of scoring. In general, however, it is possible to conclude from this section that the genetic correlation between body weight and natural ovulation rate is positive, and is probably greater than 0-4. The correlation between ovulation rate and body weight within full sib families was calculated from the sib analysis to be 0-86 and 0-85 at 6 weeks and at the time of scoring, compared to 0-45 and 0-33 for the estimates of the additive genetic correlations. The higher estimates within full sib families could be due to nonadditive genetic causes, but are much more likely to be due to the effect of the common environment within full sib families (i.e. maternal effects). The genetic correlations between induced primiparous ovulation rate and body weight were calculated to be 0-78 and 0-58 at the time of scoring and 6 weeks of age respectively. The genetic correlation between body weight and ovarian sensitivity may therefore be concluded to be positive, and of a similar order of magnitude to that between body weight and ovulation rate itself. (2) The genetic regression of ovulation rate on body weight Just as it is possible to calculate the genetic correlation between body weight and ovulation rate from the results of these experiments it is also possible to calculate the genetic change in ovulation rate which would be expected to follow, or which has followed, a unit genetic change in body weight, that is the genetic regression of ovulation rate on body weight. The genetic regression of natural nulliparous ovulation rate on body weight at 6 weeks of age and at the time of scoring was calculated from the genetic correlation multiplied by the ratio of the additive genetic standard deviations (i.e. r Ao.w x O'AO/^AW)-The regression was estimated from the results of Expt 1 to be 0-45 eggs per gram at 6 weeks of age, and 0-26 eggs per gram at the time of scoring. The realized genetic regression of natural nulliparous ovulation rate of 6-week weight was calculated from Expt 3 to be 0-48 eggs per gram, which is very close to that predicted above. Similar parameters were estimated from Expt 2 for natural and induced primiparous ovulation rate. The genetic regressions of primiparous natural ovulation rate on body weight at 6 weeks of age and at the time of scoring were estimated to be 0-34 and 0-35 eggs per gram respectively. Likewise, the genetic regressions of primiparous induced ovulation rate on body weight at 6 weeks of age and at the time of scoring were estimated to be 0-93 and 0-91 eggs per gram respectively. (3) The genetic regression of body weight on ovulation rate The genetic regression of body weight on ovulation rate is an estimate of the genetic change in body weight which would be expected from a unit genetic change in ovulation rate. As in the case of the previous two parameters its magnitude is dependent upon the additive genetic variances of each of the two characters and the genetic covariance between them. Expt 1 yielded estimates of 0-45 and 0-42 g per egg for the regression of body weight at 6 weeks and at the time of scoring on natural nulliparous ovulation rate. The corresponding estimate from the body weight selection experiment for 6-week weight was 0-53 g per egg. Again, the agreement between these two experiments is very close. The genetic regression of body weight on primiparous ovulation rate was obtained from Expt 2, by multiplying the regression of ovulation rate on body weight by the ratio of the additive variances (6 Aw 0 = 6 Aow x <T AW /(T AO ) . The regressions of body weight at 6 weeks of age and at the time of scoring on natural primiparous ovulation rate were 0-40 and 0-78 g per egg respectively, which are of the same order of magnitude as those for nulliparous ovulation rate. The corresponding regressions for induced primiparous ovulation rate were 0-18 and 0-33 respectively, the low values being a reflexion of the high variation in induced ovulation rate. (4) The phenotypic relationships between body weight and ovulation rate The phenotypic correlations between natural nulliparous ovulation rate and body weight at 6 weeks of age and at the time of scoring were both estimated from Expt 1 to be 0-46. The regression of ovulation rate on body weight at 6 weeks of age and at the time of scoring being 0-44 and 0-40 eggs per gram respectively. All relationships greater than zero are significantly different from zero (P<0-05). The corresponding parameters were calculated from Expt 2 and are summarized in Table 4. The regression of the number of eggs shed at natural oestrus on body weight is 0-4 eggs per gram, which is similar to that for nulliparous females. In addition, the correlation between body weight and natural primiparous ovulation 180 R. B. LAND rate (0-3 to 0-4) is similar to that for nulliparous females. The correlations between primiparous induced ovulation rate and body weight and the regressions of induced ovulation rate on body weight are lower than their natural counterpart. However, there is no indication of a negative relationship between ovarian sensitivity and body weight, showing that it is the absolute dose of PMS which determines the response, not the concentration. The above estimates of the relationships between body weight and primiparous ovulation rate are the pooled estimates over the course of selection, but the data were also examined for possible changes in these relationships during the course of selection. The differences between each parameter in the high and the low lines were regressed on the generation number (the regressions being constrained to pass through zero). In this way, consistent changes could be differentiated from random fluctuations. None of the regressions so calculated were significantly different from zero, nor was there any indication of a uniform change in these parameters during the course of selection. It is therefore reasonable to assume that the changes in gene frequency during selection have not affected the phenotypic relationships between body weight and ovulation rate. The phenotypic regressions of ovulation rate and body weight in the body weight selection lines have already been used to correct the mean ovulation rates of the lines to that which they would have been before selection. In general the relationships are positive and confirm the data presented earlier in the section, in addition, however, they also indicate that the phenotypic relationships differ between the lines. Part of the differences between the regression coefficients are related to differences in body weight. This was demonstrated by regressing the regression coefficient of ovulation rate on body weight within a line on the mean body weight of that line. These regressions were -0-15, -0-20 and -0-08 in the large, control and small lines respectively. Although each of these individual regressions and the pooled estimate are not significantly different from zero (P>0-05), the large, control, small and pooled regressions remove 66-3, 49-5, 6-9 and 43-3% of the total variation in the regression coefficients of ovulation rate on body weight in each of the four groups respectively. DISCUSSION All the analyses presented show that there is a positive genetic relationship between body weight and ovulation rate in the mouse; and between body weight and ovarian sensitivity-as measured by the response to PMS. Furthermore, the change in the body weight of the lines selected for natural ovulation rate (5-3 g) is greater than would have been expected if the correlation between body weight and ovulation rate was only mediated via ovarian sensitivity (1-5 g). Both components of ovulation rate (FSH activity and ovarian sensitivity) must therefore be genetically correlated with body weight. This demonstration of the correlation between body weight and the two components of ovulation rate is compatible with the changes in FSH activity following selection for body weight (Fowler & Edwards, 1960;Edwards, 1962), and the changes in ovarian sensitivity and body weight following selection for litter size (McLaren, 1962). The analyses of the phenotypic relationships between body weight and ovulation rate all indicate that the regression of natural ovulation rate on body weight at the time of scoring is about 0-4 eggs per gram, and that the regression of induced ovulation rate on body weight at the time of scoring is zero. The later result is of particular interest as it shows that larger females shed a similar number of eggs to their smaller counterparts in response to a lower concentration of exogenous gonadotrophin. This result confirms the decision to select for ovarian sensitivity on the basis of the response to a standard dose of PMS. If the response to a standard concentration had been chosen-as has been done by most workers in the fieldthe dose of PMS and the body weight of the recipient would have been fully confounded, and the response would have been biased in favour of large animals. In this situation it would have been impossible to separate the effects of body weight and dose of PMS on the number of eggs shed. The estimate of 0-4 for the phenotypic regression of natural ovulation rate on body weight is similar to one of the estimates of Fowler & Edwards (1960), i.e. 0-49, but higher than the estimates from the other four lines they examined, none of which were significantly different from zero. One possible reason for this discrepency is that in the present study the range in age of females at the time of scoring was rarely more than 2 weeks whereas in the earlier work, age at scoring varied between 6 and 25 weeks of age. The genetic regression of ovulation rate on body weight calculated from the body weight selection experiment is similar to those predicted from the sib analysis and the ovulation rate selection experiment. After five generations of selection however the phenotypic regression is declining in the lines with higher body weights. It is possible therefore that some of the alleles which produce a positive phenotypic correlation between these two traits may be becoming fixed in the larger fines. The consistency of the results of this and earlier studies leads to the conclusion that in the mouse, natural selection has failed to fix some genes which affect body weight and ovulation rate in the same direction. One reason for the maintenance of this segregation can be deduced from the results of earlier experiments. Selection for both high and low litter size (Falconer, 1960a) led to increases in body weight, ovulation rate and embryonic mortality; two way selection for body weight led to changes in ovulation rate and embryonic mortality in the same direction as selection (Fowler & Edwards, 1962); and selection for natural ovulation rate (Land & Falconer, 1969) led to changes in body weight without any changes in litter size, changes in embryonic mortality compensating for the differences. In each case the three traits, body weight, ovulation rate and embryonic mortality were all positively genetically correlated in the mouse populations concerned. It is possible therefore that the segregation of the alleles affecting body weight and ovulation rate is maintained in the populations through the pleiotropic deleterious effects of these alleles on embryonic mortality, and that the intermediate optima for body weight and ovulation rate are arte-facts resulting from the antagonistic action of natural selection on the traits themselves or those genetically correlated with them. This system may be regarded as an example of the maintenance of genetic variation in a population by the counteracting forces of natural selection on different end-products of gene action. I am extremely grateful to Professor D. S. Falconer and Dr R. C. Roberts for their guidance and encouragement during the course of this work and in the preparation of the manuscript, and to Dr H. P. Donald for the facilities to complete this work. I also wish to thank the Agricultural Research Council for a post-graduate studentship and the Ford Foundation for further financial support.

v3-fos

2018-04-03T00:00:38.028Z

1970-10-01T00:00:00.000Z

8978934

s2

Dry-heat inactivation kinetics of naturally occurring spore populations. Twenty-three soil samples were collected from areas of the United States where major spacecraft assembly and launch facilities are in operation. Soil samples were treated with ethyl alcohol, ultrasonic energy, and gross filtration. The resultant suspensions consisted of viable, naturally occurring bacterial spores and were used to inoculate stainless-steel strips. The strips were suspended in a forced air oven and assays were made at 5-min intervals for the number of viable spores. Most survivor curves were nonlinear. Subsequently, spore crops of heat-sensitive and heat-resistant soil isolates were found to have linear survivor curves at 125 C which were unaffected by the presence or absence of sterile soil particles from the parent sample. When two spore crops, one of which was heat-resistant and the other heat-sensitive, were mixed, the resultant nonlinear curves were unaffected by the presence or absence of sterile parent soil. Therefore, the survivor curves obtained originally with the soils were the result of heterogeneous spore populations rather than of protection afforded by soil particles in our test system. These results question the rationale both of assuming logarithmic death and of using decimal-reduction values obtained with subcultured standard reference spores in the derivation of dry-heat sterilization cycles for items contaminated with naturally occurring spore populations. were mixed, the resultant nonlinear curves were unaffected by the presence or absence of sterile parent soil. Therefore, the survivor curves obtained originally with the soils were the result of heterogeneous spore populations rather than of protection afforded by soil particles in our test system. These results question the rationale both of assuming logarithmic death and of using decimal-reduction values obtained with subcultured standard reference spores in the derivation of dry-heat sterilization cycles for items contaminated with naturally occurring spore populations. The National Aeronautics and Space Administration (NASA) requires that spacecraft which may impact Mars or other planets of biological interest be sterilized (NASA, Outbound Planetary Biological Contamination Controi, NASA Policy Directive 8020. 10, 7 September 1967). The problems associated with the delivery of a sterile capsule to a particular planet are manifest and cannot be fully covered in this paper so that reference is made to the more detailed reports of Bruch (2, 3), Craven et al. (5), Favero (8), Hall (11), and Light et al. (13). The basic approach that will be used to produce a sterile spacecraft involves manufacturing, testing, and assembling the hardware under rigid environmental controls, enclosing it in a hermetically sealed canister, and subjecting this unit to a terminal dry-heat sterilization cycle. Once sealed and heated, the biological barrier (canister) cannot be broken to determine that the spacecraft is in fact sterile. Therefore, sterility of the spacecraft is expressed as a low probability which must be established entirely by inference. The candidates most likely to survive the terminal sterilization cycle would be heterogeneous populations of bacterial spores. Rosebury (16) states that members of the genus Bacillus are widely distributed in nature, and, although they are frequently recovered from surfaces of the healthy human body, they are not indigenous to humans but are merely transient organisms whose ultimate origin is soil. Therefore, as a contaminated spacecraft enters its terminal sterilization cycle, the organisms of major concern with regard to thermal resistance will be bacterial spores of soil origin. Presently, dry-heat sterilization cycles for spacecraft are based upon the thermal inactivation characteristics of B. subtilis var. niger (B. globigii) spores. Craven et al. (5) have stated that many workers in the field of spacecraft sterilization technology accept the spores of this microorganism to be representative of the more heat-resistant organisms likely to be found on spacecraft. They further pointed out that a representative organism is necessary because of the magnitude in variations of thermal resistance among spore populations found in and on space hardware. In addition, the "logarithmic death rate model" of spore populations is assumed in subsequent calculations (12), since survivor curves of the standard reference organism are essentially straight-line functions on a semilog scale. Rather than employ a standard reference or-ganism, it was thought that the use of naturally occurring spore populations (i.e., mixed populations of spores employed directly without intermediate isolation and subculture on conventional laboratory media) might yield more representative and therefore pertinent information regarding the dry-heat inactivation kinetics to be encountered in actual practice. Even though methods are available for efficient removal and recovery of surface contamination (8,10), collection of spores directly from spacecraft surfaces in numbers sufficient for dry-heat resistance tests to be conducted is impossible. Assays on several unmanned spacecraft have shown the bacterial spore level to be approximately 104 to 10 per spacecraft (J. R. Puleo, personal communication). Since spores, including those found on spacecraft, are mainly of soil origin, soils collected in the vicinities of manufacture and assembly of spacecraft would be the next most logical source of naturally occurring spore populations for heat studies. The objectives of these studies were to formulate a method for obtaining spore populations from soil without the use of heat shock (4,7) or subculture and to observe the inactivation kinetics of these populations found in soil samples collected from various areas of the United States where spacecraft components are manufactured or assembled. MATERIALS AND METHODS Soil samples. One-hundred-gram amounts of each soil sample were dried in loosely capped 1-liter Erlenmeyer flasks at 50 C for 48 hr. A 100-ml amount of 95% ethyl alcohol was added to each dried soil, and the suspensions were insonated at maximum power (25 kHertz) for 30 min in an ultrasonic bath (Sonogen LTH60-3 transducerized tank; Sonogen A-300 generator; Branson Instruments, Inc., Stamford, Conn.). A single flask was placed on the center of the tank bottom with the level of the bath fluid, 0.3% Tween 80 (Hilltop Research, Inc., Miamiville, Ohio) in distilled water, slightly above the level of ethyl alcohol in the flask to ensure optimum transfer of ultrasonic energy. The temperature of the bath fluid was maintained from 4 to 20 C during the insonation period. After insonation, each suspension was filtered through a sterile linen towel to remove large particles and then stored at 4 C in a tightly capped bottle. These treatments yielded suspensions of finely dispersed soil and bacterial spores, free from viable fungi, actinomycetes, and vegetative bacteria. No isolations have been made from these suspensions other than gram-positive, sporeforming rods. Also, extended storage of bacterial spore crops in ethyl alcohol, as opposed to distilled water, has been shown to have no effect on viability or heat resistance (7). Assay system. Dry-heat inactivation kinetics of pure and mixed spore populations were determined by inoculating sterile stainless-steel strips [0.5 by 0.5 inch (1.27 by 1.27 cm); coldroll, type 302, no. 4 finish, 22 gauge] with 0.05 ml of an ethyl alcohol suspension. The strips were then dried under vacuum for 16 hr over silica gel. For each heating interval, three strips were suspended in a forced-air dry-heat oven (model no. 625, Precision Scientific Co., Chicago, Ill.) at 125 C (40.5 C). The time required to heat a strip to 125 C was 2.5 min as determined by a copper-constantan thermocouple attached to a control strip. This amount of time was added to each exposure interval. One set of samples was placed in the oven at each interval, and the temperature was monitored constantly with a recording thermometer. Immediately after removal from the oven, each strip was placed in a tube containing 10 ml of chilled (4 C), phosphatebuffered distilled water (BDW; 1) with several 3-mm glass beads and was insonated for 12 min in an ultrasonic bath. The suspensions were diluted appropriately with BDW and plated in triplicate with Trypticase Soy Agar (TSA; BBL). After the medium had solidified, 10 to 15 ml of TSA was overlaid on each plate to lessen spreading growth. One triplicate set of strips was used as a control (no heat), and six or more sets were heated for the desired times. Three uninoculated strips were processed as sterility controls, and counts of survivors were made after 24 and 48 hr of incubation at 32 C. All manipulations of sterile items, with the exception of transferring triplicate sets of strips from sterile petri plates into the oven and vice versa, were performed in class 100 horizontal laminar flow clean benches to eliminate background contamination (9). D125c values were determined from a best-fit regression line of the data points by using a least squares method. The variation of the data points around each regression line was measured by calculating a standard error of the estimate. Isolations and spore preparations. Colonies were picked randomly from pour plates of heated and unheated strips of selected soils and streaked for isolation on TSA. Pure cultures were maintained on TSA slants at 4 C. Spore preparations were made of selected isolates in the following manner. A turbid suspension of cells in sterile BDW was heat-shocked in a water bath at 80 C for 15 min. The suspension was then lightly swabbed onto TAM Sporulation Agar (Difco) supplemented with 20 ,g of MgSO4 per ml and 80 jg of CaCl2 per ml. After 2.5 hr of incubation at 32 C, growth was swabbed onto a fresh plate of medium. This procedure was performed twice. After the last incubation period, growth from the final plate was swabbed onto three plates of fresh medium and incubated at 32 C for 24 to 48 hr. Most isolates exhibited maximum sporulation at these times with only a few requiring up to 96 hr of incubation. Growth from the three final plates was harvested in approximately 20 ml of chilled BDW and insonated for 90 sec with a Biosonic III Ultrasonic Probe (Bronwill Scientific, Rochester, N.Y.) at 60% maximum intensity. The suspension was then centrifuged for 15 min at 4 C (8,590 X g; Servall SS-1, Ivan Sorvall, Inc., Norwalk, Conn.). The supernatant fluid was discarded, and the cellular debris layer of the pellet was removed by gentle washing with 5 ml of chilled BDW. The remainder of the pellet, consisting mainly of spores, was resuspended and washed twice more in the same man-574 BOND ET AL. on March 21, 2020 by guest http://aem.asm.org/ Downloaded from ner. Simple staining with crystal violet and microscopic examination were used to determine if more washings were necessary. The pellet was then washed three times with 95% ethyl alcohol and resuspended for storage at 4 C. Subsequent heat inactivation studies were performed in the same manner as described for the ethyl alcohol-soil suspensions. Model spore population. A system to simulate the survivor curve of a mixed, naturally occurring spore population was developed by using one heat-sensitive and one heat-resistant aerobic mesophilic isolate from soil sample X (Phoenix). Clean spore crops were prepared, and D12s c values of each ethyl alcohol suspension were determined in the presence and absence of sterile soil from the parent sample to determine the characteristics of the respective survivor curves. The two isolate suspensions were then mixed in a proportion similar to the heat-sensitive and heat-resistant populations in the original soil. Sterile parent soil was added to a portion of this mixture to approximate the spore per unit volume of the original soil sample (0.3 g/ml). Dry-heat assays were performed in the same manner as the comparative DI25 c survey of soils. RESULTS During preliminary testing of the heating system, the soil sample collected in Phoenix (sample X) consistently produced a nonlinear survivor curve (Fig. 1), indicating a decrease in inactivation rate with time. In general, the survival data from the 23 soil samples tested subsequently did not appear linear, and the shapes of the survivor curves were similar to that of sample X. The sample containing the most resistant spores was the one collected in Phoenix. To examine the possibility that the nonlinear survivor curves of this sample could have been due to protective effects offered by the small amount of soil particles present, various subcultured spore suspensions of heat-sensitive and heat-resistant isolates suspended in ethyl alcohol were tested in the presence and absence of sterile parent soil. Survivor curves (Table 1). Consequently, the soil per se did not seem to alter dry-heat resistance by physical protection. Also, the nonlinear survivor curves of the soil samples did not appear to result from technique-induced error since pure spore cultures of B. subtilis vat. niger, B. cereus T, B. subtilis 5230, and several soil isolates have consistently yielded linear survivor curves at 125 C in our test system. By expressing the standard error of the estimate for each line as a coefficient of variation, it was possible to compare the variation from a straight line in the soil suspension tests with the variation measured in like tests on spore isolates. These data are presented in Tables 1 and 2. It is evident that the coefficients of variation for the soil suspension tests are markedly higher than those for spore isolates, the mean value of the suspensions being more than five times the mean value of the isolates. The consistency in the shape of the 24 survivor curves for the soil suspensions coupled with the large coefficients of variation for straight lines fit to these data indicated that the survivor curves were not linear. In an effort to define more clearly the nature of the nonlinear survivor curves observed with the soil samples, a model system described above was developed by using heat-sensitive and heat-resistant aerobic mesophilic isolates from soil sample X. Clean spore crops were prepared, and survivor curves of both suspensions were found to be linear and unaffected by the presence of added soil (isolates X-1 and XA, Table 1). When the two suspensions were mixed in the presence VOL. 20, 1970 and absence of soil and tested in parallel, the survivor curves were shown to be nonlinear and unaffected by the presence of soil (Fig. 2). DISCUSSION During comparison of D125 c values of naturally occurring spore populations in soils and respective subcultured spore isolates, another factor was noted which may significantly affect the current rationale behind calculation of spacecraft sterilization cycles. No spore isolates from heated soils were obtained which exhibited resistances equal to or greater than the portion of the survivor curves from which they were isolated. Consequently, it appeared that the subculture of the naturally occurring spores significantly lowered their dry-heat resistances. Whether these losses in dry-heat resistance were due to unsatisfied nutritional requirements or differences in environmental conditions or both is not known. However, studies by many other investigators have shown that the heat stability of spores may be altered significantly by manipulation of cultural conditions (14). Lowering of dry-heat resistance by subculture has also been noted with gramnegative organisms such as salmonellae (15). It must be reemphasized here that current space-craft sterilization cycles are calculated on the basis of a standard reference spore population, i.e., B. subtilis var. niger. However, there is no evidence available to suggest that this organism or its thermal inactivation characteristics are in any way associated with actual microbial contaminants on spacecraft. Qualitative and quantitative microbiological assays conducted on many spacecraft while in residence at Cape Kennedy (Apollo and Mariner) have failed to reveal a single instance of contamination by B. subtilis var. niger. Ernst (6) reviewed the thermal inactivation kinetics of bacterial spores and pointed out several pitfalls which may be encountered in the interpretation and subsequent extrapolation of death rate constants (D values). When studying the nonlogarithmic survival of mixed spore populations in soils, he found, as our observations also indicate, that isolates from the later phases of the survivor curves were less heat-resistant than the original tests would have indicated. From this observation, he assumed a mechanistic approach by stating that the more heat-resistant spores were "undoubtedly protected in some way in the soil menstruum." Based on this assumption, he concluded that the slope of a straight line passing through the point of extinction (obtained by end-point determinations) of a population would represent the largest possible D value of the unprotected spores. The findings in our study, however, indicate that when end-point data are used and logarithmic death of a naturally occurring spore population is assumed, there can be a significant error in estimating the time required to achieve a high probability of sterility ( Fig. 3). This figure was adapted from a hypothetical curve presented by Ernst (6) in his illustration of the protective effects by soil. The controversy regarding calculation of sterilization cycles in general can be appropriately described as a classical argument between two basic schools of thought. One school explains deviations from logarithmic survival in terms of inherent heterogeneity within a population, whereas the other school attributes these deviations to factors in operation within the lethal period. With particular regard to dry-heat inactivation of bacterial spores, members of either school would expect to observe nonlinear survival when dealing with mixed populations in soil. However, it remains that such nonlinear survival has been and is today explained entirely in terms of protective effects by the soil menstruum. This philosophy continues by reasoning that, when dealing with relatively low levels of surface contamination such as encountered with spacecraft, the protective effects of soil may be neglected with concomi- (6): calculated heating times necessary to effect a 7-log reduction. tant assumption of logarithmic death. Data presented in this report indicate such an assumption to be workable provided that the probability of contamination by a particular population is not extrapolated below 100. However, in extrapolation below measurable range, for example, to 10-, the greatest possible D value of the population becomes critical and may not be accurately reflected by an end-point determination. Therefore, in dealing with sterilization of naturally occurring spore populations, it is recommended that assumption of logarithmic death in conjunction with a standard reference organism may well constitute an invalid model in certain applied situations, particularly in the field of spacecraft sterilization. ACKNOWLEDGMENT Services were provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration under contract W-13,062.

