Datasets:

claran

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modular-s2orc

like 1

2018-04-03T00:40:00.233Z

1973-02-01T00:00:00.000Z

13469923

s2

Effect of variations in conditions of incubation upon inhibition of Staphylococcus aureus by Pediococcus cerevisiae and Streptococcus lactis. The effects of pH, temperature, proportion of Staphylococcus aureus in the inoculum, various strains of effector organism, and various strains of S. aureus were examined for their influence on interactions between staphylococci and effector organisms in associative culture. In general, small changes in pH had little effect upon either growth of S. aureus or production of enterotoxin in associative culture. Inhibition of growth of S. aureus caused by effector organisms was much greater at 25 than at 30 C. Proportion of S. aureus in the inoculum greatly affected both growth of the staphylococci and production of enterotoxin. Only slight differences were found between strains of either effector organism or S. aureus which affected the interactions in associative culture. Previous work in this laboratory has shown that several species of lactic acid bacteria, particularly Streptococcus lactis and Pediococcus cerevisiae, were inhibitory to growth and enterotoxin production by Staphylococcus aureus when grown in association with S. aureus at temperatures favorable to the organisms concerned. Several reports (2)(3)(4)(5)(6)(7)(8) have indicated that certain conditions of incubation may significantly affect the influence of the competing organisms upon growth of S. aureus, but none report any effect on enterotoxin production. The investigation reported herein was conducted to determine the effect of variations in some conditions upon the ability of S. lactis and P. cerevisiae to inhibit growth and production of enterotoxin by S. aureus. MATERIALS Procedure. One-liter Erlenmeyer flasks containing 300 ml of all-purpose medium with Tween broth (Difco) were inoculated and incubated 48 hr in a thermostatically controlled gyratory shaker-incubator operated at 175 rev/min. The effect of the initial pH of 6.0, 6.5, and 7.0 on the growth of the cultures of S. aureus and the effector organisms was determined by adjusting the pH to these values with 1.0 N HCl or 1.0 N NaOH. The effect of incubation at 25 and 30 C was investigated. In trials involving the influence of temperature, pH, and species of effector organism, the broth in each flask was inoculated with 105 cells of S. aureus per ml and 105 cells of the effector organism per ml. To determine the importance of the relative proportion of S. aureus in the inoculum, the numbers of the effector organism in the inoculum were varied, giving initial percentages of S. aureus of approximately 10, 50, and 90%. This was accomplished by inoculating the broth in each flask with 106 cells of S. aureus per ml and then adding 101, 101, and 106 cells of effector organisms per ml to appropriate flasks. Several strains each of S. lactis, P. cerevisiae, and S. aureus were used to determine the extent of variations between strains of the organisms with regard to interactions in associative culture. Enumeration of S. Aureus. Staphylococcal populations were determined on prepoured mannitol salt agar spread plates incubated 48 hr at 37 C. Assay for enterotoxin. The microslide doublegel diffusion procedure of Casman and Bennett (1) was used for enterotoxin assays. The assays were performed on samples taken at 3-to 4-hr intervals during the first 24 hr of incubation. Reference enterotoxins A, C, and D, and corresponding antisera were obtained from the Food and Drug Administration, Washington, D.C. Reference enterotoxin B and anti-B were obtained from Makor Chemicals Ltd., Jerusalem, Israel. RESULTS AND DISCUSSION This investigation involved incubation of cultures over a period of 48 hr and examination of samples taken at intervals of 3 or 4 hr. To conserve space, only maximum populations of a APT broth is all-purpose medium with Tween (Difco). b None detected in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. a None detected either in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. S. aureus and minimum pH are included. These data do not represent terminal populations or pH. Table 1 illustrates the effects of pH 6.0, 6.5, and 7.0 upon growth of S. aureus and production of enterotoxin in association with S. Iactis and P. cerevisiae. At pH values of 6.0, 6.5, and 7.0, there is a trend toward greater inhibition of aNone detected in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. growth of S. aureus strain 243 as the pH decreases, and the decrease in enterotoxin production associated with the decrease in pH is particularly evident in the absence of the effector organisms. Growth of S. aureus in the presence of effector organisms was inhibited to a much greater degree at 25 C than at 30 C ( Table 2). Also there was more inhibition of toxin production at 25 C. Troller and Frazier (8) reported that maximum inhibition of staphylococci by food bacteria occurred in the pH range of 7.4 to 6.2. They also indicated that maximum inhibition of growth of S. aureus in association with other organisms occurred at temperatures of 20 to 25 C. Peterson et al. (7) reported similar findings regarding the inhibition of S. aureus by psychrophilic saprophytes. Other previous reports (3,4) also suggest that growth of S. aureus is generally inhibited to a greater degree at temperatures lower than 30 C when in association with other organisms. Table 3 includes data illustrating the importance of the proportion of S. aureus in the inoculum upon growth and production of enterotoxin. Growth of S. aureus and production of enterotoxin were most inhibited when the proportion of S. lactis or P. cerevisiae were greatest. The data indicate that the ratio of inoculum was more important when S. lactis was used as the effector organism than when P. cerevisiae was used. Similar findings regarding the influence of the proportion of staphylococci and effector organisms have been reported by (2,3,6,8). Table 4 indicates that there is little difference among strains of either S. lactis or P. cerevisiae as inhibitors of S. aureus when grown in associative culture, but the S. Iactis species was more inhibitory than the P. cerevisiae species. Similarly, all four strains of S. aureus were approximately equal in sensitivity to inhibition by the effector organisms (Table 5) as evidenced by the fact that there was approximately a 4-log reduction in maximum population of all strains of S. aureus when grown in association with S. lactis and a 1-to 2-log reduction when grown in association with P. cerevisiae. Obviously inhibition of staphylococci is enhanced by selecting conditions of incubation which are not conducive to staphylococcal growth, and inhibition of S. aureus in mixed culture is greatly enhanced when the proportion of staphylococci in the population is small. It would generally be expected that the lactic acid culture organisms in a cultured food product would greatly outnumber any staphylococci present. Since only slight differences were observed between strains of the organisms studied, strain differences which might influence interaction in associative culture probably are uncommon. Kao and Frazier (3) reported data which also indicated that strain variations are not great regarding either the ability of a species of lactic acid bacterium to inhibit S. aureus or the susceptibility of S. aureus to inhibition. McCoy and Faber (4) found 15 strains of S. aureus approximately equal in sensitivity to inhibition by various food microorganisms. It is likely, then, that data obtained by use of selected strains to determine interactions of lactic acid organisms and S. aureus in associative culture are generally representative.