v3-fos

2018-04-03T00:55:10.905Z

1970-11-01T00:00:00.000Z

27225077

s2

Quantitation of the Antigenicity and Immunogenicity of Purified Foot-and-Mouth Disease Virus Vaccine for Swine and Steers The antigenicity and immunogenicity of a purified preparation of foot-and-mouth disease virus [type A12, strain 119 (FMDV A-119)] inactivated with 6.0 mmN-acetylethylenimine at 37 C were compared in swine and steers. Three antigen doses were tested, 640, 160, and 40 ng. In accordance with findings for guinea pigs, as previously determined by dose-response curves, as little as fourfold changes in antigen in the region of the minimum effective dose produced marked differences in the serological and immune responses of swine. The minimum effective dose of antigen for antibody formation in swine and guinea pigs, as determined by mouse median protective dose (PD50) values, was 160 ng. The minimum immunogenic dose for swine was also 160 ng. The vaccinated swine were challenged with either FMDV A-119 or with heterologous subtype A24 strain Cruzeiro or type A strain A-CANEFA-1. Those immunized with 640 ng of antigen were about equally immune to the three challenge viruses; most swine having a mouse PD50 value of 2.0 or greater were immune regardless of which strain was used for challenge. In steers, the smallest dose tested, 40 ng, was satisfactory in eliciting circulating antibodies and immunity. Physical and biological tests indicated that the antigen used in the vaccine is stable for at least 9 months at 4 C. 1. Those immunized with 640 ng of antigen were about equally immune to the three challenge viruses; most swine having a mouse PD50 value of 2.0 or greater were immune regardless of which strain was used for challenge. In steers, the smallest dose tested, 40 ng, was satisfactory in eliciting circulating antibodies and immunity. Physical and biological tests indicated that the antigen used in the vaccine is stable for at least 9 months at 4 C. Foot-and-mouth disease virus (FMDV) vaccines prepared from virus grown in the BHK-21 line of baby hamster kidney cells have elicited undesirable reactions resembling delayed hypersensitivity after revaccination of cattle (12). Purification of FMDV before vaccine formulation under certain experimental conditions appears to reduce the incidence of such reactions (Bahnemann, personal communication). Moreover, the use of purified and concentrated virus in a vaccine allows potency and volume parameters to be accurately adjusted and eliminates or lessens the chances for immunological competition (6) caused by nonviral components of crude vaccines. Because of the high costs of testing FMDV vaccines in cattle and swine, antigen dose versus neutralizing antibody data are usually developed only for what is considered to be the practical immunological range in lieu of establishing an entire dose-response curve. Such curves, which allow a more comprehensive and critical assess-1 Present address: Department of Veterinary Science, University of Kentucky, Lexington, Ky. 40506. ment of vaccine potency, have, however, been established in guinea pigs for purified and concentrated, acetylethylenimine (AEI)-inactivated high-passage FMDV, type A12, strain 119 (A-119; 13,15). It was the purpose of the present work to utilize this data in helping to establish the smallest dose of a similar vaccine which elicits measurable neutralizing antibody responses in both cattle and swine as well as the doses required to induce resistance to challenge in 50% of the animals. MATERIALS AND METHODS Virus inactivation and storage. FMDV A-119 was passaged once in suckling mice, 150 times in primary calf kidney cultures, and once in BHK cells (16) derived from line 21, clone 13 of MacPherson and Stoker (11). This virus, at 2.62 mg/ml, purified as previously described (2), was inactivated with 6.0 mM AEI at 37 ± 0.5 C for 48 hr in 0.2 M NaCl, 0.05 M sodium phosphate, pH 7.5 (14). Titrations in suckling mice and plaque assays in calf kidney cultures were used to determine the rate of virus inactivation. The inactivation was a first-order reaction which extrapolated to 10-5 plaque-forming units (PFU)/ml at 48 hr. In addition, intradermalingual inoculation of 2 ml (0.65 mg/ml, 0.1 ml/site) after 48 hr of AEI treatment in each of six steers failed to demonstrate infective virus (9,14). After inactivation, appropriate concentrations of antigen were obtained by dilution in the above buffer. No attempt was made to remove, assess, or maintain the presence of AEI after the 48-hr treatment. Absorbancetemperature profiles (1) and complement fixation activities (4) of the purified virus were essentially the same before and after inactivation as well as after 9 months of storage at 4 C. Vaccination and bleeding. Vaccines containing 640, 160, and 40 ng of inactivated virus were each tested in swine, steers, and guinea pigs. Vaccines were composed of 1 ml of an appropriate dilution of the AEI-inactivated virus preparation emulsified with 1 ml of an oil adjuvant consisting of one part emulsifier (Arlacel A) and nine parts light mineral oil (Marcol 52, Esso Research and Engineering Co., Linden, N.J.; reference 5). Nine Hereford steers were vaccinated subcutaneously, midway between the base of the ear and the point of the shoulder, with vaccines prepared with antigen stored for 5 months. Thirty-six swine were injected in the dorsal surface of the ear with vaccines which had been prepared from antigen stored for 9 months at 4 C. Two groups (15 per group, 5 per dose) of female Duncan Hartley strain guinea pigs were also vaccinated subcutaneously. Group 1 was vaccinated at the same time as the steers, and group 2 was vaccinated at the same time as the swine. Blood samples were collected from the cattle and swine at 0, 7, and 28 days postvaccination (DPV) and from the guinea pigs at 7 and 28 DPV. Individual sera were prepared and stored at -10 C. Serum/virus neutralization determinations. Lowpassage tissue culture-produced FMDV [6,600 mouse median lethal dose (LDo)/ml] of the desired subtype was mixed with equal amounts of various dilutions of serum and incubated at 37 C for 1 hr. Each mixture was then injected (0.03 ml/dose) into 5-and 9-day-old unweaned Rockefeller H strain mice to detect unneutralized virus. Neutralizing capacities of sera were computed as mouse median protective dose (PDo0) values (7). Challenge of immunity of vaccinated swine. The immunity of each of the 36 vaccinated swine was challenged 28 DPV by injection with 1 ml of guinea pig FMDV-infected vesicular fluid diluted with Hanks balanced-salt solution to contain 40,000 mouse LDim of the desired strain of FMDV. This virus is highly virulent for both swine and cattle (Tables 2 and 3). The injection was given intradermally in the ventral area of the pastem of the left foreleg (3). The swine were in three groups, one of 18 animals and two of 9 animals. The group of 18 consisted of 6 swine vaccinated with each of the antigen doses, 640, 160, and 40 ng; similarly, each group of 9 contained 3 swine from each of the three antigen doses. These animal groupings were made without regard to antibody levels of the individual swine. The 18 swine were challenged with FMDV A-119, whereas one group of 9 was challenged with type A (subtype not designated) strain A-CANEFA-1 (A-CANEFA-1) and the other was challenged with type A24, strain Cruzeiro, Brazil (A24). In addition, each challenged group contained two nonvaccinated control swine, one of which was challenged along with the vaccinated swine. The swine were examined for signs of foot-and-mouth disease (FMD) at 2-day intervals for 14 days postchallenge (3). Challenge of immunity of vaccinated steers. The immunity of the vaccinated steers was challenged 28 DPV by exposure to experimentally infected steers (8). Two of five nonvaccinated control steers placed in contact with the immunized animals were inoculated intradermalingually with 10,000 mouse LD50 of FMDV A-1 19 in guinea pig vesicular fluid. The steers were examined for signs of FMD at 2-day intervals for 14 days after exposure. RESULTS Swine. Neutralizing antibody measured as mouse PD50 values in the sera of vaccinated swine is shown in Table 1 c Type A (subtype not designated) strain A-CANEFA-1. neutralization potencies of the 7 DPV sera against FMDV A-CANEFA-1 and subtype A24 were 0.4 and 0.5 PD50, respectively, or essentially the same as the sera of the nonvaccinated controls. By contrast, sera from these same swine at 28 DPV demonstrated similarly high, i.e., 2.8 to 2.4, mouse PD50 values against all three of these subtypes of type A FMDV. When the group of 18 swine, containing 6 animals vaccinated with each of the antigen doses, was challenged at 28 DPV with the homologous strain FMDV A-119, 5 of 6 vaccinated with the 640-ng dose and 3 of 6 vaccinated with the 160-ng dose were immune ( Table 2). All six swine vaccinated with 40 ng, the smallest dose used, became infected when challenged with FMDV. When challenged with FMDV A-CANEFA-1, two of three swine vaccinated with the 640-ng dose were immune, whereas all six swine vaccinated with either 160 or 40 ng were susceptible and developed frank signs of FMD. Swine vaccinated with the 640-ng dose and challenged with FMDV type A24 were immune to challenge, whereas only one of three vaccinated with the 160-ng dose and none of those vaccinated with 40 ng were immune. All six of the nonvaccinated control swine were susceptible. Steers. Serological response and immunity of steers after vaccination are shown in Table 3. At 7 DPV, mean mouse PD50 values of 1.8 from the sera of steers vaccinated with 640 ng of antigen were appreciably higher than those (ca. 1.0 PD50) of animals receiving the lower doses. By 28 DPV, the antibody levels (3.1 to 3.7 PD50) resulting from the different doses of vaccine were markedly higher than any at 7 DPV and were of a similar magnitude, regardless of vaccine dose. All vaccinated steers together with three normal steers were challenged by contact exposure to two steers experimentally infected with FMDV A-119. The two inoculated control steers developed typical FMD lesions within 48 hr, whereas the three noninoculated controls did not develop lesions for an additional 48 hr. All nine of the vaccinated steers resisted this exposure. Guinea pigs. Guinea pigs in group 1, receiving vaccine concurrently with the steers in which the 640 ng of antigen had been stored for 5 months at 4 C, developed PD50 values of 1.8 log units at 7 DPV, which increased significantly by 28 DPV to 2.6. Guinea pigs vaccinated with 160 ng demonstrated no response at 7 DPV, but did develop a measurable response (1.4 PD50) by 28 days. Those inoculated with the 40-ng dose gave no detectable response at either bleeding date. The guinea pigs in group 2, which were vaccinated along with swine with either 160 or 640 ng of antigen (stored 9 months at 4 C), developed measurable antibody responses (1.1 to 1.6 PD50) at 7 DPV; however, their PD50 values were considerably lower (0.7 log unit) at 28 DPV. As with group 1 guinea pigs, serum antibodies were not detectable in group 2 guinea pigs vaccinated with the smallest dose, 40 ng, at either 7 or 28 DPV. DISCUSSION The data provide quantitative information for the antigenicity and immunogenicity of the purified vaccine in steers and swine. A 160-ng amount of virus appeared to be the minimum effective antigenic dose in guinea pigs and swine and also to be close to the minimum immunogenic level for the latter. This dosage immunized 50% of the swine against the homologous virus type, whereas 40 ng was without effect and 640 ng protected five of six swine. By comparison, the smallest amount of antigen, i.e., 40 ng, used to vaccinate steers appeared to be in excess of the minimum effective dose. By 28 DPV, the three doses, 40, 160, and 640 ng, elicited similar serological responses in steers, all of which were immune to challenge. The higher inactivated vaccine dosage requirements for swine than for steers are in accord with the known requirements of immunizing swine during field outbreaks of FMD. The control of extensive outbreaks of FMD in swine requires a single vaccination of either monovalent Frenkel bovine tongue epitheliumor calf kidney cell-produced vaccine containing 4 to 10 times the antigen content required for cattle, or two inoculations of regular strength Frenkel vaccine spaced 2 weeks apart (17,18). In limited experimental trials, vaccine containing incomplete Freund's adjuvant imparted protection to swine at a dosage similar to that used in cattle (10). However, the circulating antibody was considerably less than in cattle. Vaccination of swine with purified-concentrated, inactivated FMDV A-119 antigen elicited serological and immunological responses to heterologous as well as to the homologous subtypes of the virus at 28 DPV with the 640-ng dose. Subtype cross-protection was not detected in serum taken at 7 DPV (Table 1), possibly indicating a higher degree of specificity for the 19S class of antibodies, the predominant species of antibody at the 7-DPV bleeding period. Table 2 indicates relatively small differences in the immunity of the vaccinated swine to challenge with the three subtypes of FMDV. Examination of swine sera revealed that individual swine possessing an FMDV A-119 mouse PD50 value of 2.0 or greater were protected against all three challenge viruses, whereas those with PD50 values of less than 1.5 developed generalized FMD. Postchallenge FMDV A-119 mouse PD50 values in swine were essentially the same (3.7 to 4.1) regardless of previous vaccine experience or strain of challenge virus (Table 1). In previous studies with this product, it was found that 10to 16-fold differences in antigen were required to produce significantly different serological responses in guinea pigs in the region of the dose-response curve above the minimum effective dose (13). In the present work, vaccines with fourfold differences in antigen content in the region of the minimum effective dose produced distinctly different antibody responses in guinea pigs as well as different antibody and immunogenic responses in swine. The purified concentrated antigen used for vaccine preparation in these experiments appeared to be stable for several months when stored at 4 C. Because of space limitations, experiments in large animals cannot always be done at one time, and thus guinea pigs were vaccinated at the same time as the swine and steers to serve as a control of the vaccine. A change in the antigen during storage should be indicated by a difference in antibody response in the guinea pigs. The PD50 values obtained with the 28-DPV sera of the guinea pigs vaccinated at the same time as the swine could possibly indicate a deterioration of the antigen not detectable by either complement fixation or the absorbancetemperature tests, both of which indicated no significant changes in the antigen during storage. The latter two tests are in accord with the good immunogenicity of the stored antigen in swine and steers.