v3-fos

2018-04-03T01:33:53.492Z

1973-03-01T00:00:00.000Z

30022269

s2

Effects of Aflatoxin on Germination and Growth of Lettuce The relative susceptibility of 30 cultivars of lettuce to inhibition by aflatoxin was studied. Seed germination was not inhibited by concentrations as high as 1,000 μg/ml in cultivar Imperial 44 or by 100 μg/ml in the remaining cultivars. Hypocotyl elongation was inhibited by 46 to 68% at a concentration of 100 μg of aflatoxin per ml. Seedlings exposed to aflatoxin did not become chlorotic. The similarity between the morphological reaction of plants to coumarin and aflatoxin suggests a common mode of action, but further studies of the physiological basis for the inhibitory reactions induced by these compounds will be necessary before such conclusions will be valid. Hypocotyl elongation was inhibited by 46 to 68% at a concentration of 100 gg of aflatoxin per ml. Seedlings exposed to aflatoxin did not become chlorotic. The similarity between the morphological reaction of plants to coumarin and aflatoxin suggests a common mode of action, but further studies of the physiological basis for the inhibitory reactions induced by these compounds will be necessary before such conclusions will be valid. Macroscopic observations show that aflatoxins affect certain plants by inhibition of seed germination (19), elongation of the hypocotyls or roots of developing seedlings, or both (5,15,16), and by interference with chlorophyll synthesis (5,19,20). Similar inhibitory activities have been attributed to coumarin (2,10) and, since aflatoxins are derivatives of coumarin, analogous modes of action have been suggested for these substances (3). The germination of lettuce seeds is particularly sensitive to coumarin inhibition. If aflatoxin and coumarin do function in a biologically analogous manner, lettuce should be equally sensitive to the mycotoxin. This is a report on the relative susceptibility of cultivars of lettuce to aflatoxin. MATERIALS AND METHODS Test organisms. Seeds of 28 cultivars of lettuce (Lactuca sativa L.) and two of cos or romaine lettuce (L. sativa var. longifolia Gam.) were obtained from W. Atlee Burpee Co., Philadelphia, Pa. The test organisms included both leaf and head lettuce (L. sativa var. capitus L.) as well as black-and whiteseeded varieties (Table 1). One cultivar, Imperial 44, was used in specific tests to determine the effect of the test system and sample size on seed germinability and the effect of aflatoxin on the growth response. Germination substrate. Seeds were germinated on a 2% washed agar substrate prepared from agar (Difco) by alternately soaking the dehydrated agar in water and 95% ethanol for 4-h periods at 25 C. After four extraction cycles, the agar was filtered and the filter cake was rinsed in situ with absolute ethanol, crumbled and air-dried on sheets of absorbent paper. Unwashed agar was unsatisfactory as a germination substrate since sufficient trace nutrients were present to support the growth of fungal contaminants which inhibited seed germination and subsequent seedling development. Filter paper could not be used as a germination substrate in these experiments since evaporation of the solvent and oxidation of the aflatoxin could change the effective concentration and result in unreliable data. Furthermore, filter paper has been reported to contain impurities which might inhibit seed germination (14). Aflatoxin production and purification. The aflatoxin mixture used in this study was produced by Aspergillus parasiticus NRRL 2999 cultured on polished rice for 7 days, as previously reported (1). Chloroform extracts of the cultures were precipitated in 10 volumes of petroleum ether (bp 20-60 C), filtered, and reextracted with methanol, and the methanolic extract was evaporated to dryness in vacuo. This extract was redissolved in a minimal volume of chloroform and reprecipitated in petroleum ether to provide a purified toxin extract. The potency of the purified toxin extract was determined on thin-layer chromatograms by visual comparison with a reference standard containing a known concentration of the four major aflatoxins obtained from L. A. Goldblatt, Southern Marketing and Nutrition Research Division, New Orleans, La. Equivalent concentrations of each aflatoxin in the purified extract and the reference standard were determined by observing the point of extinction of long-wave ultraviolet fluorescence. The purified toxin contained a mixture of aflatoxins B, and G, in a ratio of 1:1 (approximately 350 mg/g each), with only traces of aflatoxins B2 and G2 present. The remaining portion of the extract contained nonaflatoxin substances exhibiting little or no fluorescence. Preparation of the test substrates. Chloroform solutions containing 0.1, 0.2, and 0.5 mg of aflatoxin mixture per ml were prepared by serial dilution from a stock solution containing 1 mg of purified extract per ml (2.19 x 10-1 M aflatoxin). Toxic germination substrates were prepared from these solutions by adding portions of 1, 2, or 3 ml to individual test tubes to provide a concentration of 10 times the desired final concentration of aflatoxin mixture in each tube. The solvent was removed under a stream of N2 while the tubes were rotated to form an even coating of toxin in the base of each tube. Ten milliliters of melted, 2% washed agar were added to each tube to adjust the final concentration of aflatoxin mixture to the desired concentration. The tubes were plugged, autoclaved at 121 C for 10 min, rapidly cooled to 45 C, and poured into individual, sterile, flat-bottomed petri dishes (9 cm; Coming no. 3162). Substrates for experimental controls were prepared in the same manner as above but using 3 ml of pure chloroform in place of the toxin solutions. Under these conditions, the volume of agar was sufficient to dissolve the quantity of aflatoxin used in each test substrate. Although crystalline aflatoxin is heat sensitive, autoclaving did not significantly decrease the potency of the purified mixture used in this study. Test system. A preliminary study using 25, 50, 75, 100, and 150 seeds per germination plate showed that within these limits the number of seeds did not affect the degree of germination or subsequent seedling development within 120 h. A sample size of 100 seeds was chosen to simplify data collection. Seed samples were counted and distributed evenly on the surface of the test substrates. A test series contained four plates of each test substrate, and each series was repeated a minimum of four times. Seeded plates were incubated at 25 C in the dark to prevent the photodecomposition of aflatoxin and to minimize the inhibitory effect of light on the germination of light-sensitive cultivars. Plates were examined periodically, as indicated, and germination was considered positive after the radicle (root tip) had pierced the seed coat and extended at least 1 mm beyond the seed proper. The hypocotyl, that portion of the germinated seed extending from the point of maximum root hair development to the crook at the base of the cotyledons, was measured to the nearest millimeter. The mean and standard deviations were determined, and the latter fell within acceptable limits for biological data (P = <0.001). Experiments conducted. The effect of aflatoxin on the germination of seeds of Imperial 44 was determined at concentrations of 0, 25, 100, and 1,000 Mg of aflatoxin mixture per ml. The numbers of germinated seeds were recorded periodically between 12 and 48 h. The final percent germination in the controls and at each toxin concentration was recorded after 48 h. The effect of aflatoxin on seedling growth was determined at a series of concentrations between 0 and 100 ,g of aflatoxin mixture per ml. Plates were seeded with Imperial 44, four replicate plates were harvested, and the hypocotyls were measured after 48, 72, 96, and 120 h of incubation. The inhibitory effect of aflatoxin on 30 cultivars of lettuce was studied to determine the degree of aAbbreviations: H, heading; L, leaf; bs, black seeded; ws, white seeded; ua, data on type unavailable. b PG, poor germination. Both controls and treated seeds showed less than 15% germination under these experimental conditions. susceptibility of each variety to the toxin. Test substrates containing 100 Mg of aflatoxin mixture per ml were seeded with each test organism the percent germination was determined, and hypocotyl measurements were made after 68 h. RESULTS Inhibition of germination. Some seeds on each of the toxic and control substrates germinated within 18 25,1973 of aflatoxin. The maximum amount of germination, 95%, was observed on all test substrates within 48 h. Except for a slight initial lag in germination observed at a concentration of 1,000 jLg/ml, aflatoxin did not inhibit the germination of Imperial 44. Growth response. At concentrations of aflatoxin below 20 Aig/ml, elongation of the hypocotyls in seedlings of Imperial 44 was only slightly inhibited. As the concentration of toxin was increased above 25 gg/ml, there was an increasing degree of inhibition of hypocotyl elongation. After 120 h, seedlings exposed to 100 ,g of aflatoxin mixture per ml exhibited a 33% inhibition. The growth response of this cultivar of lettuce to concentrations of aflatoxin is shown in Fig. 1. Varietal susceptibility to aflatoxin. In all of the cultivars tested, the percentage of seeds germinating in the presence of aflatoxin was not significantly less than that of the controls. The seeds of three cultivars, Black-seeded Simpson, Burpee Bibb, and New York 515, exhibited poor germination in the presence or absence of toxin. Whether this reflected an inability to germinate under these test conditions or, more probably, the low viability of the specific seed samples, was not determined. These three cultivars were not studied further. (Table 1). There was no significant difference in the degree of inhibition exhibited between leaf or heading varieties or between those producing black or white seeds. When compared visually, no differences could be distinguished between the coloration of treated and control seedlings. Surprisingly, the cotyledons of the test seedlings exhibited the same degree of coloration after incubation in the dark as did comparison seedlings incubated in the light. After 68 h of incubation, the seedlings did not appear etiolated. DISCUSSION Inhibition of seed germination and seedling elongation are the two physiological effects most often associated with coumarin activity. Aflatoxin does not affect seed germination but is inhibitory to hypocotyl elongation in lettuce. In comparing the inhibitory effect of coumarin and various coumarin derivatives on seed germination in lettuce, Mayer and Evenari (9) found the derivatives to be less inhibitory than the parent compound. They postulated that the inhibitory effect of coumarin is a function of the unsaturated lactone ring of the coumarin molecule. Goodwin and Taves (6) studied the effects of coumarin and coumarin derivatives on seed germination and root growth in Avena. Some derivatives were active inhibitors of root growth but were inactive as germination inhibitors, whereas other derivatives, which were very weak inhibitors of root growth, were as active as coumarin in inhibiting seed germination. The aflatoxin molecule contains the unsaturated lactone ring structure postulated to be necessary for coumarin-like activity (9). The remaining portion of the aflatoxin molecule, however, is only distantly related to coumarin. This might explain aflatoxin's lack of inhibitory activity towards lettuce seed germination but does not explain the observation of Schoental and White (19) (7), however, were unable to induce albinism in citrus, tomato, and several legumes with toxigenic and nontoxigenic strains ofA. flavus. Schoental and White (19) observed albinism in seedlings of Lepidium exposed to 10 ,g of aflatoxin per ml. This "bleaching" phenomenon was further studied by Slowatizki et al. (20) and was proposed as a bioassay method for aflatoxin M (11). Reiss (16) observed some lightening of the coloration of Lepidium exposed to 100 gg of aflatoxin per ml but did not observe complete loss of chlorophyll. Lettuce seedlings observed in this study did not exhibit albinism at concentrations as high as 1,000 ug/ml. The relationships, if any, between the inhibitory effects of coumarin and aflatoxin on plants remain unclear. Although research on coumarin has provided much information concerning its scope of activity and its proposed mode of action, little is known about the physiological basis for aflatoxin inhibition in plants. The observed similarities in the phytomorphological effects of coumarin and aflatoxin are representative of the general type of inhibitory response characteristic of many toxic compounds. The fact that both coumarin (13) and aflatoxin (16,17) exhibit auxin-like activities at very low concentrations may support the hypothesis that they share some common physiological activities. However, many toxic compounds, such as antibiotics, exert a stimulatory effect at sublethal concentrations. On the evidence presently available, it cannot be assumed that coumarin and aflatoxin share a common mode of action. ACKNOWLEDGMENTS The technical assistance of Elena Mazzucca Manasse and Anne Sands in completing this study is gratefully acknowledged. This investigation was supported, in part, by Public Health Service research grant EF-00663 from the Division of Environmental Engineering and Food Protection.

v3-fos

2020-12-10T09:05:58.184Z

1973-01-01T00:00:00.000Z

237233090

s2

Determination of the Optimal Ammonium Sulfate Concentration for the Fractionation of Rabbit, Sheep, Horse, and Goat Antisera Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH4)2SO4 was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH4)2SO4 were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations—the first in 30% and the second in 45% saturated (NH4)2SO4. The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin. both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH,)2SO. The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin. The most frequently used method for fractionating antisera is precipitation in the cold with half-saturated ammonium sulfate. The procedure is simple and inexpensive, and the resulting crude antibody fractions from the antisera of a number of animal species have been successfully used in many areas of immunology. In this laboratory these crude fractions have been conjugated with fluorescein isothiocyanate (FITC) for use as fluorescentantibody (FA) reagents. During a series of studies designed to improve FA methodology, these crude fractions were analyzed by cellulose acetate strip electrophoresis. The results revealed that most of the fractions contained less than 50% gamma globulin and that many of them contained undesirable amounts of albumin. A survey of commercial antibacterial FA reagents also had shown a need for improved fractionation procedures (5). This need for improved fractionation coupled with the need to retain the simple and inexpensive aspects of the (NH,)2SO4 procedure led to this 26 systematic study of various concentrations of the salt for serum fractionation. (Presented in part at the Annual Meeting of the American Society for Microbiology, Minneapolis, Minn., 3 May 1971.). MATERIALS AND METHODS Antisera. The antisera used in these studies were produced in rabbits, sheep, horses, and goats against a variety of bacterial antigens including Bacillus anthracis, Bordetella bronchiseptica, Bordetella pertussis, Escherichia coli, Pseudomonas pseudomallei, Salmonella, Shigella dysenteriae, Shigella sonnei, and Yersinia pestis. Some normal sera were also used. Ammonium sulfate. A stock solution of saturated ammonium sulfate (SAS) was prepared and stored at room temperature (approximately 25 C). Working solutions of 50, 60, 70, 80, and 90% SAS were prepared (v/v) fresh as needed from the stock saturated solution. Equal volumes of these solutions and various antisera resulted in reaction mixtures of 25, 30, 35, 40, 45, and 50% SAS (6). Fractionation. The following procedure for frac-tionation was used. A volume of serum was gently stirred while an equal volume of an ammonium sulfate solution was slowly added and mixed well. The reaction mixture was set aside at room temperature for 4 hr and then centrifuged to pack the precipitated protein. The supernatant fluid was removed and stored for later analysis. The precipitate was resuspended and dissolved in distilled water to a final volume equal to the original volume of serum. For a second precipitation, the dissolved protein was gently stirred while an equal volume of an ammonium sulfate solution was slowly added. The mixture was immediately centrifuged to pack the formed precipitate, and the supernatant fluid was discarded. The precipitate was dissolved and brought to volume as before. A third precipitation was handled in the same manner. All fractions were dialyzed against frequent changes (3) ofpH 8, 0.85% NaCl solution until sulfate was no longer detected in the dialysate (7). The saline was brought to pH 8 with a few drops of 10% NaOH. A small volume of saturated barium chloride solution was added to an equal volume of well-mixed saline dialysate to check for the presence of sulfate. If no cloudiness resulted, the dialyzed fraction was considered substantially free of sulfate. Protein. Protein concentrations were measured by the Biuret method (2) with a Beckman DB spectrophotometer. Protein compositions were determined by cellulose acetate strip electrophoresis (CASE) with the Beckman Microzone (1) equipment and procedure with a slight modification. After the membranes were stained with Ponceau S, they were rinsed in 5% acetic acid until the background was white; then they were gently blotted to remove excess moisture and placed between dry blotters on a flat surface beneath a glass thin-layer chromatography plate to dry. After drying, the uncleared membranes were read on a Beckman Densitometer, model R-110, at the recommended settings. Electrophoreuis interpretation. The integrated densitometer tracings of the proteins subjected to electrophoresis were interpreted by the following method ( Fig. 1). (i) Each of these proteins, when isolated and studied in the pure state, has a peak of Gaussian configuration. Use Gaussian projections to extrapolate to the base line the right side of the gamma peak and the left side of the albumin peak. (ii) Drop perpendiculars through these projections so that an area "A" between the original tracing and the projection is equal to an area "B" inside the projection and on the opposite side of the perpendicular. Extend these perpendiculars down through the integrator trace. (iii) The integrated tracing of the proteins subjected to electrophoresis has been partitioned into three regions-gamma globulin, beta and alpha globulins, and albumin. To calculate the area in each region, add the individual counts registered for each region and obtain the relative proportion of each. Fluoresein and fluorescein to protein ratios. Fluorescein isothiocyanate was determined as protein-bound FITC by absorbance at A max (near 495 nm) in 0.1 N NaOH, and related to a pure fluorescein diacetate reference standard (9). The fluorescein to protein (F/P) ratio was calculated from the FITC and protein measurements and expressed as micrograms of protein-bound FITC per milligram of protein. Specific antibody titration. The specific staining titers of all conjugates were determined by routine FA staining procedures (10,11). The highest dilution, which gave a 4+ staining of the bacterial cells, was recorded as the specific titer. RESULTS Preliminary studies. Many of these studies were done with pools of animal sera rather than individual sera. Once a procedure was worked out it was applied to other pooled sera as well as individual antisera. The electrophoresis profiles of rabbit, sheep, horse, and goat sera pools are shown in Fig. 2. They are easily distinguished from each other, and each is very characteristic of its species. The rabbit profile is the least distinctive and is quite similar to the familiar human serum profile. The sheep gamma globulin resolves as two separate peaks. Horse serum profiles exhibit two strong peaks in the alpha globulin region. In goat serum, gamma is the dominant globulin. These features remain constant within the species, and the differences in individual animals are minor except for fluctuations in the gamma globulin concentrations, which reflect immunological status. The CASE profiles of serum fractions obtained after three precipitations of these four sera in 50% SAS are shown in Fig. 3. The numerical data extracted from Fig. 2 and 3 are presented in Table 1. These crude. fractions contained only 23 to 48% gamma globulin and as much as 9 to 21% albumin. All of the gamma globulins present in the original sera were recovered, but many other proteins were also precipitated. Rabbit studies. The CASE profiles of the first, second, and third precipitations of rabbit E. coli antisera in 50, 45, 40, and 35% SAS are shown in Fig. 4. These tracings show that, as the concentration of ammonium sulfate was decreased, the gamma globulin percentage in the profiles of animal serum fractions after three precipitations in 50% saturated precipitates was increased. Consecutive precipitations with a given concentration of ammonium sulfate did not change the general ratio of the amount of gamma globulin to the amount of beta and alpha globulins, but they did reduce the percentage of albumin in the precipitate. The third precipitation in 35% SAS gave a fraction which was 65% gamma globulin and only 1% albumin. Very little precipitate formed in 30% SAS, and its composition was not desirable. No precipitation occurred in 25% SAS. Seven other bacterial antisera of rabbit origin were fractionated by three consecutive precipitations in 35% SAS. The data from CASE of all eight antisera and their sulfate fractions are given in Table 2. Gamma globulin in the original antisera ranged from 5 to 21%, and the fractions contained from 54 to 68% gamma globulin with a maximum of 2% albumin. Sheep studies. The CASE profiles of the first supernatant fractions and the third consecutive precipitates of sheep serum in 45, 40, 35, and 30% SAS are shown in Fig. 5. With sheep serum as with rabbit serum, decreasing the sulfate concentration resulted in an in- creased percentage of gamma globulin in the supernatant fractions and third precipitates of precipitate, and repeated precipitations in a horse serum in 45, 40, 35, and 30% SAS are given SAS concentration yielded reduced per-shown