v3-fos

2020-12-10T09:04:11.094Z

1970-09-01T00:00:00.000Z

237234307

s2

Biological Assays for Two Mycotoxins Produced by Fusarium tricinctum A survey was made to detect microorganisms useful for assaying butenolide [4-acetamido-4-hydroxy-2-butenoic acid γ-lactone] and T-2 toxin [4β, 15-diacetoxy-8α-(3-methylbutyryloxy)-12,13-epoxytricothec -9-en-3α-ol]. These mycotoxins produced by strains of Fusarium tricinctum have been implicated in mycotoxicosis of livestock. Although butenolide proved to be a very weak antibiotic, assay discs containing 100 μg of this toxin inhibited Sprillum serpens NRRL B-2052, Vibrio tyrogenus NRRL B-1033, and Xanthomonas campestris NRRL B-1459. T-2 toxin had no effect on 54 bacterial strains but inhibited 6 of 11 fungi. Growth of Rhodotorula rubra NRRL Y-7222 and Penicillium digitatum NRRL 1202 was retarded by assay discs containing 4 μg of T-2 toxin. Solutions with less than 1 μg of T-2 per ml toxin were readily detected by a pea seed germination test. Germination was reduced more than 50% when seeds imbibed solutions of 0.5 μg of T-2 toxin per ml. Butenolide had no effect on pea seed germination at concentrations as high as 200 μg/ml. Toxic feeds are being associated more and more with specific fungi that cause certain mycotoxicoses in farm animals. Identification, production, purification, and biological testing of fungal metabolites suspected of being toxic have aided in elucidating the cause of many feeding problems. Metabolites toxic to animals are elaborated by strains of Fusarium tricinctum isolated from fescue, Festuca arundinacea, a valuable forage grass used as winter pasture in the Southern and Western areas of the United States. These isolates have been implicated in a disease known as fescue foot (6)(7)(8)(9). Yates et al. (8) characterized a toxic butenolide, 4-acetamido4hydroxy-2-butenoic acid y-lactone, from these strains cultured on Sabouraud agar at low temperature. Reportedly, F. tricinctum was the fungus most prominent on moldy corn, and strains of this species produced the most potent toxins of the molds isolated from toxic corn (5). Bamburg et al. (1) tested culture filtrates of hundreds of molds taken from moldy grain; the filtrates most toxic to mice were obtained from strains of F. tricinctum. Among the several mycotoxins elaborated by this fungal species, the formation of the major toxic metabolite depends upon cultural conditions and on the particular fungal strain. In laboratory culture, strains isolated from moldy grain yield diacetoxyscirpenol, T-2 toxin, and 4-desacetoxy T-2 toxin (3; J. R. Bamburg, Ph.D. Thesis, Univ. of Wisconsin, Madison, 1969). The T-2 toxin characterized as 4#, 15-diacetoxy-8a-(3-methylbutyryloxy)-12, 13-epoxytricothec-9en-3a-ol (2) has an LD.% of 4 mg/kg of body weight when administered orally to mice (1). The butenolide has an LDw of 275 mg/kg of body weight when given orally to mice and 44 mg/kg when injected into the peritoneum. An application of the culture filtrates to the skin of laboratory animals provides the simplest test for these mycotoxins (6; J. R. Bamburg,Ph.D. Thesis;E. B. Smalley et al.,in press). Toxicity is indicated by an edematous, hemorrhagic skin response, and the severity of the response is graded to aid in estimating toxin quantities (J. R. Bamburg, Ph.D. Thesis). Although thin-layer chromatography is useful in detecting T-2 toxin in partially purified extracts, small amounts of toxin recovered along with impurities are difficult to quantitate. Because of the difficulties in detecting low levels of T-2 toxin by thin-layer or paper chromatography, Bamburg (Ph.D. Thesis) developed a gas-liquid chromatographic (GLC) procedure for measuring the trimethylsilyl ether of the toxin in extracts of mold cultures and corn samples. As for T-2 toxin, a simple chemical method for quantitating butenolide in fungal extracts has not been developed; however, its presence can be detected and its relative amount estimated by infrared spectroscopy (9). Current chemical and biological tests for de-tecting butenolide or T-2 toxin are time-consuming, require expensive equipment, and with the possible exception of the GLC procedure, are only crude estimates of the toxin quantities in culture extracts. The animal skin test has proved the most useful thus far in confirming the potency of culture filtrates. Since animals were sensitive to these toxins, some other cellular system should also react to their presence. If a simple test could be developed for detecting these mycotoxins, it could aid in evaluating the toxic potential of moldy grains. Various microorganisms are used to estimate the quantity of growth inhibitors in crude extracts of many products. Because toxinsensitive microorganisms provide a quick and easy method of estimating toxin quantities, a survey was carried out to find bacteria and fungi inhibited by T-2 toxin or butenolide. In addition to microorganisms, a pea seed germination test was made because diacetoxyscirpenol, a compound with a molecular structure similar to T-2 toxin, inhibited pea stem elongation in solutions of 1 Ag/ml (4). Both butenolide and T-2 toxin reportedly have antibiotic properties. At concentrations of 10 mg/ ml, the butenolide inhibited 11 of 14 bacteria and 2 of 3 molds that were studied. Only slight activity was detected at 1 mg/ml and none at 0.1 mg/ml (9). Even though T-2 toxin was said to have weak fungistatic properties (J. R. Bamburg, Ph.D. Thesis), a definitive method and identification of test fungi were not published in support of this finding. MATERIAILS AND METHODS Survey for sensitive microorganisms. Acetone solutions of crystalline butenolide or T-2 toxin were added to antibiotic assay discs (no. 740E Schleicher and Schuell Co.) to give discs containing 50 ,ug of T-2 toxin or 200 pg of butenolide. Acetone was removed by warming the discs for 30 min at 70 C. The mycotoxins were obtained from our Industrial Crops Laboratory, the butenolide having been chemically synthesized and the T-2 toxin having been extracted from mold cultures. Bacteria and fungi from the Agricultural Research Service Culture Collection were selected for testing. Microbes were transferred into a suitable medium and incubated at temperatures permitting good growth. The bacteria were generally propagated in nutrient broth at 32 C, and the fungi were propagated on yeast-malt (YM) agar at 22 to 27C. After an incubation of 24 to 36 hr, 0.1 ml of bacterial suspension was spread over the surface of 20 ml of nutrient agar medium in a standard petri dish. The surface was allowed to dry for about 1 hr before the toxic filter discs were placed onto the surface. Heavy suspensions of 48-hr yeast cells and mold conidia were prepared in water. The fungal growth was loosened with a loop and suspended by vibrating before the 0.1 ml of cell suspension was spread onto the surface of YM agar. After a suitable incubation time, inhibition zones of sensitive microorganisms were measured. Zones recorded as + had a diameter of less than 16 mm; those recorded as + + had greater diameters. Standardized assay with sensitive microorganisms. Microorganisms indicating the greatest sensitivity to either mycotoxin were selected for testing by a standardized procedure. Rhodotorula rubra NRRL Y-7222 was used to quantitate T-2 toxin, and Spirillum serpens NRRL B-2052, Vibrio tyrogenus NRRL B-1033, and Xanthomonas campestris NRRL B-1459 served as indicators of butenolide concentrations. Slant cultures of the selected microorganisms were grown on YM agar. R. rubra was incubated for 48 hr and the bacteria were incubated for 24 hr at 32 C before assay plates were prepared. Cell suspensions were prepared by washing incubated slants and diluting the cells in YM broth to give an optical density (measured in a Spectronic-20 spectrophotometer at 600 nm) of 0.15 for the bacteria and 0.3 for R. rubra. The standardized inoculum, 0.1 ml, was added to 5 ml of melted sterile YM agar cooled to 45 C. Inoculated agar was poured into a standard lOOX, 15-mm petri dish. Warming the dishes to 50 C before pouring kept the agar from rapidly solidifying and enabled the inoculated agar to spread. Known quantities of the mycotoxins were dissolved in acetone and added by pipette to the filter paper discs (0, 25, 50, 100, and 200 pg of butenolide and 0, 2, 4, 6, 8, and 10 pug of T-2 toxin). Before being placed on the inoculated medium, the mycotoxic discs were dried at 70 C for 30 min and the zones of inhibition were measured with a ruler after incubation for 44 to 48 hr. Pea seed germination test. Little Marvel, a wrinkledseed variety of Piswn sativum, was tested for germination after imbibition of mycotoxin solutions. A measured quantity of crystalline T-2 toxin was dissolved in acetone and a sample was transferred to a 100-ml beaker. After the acetone evaporated, 50 ml of water was poured into the beaker and the small quantity of toxin dissolved in it. The butenolide was diluted with water. Duplicate tests were done by steeping 25 pea seeds overnight in the toxic solutions; the turgid seeds were then planted in wet sand and covered with moist paper toweling. After 4 days of incubation at 22 to 27 C, the seeds that had germinated were counted. RESULTS The antibiotic activity of T-2 toxin and butenolide against selected microorganisms is presented in Table 1. Butenolide concentrations of 200 jig per assay disc inhibited 9 of 54 bacteria but none of the fungi. S. serpens NRRL B-2052, V. tyrogenus NRRL B-1033, and X. campestris NRRL B-1459, as indicated by the diameter of the inhibition zone, were more sensitive to butenolide than the other bacteria. In a standardized assay, 50 ,ug of butenolide noticeably inhibited each bacterium; discs bearing 100 and 200 ,ug gave clear zones with increasing diameters (Table 2). T-2 toxin was not bacteriostatic at a concentration of 50 ,ug per disc but was toxic to 6 of 11 fungi (Table 1). R. rubra NRRL Y-7222 and Penicillium digitatum NRRL 1202 exhibited larger inhibition zones than the other fungi. Discs with 4 ug of T-2 toxin caused a detectable fungistatic activity against R. rubra, and zone diameters of more than 20 mm around discs with 8 and 10 ,ug of toxin were recorded ( Table 2). DISCUSSION Neither the microorganisms nor the pea seeds provide an indicator for measuring low levels of butenolide. A standard 12.7-mm filter disc with 0.1 ml of toxic filtrate would not inhibit any of the indicator bacteria if the filtrate contained less than 0.5 mg of butenolide per ml. Despite their tolerance to this mycotoxin, S. serpens, V. tyrogenus, and X. campestris could be useful in studying the effects of butenolide in a living system. In contrast to butenolide, T-2 toxin was fungistatic and phytotoxic but was not active against bacteria. T-2 toxin was fungistatic at concentrations comparable to those causing an inflammatory skin response of animals receiving topical doses. Toxic solutions containing more than 40 Mug/ml were detectable with R. rubra as the indicator fungus. Despite our emphasis on finding toxin-sensitive microorganisms, the biological test most easily standardized and requiring the lowest concentration of T-2 toxin was the pea germina- tion test. Germination was inhibited by 50, 64, and 90% when seeds were soaked overnight in solutions containing only 0.5, 1.0, and 2.0 ,ug of T-2 toxin per ml, respectively. The effect of T-2 toxin on biological agents suggest that a combination of animal, plant, and microbial test may provide a simple, although presumptive, means of estimating its quantity in culture filtrates or extracts of feeds molded with F. tricinctum.

v3-fos

2020-12-10T09:04:16.634Z

1970-02-01T00:00:00.000Z

237232762

s2

Effects of N2-O2 and CO2-O2 Tensions on Growth of Fungi Isolated from Damaged Flue-Cured Tobacco Ten fungi, Aspergillus niger, A. flavus, A. ochraceus, A. ruber, A. repens, A. amstelodami, Alternaria tenuis, Penicillium brevi-compactum, Cladosporium herbarum, and Chaetomium dolicotrichum, were isolated from moldy flue-cured tobacco and grown in various mixtures of N2-O2 or CO2-O2. A 1 to 5% concentration of O2 in an N2 atmosphere caused the greatest change in growth of the nine species, and a 10 to 20% concentration of O2 for A. flavus. All species, except A. amstelodami and A. ruber, grew faster in air than in mixtures containing 10% O2. High O2 concentrations generally inhibited furrow production in the mycelial mats. In an atmosphere of 5 to 40% O2 in the N2 atmosphere, furrows formed in mycelial mats between 5 and 40% O2 in the species except for A. ruber, A. repens, and A. amstelodami, which produced none in any concentration. As O2 decreased below 20%, spore production was progressively decreased, colony color faded to white, and cleistothecia formation was suppressed. In CO2-O2 mixtures radial growth of all species increased with each quantitative decrease of CO2. All species except A. niger grew faster in air than in 10% CO2. In contrast to N2-O2 mixtures, the fungi formed furrows, sporulation and cleistothecial formation were suppressed, and colony color changed to white in higher O2 concentrations. Fungi deteriorate stored agricultural products. The storage fungi, mostly species of Aspergillus and Penicillium, invade seeds of corn and wheat when the moisture content is above approximately 12%. These fungi may decrease germination, discolor embryos, increase fatty acids, produce toxins, and cause heating of stored grains (1). According to Johnson (4), Suchsland in 1891 isolated bacteria from sweating tobacco and inoculated them back to tobacco. Other reports prior to 1950 of molds of stored tobacco are reviewed by Wolf (13), Lucas (5) and Lukic (6). Welty and co-workers (10)(11)(12) reported 73% of the fungi isolated from moldy flue-cured tobacco to be Aspergillus and Penicillium, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isolated more frequently from undamaged tobacco. Immediately after redrying, tobacco is pressed into bales or hogsheads and stored up to two years. Because towards the possible effects of ambient gas composition on the stored tobacco leaves. Particular attention is directed towards possible effects of low oxygen and high carbon dioxide concentrations on the growth of fungal species that commonly infest flue-cured tobacco leaves (9). The objectives of this investigation were to measure the effects of atmospheres containing various ratios of N2 to 02 and CO2 to 02 in a closed system on the radial growth and colony characteristics of 10 fungal species isolated from moldy flue-cured tobacco. MATERIALS AND METHODS The fungi, isolated from moldy flue-cured tobacco (10) with several infested grains and grown for 1 week in the laboratory. Two 5-mm discs were cut from each sporulating colony and placed in 5 ml of sterile water in a test tube, the tube was shaken, and the spore suspension was poured into the petri dishes. Melted Czapek's medium reinforced with 34 g of agar per liter was added to these dishes to a depth of approximately 5 mm and mixed with the spore suspension. After 12 hr of growth at room temperature, 5-mm discs were cut from these plates and one disc was transferred to the center of each test plate, with the disc surface bearing germinated spores placed upon the agar surface. Mycelial discs of A. tenuis and C. dolicotrichum were cut from the colonies growing on Czapek's medium. One plate of each of the 10 fungi was placed at random in a stack of 10 plates, taped together, placed in a 9.5-liter desiccator, and sealed. In studies with 5% or more of 02 in the N2 atmosphere, the desiccators were filled with the desired gas mixtures by evacuating them repeatedly and flushing with either N2 or O2 depending upon which gas was in greater concentration in the mixture. In the 50:50 mixture, N2 was used. During the final evacuation, the pressure was decreased with N2 or O2, allowing 0.76 cm on a mercury column for 1% of the gas desired. The other gas was then added until the mixture reached atmospheric pressure. Mixtures containing 5% 02 or less in N2 were commercially mixed and analyzed. In the 100% N2 treatment, 40 ml of alkaline pyrogallol was placed in the desiccator to absorb any trace of remaining 02. A beaker containing 30 ml of 20% KOH was placed in the desiccator to absorb evolved CO. In the closed-system air control, the desiccator was similarly evacuated and filled with air. Each gas mixture was replicated in five desiccators, and the closed-system air control was replicated in three desiccators. Two gas mixtures and one air control consisting of 13 desiccators composed a test. To measure growth when O and CO2 were not limiting, the fungi were grown in air flowing at 1 liter per hr. The organisms were grown at 25 C in continuous white fluorescent light. After 1 week of growth, two horizontal diameters of each colony were measured in millimeters. These values were averaged and analyzed statistically to determine significant growth differences. The initial and final compositions of various mixtures in two desiccators were analyzed with an Orsat-type gas analyzer (Fisher Scientific Co., Pittsburgh, Pa.). RESULTS Influence of N2-02 mixtures on fungal growth. The comparative growth of the 10 fungi in atmospheres of N2-02 is summarized in Fig. 1. Aspergillus flavus, A. niger, A. ochraceus, Cladosporium herbarum, and Penicillium brevicompactum did not show any significant increase in growth above 40% 02. A. amstelodami reached maximal growth in 30% 02. Alternaria tenuis reached maximal growth in 50% 02 and decreased in higher concentrations. Below 30% 02 all fungi, except A. niger at 20% 02, showed significant decreases in growth with each decrease in 02 concentration and none grew in the absence of 02. Nine species showed the greatest change in growth in the nitrogen atmosphere containing 1 to 5% 02 and A. flavus at 10 to 20% 02. The 10 species can be divided into two groups on the basis of their growth at 0.5% 02: (i) Alternaria tenuis, Aspergillus flavus, A. niger, A. ochraceus, and Penicillium brevicompactum, which grew in 0.5% 02; (ii) and A. amstelodami, A. repens, A. ruber, and Cladosporium herbarum, which did not grow in 0.5% 02, and Chaetomium dolicotrichum, which was inhibited in 1% 02. All species except A. niger grew more extensively in 30% 02 and above than their corresponding closed-system air control. However, when the growth of the 10 species was compared with that of the continuousairflow control, growth of the control was equal to the maximal growth of the fungi in the most favorable N2-02 mixtures. Influence of N2-02 mixtures on fungal morphology. In addition to differences in growth, the fungi exhibited changes in colony morphology in the N2-02 mixtures. In general, furrows in the mycelial mats were produced in the presence of 5 to 40% 02. Spore production was progressively reduced when the 02 concentration was 20% or less and ceased below 1.0% 02 concentration, except for A. amstelodami and Cladosporium herbarum, which produced mature spores in all mixtures. The colony color generally faded to white as 02 concentration decreased except for Alternaria tenuis and Chaetomium dolicotrichum, in which no color change occurred. Cleistothecial formation also was suppressed in 02 concentrations below 10% in the three species-Aspergillus ruber, A. repens, and A. amstelodami-which produced cleistothecia. Influence of C02-02 mixtures on fungal growth. The comparative growth of the 10 fungi in atmospheres of C02-02 is summarized in Fig. 2. In general, radial mycelial growth of all species increased with each quantitative decrease of CO2. At CO2 concentrations higher than 50%, the mycelial growth of all the fungi fell into two general patterns. Chaetomium dolicotrichum, A. amstelodami, A. ruber, A. repens, and Cladosporium herbarum were C02-sensitive, whereas A. niger, A. flavus, A. ochraceus, Alternaria tenuis, and Penicillium brevi-compactum were C02-tolerant. The radial mycelial growth in air in closed systems was faster than in CO2 concentrations of 10% or higher, with the exception of A. niger whose mycelial growth in 10% CO2 equalled that in air. In 100% 02 these fungi grew faster than their corresponding closedsystem air controls. The mycelial growth in flowing air exceeded that in all gas mixtures with the exception of A. niger, A. flavus, and A. ochraceus in pure O2. Influence of C02-02 mixtures on fungal morphology. The different C02-02 mixtures also affected colony morphology. In contrast to the effect in N2-02 mixtures, furrowing generally occurred in the CO2 atmosphere at higher 02 concentrations; sporulation was suppressed, e.g., A. ochraceus in 50%, A. ruber and A. repens in 30%; colony color faded to white; and cleistothecial formation was suppressed in A. ruber, A. repens, and A. amstelodami. DISCUSSION The above results agree with those of Peterson et al. (7), who investigated the influence of 02 and CO2 concentrations on stored grain and found that the fungal growth and germ damage gradually decreased as the 02 concentration was lowered. Stotzky and Goos (8) reported that CO2 and N2 completely inhibited the growth of soil microbiota as well as A. niger, A. flavus, Alternaria species, and Penicillium species and that 95% CO2 completely inhibited growth of these fungi in soil, whereas in the present study growth occurred in this mixture. As in the present study, the inoculum was allowed to germinate before the test was started; this initial advantage in growth, in conjunction with availability of nutrients in soil vs. Czapek's medium, may be responsible for the differences between the two investigations. In analyzing the C02-02 test data, it is difficult to determine whether increasing C02 or decreasing 02 of the initial mixture altered the growth of these fungi. However, when these data are compared with the N2-02 data, there is little doubt that the C02 is the important inhibitor of growth. Although oxygen is essential for fungus growth, the minimum concentration for satisfactory growth is rather low. In general, only 02 concentrations below 20% inhibited growth of the fungi. All species that were inhibited with high C02 and low 02 mixtures resumed normal growth after exposure to air. Thus, high concentrations of CO2 and low concentrations of 02 are only fungistatic. 02 and C02 altered furrow development in 7 of the 10 fungi. In the N2-02 test, high 02 reduced or eliminated formation of straight furrows. The disappearance of the furrows at the lower 02 levels was associated with reduced growth. In the C02-02 test, furrow formation was affected by both 02 and C02. The concentration of C02 had a marked effect on furrow formation and overcame the inhibiting effect of 02-The colony color of the eight species that produced spores was directly related with the amount of colored spores, pigmented mycelium, and cleistothecia. These characteristics were in turn influenced by different levels of 02 and C02. In the N2-02 mixture, when 02 became a limiting factor, some of the conidial heads that were formed were devoid of color. In general, the growth was composed entirely of vegetative mycelium at the lowest 02 concentrations permitting growth, as Follstad (2) reported for his isolates of A. tenuis and Cladosporiwn herbarum. Aspergillus flavus produced extensive vegetative mycelium at high 02 levels, which may be a result of an early physiological maturity of the culture. In the COr-02 tests, A. ochraceus cultures became vegetative at 60% CO2 and the others before their growth was inhibited. Both low 02 and high C02 tensions, therefore, affect conidial head formation and its coloration. The effect of the 80% N2-20% 02 mixture was similar to the effect of air. There were more colorless conidial heads in the A. niger and A. flavus cultures at 80% N2-20% 02 than were present in the corresponding air control. APPL. MICROBIOL. Tobacco leaves are a natural substrate for many microorganisms and are exposed to soil and air contaminants from the time the seeds germinate until the cured leaves are made into cigarettes. Few, if any, tobacco leaves are devoid of leaf spots at the time of harvest. In addition, cured tobacco leaves are highly hygroscopic, and the moisture content of tobacco therefore is often favorable for the growth of contaminating fungi and bacteria. Many of these organisms are killed by heat during flue-curing but, inevitably, as the tobacco is handled, the leaves become recontaminated. If tobacco is maintained at moisture contents below approximately 12%, there is little danger of the tobacco becoming moldy. Since flue-cured tobacco, as it is offered for sale today, may range in moisture content from 12% to 25% or higher, it must be redried to below 12% moisture to prevent or reduce fungal growth in storage. However, if such levels of moisture cannot be maintained during storage, the results from the present study indicate that tobacco might be stored under high CO2 or low 02 mixture of gases that preclude growth of fungus contaminants that frequently damage the stored leaf. This practice is used successfully in Argentina and France where huge bulks of grain are stored in CO2 (3).