in Fig. 6. Decreasing the sulfate concencentages of albumin. The third precipitation in 35% SAS gave a fraction containing 68% tration had the same general effect on horse serum as that described for rabbit and sheep sera. At 35% SAS some gamma globulin remained in the supernatant fraction, but the third precipitate still contained a high percent-age of beta and alpha globulins. Lowering the concentration of SAS to 30% left a large amount of gamma globulin in the first supernatant fraction but gave a third precipitate which was 83% gamma globulin with no albumin. Goat studies. CASE profiles of goat E. coli antiserum fractions are shown in Fig. 7. Decreasing the sulfate concentration caused a greater change in the composition of the first precipitate of goat antiserum than it had with any of the other three animal sera. The first precipitate in 30% SAS contained 71% gamma globulin and only 6% albumin. No precipitate formed when a second sample of 60% SAS was used. When a 45% concentration of SAS was used to refractionate the 30% SAS precipitate, a fraction containing 81% gamma globulin and only 1% albumin was obtained. Recommended optimals. The CASE profiles of the improved fractions from rabbit, sheep, horse, and goat sera, obtained by using the optimal ammonium sulfate concentrations for precipitation, are shown in Fig. 8. Rabbit and sheep sera were handled in the same way with three consecutive precipitations in 35% SAS. Horse serum required three precipitations in 30% SAS to effectively remove its high percentage of beta and alpha globulins. Goat serum required precipitation in 30% SAS followed by precipitation in 45% SAS. Under these conditions the percentage of gamma globulin recovered from the sera of rabbits, sheep, and goats was above 80%, and from that of horses, slightly less than 50% (Table 3). Refractionation. Crude globulin fractions of rabbit antisera which had been prepared several years earlier by three precipitations in 50% SAS were subjected to a single precipitation in 35% SAS. The improvement was demonstrated by CASE profiles (Fig. 9). Several FA conjugates for Salmonella and Shigella detection have also been improved by a single precipitation in the optimal ammonium sulfate concentration appropriate for the animal species involved. The data for one of the Shigella conjugates are shown in Fig. 10. Refractionated conjugates were dialyzed in pH 7.6 phosphatebuffered saline (PBS) for sulfate removal. Since PBS reacts with saturated BaCl2 to form a precipitate, BaCl2 could not be used to check for the presence of sulfate; therefore, the dialysis time was extended to insure complete removal of the sulfate. Both globulins and conjugates were composed of considerably less albumin and beta-alpha globulins after one optimal precipitation. The Shigella conjugate lost 30% of its total protein, but only 7% of its gamma globulin. Although its FITC concentration was reduced 55% and the F/P ratio 36%, the specific antibody titer did not change. 9. Improvement in crude globulins from Shigella rabbit antisera after one precipitation with the optimal (NH4)2SO4 concentration. The crude globulin (a) was obtained after three precipitations in 50%o saturated (NH4)2SO4. The improved globulin (b) was obtained after one additional precipitation of the crude globulin, using 35% saturated (NH4)2SO4. the adjusted pH 7.2 reagents and unadjusted pH 5.8 reagents. After three consecutive precipitations under the six different conditions, the three fractions from the pH 7.2 sulfate solutions were practically identical to those obtained with the pH 5.8 reagents ( Table 4). Effect of dilution. Some rabbit B. bron: chiseptica antiserum was also used to study the effect of dilution of serum prior to fractionation. Equal volumes of the original antiserum and of a 1:2 and 1:10 dilutions were each precipitated three times in 40% SAS. The final fractions had practically identical CASE profiles ( Table 5). Effect of temperature. To examine the possible influence of room temperature variations, we prepared and stored saturated solutions of ammonium sulfate at 20, 25, and 30 C. At each temperature, 70% SAS was prepared and used to fractionate rabbit B. bronchiseptica antiserum. The fractions obtained after three precipitations in 35% SAS at each tem-34 APPL. MICROBIOL. _ -, _, perature were practically identical (Table 6). DISCUSSION For most immunological applications, the purpose of serum fractionation is to separate and recover the gammaor immunoglobulins. Except for the column fractionation methods and even as a preparative step for these techniques, the most widely used method of serum fractionation is precipitation in 50% SAS. Three precipitations are usually recommended to free the material of hemoglobin, but frequently the adequacy of the fractionation in terms of the protein composition of the final product is not determined. When rabbit, sheep, horse, and goat sera were fractionated by three precipitations in 50% SAS, all of the final fractions (Fig. 3) contained more beta and alpha globulins and more albumin than gamma globulin. By using the optimal concentration of (NH4)3SO4 for a given animal species of serum, all of the final fractions (Fig. 8) contained predominantly more gamma globulin than any other protein with very little or no albumin. More than 80% of the gamma globulins present in the original rabbit, sheep, and goat sera were recovered by the improved procedures. The results with horse serum were not as satisfactory. After the serum was precipitated c Saturated (NH4)2804-three times in 30% SAS, the high percentage of beta and alpha globulins was reduced, but only 44% of the gamma globulin was recovered. This fraction was 83% gamma globulin and contained no albumin. Approximately 60% of the horse gamma globulin was recovered after only two precipitations in 30% SAS, and, after that step, the fraction contained 74% gamma globulin with only 1% albumin. The recovery of a higher percentage of gamma globulin after two 30% SAS precipitations makes the second precipitate as desirable as the third in 30% SAS, unless the elimination of all albumin is a major objective. Using 35% SAS, we selectively precipitated rabbit gamma globulin and recovered a high percentage of it regardless of the percentage in the original antiserum. The gamma globulin concentrations (5 to 21%) in the eight rabbit antisera reported in Table 2 were not correlated with the percentage of gamma globulin obtained in the product after fractionation. The gamma globulin recovered from these antisera ranged from 84 to 91% of the original concentration. These serum fractionation procedures were reliable under variable reaction conditions. At the values tested, the composition of the fractions was not altered by the pH of the ammonium sulfate solutions, probably because the buffering capacity of serum kept the pH of the mixture within a very narrow range. However, diluted serum has less buffering ability, and, if it is fractionated with ammonium sulfate that has not been neutralized, careful attention should be given to pH to avoid acid reaction conditions. No differences were noted in the composition of fractions obtained by precipitation of rabbit antiserum with 35% SAS at 20, 25, and 30 C; this finding indicates that variations in temperature within these limits had no effect. Because the molarity of SAS varies with temperature, no attempt has been made to express concentration in molar equivalents, but the SAS used at approximately 25 C was approximately 4.09 M. The data indicated that the composition of fractions prepared from undiluted serum was approximately the same as the composition of fractions prepared from serum diluted either 1:2 or 1: 10. The reliability of these procedures depends upon proper technique. Ammonium sulfate solutions must be slowly added to serum while it is gently stirred. When sulfate solutions are poured into sera and the reaction mixtures are shaken or stirred vigorously, the final fractions contain considerably more albumin and beta and alpha globulins than do properly prepared fractions. Refractionation of very crude 50% SAS globulins with a single precipitation in the optimal SAS concentration for the particular animal species greatly improved the composition of the fractions. Most of the albumin and a large portion of the beta and alpha globulins were effectively removed. Crude FITC conjugates were also successfully refractionated with optimal SAS concentrations. Only very small amounts of the labeled gamma globulins were lost, and the specific titers did not change. Approximately one-third of the total labeled proteins were removed, and the FITC concentrations were reduced more than 50%. Because of the direct relationship between FITC concentration and nonspecific staining (4), the improved conjugates should exhibit less nonspecific staining than before. The large amount of FITC removed by reducing the percentage of labeled beta and alpha globulins and eliminating the labeled albumin reflects the high avidity of these proteins for FITC (8); it also illustrates the need for employing optimum procedures for fractionation before the antibody is labeled with a fluor.