v3-fos

2017-09-12T18:48:44.673Z

2011-12-06T00:00:00.000Z

32637058

s2

An Assessment of Microbiological Quality of Some Commercially Packed and Fresh Fruit Juice Available in Dhaka City: A Comparative Study Fruit juices are regarded as the most preferred non-alcoholic beverage worldwide to all age groups. In general, microbial growth in fruit juice is restricted by using preservatives and also through other environmental factors during production. Still, the presence of some indicator organisms in fruit juice is responsible for causing food-borne diseases and even death. So, from public health point of view, it is quite important to know the microbiological quality of the fruit juices available in the market. Primary objective of this study was to assess the microbial quality of fresh and commercially packed available juices collected from different locations of Dhaka city. A total of six fresh juice and nine commercially packed juice samples were collected. Standard culture techniques were followed to assess total viable count (TVC), total staphylococcal count (TSC), total Bacillus count (TBC) and total fungal count (TFC) on different culture media. The TVC varied from the range of 10 to 10 cfu/ml with the highest of 2.4 x 10 cfu/ml. A large number of staphylococci and Bacillus was also found from several samples. Total coliform and fecal coliform was found in six and five (out of fifteen) samples, respectively. Among total coliforms, Klebsiella, Enterobacter along with E. coli were detected. From all the assessment it was determined that the microbial quality of commercially packed juice was fairer than that of fresh juice collected from local market. An Assessment of Microbiological Quality of Some Commercially Packed and Fresh Fruit Juice Available in Dhaka City: A Comparative Study In recent years, the increasing consumer awareness has emphasized the need for microbiologically safe food. Since the human food supply consists basically of plants and animals or products derived from them, it is understandable that our food supply can contain microorganisms in interaction with the foods. When the microorganisms involved are pathogenic, their association with our food is critical from a public health point of view. Serious health hazards due to the presence of pathogenic microbes in food can lead to food poisoning outbreaks (1). Freshly extracted fruit juices, which have always been considered as healthful drink, may not always be safe owing to the heavy load of microbes (2). In a situation where storage of juice is indispensable may be for a short duration, then the use of simple processing techniques soon after its extraction is essential for improving the keeping quality to ensure it's safety, nutritive quality and acceptability while consumption. Preservation of fruit juice by pasteurization, refrigeration and sterilization are popular methods used to attain microbiological stability by destroying pathogenic microorganisms and to preserve the color, aroma and taste of fresh juice (3). As indicated, the sources of fruits that can be made into juices are nearly limitless in plants and the plant parts appropriate for juice. However, the fruit producers and juice processors must adhere to some very important guidelines in fruit selection and juice manufacture (4). Environmental sources of contaminating organisms of juices are carefully considered as these microbes invade the drink preparations during processing, packaging and handling (5,6). The major ingredients of the juice such as water, sugar, natural fruit pulp etc may also carry some microbial contaminants. Food-borne illness is commonly caused by certain bacteria or their toxins, which are poisonous proteins produced by these bacteria (7). The most common food borne pathogenic bacteria are Bacillus cereus, Clostridium botulinum, Escherichia coli, Shigella spp., Salmonella spp., Vibrio parahaemolyticus, Staphylococcus aureus, Campylobacter jejuni, Streptococcus pyogenes, Mycobacterium bovis, Listeria monocytogenes etc. In pregnant women, the fetus is most heavily infected, leading to spontaneous abortion, stillbirths, or sepsis in infancy (8). Contamination of juices with pathogenic microorganisms such as E. coli and Salmonella spp. has caused numerous illness and even some fatalities (9,10). The fruit producers and juice processors must adhere to some very important guidelines in fruit selection and juice manufacture. The juices contain mainly water, sugar, preservatives, color, fruit pulps and other additives ( Table 1). The presence of microbial contaminants in water, sugar and pulp may cause spoilage of the drinks or gastrointestinal diseases to the consumers (4). Industrially processed fruit juices were investigated for some physicochemical tests. They apply chemical preservatives that can inhibit all types of microbial growth. It is well known that the manufacturers commonly use Sulphur Dioxide (SO 2 ) and benzoate as preservatives in processed fruit juices. Sulphur dioxide and benzoate can significantly damage the vegetative cells. The sulphites inhibit yeasts, moulds and bacteria and are most effective as inhibitors of browning in foods (8). They also reduce the number of growth of microbes and increase the shelf life of juice products. The effect of storage temperature has also been reported in various research papers. At 4 °C nutritional values of juice were longer than 25 days in all system tested (12). In another study, while the refrigeration condition was able to stabilize Rosselle fruit flavored drinks for a week, the freezing condition was able to preserve the drinks for more than a week (13). The focus of this study was to compare the quality of fresh and commercially available fruit juices by assessing their microbial load and the presence of pathogenic bacteria. MATERIALS AND METHODS Sample collection. Fifteen samples were collected, of which 6 fresh juice samples were collected from 5 different locations of Dhaka city and other 9 juice samples were commercially packed juices available in market. The table of collection sites are given below ( Table 2). Enumeration of total bacteria. A total of 1 ml of sample was transferred with a sterile pipette to the tube containing 9ml of ringer solution to make 10 -1 dilution. In such a way, dilution upto 10 -6 was made. If in any sample countable number of colonies were detected at 10 -6 dilution, further dilution of same item were prepared and tests were repeated using ringer solution. All colonies on the dishes were counted using a colony counter and following back calculating the actual numbers of bacteria. Isolation and identification of coliform. For presumptive identification of coliform bacteria, samples were inoculated onto MacConkey agar media. The isolates were then inoculated in Brilliant Green Lactose Bile (BGLB) broth followed by streaking on Eosine Methylene Blue (EMB) agar plate. Indole test, Methyl red test and Citrate utilization test were done to differentiate among groups of coliform bacteria. All media were prepared in the laboratory according to the manufacturer's instruction. After inoculation of the samples, plates were incubated at 37 °C and colonies were obseved after 24 hours. RESULTS Microbial load in fresh juice samples. The mean bacterial count in fresh juice samples was 1.14x10 4 cfu/ml. The total viable count varied with different types of juices that ranged from 3.2x10 2 to 2.4x10 4 cfu/ml. Similarly, we found mean staphylococcal count as 7.2x10 3 cfu/ml with the range from 0 to 2.5x10 4 cfu/ml. In case of total Bacillus count, we found that the mean count was 7.2x10 3 cfu/ml and it ranged from 0 to 2.5x10 3 cfu/ml in different fruit juice samples (Fig. 1). Microbial load in commercially packed juice sample. The mean bacterial count in commercially packed juice samples was 1.8x10 3 cfu/ml. The total viable bacterial count varied with types of juices as well as different brands of same type of juices that ranged from 3.1x10 2 to 3.2x10 3 cfu/ml. Likewise, we found mean Staphylococcal count as 4.9x10 2 cfu/ml with the range from 0 to 2.3x10 3 cfu/ml. Moreover, mean Bacillus count was found 7.3x10 2 cfu/ml and it ranged from 1x10 2 to 2.0x10 3 cfu/ml in different types and brands of fruit juice samples ( Fig. 1 & 2). Total fungal count. The total fungal count was observed on potato dextrose agar (PDA) media. The single medium used for the enumeration of fungi showed a little range of growth from 2.3 x 10 2 to 9.0 x 10 6 cfu/ml (Table 3). Identification of the total coliform. The isolated coliforms were further inoculated in different biochemical media for the differentiation among the groups. On the basis of those tests, the bacteria were identified to the genus level. The result is given in the following table (Table 5). Isolation of the total coliform and fecal coliform bacteria. In the confirmatory test for the presence of coliform bacteria in the fruit juice, the samples were inoculated on the EMB agar media (Table 4). DISCUSSION Fruit juices are very popular among the people of all ages around the world. Also in Bangladesh, ready to eat packed fruit juice is becoming more and more popular as they are usually tastier than soft drinks. Advantage of packed fruit juices is that these are convenient to carry and can be kept for a considerable amount of time (14,15). Fruit juices by their very nature contain various organisms. Clearly, many of these microorganisms will be harmless yeasts & saprophytic bacteria. By detecting this bacterial load in the juice, it apparently gives an idea about the quality of the sample. Therefore, aerobic plate count or total counts of some popular juice were carried out in the present experiment. Total viable bacterial count in most of the fresh juice samples was higher than the commercially packed juice, as the highest count was found as 2.4x10 4 cfu/ml and 3.2x10 3 cfu/ml in fresh and packed juice, respectively. The scenario of total staphylococcal and Bacillus count was also similar. Though most of the juice samples showed equal or slightly higher count than the permitted count (11), these were unfavorable for consumption because six of them showed the presence of coliform. The presence of coliform in fruit juice is not allowed by safe food consumption standard (14). Coliforms were identified as E. coli, Enterobacter and Klebsiella, which can produce many systematic infections after consumption. The fungal count was also much higher in many samples and varied from sample to sample. Though the fungal isolates were not identified, colony morphology and color showed the probability of the presence of pathogenic strain and could produce mycotoxins in juice samples. The comparative study anticipated the safe consumption of commercially packed juice than the freshly packed juice marketed locally. This might be the reason of using automated machine and also some preservatives during fruit juice processing. But some preservatives of higher concentrations can be harmful for our health. Therefore studies on preservative concentrations should also be carried out. The juices that are mishandled and mistreated during preparation or storage generally cause most outbreaks. The disease agents spread by juice like drink not only harm large groups of people but also sometimes result in serious disability and death. The lack of knowledge in cleaning, safe fruit juice preparation and contamination sources can contribute to the contamination process unknowingly to the people working in fruit juice industries. It is therefore, essential for the people who handle and prepare juices, to be properly trained in safe fruit handling technique. The microbiological quality of processed fruit juices is the most important aspect to be taken care of by the manufacturer. Negligence in this area may result in serious contamination that ultimately represents a low quality product to the consumers. As these unwanted unhygienic conditions are usually due to the lack of knowledge and unawareness to the fundamental sanitary principles, it is preventable by proper training and monitoring. The government authorized institute (like BSTI) should take intensive investigation to control the microbial and chemical quality of the juices as well as the public awareness about the adulterated fruit juices should be increased.

v3-fos

2020-12-10T09:04:12.230Z

1970-02-01T00:00:00.000Z

237231658

s2

Bacterial, Fungal, and Actinomycete Populations in Soils Receiving Repeated Applications of 2,4-Dichlorophenoxyacetic Acid and Trifluralin Soil samples were collected from an untreated plot and plots receiving repeated applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and α,α,α-trifluoro-2, 6-dinitro-N,N-dipropyl-p-toluidine (trifluralin); they were then plated on media specific for bacteria, fungi, and actinomycetes. The actinomycete colony count in the trifluralin-treated plot was greater than the control, but the same as the control in the 2,4-D-treated plot. The bacterial count was lower in both treated plots. Fungal colonies in the trifluralin-treated plots were greater than the control, but not different from the control in the 2,4-D-treated plot. "' Significant changes in soil microbial populations by field rate applications of herbicides have rarely been demonstrated (1,2,4,5); however, whole populations or only bacteria were examined in these studies. This report presents data from a survey of the population of actinomycetes, bacteria, and fungi in soil plots which received repeated applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and a,a,atrifluoro -2,6 -dinitro -N,N-dipropylp-toluidine (trifluralin) over a period of years. Soil samples (25 g) were collected from two herbicide residue plots and a control plot on sandy loam soil. The mycin agar (RBSA) were used to enumerate fungi (8). Starch-caseinate agar with and without 50 jig each of Nystatin (fungicidin) and Actidione (cycloheximide) per liter was used to enumerate the actinomycetes (6, 10). The two antibiotics were used as antifungal agents and were added to the cooled media after autoclaving at 15 lb of pressure for 15 min. The more selective and less selective media were used in each case to reduce the error in counting arising from the selectivity of the media in question. Bacteria were enumerated on sodium albuminate agar (6). Portions (1 ml) from each dilution of each sample were placed into five different plates with approximately 18 ml of either VDYA, RBSA, starchcaseinate with antibiotics, starch caseinate without antibiotics, or sodium albuminate. An appropriate control for each medium was prepared. After inoculation, the cultures were incubated for 7 days at 28 C. Fungal counts were made after 4 days of incubation; bacterial and actinomycete counts were made after 7 days. Soil samples taken from the trifluralin-treated plots showed an 89 % increase in the actinomycete population as compared to the control (Table 1). There was no significant difference in the numbers of actinomycetes from the control and the 2 ,4-Dtreated plots. The bacterial population (Table 1) a Treatment means were calculated from two samples per treatment plated in quintuplet for each media and analyzed by the least significant difference value between treatment means. b Significant at 5% level. C Significant at 1% level. was 50 and 46% less in soil samples taken from the trifluralin-and 2,4-D-treated plots, respectively, than from the control. The fungal colonies from the trifluralin-treated plots were 81 % less than from the control. No significant difference in fungal counts was noted between samples from the 2,4-D-treated and control plots. The results presented here may be attributed either to a direct effect of the herbicide on the microorganisms or an effect on the relationships of the bacteria, actinomycetes, and fungi. The lower fungal population in the trifluralin-treated plot may be the result of a combination of factors. It is possible that actinomycetes may increase in numbers because of their ability to metabolize the chemical. This could result in severe competition for nutrients, in which the fungi are unsuccessful. It has been demonstrated that actinomycetes and bacteria often produce fungistatic substances which may reduce the observed numbers of fungi over a long period of time (3,7,9). Another factor involved could be direct inhibition of fungal growth by trifluralin. 2,4-D appeared to exhibit an inhibitory effect on the total bacterial population. From the data presented, it is evident that the microfloral populations in herbicide-treated soil have been altered. A change in the normal soil microfloral balance may be either beneficial or harmful to soil fertility. Thus, it seems that a more critical evaluation of the effects of soil-applied herbicides on microbial populations is needed.