v3-fos

2020-12-10T09:04:12.846Z

1973-06-01T00:00:00.000Z

237233870

s2

Recovery of Bacteriophage from Contaminated Chilled and Frozen Samples of Edible West Coast Crabs Edible West Coast crabs (Cancer magister and C. antennarius) were contaminated with bacteriophage and then held in a chilled or frozen state. Results indicated a significant survival of virus regardless of storage conditions. It has been demonstrated that viruses and other pathogenic microorganisms, when present in food products, can withstand freezing or chilling for a considerable length of time (1,2,3,7,8,9,11,12,13). It has recently been shown that edible crabs can also become contaminated with viruses (4,5). However, it is not known to what extent viruses survive in these animals when they are kept in a chilled or frozen state. Therefore, a preliminary investigation into this problem has been initiated in our laboratory. Two separate series of experiments were conducted. In the first study, edible West Coast crabs Cancer magister and C. antennarius, which have a carapace width of 13 to 16 cm, were exposed to coliphage T4 (5.0 x 10' plaqueforming units [PFU]/ml of seawater) for 24 h. These viruses were chosen as models because the bacteriophage have been used by other workers to study viral uptake and persistence in shellfish. (6) Furthermore, Kott et al. (10) have shown the coliphage to be as resistant to the marine environment as are enteroviruses. Therefore, it was felt that coliphage T4 would be a suitable model for the present studies. After contamination, the crabs were divided into two equal lots, dipped in a 1% hypochlorite solution to inactivate any viruses adhering to the carapace surfaces, and then washed in distilled water. One lot was sealed in polymylar pouches, six crabs per pouch, and placed in a refrigerator set at 8 C. The second lot was processed by being boiled in water containing 0.5 g of NaCl per liter for 20 min at 100 C. This lot was then sealed in polymylar pouches, six crabs per pouch, and refrigerated. The crabs were assayed for virus content at 0, 24, 48, 72, 96, and 120 h. Samples were readied for assay by preparing 10% (wt/vol) homogenates of tissue (crab muscle, digestive gland, and blood), with nutrient broth as a diluent. All homogenates were blended for 2 min at 6,500 rpm in a Waring blendor. After clarification, serial decimal dilutions were prepared in sterile, isotonic saline, and the samples were.assayed for virus by being plaqued in nutrient agar (pH 7.6; 0.0025% CaCl2) containing approximately 108 host cells per ml. The host cells, Escherichia coli B, were propagated in 250-ml flasks of nutrient broth at 37 C. The plates were incubated at 37 C for 12 h. In the second study, the crabs were allowed to be contaminated for 24 h in seawater containing 4.6 x 104 phage PFU per ml. Samples were assayed for viral content and found to contain 3.6 x 104 phage PFU per g. On a per-unit weight basis, this represented a 78% uptake of the virus by the crabs. This uptake was significantly higher than that observed in a study of enterovirus uptake by shore crabs (4). In this previous study, viral uptake at 24 h was found to be only 32%. However, this difference in accumulation probably reflects the size difference existing between the species of test animals. The remaining animals were sanitized, washed in distilled water, and divided into two equal lots. One lot was sealed in polymylar pouches, four crabs per pouch, then frozen and stored at -20 C. The second lot was processed by being boiled for 20 min, sealed in polymylar pouches, and then frozen and stored as above. Samples to be tested were allowed to thaw to room temperature before being assayed. Assays were conducted at 0-, 1-, 20-, and 30-day intervals. 1020 In all studies, the zero-hour samples represented the titer of viruses existing in the crabs after either contamination or processing. The results of the chilling experiments using contaminated unprocessed and processed (boiled) crabs are shown in Fig. 1. A gradual decrease in virus titer was observed in all samples during the entire test period. However, after 120 h 7.0 x 101 virus PFU per g were still recovered from the unprocessed crabs, and 45 virus PFU per g were recovered from the processed samples. On a per-unit weight basis, these represent approximately 29 and 40% of the virus contained per gram of respective tissue sample. The studies were discontinued after 120 h because of the rapid decomposition of the unprocessed crabs. The results of the freezing studies are presented in Table 1. There was a decrease in virus titer during the entire period of this experiment. However, the decrease was gradual; after 20 days of storage, 1.5 x 104 virus PFU per g from the frozen unprocessed crabs and 1.4 x 102 virus PFU per g from the processed samples were recovered. This represents survival or recovery rates of approximately 42 and 55%, respectively. At the end of the study (30 days), 1.2 x 104 virus PFU per g were still found to be present in the unprocessed samples, and 45 PFU per g were recovered from the processed samples. The results reported are preliminary observations. Results obtained by other workers have 5. indicated a long-term persistence of human enteroviruses in chilled and frozen foods. Lynt (12) observed that type 1 poliovirus and types B1 and B6 coxsackievirus would survive for 1 month in chilled foods and up to 5 months in frozen products. These findings were confirmed by Heidelbaugh and Giron (8) and substantiated by DiGirolamo et al. (3). However, our findings suggest that the rate of virus inactivation in crabs may be more rapid than that which occurs in other food products. Possibly this increased rate of inactivation was due in part to the thawing process and in part to autolytic reactions in the crab, either of which may have had an effect on the virus used. LITERATURE CITED