v3-fos

2018-04-03T06:08:15.718Z

1970-05-01T00:00:00.000Z

45689388

s2

Bacteriological examination of commercial precooked Eastern-type turkey rolls. Studies were conducted to ascertain the bacteriological condition of commercially cooked Eastern-type (foil-wrapped-oven roasted) turkey rolls during processing and storage. After 2 weeks at 5 C, numbers of aerobes on the surface of rolls, in slices, and in whole rolls reached levels of from 1 to 10 million per cm(2) or per g. In stored whole rolls, coliform and enterococcus counts ranged, respectively, from about 10,000 to more than 1 million per g and from < 100 to more than 1 million per g. Postcooking processing operations in two plants did not significantly affect the total count of turkey rolls. Eight of 28 rolls obtained after handling and packaging contained coagulase-positive staphylococci. A variety of precooked poultry products are marketed for home and institutional consumption. Among the most popular of these items are precooked turkey rolls. Federal regulations (5) require that precooked turkey rolls be heated to an internal end-point temperature of 160 F (71.1 C). That such a procedure is effective in destroying salmonellae and coagulase-positive staphylococci in turkey meat has been demonstrated (3,6). The majority of ready-to-eat turkey rolls are cooked either by enclosing the raw roll in a fibrous moisture-proof casing and cooking in a hot-water bath, or by wrapping the roll in aluminum foil and oven roasting to the required endpoint temperature. The casing used in the former method is not removed and usually serves as the final package, so that additional handling of the meat is not required. Bacteriological studies relating to this type of roll have been reported (2, 3; G. A. N. da Silva, Bacteriol. Proc., p. 12, 1967). With the latter type, often referred to as the Eastern-type (4), plant employees remove the foil after cooking and insert the roll into a plastic casing which is closed at one end. The natural juices from cooked rolls are combined with spices and gelatin (according to the formulation of each plant) and the mixture is heated to a minimum of 71.1 C. A portion of this heated mixture is then added to the casing, containing the roll, after which a vacuum is drawn and the casing closed at the other end. Kinner et al. (4) showed that one of the major sources of bacteriological contamination of the Eastern-type roll is the natural juice-gelatin-spice mixture which is added before final packaging. They also showed that such mixtures should be heated to an end-point temperature of 82 C to reduce significantly numbers of coliforms and enterococci and to 93 C to reduce significantly the total bacterial count of these mixtures. The objective of this study was to evaluate the bacteriological condition of commercial Easterntype rolls immediately after processing and after various periods of refrigerated storage. Both intact packaged rolls (the form in which they would be stored by processors, distributors, or institutional users) and rolls from which slices were periodically removed during storage (to simulate a delicatessen or household practice) were evaluated. MATERIALS AND METHODS Stored roll studies. Packaged Eastern-type turkey rolls, each weighing between 5 and 7 lb (2.27 and 3.17 kg), were obtained from each of three commercial plants (A, B, and C). The rolls were from lots that had been processed 2 days previously and then held in the plant coolers. The rolls were placed in crushed ice in an insulated container and transported to the laboratory where they were placed in a laboratory refrigerator at 5 C. These rolls were examined bacteriologically as follows. (i) At intervals of 1, 4, 8, 11, 15, 18, 22, and 25 days, during storage at 5 C, two surface swabs were made on each of two rolls from each of the three plants. The same two rolls from each plant were sampled throughout the storage period, with different areas of the rolls swabbed on each sampling day. This was done by aseptically cutting, for each area, a flap in the casing of the roll of sufficient size to accommodate a sterile cardboard circular template which circumscribed an area of 12.3 cm2. After swabbing this area for 30 sec with a cotton swab moistened with 0.1% peptone solution, the flap was on March 21, 2020 by guest http://aem.asm.org/ Downloaded from VOL. 19, 1970 EASTERN-TY closed and sealed with transparent tape. Serial decimal dilutions were then made and plated on plate count agar and the plates were incubated at 20 C for 72 hr. (ii) Six additional rolls, two from each of the three plants, were removed from the refrigerator at 5 C after 2, 16, and 30 days of storage. Each roll was cut approximately in half by using an electric knife with a sterile blade. One-half of each roll, with casing removed, was placed in a previously autoclaved large stainless-steel blendor jar. Sterile distilled water, equal to the weight of the half roll, was then added to the jar and the roll was blended with the water at low speed for 4.75 min. During this period, the blendor was turned off for 30 sec every 15 sec to avoid overheating the blendor motor and the sample. Approximately 1 pint (473.2 ml) of the resulting homogenate was then poured into a sterile Mason jar and the remainder was discarded. Without rinsing the jar, the second half of the roll, with an equal weight of sterile distilled water, was blended in a similar manner and a pint of the homogenate was placed in another sterile Mason jar. Serial decimal dilutions of each of these homogenates prepared in 0.1% peptone water were plated on Plate Count agar (incubated at 20 C for 72 hr) for total count, on Violet Red Bile agar for coliforms (18 to 24 hr at 35 to 37 C), on M-Enterococcus agar for enterococci (48 hr at 35 to 37 C), and on Sulfite Polymyxin-Sulfadiazine agar for Clostridium perfringens (24 and 48 hr at 37 C in anaerobicjars). Salmonella determinations were carried out on 50 g of each of the homogenates. Sufficient extra strength selenite cystine broth was added to yield a mixture equivalent to 25 g of undiluted turkey roll meat to 225 ml of "normal" selenite broth. After incubating this mixture for 18 to 24 hr at 37 C, loopfuls were streaked on Brilliant Green Sulfa, Bismuth Sulfite, and SS agars. After incubation at 37 C for 18 to 24 hr, typical Salmonella colonies were picked from these plates and transferred to Triple Sugar Iron and Lysine Iron agar slants. All cultures showing Salmonella reactions after 24 hr at 37 C were tested with Salmonella polyvalent "O" and Spicer-Edwards "H" antisera. (iii) Two sequential slices of approximately equal thickness (3 to 4 mm) were removed from each of two additional rolls from each plant after 1, 4, 8, 11, 15, 18, 22, and 25 days of storage at 5 C. After sampling, the remainder of each roll was placed in a polyethylene bag and returned to the refrigerator at 5 C until the next sampling day. Each slice was weighed and, with an equal weight of sterile distilled water, blended in a Waring blendor for 2 min at low speed. Serial decimal dilutions of this homogenate were prepared and plated on Plate Count agar, and the plates were incubated at 20 C for 72 hr. Two slices from a roll on each sampling day were identified as "outer slice" and "inner slice." Additional examinations. Employing the homogenized roll procedure described above, an additional 42 rolls obtained periodically from two plants over a 2-month period were examined (on the day after processing) for total counts, salmonellae, and C. perfringens. Coagulase-positive staphylococci in these rolls were also determined by the method of Baer (1). Fourteen of the rolls were obtained immediately after cooking, 14 were obtained immediately before the addition of the juice-spice-gelatin mixture, and 14 were "final packaged" rolls. RESULTS AND DISCUSSION Total counts of the surface of the ready-to-eat, foil-roasted rolls at various intervals during storage at 5 C are shown in Fig. 1. Counts of all rolls were relatively low after the first 4 days of laboratory refrigerator storage. After 11 days (plant and laboratory holding), five of the six rolls had at least one swab count in excess of 106/cm2, and, by the 14th day, counts of all rolls were in the range of 106 to 107/cm2. After 18 days, counts remained relatively constant at 107 to 108/cm2. An analysis of variance of the swab bacterial counts revealed no significant difference among the rolls from the three plants nor between rolls within plants. With minor exceptions, the differences on any particular sampling day between swab counts on a roll were within the range of experimental error. Only rarely were differences greater than 1 log observed. This suggests that contamination is relatively uniform over the entire surface of this type of roll. Total, coliform, and enterococcus counts of rolls stored for 2, 16, and 30 days are shown in Table 1. In all cases, counts between halves of the same rolls were in excellent agreement. No salmonellae or C. perfringens were detected in any of the rolls sampled. The rate of increase in numbers of bacteria per gram of turkey slices during storage approximated that of the swab counts of the packaged roll (Fig. 2). Total counts of the outer slice were significantly greater than those from the inner slice, although the magnitude of these differences was not very great until late in storage. Since the slices included a portion of the external surface of the roll, possibly only bacteria on the external surface were determined. The higher counts of the "outer" than of the "inner" slice might be attributed to exposure of a larger surface area which would have encouraged growth of obligate aerobes transferred from the outside of the roll to the cut surface during slicing. Growth of such aerobes in the inner portion of the roll, however, where the oxygen tension is obviously much lower, would have been restricted. In a few instances, the presence of slime on the exposed surface of cut rolls stored for 3 weeks was noted. Significant off-odors were not detected, however. Additional examinations. No significant differences were found in total counts among cooked rolls obtained at the three stages of processing. The majority of rolls obtained at the three stages had counts of less than 5000/g. Salmonellae were found in 2 of 14 rolls taken directly from the oven but not in rolls obtained at the later two stages. Coagulase-positive staphylococci were found in 8 of the 28 rolls obtained at the later two stages and in 1 roll obtained directly from the oven. C. perfringens was not detected in any of the rolls examined. Results of these examinations indicate that initial bacterial contamination of the Easterntype turkey roll is relatively low. Numbers of FIG. 2. Bacterial counts of "outer" and "inner" slices of "ready-to-eat" Eastern-type turkey rolls. Sequential pairs of slices were periodically removed from stored turkey rolls. Values are means of three slices, one from each roll, from each of three plants. bacteria may increase substantially, however, during refrigerated storage at 5 C in a matter of 2 to 3 weeks. Holding temperatures employed by processors usually range from about 34 to 40 F (1.1 to 4.4 C). At delicatessen counters, however, temperatures may be nearer to 45 to 50 F (7.2 to 10 C) so that extensive growth of bacteria as demonstrated here could occur in a relatively short period of time, with the possible development of slime. The occasional finding of salmonellae and coagulase-positive staphylococci in this type of roll emphasizes the need for continuous in-plant application of effective temperature controls both with respect to cooking of the roll itself and of spice-juice mixtures added to the rolls. Stringent adherence to plant sanitation principles and practices during handling and packaging is also necessary to minimize contamination of the cooked product with food-borne pathogens.

v3-fos

2018-04-03T02:31:29.442Z

1970-06-01T00:00:00.000Z

34042743

s2

Use of 'a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef' A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 N HCI and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which re- quires more than 2 ml of 0.02 N HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min. Although the degree of freshness or spoilage of meats is often evaluated by plate counts, it is known that spoilage is not the result of bacterial numbers per se but is caused by biochemical changes brought about by the growing flora. Investigations on the mechanism of spoilage of fresh refrigerated meats over the past several years have led to the proposal of a number of techniques for assessing its presence and extent. Among these are techniques based upon the phenomena of extract-release volume (ERV), water-holding capacity (WHC), meat swelling, and viscosity (1,2,5,6). All four of these are based primarily on changes in hydration capacity of meat proteins, which is lowest for fresh meat but gradually increases as spoilage occurs. Although the pH of fresh beef is around 5.6 to 5.8 and gradually increases to as much as 8.5 when beef becomes putrid, the increase from freshness to incipient spoilage generally does not exceed 0.3 to 0.5 of a pH unit. This, along with the fact that the change is usually not uniformly distributed in a meat sample, makes direct pH measurements unsuitable for the purpose of detecting incipient spoilage. Also, beef is often judged as spoiled without any noticeable pH changes The present report describes a more direct 1 technique for detecting incipient beef spoilage by accurate titration of the basic (alkaline) substances or functions produced in beef by the spoilage flora. The technique employs the measurement of the quantity of dilute acid required to bring beef homogenates to pH 5.00. The volumes of acid have been correlated with log bacterial numbers, ERV, and pH on beef from different sources and in different stages of spoilage. MATERIAL3 AND METHODS Titrations were carried out by blending 10-g samples of beef in 100 ml of deionized water for 2 min and filtering through cheesecloth to eliminate connective tissue. Duplicate samples of the homogenate containing 2 g of meat each were titrated with 0.02 N HCI by using an autotitrator (model iT-T and ABU1, Radiometer, Copenhagen). The amount of acid required to bring the homogenates to pH 5.00 was recorded. The initial pH of homogenates was read simultaneously on the titrator. The relationships between titration and spoilage, aging, and fat content were studied employing semitendinous (ST) muscle. To study the effect of spoilage on titration values, 15-g samples of ground muscle were stored at 5 C in small beakers covered with aluminum foil. Log bacterial numbers, pH, and titration values were determined on the stored meat at 2-day intervals. For the study of aging, 15-g samples of meat were stored at 5 C in gas-impermeable plastic bags as previously described (3). The effect of fat on titration values was determined by adding 902 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from known quantities of beef fat to fat-free ST ground muscle followed by immediate titration. ERV values, log bacterial numbers, and percentage of fat were determined as previously described (1). RESULTS AND DISCUSSION The relationship between log bacterial numbers, pH, and titration values in fresh ground ST muscle undergoing spoilage at 5 C is presented in Fig. 1. During spoilage, log bacterial numbers, titration values, and pH showed a marked increase from the 2nd day of storage to the 10th; spoilage was detected on the 5th day, at which point the log bacterial count was 9.1 /g, the titra- FiG. 1. Relationship between bacterial nwnbers, pH, and titration values on ground semitendinosus muscle held at 5 C for 10 days. Beef stored in beakers and covered with aluminum foil underwent spoilage in the usual manner, whereas spoilage was delayed in beef stored in gas-impermeable plastic bags. 10 ui 9 z 0 0 8 7 tion volume was 2.9 ml, and the pH was 6.3. Beef stored in plastic bags, however, showed only a slight increase in both pH and titration values after 10 days of storage when the log bacterial count did not exceed 8.90/g. This meat was judged acceptable, even at the end of this 10day holding period of 5 C. With respect to titration volume of ground beef, the method of sampling may affect results. Since ground beef spoilage at refrigerator temperatures is largely due to surface growth, surface samples would yield higher titration volumes than those taken from the interior. To minimize this difference, the entire batch of ground beef [1 to 1.5 lb (453 to 679 g) portions] was thoroughly mixed by use of spatula, followed by the removal of test samples in a random manner. Replicate samples from ground beef treated in this manner gave results with low degrees of variation. When beef cuts are to be tested, both surface and interior portions should be mixed as for ground beef. The effect of mixing surface samples with interior samples, where there are generally fewer bacteria, is to dilute the generally higher level of titration substances present in surface samples and to neutralize any microbially produced organic acids that may be present in subsurface portions. A pattern similar to that presented in Fig. 1 is presented in Fig. 2, employing retail-store stew beef which was ground in the laboratory. Statistically, the correlation coefficients (r) between titration values and pH and between titration values and log bacterial numbers were significantly above the 1% level; however, between titration values and ERV, r was significantly above the 2% level. The effect of fat content on titration is presented in Table 1, from which it can be seen that mean values for four replicates decreased from 1.73 ml to 0.91 ml as the percentage of fat increased to 50. It was concluded from these findings that fat alone does not contribute to the acid-titratable groups in fresh beef, although there is some evidence that fat may affect titratable functions as beef undergoes spoilage. In an effort to determine the performance of titration on market meats, hamburger meat was obtained from 23 retail chain stores, and titration values, log bacterial numbers, ERV, pH, and fat content are presented in Table 2. Titration values, log bacterial numbers, ERV, and pH are given at freshness (day of purchase) and at onset of spoilage. The titration volumes of fresh hamburger meats ranged from 0.53 to 2.15 with a mean value of 1.32 i 0.36, whereas values at the onset of spoilage ranged from 1.34 to 3.41 with a mean of 2.58 i 0.56. The average time for the onset of spoilage was 5 days, with a range of 2 to 7 days at 5 C. With respect to its degree of sensitivity to the changes that occur when fresh beef undergoes spoilage, the titration volume increased by 95.4% from freshness to spoilage, based on mean values. The per cent increase with respect to bacterial numbers was 24.8, with the mean log number for fresh beef being 7.41 ± 0.61 and 9.25 i 0.56 at the first signs of detectable spoilage. With respect to ERV, the per cent increase was 44.1; the mean at freshness was 34, whereas the mean value at spoilage decreased 7.0 7.0 6.3 6.5 6.7 6.5 6.6 6.3 6.4 6.3 6.7 6.8 6.5 6.5 6.9 6.8 6.9 to 19. The r between values for titration, log bacterial numbers, and ERV at the two different times is very highly significant (P < 0.001). In an effort to determine the titration value of ground beef at the onset of spoilage, titration volumes were determined on the 23 samples of retail-store hamburger and related to the previously established ERV of 25 and log bacterial numbers of 8.50/g (1). These values are presented in Table 3. Employing an ERV of 25, the corresponding titration values ranged from 1.33 to 2.68 with a mean of 2.01 i 0.37, whereas the mean was 2.10 + 0.35 when a log bacterial number of 8.50/g was employed as reference. On the basis of these findings, ground beef that requires in excess of 2 ml of 0.02 N HC1 for titration of 1 g to pH 5.00 may be expected to be in some state of microbial spoilage. Of the 23 retailstore samples, only two required more than 2 ml of acid for titration at freshness (no. 13 and 21, Table 2). It may be noted further from Table 2 that sample 13 had a titration value of 2.15, a log bacterial count per gram of 7.91, but an ERV of 25 at freshness; whereas sample 21 had a titration value of 2.05, a log bacterial count of 8.32, and an ERV of 29 at freshness. Both of these samples of meat were apparently undergoing incipient spoilage at the time of purchase. A simplified technique for the rapid detection of spoilage in ground beef can be achieved by adding 2 ml of 0.02 N HCl/g of meat to the blended and filtered homogenate and checking the final pH of the homogenate. Using this method, when the pH is >5.0, the meat may be presumed to be in some state of incipient spoilage. Although the identification of the basic substances that are titrated by this technique is not known at this time, all available evidence suggests that they are microbially produced and their appearance is time-dependent. When fresh beef was inoculated with meat spoilage flora to log 8.5 to 9.0/g and tested immediately, titration values remained low and no signs of spoilage resulted. The homogenizing step, along with the constant stirring that accompanied titration, suggests that the substances in question are mainly nonvolatile. In a previous report from this laboratory (4), amino sugar complexes were shown to increase along with bacterial numbers and hydration capacity. The possibility exists that these compounds are at least partly responsible for the increased amounts of acid necessary to lower the pH of spoiling beef. Further research towards identification of the basic functions and their role in meat spoilage is in progress.

v3-fos

2020-12-10T09:04:12.503Z

1970-01-01T00:00:00.000Z

237232929

s2

Effect of Added Moisture on the Heat Resistance of Salmonella anatum in Milk Chocolate The heat resistance of Salmonella anatum in milk chocolate at a processing temperature (71 C) was greatly decreased by adding 1 to 4% moisture. the heat resistance of salmonellae in dried egg white is 600 to 700 times higher than in liquid egg whites. Ingredients such as sucrose can act to remove available water. Foster (1) reported at least a 10-fold increase in the heat resistance of Salmonella typhimurium when the sucrose concentration was increased from 5 to 40% at 60 C. Since one of the major differences between milk chocolate and other food products is the low moisture content, this study was made to determine the effect of additional moisture on the heat resistance of salmonellae in milk chocolate at a practicable processing temperature. At the temperature chosen for this study, 71 C, the physical properties of milk chocolate are not seriously altered. Salmonella cultures isolated from chocolate products were obtained from the Food and Drug Administration. S. anatum (FDA no. 3989) was found to be the most heat-resistant strain in milk chocolate of our collection; therefore, it was used in this study. To avoid altering the moisture content of the chocolate and to prevent osmotic shock of the cells upon inoculation, lyophilized cultures were used for inoculation. Lyophilization was accomplished by centrifuging 500 ml of a 24-hr nutrient broth culture, resuspending the cells in 40 ml of sterile skim milk, freezing, and drying the cells The log1O of the number of surviving cells was plotted as a function of time. The survivor curves were extrapolated to obtain the D value (time required to reduce the population by 1 log cycle). The D value was then plotted as a function of percentage of added moisture. The effect of added moisture on the D value of S. anatum in milk chocolate at 71 C is shown in Fig. 1. A dramatic decrease in the D value was evidenced with 2.0% added moisture, reducing the D value from 20.0 hr to 4.0 hr. D values decreased as the level of added moisture increased. However, as illustrated in Fig. 1, the change per increment of moisture was especially pronounced at the 2.0% level and below. In no instances were survivors detected after heating periods longer than the calculated D value with low initial inocula. With high initial inocula (105 cells per g of chocolate), when the chocolate was heated for periods of time greater than that calculated to assure a salmonellae-free product, viable cells were detected. A progressive loss of moisture occurred when the chocolate was heated, with the greatest loss occurring early in the heating period. For example, with 2.0% added moisture, the moisture was reduced from 3.72 to 2.50% during 2 hr of heating at 71 C, as shown in Table 1. This moisture loss may be offered as a possible explanation as to why survivor curves with large initial inocula showed a "tailing off" effect. Since only one strain of Salmonella was employed in this study, caution should be used in applying these results to all situations involving contamination of milk chocolate. With low levels of contamination, the results indicated that a short-time heating process with additional moisture could be used for recovering milk chocolate contaminated with salmonellae. By continuously replacing moisture, it might be possible to render chocolate salmonellae-free, even with relatively high levels of contamination. This investigation was sponsored by the Chocolate Manufacturers' Association of the US.A.

v3-fos

2014-10-01T00:00:00.000Z

1970-01-01T00:00:00.000Z

13473895

s2

Biochemical and morphological comparison of plasma membrane and milk fat globule membrane from bovine mammary gland. Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents. INTRODUCTION The flow of fat through acinar cells of mammary tissue is very substantial . Small fat droplets form near the basal lamella and migrate toward the apical region of the cell as they mature (3) . These globules range in size from 1 to 8 µ and average 3-4 ,z in diameter. Such globules often approach the smaller dimensions of the cells in which they originate (28) . How such large droplets are transported from the cells is a challenging question . Bargmann ABSTRACT Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids . The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components . j3-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane . Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes . Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources . Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material . Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents. mary tissue that the milk fat droplets are progressively enveloped by the plasma membrane and ultimately pinched off into the alveolar lumen . Electron micrographs of bovine mammary tissue are also suggestive of this process (12) . The Bargmann-Knoop hypothesis predicts direct derivation of the milk fat globule membrane (MFGM) from the plasma membrane . Patton and Fowkes (28) presented biochemical evidence in support of a role for the plasma membrane in enveloping the fat globule along with a rationale for the biophysics of the process . These authors explained the envelopment of the droplet as being due to London-van der Waals attractive forces estimated to be several atmospheres at distances between membrane and droplet of less than 20 A. While it has been suggested that the MFGM consists of skim milk proteins adsorbed on the fat globule surface (19, 37), a number of findings are not consistent with this view . Fat globule membrane proteins differ in amino acid composition, in physical properties (15,25), and immunologically (7, 24) from all but a minor protein component of skim milk . The lipids of the MFGM are largely polar lipids and sterols in contrast to those of the core fat, which consists principally of triglycerides (18,36) . Several enzymes generally associated with plasma membranes are found in MFGM (1,9) . Other authors propose that the fat globules in milk are surrounded by a continuous protein layer to which lipoprotein particles are adsorbed (13,14,22,34) . Brunner (5) has reviewed a number of lines of evidence which suggest that the MFGM differs at least morphologically from typical plasma membranes . Knoop, as cited by Brunner (5), did not detect a typical dark-lightdark pattern in the membrane surrounding the fat globule . Instead, she proposed that the cell membrane is quickly dissociated in the milk serum and that a new layer is formed for physicalchemical reasons . We offer here comparative biochemical and morphological evidence suggesting that MFGM is derived from the plasma membrane with accompanying structural rearrangement of the membrane constituents . Tissue and Fat Globule Fractionation Milk and mammary tissue were obtained from several cows (Holstein) and one goat (Alpine-Toggenburg cross) . Immediately before death, milk samples were drawn from the same gland from which mammary tissue was obtained . Milk samples were extracted to recover total lipids within 10 min after milking . Other portions of the milk were immediately cooled in ice and transported to the laboratory for MFGM isolation . Immediately postmortem, the udder was removed and mammary tissue recovered . Tissue was cut into thin slices and held in ice-cold 0 .25 M sucrose during transportation to the laboratory (approximately 1 .5 hr) where subsequent operations were performed at 2°C . Tissue was cut into approximately 1 cm' pieces that were washed with cold 0.25 M sucrose solution. Adipose and connective tissue were excluded insofar as possible . Cow and goat mammary tissue (30 g wet weight) were placed in 150 ml of 0 .25 M sucrose and homogenized with a Polytron 20 ST homogenizer (Kinematica, Lucerne, Switzerland) at lowest speed for 3-5 min . Debris, mitochondrial, microsomal, and postmicrosomal supernatant fractions were obtained from these homogenates according to Patton et al . (29) . The floating lipid layer formed at various stages in the sedimentations was discarded . Plasma membrane fractions were obtained from cow mammary tissue by a method based on the procedure of Neville (23) as modified by Emmelot et al . (11) . Approximately 100 g of mammary tissue were homogenized in 1 mm potassium bicarbonate with the Polytron homogenizer . This homogenate was stirred at low speed for 5 min in 600 ml of bicarbonate solution, filtered through cheesecloth, and then centrifuged at 120 g for 10 min at -2°C to remove intact cells, debris, and nuclei . The supernatant was centrifuged at 1500 g for 10 min, and the resultant supernatant was carefully removed by aspiration and discarded . The friable upper portion of the pellet was resuspended in bicarbonate buffer . The tightly packed portion of the pellet was discarded . To eliminate gross mitochondrial contamination of the resultant preparation the above step was repeated 5-7 times . Plasma membrane was isolated from the final fraction by sucrose density gradient centrifugation as described (11) . Plasma membrane was collected at the 1 .16-1 .18 density interface . Only those plasma membrane fractions judged to be greater than 80 0/0 pure by electron microscopic examination were subsequently analyzed . Fat globules were recovered by centrifuging milk at 40,000 g for 1 hr at 5°C, washed with 0 .9 0 70 saline, and subjected to one cycle of freezing and thawing (6,18) . Membranes were collected by suspending the ruptured globules in warm (35°C) 0 .9 0/0 saline and centrifuging at 40,000 g for 1 hr at 5°C . The pellet was recovered and subjected to two resuspensioncentrifugation cycles. Nascent fat droplets were recovered from tissue homogenates by collecting the lipid layer rising to the top during the first centrifugations used for recovery of plasma membrane . To free the droplets of adhering membranous fragments, they were washed with 1 mm bicarbonate, recovered by centrifugation at 40,000 g for 1 hr, subjected to one cycle of freeze-thawing, and again recovered from bicarbonate buffer as above . Lipid Recovery and Analysis Milk and total tissue lipids were extracted as described previously (26) . All solvents used were of reagent grade quality and were redistilled before use . Silicic acid column chromatography was used to KEENAN ET AL . Comparison of Membranes from Bovine Mammary Gland 81 separate polar from neutral lipids of the milk and total tissue (16) . Portions of the MFGM and cellular fractions were suspended in sucrose solution and extracted 3 or 4 times with several volumes of chloroform :methanol (2 :1, v/v) . The chloroform-rich layers were combined and evaporated to dryness in vacuo or under a stream of nitrogen at room temperature . Lipid residues were immediately weighed, redissolved in an accurately measured volume of chloroform (50 ul/mg lipid), and stored at 2°C in sealed vials until analyzed . Polar lipids were separated by thin-layer chromatography on 500-µ layers of silica gel HR (Brinkmann Instruments, Inc ., Westbury, N .Y .) . One-dimensional separations were accomplished in the solvent system chloroform : methanol : water (70 :22 :3, v/v/v) . Twodimensional separations were performed in the solvent systems described by Parsons and Patton (26) . Spots were detected on plates by exposure to iodine vapors, recovered and analyzed for phosphorus by the procedures of Rouser et al . (32) . Identity of the polar lipids of milk and mammary tissue has been established previously (10,26) . Additional evidence for the identity of lipid components was gained through the use of selective spray reagents and by cochromatography of lipid extracts with authentic reference compounds (Applied Science Labs ., Inc ., State College, Pa.) . Identity of cerebrosides was established by chohromatography with authentic cerebrosides and anthrone-positive reactions (31) of the material recovered from thin-layer plates . Neutral lipids were separated on 500-µ silica gel G plates in the solvent system petroleum ether : ethyl ether : acetic acid (90 :10 :1, v/v/v) . ,l3-carotene was separated from other components of the lipid extracts on silica gel G plates developed in hexane :benzene (4 :1, v/v) . In addition to thin-layer mobility, identity of f3-carotene was established by comparing its ultraviolet spectrum (in hexane) to that of authentic 3-carotene . Fatty acid composition of the lipid classes recovered from thin-layer plates was determined by gas chromatography of the methyl esters . Lipid components were revealed by brief exposure to iodine vapors, recovered and transmethylated by previously reported methods (29) . Visualization by exposure to iodine vapors was more sensitive and, as employed by us, yielded the same fatty acid composition as when the lipids were revealed by spraying with 2,7-dichlorofluorescein . Methyl esters were separated on a 2 .4 m by 0 .6 cm column packed with an ethylene glycol succinate polyester and operated isothermally at 170 or 190°C in an Aerograph (Varian Aerograph, Van Nuys, Calif.) Model 1520 gas chromatograph equipped with flame ionization detectors. Quantification of standard methyl ester mixtures (Supelco, Inc ., Bellefonte, Pennsylvania) indicated that the major components (greater than 5%) were being analyzed with a relative error of less than 5% . 82 THE JOURNAL OF CELL BIOLOGY • VOLUME 44,1970 Protein Analysis Portions of the plasma membrane and MFGM pellets were dissolved in a phenol, acetic acid, water, and urea solution and subjected to polyacrylamide gel electrophoresis (35) . Electron Microscopy For negative staining, portions of the plasma membrane and MFGM preparations were mixed with equal volumes of deionized water and 2% phosphotungstic acid (neutralized to pH 6 .8 with KOH), spread on carbon-stabilized, collodion-coated grids and air-dried. Portions of the same preparations were prepared for sectioning by fixation in 2% glutaraldehyde, postfixation in 170 OsO" dehydration in an ethanol-acetone series, and embedding in an Epon-Araldite mixture (8) . To insure adequate sampling, bands were collected from sucrose gradients by centrifugation by use of a swinging bucket rotor so that sedimentation was exactly perpendicular to the axis of rotation . To insure serial sections through all strata of sedimented material, sectors of the embedded pellets taken from near the pellet center were mounted in the microtome with the axis of sedimentation parallel to the plane of sectioning . Sections were then examined at intervals perpendicular to the axis of centrifugation . Specimens were observed and were photographed in a Philips EM 200 electron microscope. RESULTS Plasma membrane fractions of high purity were obtained from lactating bovine mammary tissue . Mitochondria and collagen fibrils were the only consistent contaminants observed . The fractions isolated from bovine mammary gland were adjudged to represent plasma membrane on the basis of morphological criteria . Fragments consisted of large sheets of membranes that were interconnected through junctional complexes that are also present in situ (12,20) . In thin section, high magnification electron micrographs (not shown) show the typical triple-layered structure that clearly distinguishes these membranes from endoplasmic reticulum membranes, which show a globular appearance in thin section (4) . The overall width of the membrane element was about 80-100 A . Thinner membranes (50-60 A), characteristic of the endoplasmic reticulum and nuclear envelope, were not observed in our preparations . Ribosomes were absent and, except for an occasional mitochondrion, the presence of other intracellular organelles could not be demonstrated in the plasma membrane preparations by electron microscopy either in negative stain or in thin section . After negative staining the membranes appeared as large collapsed and distended sacs embedded in an amorphous film of phosphotungstate . The membrane surfaces showed a fine granular structure on the surface with smooth edges, features characteristic of plasma membrane isolated from other sources (4) . These morphological markers, especially in negative contrast, allow one to readily distinguish the plasma membrane vesicles from other intracellular membranes and membrane fragments (4) . The degree of contamination by cytoplasmic material contained within the vesicles was not assessed but was assumed to be minimal, for the reasons discussed by Benedetti and Emmelot (4) . Approximately 12-15 mg of dry plasma membrane fraction was obtained from 100 g wet weight of mammary tissue . The major pigmented material was #carotene as identified by its identical mobility with authentic //3-carotene on thin-layer plates . The ultraviolet absorption spectra from 300 to 600 my for both sample and reference material were identical with a single maximum at 440 mµ and a shoulder at 465 mµ. Milk fat globule membranes, released and purified from isolated milk fat globules, appeared homogeneous but electron microscopic examination showed them to contain small quantities of microsome-like membrane fragments . The yield of MFGM was 12-16 mg dry weight/100 ml milk . Two-dimensional thin-layer chromatography of membrane lipids in solvent systems that separate all known polar lipids of milk and mammary tissue revealed the same qualitative distribution in both MFGM and plasma membrane (Fig. 1) . Both preparations appeared to contain the same relative proportions of cerebrosides . Mono-and dihexose cerebrosides were not observed in the lipids of cell fractions from the goat and appeared to be concentrated in the plasma membrane from bovine preparations. Detailed analyses of the phospholipid distribution of two plasma membrane fractions and the corresponding MFGM fractions revealed a remarkable similarity in distribution of all five components (Table I) . Other mammary tissue cell fractions did not show this distribution (Table II ; 28) . Comparing polar lipids from cell components, milk and total mammary tissue from the cow (Table II), sphingomyelin (relative to total lipid phosphorus) was higher in milk than the other cell fractions analyzed (Table II) . With goat mammary tissue, important differences in phospholipid distribution were noted when comparing milk and tissue fractions (Fig . 2) . In contrast, the relative sphingomyelin content of plasma membrane was high and sufficient to account for the relative amounts of sphingomyelin observed in milk (Table II), if direct origin of MFGM from plasma membrane is assumed . Disc electrophoresis of MFGM and plasma membrane total protein on polyacrylamide gels revealed an identical major protein component in each fraction (Fig . 3) . The only demonstrable difference in the two patterns was the presence of one additional minor band in the MFGM fraction (identified by the arrow in Fig . 3) . All phospholipids were observed to have the same principal (greater than 2 %) fatty acids when comparing plasma membrane and MFGM (Fig . 4) . Although not drastic, some variation in fatty acid composition was observed . The plasma membrane fraction analyzed was estimated to contain about 15% mitochondria whereas microsome-like material contaminated the MFGM preparations . Comparisons of the fatty acid composition of the cholesterol esters showed a much closer similarity between plasma membrane and MFGM (Table III) . Palmitate and oleate were the only acids that differed appreciably between the two fractions . Even the comparative levels of the minor acids showed this homology . Cholesterol ester fatty acids from other cell fractions were not similar to those of MFGM . Nascent fat droplets (the cell cream which rises to the top on the first centrifugations) were observed to contain small but significant amounts of phospholipid . When separated from the bulk of the neutral lipid on silicic acid columns, polar lipids constituted about 0 .5-1 % of the total fat . The phospholipid distribution of these droplets was different from that of the MFGM and plasma membrane (Table I) away from the fat during staining (Fig . 10) . In thin sections of MFGM, short rodlike profiles were observed (Fig. 8) . Thin sections of plasma membrane contained large and small vesicles with undulating profiles (Fig . 6) . The vesicular nature of the isolated plasma membrane fraction was evident in both negatively stained (Fig . 5) and sectioned material (Fig . 6), but was most clearly demonstrated by occasional preparations where membrane blebbing resulted in the formation of many small vesicles still attached to a large fragment (Fig . 9) . DISCUSSION Detailed analyses of phospholipid constituents when comparing plasma membrane and milk fat globule membrane (MFGM) of bovine mammary gland reveal a similar distribution of all components. This is strongly suggestive of direct plasma membrane origin of MFGM . However, when comparing plasma membrane and MFGM with other cell fractions from mammary gland, major differences are evident. The sphingomyelin contents of milk, MFGM, and plasma membrane are Membrane from the Lactating Cow differences. Exchange between fatty acids of lipids of the MFGM and those of the underlying core fat is likely, and there is no reason to suppose that plasma membrane lipids are uniformly distributed within the membrane . For example, fatty acids of the apical portion of the plasma membrane specifically involved in the formation of MFGM might differ from those of the remainder of the membrane by virtue of its more rapid turnover in bulk . In any event, the observed differences in fatty acids were not great enough to preclude origin of MFGM from plasma membrane . Perhaps a more critical comparison is the cholesterol ester fatty acid composition, since these constituents have been found to replenish their fatty acids only very slowly, if at all, in milk after secretion (T. W . Keenan and S . Patton, Cholesterol esters of milk and mammary tissue. In preparation) . This comparison, when made in some detail, revealed a remarkable homology of cholesterol ester fatty acids between plasma membrane and MFGM. Finally, the disc gel electrophoretic patterns of plasma membrane and MFGM are similar, each having a single major protein component . A similarly migrating major protein component is also characteristic of plasma membrane from rat liver . This band is not observed as a major component in patterns obtained for Golgi apparatus, endoplasmic reticulum, and mitochondrial preparations from rat liver and carried out in a manner identical to that used for the preparations reported here (W . Yunghans, T. W. Keenan, and D . Morre, in preparation) . The oil recovered on lysis of the milk fat globules (core fat of the globule interiors) is extremely poor in polar lipids. This is in contrast to the nascent fat droplets (cell cream) that contain small but significant amounts of phospholipids . Nascent fat represents droplets in all stages of synthesis, whereas core fat is predominantly completed product. For example, nascent fat is rich in phosphatidyl-and lysophosphatidyl choline, compounds that have been suggested to be intermediates in the formation of milk triglycerides (30) . Electron microscopic examination has shown that the nascent fat droplets do not possess a discernible membrane (2, 3) . However, there is no evidence to rule out the possibility that their sur-* Number before the colon gives the number of carbon atoms ; number after the colon gives the number of double bonds . nearly equal and at least two times that of the total tissue and higher than that of other cell fractions on a lipid phosphorus basis. The distribution of the major phospholipids in total milk and the MFGM is virtually identical (18,21,26,27) but different from that of other cell fractions . The sphingomyelin content of milk is largely derived from MFGM, and its content in plasma membrane is sufficiently high to account for that present in MFGM . On analysis of Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions from rat liver (T. Keenan and D . Morre, unpublished observations), only plasma membrane was found to have the uniquely high sphingomyelin content characteristic of MFGM and plasma membrane from the bovine . Although the close correlation in sphingomyelin content is most striking, the distribution of other phospholipids of rat liver plasma membrane is similar to that of MFGM and milk (18,21,26 Preparations contained vesicles of many sizes or long undulating membrane profiles with occasional desmosomes and junctional complexes to identify them as plasma membrane fragments . Material, the composition of which is unknown, is seen adhering to junctional complexes . Tangential cuts through large membrane fragments cause a loss of clear membrane images and give the appearance of "smudged profiles ." X 40,000 . FIGURE 7 Milk fat globule membrane fraction in negative contrast after staining with PTA . The membranes have an irregular surface but appear smooth with no evidence of a granular or fibrous texture . Much of the surface appears to repel the stain. The margins end abruptly lending a platelike aspect to individual fragments (compare with Fig . 5) . X 21,000. Fig . 6) . The membrane binds stain in a manner that causes the formation of small electron-opaque grains that resist washing of the section . X 22,000 . 90 THE JOURNAL OF CELL BIOLOGY . VOLIIME 44, 1970 faces are bounded by polar lipid and/or protein layers (3) . Our findings of phospholipids in the nascent fat droplet fraction support this suggestion. Among the components absent from the nascent fat but plainly evident in milk fat are the carotenoids. Lipids of the plasma membrane are a deep orange-yellow color, while lipids of the cell cream (nascent fat) are nearly colorless. White et al . (40) found that 0-carotene, the major carotenoid of milk, is distributed in milk fat globules in direct relation to surface area. In the present study, /3carotene was the major pigmented material encountered in bovine plasma membrane, and the plasma membrane origin of MFGM would explain the origin of ,B-carotene in milk . The qualitative and quantitative biochemical similarities of plasma membrane and MFGM are not reflected in their morphologies as revealed by the electron microscope . Electron micrographs of the isolated cell fractions presented here and in vivo studies (2, 3,12,17,20,33,38,39) show their morphologies to be different . The early observation of Bargmann and Knoop (3) that milk fat droplets are progressively enveloped by the plasma membrane and ultimately pinched off into the alveolar lumen has since been confirmed by a number of investigators (2, 12, 20, 38, 39) . Electron micrographs presented by these authors indicate that the structural integrity of the plasma membrane is maintained during envelopment of the fat droplet. Although there are conflicting views as to whether small amounts of cytoplasm are constricted along with the droplet, it is clear that large amounts of cytoplasm are not released with lipid droplets (2, 3, 20) . The mechanism of lipid secretion is highly efficient in that it does not permit mitochondria to pass into the gland lumen (29) . Electron microscope-radioautographic studies of Stein and Stein (33) show that small fragments of the rough endoplasmic reticulum, which appears to be the site of elaboration of lipid droplets, adhere to the fat globule after secretion. These fragments may be the source of the microsome-like membrane fragments observed in MFGM preparations in the present study . Whereas other membranes (including plasma membrane) tend to form vesicles when disrupted during tissue homogenization, the MFGM shows REFERENCES 1 . BAILIE, M. J ., and R . K. MORTON . 1958 . Comparative properties of microsomes from cow's milk and mammary gland . I . Enzymatic no tendency to vesiculate . MFGM is present in the isolated preparations as relatively rigid plates, whereas the bovine plasma membrane exhibits the highly deformable and vesicular morphology characteristic of plasma membranes from other sources (4) . Differences in staining and thickness of the membrane also exist for the isolated preparations but will require a more detailed study, along with more knowledge of lipid : protein ratios, to assess their significance . At present, the differences in gross morphology are sufficient to emphasize the point that if MFGM is derived largely from plasma membrane without quantitative or qualitative changes in membrane constituents, the process must at least involve a rearrangement of constituents within the membrane . Although our results are not in themselves proof, when they are taken together with information from in vivo studies (2, 3,12,20,38,39) there is little doubt that MFGM originates directly from the plasma membrane . Direct contribution of the phospholipids, cerebrosides, cholesterol esters, j3-carotene, and major protein constituents of MFGM by plasma membrane is indicated. Structural rearrangement orchangesin the lipid : protein ratio would then be necessary to account for the morphological differences between MFGM and plasma membrane . From a purely physical standpoint, it is reasonable to assume that the plasma membrane, which is relatively stable in the predominantly aqueous environment of the cytoplasm, would undergo a rearrangement when it comes in contact with the less polar, more hydrophobic fat droplet . The London-van der Waals attractive forces between the droplet and membrane appear to be sufficient to effect the reorientation of membrane constituents (28), but a more precise understanding of the nature of the reorientation must await further study.