v3-fos

2020-12-10T09:04:16.751Z

1973-02-01T00:00:00.000Z

237231843

s2

Effect of Variations in Conditions of Incubation upon Inhibition of Staphylococcus aureus by Pediococcus cerevisiae and Streptococcus lactis The effects of pH, temperature, proportion of Staphylococcus aureus in the inoculum, various strains of effector organism, and various strains of S. aureus were examined for their influence on interactions between staphylococci and effector organisms in associative culture. In general, small changes in pH had little effect upon either growth of S. aureus or production of enterotoxin in associative culture. Inhibition of growth of S. aureus caused by effector organisms was much greater at 25 than at 30 C. Proportion of S. aureus in the inoculum greatly affected both growth of the staphylococci and production of enterotoxin. Only slight differences were found between strains of either effector organism or S. aureus which affected the interactions in associative culture. Previous work in this laboratory has shown that several species of lactic acid bacteria, particularly Streptococcus lactis and Pediococcus cerevisiae, were inhibitory to growth and enterotoxin production by Staphylococcus aureus when grown in association with S. aureus at temperatures favorable to the organisms concerned. Several reports (2)(3)(4)(5)(6)(7)(8) have indicated that certain conditions of incubation may significantly affect the influence of the competing organisms upon growth of S. aureus, but none report any effect on enterotoxin production. The investigation reported herein was conducted to determine the effect of variations in some conditions upon the ability of S. lactis and P. cerevisiae to inhibit growth and production of enterotoxin by S. aureus. MATERIALS Procedure. One-liter Erlenmeyer flasks containing 300 ml of all-purpose medium with Tween broth (Difco) were inoculated and incubated 48 hr in a thermostatically controlled gyratory shaker-incubator operated at 175 rev/min. The effect of the initial pH of 6.0, 6.5, and 7.0 on the growth of the cultures of S. aureus and the effector organisms was determined by adjusting the pH to these values with 1.0 N HCl or 1.0 N NaOH. The effect of incubation at 25 and 30 C was investigated. In trials involving the influence of temperature, pH, and species of effector organism, the broth in each flask was inoculated with 105 cells of S. aureus per ml and 105 cells of the effector organism per ml. To determine the importance of the relative proportion of S. aureus in the inoculum, the numbers of the effector organism in the inoculum were varied, giving initial percentages of S. aureus of approximately 10, 50, and 90%. This was accomplished by inoculating the broth in each flask with 106 cells of S. aureus per ml and then adding 101, 101, and 106 cells of effector organisms per ml to appropriate flasks. Several strains each of S. lactis, P. cerevisiae, and S. aureus were used to determine the extent of variations between strains of the organisms with regard to interactions in associative culture. Enumeration of S. Aureus. Staphylococcal populations were determined on prepoured mannitol salt agar spread plates incubated 48 hr at 37 C. Assay for enterotoxin. The microslide doublegel diffusion procedure of Casman and Bennett (1) was used for enterotoxin assays. The assays were performed on samples taken at 3-to 4-hr intervals during the first 24 hr of incubation. Reference enterotoxins A, C, and D, and corresponding antisera were obtained from the Food and Drug Administration, Washington, D.C. Reference enterotoxin B and anti-B were obtained from Makor Chemicals Ltd., Jerusalem, Israel. RESULTS AND DISCUSSION This investigation involved incubation of cultures over a period of 48 hr and examination of samples taken at intervals of 3 or 4 hr. To conserve space, only maximum populations of a APT broth is all-purpose medium with Tween (Difco). b None detected in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. a None detected either in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. S. aureus and minimum pH are included. These data do not represent terminal populations or pH. Table 1 illustrates the effects of pH 6.0, 6.5, and 7.0 upon growth of S. aureus and production of enterotoxin in association with S. Iactis and P. cerevisiae. At pH values of 6.0, 6.5, and 7.0, there is a trend toward greater inhibition of aNone detected in direct assay or in sample concentrated 10-fold by lyophilization and rehydration. growth of S. aureus strain 243 as the pH decreases, and the decrease in enterotoxin production associated with the decrease in pH is particularly evident in the absence of the effector organisms. Growth of S. aureus in the presence of effector organisms was inhibited to a much greater degree at 25 C than at 30 C ( Table 2). Also there was more inhibition of toxin production at 25 C. Troller and Frazier (8) reported that maximum inhibition of staphylococci by food bacteria occurred in the pH range of 7.4 to 6.2. They also indicated that maximum inhibition of growth of S. aureus in association with other organisms occurred at temperatures of 20 to 25 C. Peterson et al. (7) reported similar findings regarding the inhibition of S. aureus by psychrophilic saprophytes. Other previous reports (3,4) also suggest that growth of S. aureus is generally inhibited to a greater degree at temperatures lower than 30 C when in association with other organisms. Table 3 includes data illustrating the importance of the proportion of S. aureus in the inoculum upon growth and production of enterotoxin. Growth of S. aureus and production of enterotoxin were most inhibited when the proportion of S. lactis or P. cerevisiae were greatest. The data indicate that the ratio of inoculum was more important when S. lactis was used as the effector organism than when P. cerevisiae was used. Similar findings regarding the influence of the proportion of staphylococci and effector organisms have been reported by b Samples concentrated 10-fold by lyophilization and rehydration. other investigators (2,3,6,8). Table 4 indicates that there is little difference among strains of either S. lactis or P. cerevisiae as inhibitors of S. aureus when grown in associative culture, but the S. Iactis species was more inhibitory than the P. cerevisiae species. Similarly, all four strains of S. aureus were approximately equal in sensitivity to inhibition by the effector organisms (Table 5) as evidenced by the fact that there was approximately a 4-log reduction in maximum population of all strains of S. aureus when grown in association with S. lactis and a 1-to 2-log reduction when grown in association with P. cerevisiae. Obviously inhibition of staphylococci is enhanced by selecting conditions of incubation which are not conducive to staphylococcal growth, and inhibition of S. aureus in mixed culture is greatly enhanced when the proportion of staphylococci in the population is small. It would generally be expected that the lactic acid culture organisms in a cultured food product would greatly outnumber any staphylococci present. Since only slight differences were observed between strains of the organisms studied, strain differences which might influence interaction in associative culture probably are uncommon. Kao and Frazier (3) reported data which also indicated that strain variations are not great regarding either the ability of a species of lactic acid bacterium to inhibit S. aureus or the susceptibility of S. aureus to inhibition. McCoy and Faber (4) found 15 strains of S. aureus approximately equal in sensitivity to inhibition by various food microorganisms. It is likely, then, that data obtained by use of selected strains to determine interactions of lactic acid organisms and S. aureus in associative culture are generally representative.