v3-fos

2020-12-10T09:04:11.453Z

1970-12-01T00:00:00.000Z

237229345

s2

Stimulation of Malo-Lactic Fermentation in Eastern Grape Musts Induced malo-lactic fermentation was stimulated in Eastern grape musts by the addition of a new fermentation enhancer product. terial conversion of the inherent malic acid of a wine to lactic acid and carbon dioxide. MLF is initiated by certain strains of lactic acid bacteria at some time after the alcoholic fermentation. The primary effects of this secondary fermentation are a natural deacidification of the wine and an apparent improvement in flavor due to unknown biochemical changes associated with the bacterial metabolism (2). A major problem in producing quality table wines in the Eastern United States is overcoming the high natural acidity of the grapes. Several artificial methods of reducing acidity are currently used, but all are somewhat detrimental to wine quality. Therefore, the stimulation of MLF appears as an attractive solution to this problem. Unfortunately, the initiation of MLF in Eastern table wines is not predictable. In years when acidity of the grapes is high and MLF would be most beneficial, the low pH inhibits the bacteria responsible for the fermentation (3). Also, hotpressed musts (crushed grapes pressed after heating to 62 C) which are often used for red table wine production in the East are probably less susceptible to MLF. Reports have indicated that grapes fermented "on the skins" are more susceptible to MLF than musts pressed at crush (2). This report is concerned with preliminary studies testing a new product as a potential stimulant to induced MLF in Eastern grape musts. The product, termed a bioenhancer, was developed by CPC International, Inc. (Argo, Ill.), and is currently being tested as a stimulant in fermented food systems. Four hot-pressed grape musts (Table 1) dilution bottles equipped with water seals. Two bioenhancers (code no. 11 and 12) were added to each must, except controls, in a concentration of 0.20% (w/v) on a dry-weight basis. The sugar content of each must was adjusted to 220 Brix with sucrose, and they were pasteurized at 80 C for 15 min. After cooling, each lot was treated with 50 ug of sulfur dioxide per ml and equilibrated overnight. After the addition of a 1% yeast inoculation (Montrachet no. 522), each lot was inoculated with a 1 % culture of Leuconostoc b Mixture of 50% "Concord" and 50% "Couderc 7120." c Mixture of 50% "Concord" and 50% "Couderc 7120." Acidified with 6 N HCI to pH 3.0. citrovorum (ML 34) grown in grape juice as described by Kunkee (2). MLF was followed by paper chromatographical analysis of the organic acids (2) as the wines were incubated at 21 C. Completion of the fermentation was based upon the complete disappearance of malic acid on the chromatograms. The results recorded in Table 2 demonstrated that the bioenhancers had a profound effect on stimulation of MLF. In every case, MLF was complete at least twice as rapidly as the corresponding control lot. The bioenhancers were even effective at pH 3.0 where no MLF was ob- served in the control lots after 120 days. In addition, both bioenhancer preparations appeared to be equally effective to stimulate MLF. The results of this study indicate that the bioenhancers possess a great deal of potential as agents for the stimulation of MLF. However, much additional experimentation will be necessary to demonstrate that these agents can be effective under normal commercial cellar practices and also have no detrimental effects on the wine quality. We thank E. M. Mikolajcik and S. A. Watson for their assistance.

v3-fos

2020-12-10T09:01:29.155Z

1970-01-01T00:00:00.000Z

235366880

s2

Antimicrobial Action of Some Citrus Fruit Oils on Selected Food-Borne Bacteria The antimicrobial properties of essential oils, terpineol, and orange oil, in particular, varied according to the type of bacteria tested. Terpineol and other terpeneless fractions of citrus oils appeared to have greater inhibitory effect on food-borne bacteria than the other citrus oils or derivatives. Gram-positive bacteria were, in general, more sensitive to essential oils than gram-negative bacteria. Terpineol extended the shelf life of commercially pasteurized skim milk, low-fat milk, and whole milk for more than 56 days at 4 C. Orange oil extended the shelf life of skim milk and low-fat milk for the same period. The antimicrobial properties of essential oils, terpineol, and orange oil, in particular, varied according to the type of bacteria tested. Terpineol and other terpeneless fractions of citrus oils appeared to have greater inhibitory effect on food-borne bacteria than the other citrus oils or derivatives. Gram-positive bacteria were, in general, more sensitive to essential oils than gram-negative bacteria. Terpineol extended the shelf life of commercially pasteurized skim milk, low-fat milk, and whole milk for more than 56 days at 4 C. Orange oil extended the shelf life of skim milk and low-fat milk for the same period. The use of antimicrobial agents to kill bacteria in processed food or at least to inhibit growth of bacteria could, in conjunction with the prevention of contamination and adequate detection methods, help control the wholesomeness of food. As early as 1924, Schobl (21) and Schobl and Kusama (22) compared the disinfecting power of chaulmoogra oil with that of vegetable and animal oils. Maruzzella and co-workers (8)(9)(10)(11)(12)(13) extensively studied the potential of essential oils as antimicrobial agents in perfumes and cosmetics or against wood pathogens. Piacentini (18) reported that essential oils of bergamot, orange, and lemon are more antiseptic than phenol. Hahn and Appleman (4) studied the role of orange oil in the microbiological quality of frozen orange concentrate, whereas Murdock and Allen (14) showed that the addition of orange oil or d-limonene increases the preservative properties of sodium benzoate. Marth (7) reviewed the literature on the potential use of essential oils in the inhibition of Salmonella, whereas Nagy and Tengerdy (15,16) and Oh et al. (17) studied their effect on rumen bacteria of sheep and deer. Pirie and Clayson (19) found that liquid seasonings containing emulsified essential oils have little or no antibacterial action, because partitioning between the oil and aqueous phases reduces the concentration of the antiseptic constitutent of the essential oil. Essential oils are used for flavoring in food and are generally accepted by the Food and Drug Administrations as additives in certain type of foods. Gunther (3) reported that, because of the complexity of essential oils, no general statement can be made as to their antimicrobial properties. Furthermore, Subba et al. (24) and Dupaigne (1) showed that the degree of inhibition of bacterial growth by citrus oils varies considerably with the bacteria tested. According to Ingram et al. (5), an ideal antimicrobial agent in food should be effective, not only against food-poisoning and food-infecting species, but also against spoilage organisms. This paper reports the antimicrobial properties of some citrus oils and derivatives on a number of common food-borne bacteria. Furthermore, the use of citrus oils in fluid milk products to increase their shelf life also will be discussed. Essential oils were dispersed aseptically in sterile nutrient broth by sonic treatment for 10 min in a Sonifier Cell Disrupter (Branson Instruments, Inc., Melville, N.Y.) and then dispensed in 9.9-ml portions into screw-cap tubes. A 0.1-ml amount of a 24-hr culture was added to tubes containing the oil-nutrient broth mixture and to tubes with nutrient broth only; all were mixed with a Vortex mixer. This gave an initial inoculum of approximately 107/ml. The tubes were incubated either at 35 to 37 C or at 20 C, depending on the type of bacteria tested, and enumerated on plate count agar (PCA), unless specified otherwise, after 24 and 48 hr of incubation. Plates were incubated for 24 to 48 hr at 37 C or for 48 to 72 hr at 20 C. The oilnutrient broth mixture was also tested for sterility during the test period. MATERIALS AND METHODS Calculation of antimicrobial property of essential oils. The effect of essential oils on growth was calculated as a percentage of growth inhibition by comparison between growth of bacteria in nutrient broth and growth of the same bacteria in nutrient broth and oil mixture. When the growth inhibition was 100%, we calculated a percentage of reduction of initial load by comparison between the original inoculum and the count in the nutrient broth and oil mixture. RESULTS AND DISCUSSION Inhibition in solid medium versus liquid medium. Antimicrobial properties of essential oils have been studied almost exclusively by the filter paper-disc method (8-13, 23, 25). Recently, Subba et al. (24) measured the bacteriostatic properties of citrus oils by incorporation of the oils in a solid agar medium and calculating the inhibition by comparison with the growth of bacteria in the same medium without added citrus oil. We incorporated 1,000 Aliters of orange oil per liter in the plating agar and inoculated the agar with cultures of Salmonella oranienburg, S. montevideo, S. typhimurium, S. heidelberg, and S. senftenberg (775W). Calculation of percentage of inhibition of growth showed inhibition in the 40 to 50% range. When the orange oil was incorporated in nutrient broth and the mixture was inoculated with the same cultures as above and incubated for 24 hr at 37 C, inhibition of growth as measured by PCA was over 90 %. Concentrations of essential oil. Essential oils impart a flavor to food, the intensity of which increases with increases in concentration. Taste panel tests showed that a maximal concentration of 1,000 ,uliters of orange oil per liter in skim milk was acceptable. Data not shown here indicated that increasing the concentration of orange oil from 1,000 to 10,000 ,uliters per liter increased the growth inhibition effect of orange oil against S. senftenberg (775W) and Escherichia coli but not against Staphylococcus aureus and Pseudomonas species (no. 18) isolated from refrigerated pasteurized milk. Decreasing the concentration of orange oil from 1,000 to 10 ,lliters per liter reduced its growth inhibition effect against these four bacteria. Terpineol at 1,000 uliters per liter completely inhibited the growth of all four bacteria and, with the exception of Pseudomonas species (no. 18), totally reduced the initial inoculum. Increasing the concentration of terpineol to 10,000 ,uliters per liter did not further affect the inhibition of growth or the reduction of initial inoculum. On the other hand, reduction of the concentration of terpineol to 600 ,uliters per liter a Percent reduction of initial bacteria load was calculated when per cent growth inhibition was 100. bd-Limonene (Eastman) was purchased more than 4 years ago. d-Limonene (G & G) was obtained recently. reduced drastically the growth inhibition of the four bacteria. A concentration of 1,000 ,uliters of essential oils per 1 liter of solution was used in all our subsequent experiments. Size of inoculum. Variations in the initial inoculum of from 104 to 108 bacteria of S. senftenberg (775W), E. coli, and S. aureus did not change the percentage inhibition of these cultures by 1,000 ,uliters of orange oil per liter. With Pseudomonas species (no. 18), the inhibition of growth by orange oil increased from 30 to 90% when the inoculum was increased from 104 to 108. The possibility of an optimal concentration of bacteria for a given amount of oil could explain the increase in growth inhibition. An initial inoculum of approximately 107/ml was selected for the remainder of the study. Effect of various essential oils and derivatives. Growth of S. aureus was completely inhibited by all of the citrus oils and derivatives tested, with the percentage of reduction of initial inoculum ranging from 100% for the terpeneless citrus oils and terpeneless derivatives to a low of 67% for lemon oil (Table 1). On the other hand, only terpineol reduced the initial inoculum of Pseudomonas species (no. 18). Inhibition of growth of Pseudomonas species (no. 18) ranged from 87% for orange oil to 100% for terpineol. The terpeneless fractions of orange, lemon, and lime considerably reduced (86 to 100%) the initial inoculum of S. senftenberg (775W), E. coli, and S. aureus. The d-limonene that had been stored for more than 4 years showed greater activity against S. senftenberg; this confirms Zukerman's report (26) that oxidized d-limonene is more inhibitory than the freshly distilled product. However, in our study, artificially oxidized orange oil had no more inhibitory effect on bacteria than fresh orange oil. Antimicrobial effects of orange oil and terpineol on pure cultures of bacteria: SalmonelLa group. Inhibition of growth by 1,000 l.liters oforange oil per liter ranged from 84% for S. enteritidis to 99% for S. gallinarium and S. norwich. Incubation for 48 hr showed no increase in inhibition over the 24-hr results. Orange oil did not reduce the initial bacterial load in any case. On the other hand, terpineol completely inhibited the growth of 24 serotypes of Salmonella tested and also totally reduced the initial inoculum. Enterobacteriaceae other than Salmonella. Orange oil was bacteriostatic for all other tested Enterobacteriaceae. Inhibition of growth ranged from 88 % against Aerobacter aerogenes to 100% against Alcaligenes faecalis and against 2 strains of the 12 E. coli tested. Orange oil also caused a reduction of the initial inoculum of those two strains of E. coli and completely killed the initial inoculum of A. faecalis. Inhibition of growth in the Proteus group was approximately 95 %. As with the Salmonella group, terpineol completely inhibited the growth of all tested bacteria and totally reduced their initial inoculum with the exception of Serratia marcescens, against which the effect was 89% inhibition of growth. Pseudomonas and gram-negative food spoilage organisms. Orange oil in all cases inhibited the growth ofbacteria (10 to 100 %); the initial inoculum of one strain of P. fluorescens was completely killed, whereas a 75% reduction occurred in the initial inoculum of a P. aureginosa. Terpineol inhibited the growth of bacteria in various degrees (94 to 100%). Terpineol was less effective in reducing the initial bacterial load of Pseudomonas species and Achromobacter, as well as that of most of the gram-negative spoilage psychorphilic organisms isolated from milk and dairy products, than against the Enterobacteriaceae. Gram-positive bacteria. Orange oil completely inhibited the growth of various strains of S. aureus. The reduction of initial inoculum was more variable and ranged from 0 to 100%. Orange reduced the initial inoculum of Bacillus subtilus by 100% and B. cereus by 98%. Terpineol completely killed the initial inoculum of all tested gram-positive bacteria. Use of essential oils to extend the shelf life of milk. Fruit-flavored milk products are not new. Gudnason et al. (2) indicated that their acceptability is good. Some, like Gudnason et al. (2), have used syrup and commercial fruit juice concentrate for their fruit-flavored milk; others like Kosikowski (6) have used fruits or fruit concentrate as flavoring for buttermilk. We established earlier in this paper that essential oils such as orange oil or terpineol dispersed in a liquid medium inhibit the growth and even, in some cases, reduce the initial bacterial load of a variety of food-borne pathogens and spoilage organisms. The possibility of a new type of fluid milk product flavored with essential oil and with extended shelf life was tested. Commercially pasteurized fluid milk products, purchased from retail outlets in the Beltsville, Md. area, were homogenized with terpineol to a final concentration of 1,000 ,liters per liter and stored in sterile containers for 52 days at 4 C. At regular intervals, the bacterial population was estimated by plating on PCA and incubating at 20 C for 72 hr. The effect of terpineol on fluid miilk products stored at 4 C varied with the fat content of the product tested. For skim milk, the difference in viable count between milk with and without terpineol after 42 days of storage was 7 log cycles; for low butterfat milk (2% B.F.), the difference was 4 log cycles; for whole milk (3.5% B.F.), the difference was 3 log cycles; finally, there were no differences in bacterial estimate for half cream-half milk (12% B.F.) and for chocolate milk. When orange oil was mixed with various milk products to a final level of 1,000 ,uiters per liter and then incubated at 4 C for more than 52 days, the differences in counts between the control and the samples were of a lower magnitude than those obtained with terpineol. For instance, no difference was obtained in counts for whole milk (3.5 % B.F.). One or two log differences were obtained for skim milk or 2% B.F. fluid milk. As with terpineol, no difference was present in chocolate milk. Taste panel evaluation of the milk products after 16 days of storage at 4 C indicated that the milk products with orange oil were favored over those without the oil. Whole milk and chocolate milk, with or without orange oil, curdled on the 28th day and were removed from the storage test. All the other milk products tested after 28 days were categorized as "spoiled" when stored without orange oil, whereas those with orange oil were judged "acceptable." After 60 days at 4 C, skim milk mixed with orange oil did not present characteristic signs of spoilage. After 73 days of storage, a slight odor described as "stale" or "unclean" was detected. Terpineol, although more effective than orange oil in controlling bacterial population of commercially pasteurized milk, gave the milk a strong "medicinal" flavor which was not acceptable to our taste panel. A large number of essential oils and derivatives were dispersed in skim milk and presented to our panel, who selected only orange, lemon, and grapefruit oils as "acceptable." Comments indicated that the traditional "chalky" taste of skim milk was improved by the addition of these oils. The mechanism of the antimicrobial effect of essential oils is not known, although lipid solubility and surface activity of the oils at the surface of the bacteria have been implicated (15,20). The addition of citrus oils to milk, in addition to improving the shelf life, offers the possibility of introducing a wide range of new flavors to milk products.

v3-fos

2018-04-03T01:17:54.468Z

1970-08-01T00:00:00.000Z

23868042

s2

Mycotoxin from a blue-eye mold of corn. High-moisture yellow dent corn became heavily molded by Penicillium martensii after storage for 6 months at 1 C. Mice ingesting corn molded by P. martensii died within a few days. The toxin was isolated and identified as penicillic acid. Large quantities of the toxin accumulated over a 3-month period on artificially inoculated corn incubated at temperatures between 1 and 15 C. At higher temperatures, the toxin disappeared within 45 days. High-moisture yellow dent corn became heavily molded by Penicillium martensii after storage for 6 months at 1 C. Mice ingesting corn molded by P. martensii died within a few days. The toxin was isolated and identified as penicillic acid. Large quantities of the toxin accumulated over a 3-month period on artificially inoculated corn incubated at temperatures between 1 and 15 C. At higher temperatures, the toxin disappeared within 45 days. Recent changes in agricultural technology have resulted in the widespread practice of harvesting high-moisture corn by picker-sheller. The corn frequently has a moisture level in excess of 20% and has sustained considerable physical damage. Such conditions are ideal for rapid molding. After harvesting, the corn must either be dried to a moisture level safe for storage or be ensiled and used for animal feed. Several commercial firms in this country and abroad have considered refrigerated storage of high-moisture corn to be an attractive alternative to drying or ensilng (6). This method of preservation allows the corn to be used for feed, or it can be channeled to the milling industry. Even under refrigeration, however, corn is known to mold, and guidelines for cold storage have been proposed (13). During storage of high-moisture yellow dent corn for 6 months at 1 C, the mold flora shifted, and Penicillium martensii Biourge predominated. This paper reports the isolation and identification of a mycotoxin from corn molded by P. martensli NRRL 3612 and the temperatures at which toxin production occurs. MATERIALS AND METHODS Source of corn from which P. martensii was isolated. This corn was a commercial single-cross yellow dent grown on the Agricultural Engineering farm, University of Illinois, Urbana. The corn was planted on 5 June and harvested by picker-sheller on 29 October 1968. The moisture level at harvest was 25% and was determined electrically and by oven drying. Microbiological exanination of corn. Fifty-gram samples of corn were shaken vigorously in 450 ml of sterile distilled water containing 25 g of sand, and serial dilutions were then made. Bacterial counts were I Presented in part at the 70th Annual Meeting of the American Society for Microbiology, Boston, Mass., 26 April-i May 1970. determined on plate count agar containing 30 1g of Acti-Dione per ml (Upjohn; cycloheximide) and mold counts on yeast extract-tryptone agar (5) containing 100,g of Achromycin per ml (Lederle; tetracycline KCI). Bacterial colonies were counted after incubation for 3 days at 32 C; molds and yeasts, after incubation for 5 days at 28 C. Animal toxicity tests. Molded corn was extracted as described below, and, after solvent removal, the residue was dissolved in propylene glycol. The extracts were tested on mice by intraperitoneal injection. Fermentation and toxin isolation. Sizable quantities of the toxin were obtained from a liquid medium. Fifteen liters of Czapek-Dox broth (9) supplemented with 0.5% yeast extract was distributed into 30 Fernbach flasks (2.8 liter), autoclaved, inoculated with an aqueous spore suspension of P. martensii, and incubated statically for 12 days at 25 C. The mycelium was removed by filtration, and the supernatant was concentrated to 0.5 liter in a vacuum evaporator. The concentrated supernatant was extracted twice by using 1 liter of chloroform for each extraction. The chloroform extracts were combined, and most of the solvent was removed by flash evaporation. After the residual oily liquid was added to 20 volumes of pentanehexane, the precipitate was recovered by filtration and then redissolved in boiling water. On cooling, white crystals precipitated. These were recovered, air-dried, and recrystallized from benzene twice, as fine needleshaped crystals (total recovery about 130 mg). Later, considerably better yields were obtained by the use of Raulin-Thom medium (4) in place of Czapek-Dox supplemented with yeast extract. Toxin identification. The molecular weight and elemental formula of the toxin were determined with a Euclid high-resolution mass spectrometer. Melting points were obtained with a Mettler FPI melting point apparatus, and excitation and emission spectra were recorded with an Aminco-Bowman spectrophotofluorometer. Ultraviolet absorption spectra were determined with a Beckman DB-G spectrophotometer. Production of toxin on corn. Fifty-gram quantities of corn were placed in 300-ml Erlenmeyer flasks with 100 ml of distilled water and autoclaved for 15 min at 121 204 MYCOTOXIN FROM BLUE-EYE MOLD C. After autoclaving, the excess water was decanted, and the flasks, stoppered with cotton plugs, were then autoclaved for 20 min. Each flask was inoculated with 1 ml of spore suspension made by suspending the spores of P. martensii from a 10-day-old slant in 50 ml sterile distilled water. The flasks were incubated at -4, 1, 5,10,15, 20, 25, 30, 32, 35, and 37 C. Isolation and assay of toxin. The toxin, penicillic acid, was assayed fluorodensitometrically by the method of Ciegler and Kurtzman (in preparation). Briefly, the method involves thin-layer chromatography of the unknown with known amounts of standard on silica gel (solvent, chloroform-ethyl acetateformic acid, 60:40:1, v/v) followed by exposure of the plate to concentrated ammonia. Penicillic acid is excited at 350 nm and fluoresces at 440 nm. The degree of fluorescence was determined with a Photovolt model 530 densitometer equipped with an automaticscanning thin-layer plate stage and a recorder equipped with an integrator. A standard curve is prepared for each analysis, Beer's law being followed between 1 and 9 ,ug of penicillic acid. The concentration of unknown is determined from the standard curve, taking into account the dilutions involved. For most assays, 50 g of molded corn was extracted with 250 ml of chloroform-methanol (90:10, v/v) in a Waring Blendor for 3 min. The first 50 ml of solvent, after recovery by filtration through anhydrous sodium sulfate, was analyzed for penicillic acid. RESULTS Yellow dent corn with a moisture level of 25 % was stored for 6 months at 1 C in sealed screwcap l-quart (ca. 1.1 liters) glass jars. Before storage, the penicillia numbered about 8,000/g, but after storage the count increased to 1,800,000/g (Table 1). Although several species of Penicillium were present before storage, only one species, P. martensii, was recovered after storage. Initially, P. martensii caused blue-eye of the kernels but eventually spread over the surface of the entire grain. Intraperitoneal injections of extracts of corn molded by P. martensii were fatal to mice in a (2) Authentic penicillic acid. matter of minutes. Mice fed the molded corn died in 3 to 5 days. The toxin was identified as penicillic acid by analyzing both authentic penicillic acid and the isolated product. High-resolution mass spectroscopy gave m/e 170.06 and an elemental formula of C8H1004 (Fig. 1). The melting point was 84.2 to 84.8 C with no depression on admixture with authentic penicillic acid. The ultraviolet absorbance in methanol showed a single peak at 221 nm with shifts to 224 nm on acidification with 0.02 N HCI and to 293 nm in 0.02 N NaOH (Fig. 2). After reaction with ammonium hydroxide, the keto form of the acid fluoresces with excitation at 350 nm and emission at 440 nm. The toxin was co-chromatographed with authentic penicillic acid in several solvent systems and both compounds, after reaction with phenylhydrazine in ammonia, gave identical excitation spectra (Fig. 3). Production of penicillic acid on corn by P. martensii was favored by low temperatures. Greatest production (12.7 mg/g) was at 5 C after 88 days, but nearly two-thirds of this amount occurred at 1 and 10 C ( Table 2). After 83 days, penicillic acid was also detected at -4 C. Considerable synthesis occurred at 15 and 20 C, but the toxin disappeared after 45 and 90 days, respectively. Only small amounts of toxin were detected at 30 and 32 C, and there was no growth or toxin production at 35 C. DISCUSSION Blue-eye is a storage disease of corn caused by several species of Penicillium. These molds grow over the embryo but under the seed coat. When sporulation occurs, a blue or blue-green color appears over the embryo because of the color of the spores. Koehler (7) reported P. notatum Westling, P. viridicatum Westling, P. palitans Westling, and P. cyclopium Westling to cause blue-eye; Semeniuk and Gilman (11) list P. expansum Link; and Semeniuk (10) added P. rugulosum Thom and P. chrysogenum Thom. Our report is the first of blue-eye being caused by P. martensii. However, the similarity of P. in it being confused with the latter. Penicillic acid was first isolated by Alsberg and Black (1) from P. puberulum Bainier. This culture, isolated from corn, produced sufficient penicillic acid on Raulin's medium to be fatal to mice and guinea pigs. Murnaghan (8) considerably expanded this early work in his studies of the pharmacology of penicillic acid. The intravenous LD50 of mice was 5 mg/20 g, and the mean lethal dose when given orally was 12 mg/ 20 g. Penicillic acid had a digitalis-like action on the heart of the frog, the rabbit auricle, the perfused heart of the cat, and a very weak action in heart-lung preparations of the dog. A dilator action on systemic blood vessels was also found and included the coronary and pulmonary vessels. In our investigation, the greatest accumulation of penicillic acid on corn inoculated with P. martensii occurred at temperatures of 10 C and below. Production at 15 and 20 C was also high, but the toxin disappeared within 45 and 90 days, respectively. Temperatures above 25 C were decidedly unfavorable for production. Several interpretations can be made of these data. Degradation of toxin at the higher temperatures may result from a nonspecific autocatalytic process or may be enzymatic; at lower temperatures, the rate of production may exceed that of degradation, or the degradation process, whether nonspecific or enzymatic, may not function. This facet of our data requires additional experimentation. Mycotoxins produced on grains at low temperatures have previously been reported and became a serious problem in the Soviet Union in the early 1930's (3). A human disease now known as alimentary toxic aleukia (ATA) became widespread as a result of the consumption of moldy grain which had overwintered in the field. Various species of Fusarium and Cladosporium were apparently responsible for ATA, with greatest toxin production occurring at -10 to 0 C. Similarly, greatest production of T-2 toxin by Fusarium tricinctum (Corda) Sacc. emend. Snyder and Hansen, which has been associated with moldy corn toxicoses, occurred at low temperatures (2). The importance of penicillic acid as a mycotoxin on high-moisture corn stored at low temperatures must be further assessed by additional field studies and animal feeding trials. Furthermore, toxin production may not be limited to P. martensii. Other fungi producing blue-eye, such as P. chrysogenum, P. palitans, and P. rugulosum, have been shown to grow at 0.5 C (12). Their toxin-producing ability at various temperatures is currently under study in our laboratory.

v3-fos

2018-04-03T05:24:16.963Z

1970-01-01T00:00:00.000Z

42840504

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Survey of Tall-Fescue Pasture: Correlation of Toxicity of Fusarium Isolates to Known Toxins Several aspects of fescue foot in cattle suggest that this disease is caused by fungi growing on fescue grass. Certain fungi isolated from winter pasture yield toxins when grown on synthetic medium. Most of these toxin producers belong to the genus Fusarium. All but 1 of the 21 toxic and 7 questionably toxic Fusarium isolates produce either 4-acetamido-4-hydroxy-2-butenoic acid γ-lactone, or 4β, 15-diacetoxy-8α-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3α-ol, or both. Several aspects of fescue foot in cattle suggest that this disease is caused by fungi growing on fescue grass. Certain fungi isolated from winter pasture yield toxins when grown on synthetic medium. Most of these toxin producers belong to the genus Fusarium. All but 1 of the 21 toxic and 7 questionably toxic Fusariwn isolates produce either 4-acetamido4hydroxy-2-butenoic acid 'y-lactone, or 43,B15-diacetoxy-8ac-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3a-ol, or both. Tall fescue (Festuca arundinacea Schreb.) pasture, often used as a winter forage, occasionally causes a disease known as fescue foot in grazing cattle. The sporadic, seasonal, and regional occurrence of this disease suggests that it is caused by a fungus or fungi growing on the grass (4). Keyl et al. (2) isolated Fusarium tricinctum NRRL 3249, which was toxic in both a rabbit skin and mouse assay. When cultured in the laboratory on natural or synthetic media, this fungus produced at least three toxins: 4-acetamido-4-hydroxy-2-butenoic acid 'y-lactone (I), 4,B, 15-diacetoxy-8a -(3-methylbutyryloxy)-12 ,13epoxytrichothec-9-en-3a-ol (II), and a third not yet identified (3). In 1967, grass was collected from a fescue pasture in Missouri where 11 out of a herd of 100 cattle were severely affected with fescue foot. Samples were taken for mycological examination from six different areas within this pasture, plus three other samples from nearby fields. One of the three was orchard grass (Dactylis glomerata L.), intended as a control, and the other two were tall fescue samples from a farm having a history of being toxic each year. Since there were no cattle on this second fescue pasture, we do not know if the grass was toxic at the time of sampling. The 200 fungal isolates obtained from these nine grass samples represented 19 genera, the most abundant being Fusarium, Epicoccum, Cladosporium, and Alternaria (4). Of these 200 isolates previously tested, culture extracts of 25 were toxic to mice; 23 of the toxic extracts were from isolates of the genus Fusarium. Another 29 of the fungi were questionably toxic, and 9 of these were from the genus Fu-sarium. Three of the toxic Fusarium strains were isolated from orchard grass (4). The present study was undertaken to see whether toxins I and II accounted for toxicity of the Fusarium isolates and to confirm their toxicity to the mouse. MATERIALS AND METHODS Toxins I and H were detected qualitatively by thinlayer chromatography (TLC) of fungal extracts. Ethyl acetate extracts of Sabouraud's agar cultures were spotted on Silica Gel G plates and developed, by Honegger's sandwich method (1), with ethyl acetate-toluene (3:1). After the plates were air-dried, they were sprayed with concentrated sulfuric acid and heated at 125 C. Toxin I has an RF of about 0.2 and forms a tan to brown trailing spot. Toxin II has an RF of about 0.6 and forms an oval spot that varies in color from green to dark grey. The detection limits of pure compounds I and II are of the order of 10 ,g. The 32 Fusarium strains, reported as either toxic or questionably toxic (4), were regrown at 15 C on Sabouraud's agar from lyophilized cultures prepared shortly after isolation. F. tricinctum NRRL 3249 and NRRL 3299 (strain T-2 given us by E. B. Smalley, University of Wisconsin), which usually produce toxins I and II, were also included. Extracts of the cultures were made by steeping the diced agar in ethyl acetate for several days. The extracts were concentrated, and samples were evaporated to dryness under reduced pressure. The residues were suspended in Ringer's solution, and each suspension was assayed in two male white mice (25 a 5 g). Each mouse received a single intraperitoneal injection equivalent to extract from 0.1 of a petri plate (about 2.5 ml of agar). The criterion of toxicity was death of both mice within 4 days. The toxicity was recorded as questionable if only one of the two mice died or both mice became sick during this time. 103 RESULTS AND DISCUSSION The results of the mouse assay (Table 1) agreed well with the previous assay (4). Of the 32 Fusarium strains retested, 22 gave the same results. Of the 10 assays which differed, six were changes from toxic to questionably toxic or vice versa, and two were changes from questionably toxic to nontoxic. Only two samples changed from toxic to nontoxic. These changes may reflect the heterokaryotic nature of the strains. The ethyl acetate extracts were examined by TLC for toxins I and II (Table 1). Sometimes infrared spectroscopy was used to confirm the presence or absence of toxin I (4). In all of the toxic cultures toxin I or toxin II, or both, were present. In those cultures which were nontoxic, neither of these two compounds could be detected. In one questionably toxic culture, number 14, neither toxin I nor II could be detected. The three toxic Fusarium isolates that came from orchard grass, 5, 17, and 18, contained toxins I and II just as did the isolates from tall fescue. Detection of toxins I and II does not rule out the presence of other toxins in any or all of the toxic or questionably toxic strains. All except the first three entries in Table 1 are arranged in order of increasing color density of the pigments which diffuse into agar. The data seem to indicate that pigmentation and toxicity per se are not related; neither is pigmentation nor yield of extractable material. However, pigmentation may be related to which toxin is produced. None of the dark purple cultures produced any of toxin II, although most produced toxin I.

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