v3-fos

2020-12-10T09:04:16.779Z

1973-11-01T00:00:00.000Z

237233948

s2

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species. which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species. Propionic acid is a major end product of the fermentation of plant polysaccharides by the rumen microbial population. Pure cultures of certain predominant rumen bacteria produce propionate directly from carbohydrates other than cellulose. For example, Selenomonas ruminantium and Megasphaera elsdenii both produce propionate from carbohydrates and lactate, the former by the succinate or randomization pathway (13) and the latter by the acrylate pathway (2). Several important species of rumen bacteria produce succinate as a major, pure-culture product of carbohydrate fermentation. Ruminococcus flavefaciens and Bacteroides succinogenes, two of the three major cellulolytic species in the rumen, produce succinate. Succinate, however, does not accumulate in the rumen ecosystem, but it is known to be produced and rapidly decarboxylated to propionate in the rumen. Quantitative studies have shown that succinate is a major precursor of propionate in the rumen (3). A likely explanation for the conversion of succinate to propionate is that a species like S. ruminantium decarboxylates succinate produced by other rumen organisms. Resting cell decarboxylation of succinate to propionate and CO, by bacteria that use the succinate pathway for production of propionate from carbohydrates or lactate is well established (10,11). S. ruminantium presumably would obtain energy for growth in the rumen by the conversion of carbohydrates to propionate, and the grown cells would then carry out what could be considered a resting cell decarboxylation of succinate, produced by other organisms, to propionate and CO2. The purpose of this study was to obtain experimental evidence for the decarboxylation of succinate produced by rumen cellulolytic bacteria when they are grown together with S. 789 ruminantium. Initial studies were carried out by co-culturing B. succinogenes and S. ruminantium in media containing glucose or cellobiose, sugars that both species use as an energy source. It was subsequently found that S. ruminantium, a non-cellulolytic species, can grow together with the cellulolytic B. succinogenes on a medium containing cellulose as an energy source. B. succinogenes provides S. ruminantium with an energy source from cellulose, and the latter organism decarboxylates succinate produced by the former organism. The net result is a two-species cofermentation of cellulose to propionate, acetate, and CO. The results of these fermentation interaction studies provide evidence for the explanation of the mode of conversion of succinate to propionate in the rumen discussed above. MATERIALS AND MEFHODS Organisms and cell growth. B. succinogenes S-85 and S. ruminantium HD4 were used. Independent and combined cultures were usually grown at 37 C in 10 ml of medium in 18 by 150 mm rubber-stoppered test tubes. The atmosphere was CO, freed of trace amounts of 0, by passing over heated copper filings. A complex medium (4, 6) was slightly modified and used for routine transfers of both organisms. It was made by first adding the following ingredients and distilled H,0 to a final volume of 93 ml: Trypticase, 0.5 g; yeast extract, 0.1 g; dithiothreitol, 0.054 g; glucose, 0.2 g; cellobiose, 0.2 g; starch, 0.2 g; clarified rumen fluid, 20 ml; 4 ml each of minerals no. 1 (0.6% K,HPO,) and no. 2 (0.6% KH,PO0, 0.6% (NHJ),SO4, 1.2% NaCl, 0.24% MgSO4.7H,0, 0.16% CaCl, *2H20); and 0.1 ml of 0.1% resazurin. After adjusting the pH to 6.5 and autoclaving at 15 lb/in' for 15 min under CO, in a sealed flask, 5 ml of sterile 8% Na,CO, and 2 ml of sterile 2.5% cysteine-hydrochloride were added. The medium was then tubed under O,-free CO, for use. The procedures were essentially those previously described (4)(5)(6). In most experiments, the same medium was used except for the use of glucose, cellobiose, or cellulose as energy sources as indicated. A defined medium was used for some experiments, which was the same as the complex medium, except for the omission of rumen fluid, Trypticase, and yeast extract and the addition of vitamins, isobutyric, isovaleric, 2-methyl-butyric, and n-valeric acids as previously described (16). All cultures were incubated on a reciprocal shaker at 120 strokes per min. Direct counts. A Petroff-Hauser chamber was used. It was possible to enumerate both species in combined cultures because of their distinctly different morphologies. Manometric experiments. Cells for manometric analysis were grown for 24 h at 37 C in 100 ml of the complex medium, with 0.2% each of cellobiose and glucose, under an atmosphere of CO,. The cells were harvested by centrifugation (12,000 x g) for 5 min under CO, by using screw cap tubes and were suspended in approximately 5 ml of an anaerobic mineral salt dilution buffer. The dilution buffer previously described (16) was used, but was modified to delete the glucose and sodium sulfide and to contain 0.01 M dithiothreitol. Double-sidearm Warburg vessels (15 ml total volume) were used. The reaction mixtures contained 50 mM potassium phosphate buffer, at pH 6.5, approximately 1010 cells per flask, 2 jug of biotin per ml, and 10 mM sodium succinate in a final volume of 2.9 ml. The succinate, in 0.3 ml, was tipped in from one sidearm to start the reaction. After 40 min, the reaction was stopped by the addition of 0.1 ml of 6 N H,SO4 from the second sidearm, and the flasks were shaken for an additional 10 min to release and measure dissolved CO,. After centrifugation, the supernatant solutions were analyzed for acids (see below). Incubations were at 37 C in an atmosphere of argon. Fermentation analyses. Cellobiose was determined by the ferricyanide reduction method of Park and Johnson (12). Glucose was determined with glucose oxidase as described in Bulletin 510 of the Sigma Chemical Co. Culture supematant solutions were clarified by the Somogyi procedure (15) prior to analysis for glucose or cellobiose. The cellulose used was ball milled Whatman no. 1 filter paper in a 2% (wt/vol) aqueous slurry as described by Hungate (8). The concentration of the slurry was determined gravimetrically, and complete cellulose disappearance from cultures was estimated by microscopy observation of the disappearance of the cellulose particles. For fermentation acid analysis, 2 ml of culture supernant solution was acidified with 0.1 ml of 6 N H,SO, and centrifuged for 15 min at 15,000 x g to remove any precipitate. The silicic acid column and methods of Ramsey (14) were modified for batch collection (9). The solvents and elution order were (in milliliters) benzene, 56; CHCl,, 100; 1% tert-butanol (t-B) in CHCl, (C), 100; 2% t-BC, 200; 5% t-BC, 250; and 8% t-BC, 180. All solvents were equilibrated with HSO and used at room temperature. Samples were collected in graduated cylinders with 10 ml between batches to check for any trailing. The collection schedule was designed to obtain butyrate in the first 95 ml, propionate in the next 55 ml, acetate in the next 70 ml, formate in the following 240 ml, lactate in the next 165 ml, and succinate in the final 180 ml. The acids were titrated to a phenolphthalein end point by using 0.01 N ethanolic KOH. RESULTS Decarboxylation of succinate by S. ruminatium. Before carrying out studies with combined cultures, experiments were performed to determine if S. ruminantium decarboxylates succinate to propionate and CO,. The results in Table 1 show that resting cells decarboxylate succinate to propionate and CO,. In another experiment, B. succinogenes was grown for 48 h in the complex medium with cellobiose. A 24-h culture of S. ruminantium was then centrifuged aseptically in a CO2 atmosphere, the cells were resuspended in the 48-h B. succinogenes culture, and the tubes were incubated for an additional 24 h. Succinate, but no propionate, was present in the B. succinogenes culture, and propionate, but no succinate, was present after incubation of the culture with the added S. ruminantium cells (Table 2). This experiment also showed that S. ruminantium decarboxylated succinate to propionate and that changes in the medium caused by growth of B. succinogenes did not prevent the decarboxylation. Identical results were obtained when glucose was the energy source for B. succinogenes. Concurrent fermentation of cellobiose or glucose by B. succinogenes and S. ruminantium. The question of whether both organisms could grow together and carry out a combined fermentation of carbohydrate to propionic acid was examined. When cellobiose or glucose are used, the two species are competing for energy source. If competition is significantly skewed in the direction of B. succinogenes, no significant growth of S. ruminantium will take place in the combined cultures, and the fermentation would essentially be the same as the independent B. succinogenes fermentation. If competition for substrate is strongly in favor of S. ruminantium, the fermentation would be the same as the independent S. ruminantium fermentation and the presumptive competitive cofermentation by the two species. Because of the inability to distinguish between an independent S. ruminantium fermentation and a truly competive cofermentation simply on the basis of product formation, the contribution of the individual species to the cofermentation process was estimated. This was done by determining cell numbers of each species in the combined culture and calculating the expected amounts of products produced by each species from their respective per cell activities in independent, single-species fermentation. Table 3 shows the results of independent and combined fermentations of cellobiose, and Table 4 shows the results obtained when glucose was the energy source. It can be seen that succinate, but no propionate, was produced by B. succinogenes alone and that propionate, but no succinate, was produced by S. ruminantium alone. In the combined cultures, propionate but no succinate was found, although significant amounts of succinate would have been expected on the basis of the independent activity of the concentration of B. succinogenes found in the combined cultures. The results strongly suggest that the species use cellobiose or glucose at similar rates when they are co-cultured under the conditions of these experiments. This results in a combined fermentation of cellobiose or glucose to propionate, acetate, and CO, without succinate accumulation. The amount of propionate formed in the combined cultures was significantly greater than the amount expected on the basis of the amount of S. ruminantium present and was also greater than the amount expected on the basis of the estimated amount of succinate produced by B. succinogenes in the combined cultures. A possible reason for the larger than calculated amount of propionate obtained in the combined cultures has not been definitely established, but the discrepancy may be due to differences in product formation by B. succinogenes in single and combined cultures. Relatively good carbon recoveries were obtained in fermentation balance studies with the single S. ruminantium and the combined B. succinogenes-S. ruminantium fermentations, but not with B. succinogenes alone. In the single S. ruminantium fermentation, the only products were propionate, acetate, CO2 (calculated as equal to acetate), and small amounts of lactate. The combined fermentation yielded only propionate, acetate, small amounts of formate, and succinogenes alone produced succinate, acetate, and small amounts of formate, but significant amounts of carbon disappeared that could not be accounted for by the products or calculated CO2. Table 5 shows a comparison of fermentation balances for glucose. Similar results were obtained when cellobiose was used. These results suggest that either an unidentified product is produced by B. succinogenes alone which can be converted to propionate by S. ruminantium or that co-culturing of S. ruminantium and B. succinogenes prevents the formation of the unidentified compound by B. succinogenes. Fermentation of cellulose. B. succinogenes used cellulose as an energy source and fermented cellulose in the complex medium to succinate, acetate, formate, and CO, (Table 6). S. ruminantium grew only slightly in the same medium without degrading cellulose, but good growth of S. ruminantium was-obtained when it was co-cultured with B. succinogenes on the cellulose medium. No succinate was produced in the combined fermentation, and cellulose was fermented to propionate, acetate, and CO2 ( Table 6). As shown in Table 6, similar results were obtained when a defined medium was used, except that the base growth of S. ruminantium alone was eliminated. The carbon recovered in the synthetic medium (assuming CO2 equal to acetate minus formate) represented 94 and 110% of the original cellulose carbon for B. succinogenes alone and the mixture of B. succinogenes and S. ruminantium, respectively. There is probably some inaccuracy in the original cellulose concentration because the cellulose was pipetted from a suspended slurry and the actual concentrations in the fermentation media were not measured. It appears, however, that most of the carbon of the cellulose was recovered in the indicated products. When grown alone, the amount of B. succinogenes cells per milliliter of synthetic medium was 6.5 x 108, and the respective concentrations of cells in the mixed culture were 4.4 x 108 for B. succinogenes and 1.0 x 108 for S. ruminantium. The combined cultures were serially transferred in the synthetic medium at 72-h intervals by using 0.5% inocula, and the combined culture fermentation of cellulose to propionate, acetate, and CO2 was maintained through at least seven serial transfers. DISCUSSION These experiments show that it is highly likely that the conversion of succinate to propio- nate in the rumen is carried out by bacteria that form propionate via the succinate pathway. S. ruminantium is probably a major factor in the conversion although, under certain circumstances, other species such as Veillonella alcalescens in the sheep rumen (10) may play a similar role. Dehority reported that high concentrations of rumen fluid caused the succinateproducing B. ruminicola to produce small amounts of propionate (7), but it was subsequently shown that the propionate is formed by the acrylate pathway (17). It is, therefore, highly unlikely that B. ruminicola is capable of decarboxylating succinate. The rate of succinate decarboxylation by resting cells of S. ruminantium was about 13.0 #mol per h per 1010 cells. The rate of conversion of succinate to propionate by bovine rumen contents was measured by Blackburn and Hungate (3) and was found to be approximately 1.6 pmol per h per g of rumen contents. By using the resting cell rate determined in these experiments it would have taken approximately 1.2 x 109 selenomonads per ml to account for the turnover number reported by Blackburn and Hungate. It is not possible to directly extrapolate from the cell suspension decarboxylating activity to the activity of the selenomonads in the ecosystem because of the differences in the conditions for succinate decarboxylation. The rate of succinate decarboxylation by cell suspensions leaves the question of whether bovine rumen selenomonads can account for all of the ecosystem conversion of succinate to propionate an open one. We estimate the cell suspension decarboxylating activity (on a dry-weight basis) of S. ruminantium HD4 to be about 87 times greater than that reported for propionibacteria (11), but only one-third of that reported for Veillonella (10). We suggest that the model presented in Fig. 1 is a fairly accurate representation of the microbial interactions that result in propionate tion pathway when starch or soluble carbohydrates are fermented in the ecosystem in addition to the interactions depicted in Fig. 1. S. ruminantium, depending on the strain, can ferment starch, lactate, and a variety of soluble carbohydrates to propionic acid directly. Nonstarch fermenting strains could feed off starch breakdown products, either sugars or lactate produced by starch-fermenting organisms. Microbial interactions that lead to propionate formation from starch and soluble sugars are probably more complex than those interactions involved in propionate formation from cellulose. The spin-off of carbohydrate from cellulose by major cellulolytic rumen bacteria to non-cellulolytic major rumen species has been logically assumed to be a significant means of providing energy to the latter species. To our knowledge, however, the present experiments represent the first direct demonstration of this type of interaction. The interaction between B. succinogenes and the HD4 strain on cellulose was duplicated with other selenomonas strains, both lactate and nonlactate-fermenting strains, and the results were essentially the same as with the lactate-fermenting HD4 strain. R. flavefaciens has also been substituted for B. succinogenes in the cellulose system with the HD4 strain with essentially similar results.

v3-fos

2018-04-03T02:32:06.852Z

1973-09-01T00:00:00.000Z

34015452

s2

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar. During the past decade, Hawaii has reported the highest incidence of human salmonellosis to the Center for Disease Control in Atlanta, Ga. (4). Two surveys, conducted in 1960 to 1962 and 1967, on the epidemiological aspects of salmonellosis in Hawaii established that food products played a significant role as the source and in the transmission of salmonellae (2,3,5). A Salmonella surveillance project supported by the U.S. Department of Health, Education, and Welfare from January 1970 to April 1971 investigated the sources and transmission of salmonellae in food products, animal feeds, market equipment, and abattoir environments in Hawaii. A total of 15,071 specimens was examined during this period. Of this number, 11,226 were food samples. Food and food products examined included powdered food prod. ucts, eggs and egg products, and carcasses and viscera of beef, poultry, and pork of intrastate, interstate, and foreign origin. In conjunction with this project, the effectiveness and selectivity of Brillian Green (BG) and Hektoen enteric (HE) agar media for isolating salmonellae from food products were compared. MATERIALS AND METHODS Sampling procedures. The cotton swab method was used in obtaining carcasses and viscera samplings of beef, pork, and poultry. All the meat samplings were obtained by swabbing an area 4 by 4 inches (10.16 by 10.16 cm [2]). In instances where the surfaces of the meat products were semidry, the swabs were moistened with sterile normal saline prior to sampling. A sampling thus obtained was placed directly into a test tube containing 8 ml of Tetrathionate Brilliant Green (TBG) enrichment broth. The inoculated specimen was immediately delivered to the laboratory, assigned a number, and placed in a 37 C incubator. Laboratory methods. Isolation procedures for foods, feeds, etc., recommended by the Center for Disease Control were followed (1). After 18 to 24 h of incubation, inoculated TBG broth was streaked onto BG and HE agar plates. The BG and HE agar plates were examined after overnight incubation for the presence of Salmonella-like colonies. Three colonies were picked from each suspicious plate and inoculated into triple sugar iron agar slants and incubated at 37 C. Cultures exhibiting Salmonella-like reactions (mainly alkaline slant, acid, and gas butt, with or without H1S formation) were checked for urease activity by the rapid urea test (a 1: 20 dilution of urea and buffer solution). Urease-negative cultures were inoculated into a series of biochemical test media, including 1% sucrose, mannitol, lactose, and salicin broths, tryptone broth, 10% lactose agar slant, Simmons citrate agar slant, and motility test medium. Cultures showing biochemical reactions characteristic of the genus Salmonella were checked with Salmonella polyvalent somatic and grouping sera and submitted to the State's Regional Salmonella Typing Center for definitive identification. Various brands of dried milk, instant breakfasts, and powdered haupia (coconut pudding) were examined for Salmonella contamination. For each sampling, 100 g were reconstituted in a 2-liter flask with 1,000 ml of sterile distilled water containing 20 ml of a 0.1% aqueous BG solution (a 1: 50,000 concentration). The inoculated broth was incubated at 37 C. After 24 h of incubation, 10 ml of the primary broth was transferred to a 250-ml flask containing 100 ml of TBG broth and reincubated. Loopfuls of the primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. A dozen eggs was considered as a single specimen. Each dozen eggs was cracked manually (by using a sterile glove for each specimen) into a 2-liter flask containing 1,000 ml of nutrient broth. The inoculated broth was incubated at 37 C. After 24 h, 10 ml of the primary broth was transferred to a 250-ml flask containing 100 ml of TBG broth and reincubated. Loopfuls of the primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. Each 30 g of egg noodle specimen was inoculated into a 250-ml flask containing 100 ml of nutrient broth. After 24 h of incubation, 1 ml of the primary broth was inoculated into a test tube containing 9 ml of TBG broth and reincubated. The primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. RESULTS Effectiveness of BG agar and HE agar media. A comparative study of BG and HE agar media was undertaken from January 1970 to April 1971 to determine their effectiveness for isolating salmonellae from food products. Of 11,226 food samplings, 1,662 (or 14.9%) were positive for salmonellae on one or both isolation media (Table 1). Although BG agar detected significantly more specimens positive for salmonella (1,475 of 11,226) than did HE agar (1,315 of 11,226), and the difference is significant (XI = 10.47, p < 0.005), there were 187 positives that would not have been detected if HE agar were not used as the second plating media and 347 if BG had not been used. Table 2 summarizes the incidence of Salmonella in the food products examined during the study. Eggs and egg products. Of the 200 dozen (from 7 farms) local cracked eggs, 4 (or 2.0%) were positive. Salmonella infantis was isolated from three of the four dozen eggs which were positive. From 236 dozen local whole eggs (from 9 brands), 3 (or 1.3%) were positive. S. cerro was isolated from the two dozens of eggs which were positive. From the 103 mainland whole eggs (from 2 brands) examined for Salmonella, no positives were isolated. No positives were isolated from the 14 local egg noodle samplings. Powdered food products. A total of 101 powdered products was examined for Salmonella, with negative results. Samples included 74 (from 7 brands) dried milk, 25 (from 3 brands) instant breakfasts, and 2 (from 1 brand) haupias (coconut puddings). GOO, CHING, AND GOOCH Bovine carcasses and viscera. From a total of 860 beef samplings, only 1 (0.5%) positive was isolated from 211 foreign beef carcasses. S. infantis was isolated on HE agar medium. No salmonellae were isolated from the local and mainland beef samplings, which consisted of 370 (354 carcasses and 16 tripe) and 279 (253 carcasses and 26 tripe) specimens, respectively. The beef samplings were obtained from two meat companies, one sausage factory, and four supermarkets. Pisces carcasses. Approximately 77 island "reef' fishes were examined for salmonella contamination. BG and HE agar media failed to detect the presence of salmonella on fish carcasses. The fish samplings were obtained from one open market and a supermarket. Poultry carcasses and viscera. Of 3,713 chicken carcasses and viscera, 3,526 were of local and 187 were of mainland origin. Out of 2,927 local chicken carcasses, 65 (or 2.2%) were positive. BG agar isolated 59 (90.7%) positives from the carcasses and HE agar yielded 16 (24.6%) positives. From 599 local chicken viscera, 8 (or 1.3%) were positive. BG agar isolated five (62.5%) positives from the viscera and HE agar isolated four (50%) positives. The predominant Salmonella serotypes isolated were S. heidelberg, S. typhimurium, and S. saint paul. The chicken specimens were obtained from four local abattoirs and two supermarkets. No positives were found in the 62 mainland chicken carcass samplings. Of 125 mainland chicken viscera, 2 (or 1.6%) were positive. The two serotypes isolated were S. typhimurium and S. derby. They were isolated from BG agar medium. The mainland samples were obtained from one sausage factory, three supermarkets, and one drive-in restaurant. Porcine carcasses and viscera. A total of 5,922 pork carcasses and viscera was examined for salmonellae. Salmonellae were isolated from 234 (or 9.2%) of the 2,543 local pork carcasses and 1,30Y7 (or 56.9%) of the 2,296 local hog viscera. A 3-month viscera pasteurization study was carried out in the hog slaughterhouse from September to December, 1970, by utilizing methods described by Paul Yoder (personal communication). Prior to the study, the contamination percentage ranged from 70 to over 90. Due to the pasteurization treatment of all hog viscera before marketing, the degree of contamination fell considerably. A total of 170 hog viscera was subjected to pasteurization, of which 9 (5.3%) yielded salmonella. S. derby and S. anatum were the two serotypes that survived a few pasteurization treatments. The predominant serotypes isolated were S. derby, S. ana-tum, and S. typhimurium. The local pork carcass samplings were obtained from 1 abattoir, 3 processing pork plants, and 16 supermarkets. The hog viscera were sampled from one local abattoir. From 891 mainland pork carcasses, 38 (or 4.3%) were positive. S. typhimurium and S. derby were the predominant serotypes isolated. No salmonellae were recovered from 192 mainland viscera samplings. The mainland pork samplings were obtained from three supermarkets and one restaurant. Table 3 summarizes the distribution of Salmonella serotypes isolated from the various food products. A total of 28 serotypes was isolated. Of the 15 serotypes frequently isolated, only 1 (S. worthington) was isolated more frequently on HE agar medium, but the difference was not statistically significant. Selectiveness of BG agar and HE agar. To compare the selectiveness of BG and HE agar media, a 2-week study was conducted from 1-15 April 1971. A total of 410 food specimens was examined. Of this number, three specimens were positive for Salmonella on BG and four on HE agar media. There was a remarkable difference in the number of nonlactose-fermenting (Proteus, Pseudomonas, Paracolons) subcultures made from BG agar medium (92 subcultures) and HE agar medium (11 subcultures) ( Table 4). This study demonstrated that HE agar medium is far more selective than BG agar medium. DISCUSSION An ideal selective medium for the isolation of Salmonella from various kinds of specimens should be inhibitory against the rapid lactosefermenting coliforms and other nonlactose-fermenting bacteria such as Proteus, Pseudomonas, Citrobacter, etc., and expedite the identification of Salmonella by eliminating the bacteriological examination of these extraneous colonies. On HE agar medium, Salmonella colonies appear as transparent blue-green colonies, with or without black centers (H,S production). The size of the colonies ranged from 0.5 to 2 mm. HE agar medium inhibited most of the coliforms and other nonlactose-fermenting bacteria, thereby facilitating the identification of Salmonella from food products. BG agar medium supported the growth of most of the nonlactose as well as the rapid lactose-fermenting bacteria. On BG agar medium the Salmonella colonies ranged from 0.5 to 1 mm in size. The other nonlactose fermenters as well as the Salmonella colonies appear as transparent red colonies, with or without H2S formation. Therefore, more suspicious BG agaf plates than HE agar plates were selected out for picking, and this may account for the higher number of Salmonella isolates from BG agar. In conclusion, BG agar merits consideration as an isolation medium for food surveys because of its effectiveness in isolating salmonellae. HE agar, although not as effective, was far more selective, and its use would require less effort and supplies to eliminate Salmonella-like isolants. The epidemiological implications of survey results are presented and discussed in a companion paper.

v3-fos

2020-12-10T09:04:12.710Z

1973-05-01T00:00:00.000Z

237232605

s2

Interacting Effects of pH, Temperature, and Salt Concentration on Growth and Survival of Vibrio parahaemolyticus Thermal resistance and minimal pH and temperature conditions for growth of Vibrio parahaemolyticus in artificial media containing 3 and 7% sodium chloride were studied. Growth was observed at pH 4.8 and at 5 C. Vibrio parahaemolyticus was demonstrated to grow at 22 and 42 C, at pH 5 to 11, and at NaCl concentrations of 1 to 7% (4). Sodium chloride was demonstrated to have a protective effect on the viability of V. parahaemolyticus in Trypticase-soy-broth (TSB; BBL) held at 48 C for 40 min or at -18 C for up to 30 days, and fish homogenate was protective when compared to TSB (1). The organism was shown to be very sensitive to pH values below 6.0, but not to pH values ranging from 6 to 10 in shrimp homogenates (5). Some survivors were noted after heating of homogenates at 60 or 80 C for 15 min. Temmyo (3) reported that V. parahaemolyticus was killed after heating at 55 C for 10 min or at 60 C for 5 min in peptone-water. The present study was designed to establish minimal initial pH values at which six strains of V. parahaemolyticus would grow in an artificial, liquid medium. Six strains of V. parahaemolyticus (107914, 8700 04:K11, 284-72, 0, T-3765-1 03:K7, and 4750 02:K3) were activated by transfer to either TSB containing 3% NaCl or TSB containing 7% NaCl and incubated on a rotary shaker at 37 C. The broth containing either 3 or 7% NaCl was adjusted to pH values ranging from 4.7 to 8.0 in 0.1-unit increments by adding HCl or NaOH and dispensed in 10-ml samples in screw-cap tubes. Changes in pH values were measured after sterilization. Ten tubes of each pH and NaCl combination were tempered at 2, 5, 9, 13, 21, and 30 0.2 C in walk-in incubators. A standardized loopful of an 18-h culture grown in TSB with 3% NaCl was transferred to tubes containing tempered TSB with 3% NaCl at various pH values; similarly, V. parahaemolyticus was cultured in TSB with 7% NaCl before its growth was tested in TSB with 7% NaCl at various pH values. Tubes were visually examined for up to 6 days of incubation, and results were recorded as extensive, moderate, or negative. The viable number of organisms contained in the loopful of inoculum was determined to be 107, thus resulting in an initial population of 106 in the 10 ml of test medium. This level of organisms produced no visual change in turbidity and was judged to have had no increase in cell numbers after 6 days of incubation. A population of approximately 107 to 5 x 107 was recorded as moderate growth, and populations exceeding 5 x 107 were considered extensive. These populations had been determined previously by plating on Trypticase-soy-agar (TSA). Thermal resistance of V. parahaemolyticus was studied. A 0.3-ml portion of a 24-h culture was dispensed into tubes containing 19.7 ml of TSB with 3% NaCl which had been tempered at 53 ± 0.2 C (128 F) in a water bath. Heating menstrua had been adjusted to pH values ranging from 5.0 to 8.0 in 0.5-unit increments. Samples were withdrawn, and dilutions were made immediately by using a 3% NaCl in water diluent before plating in TSA with 3% NaCl tempered at 45 C. Recovery was at 37 C, and counts were made after 24 h of incubation. Two separate experiments were performed in duplicate. Table 1 shows the minimum pH values at which V. parahaemolyticus was observed to grow. Values are listed regardless of the number of tubes out of 10 which showed increased population. Extensive growth at 5 C was not observed at the pH values examined; no growth was observed at 2 C. Growth at 5 and 9 C was only at an alkaline pH. There was a tendency for growth at lower pH's as the incubation temperature was increased, regardless of NaCl concentration. With the exception of strain 4750, V. parahaemolyticus was demonstrated to grow at lower pH values in the TSB with 3% NaCl than in TSB with 7% NaCl. Strains 107914 and 8700 both grew in TSB with 3% NaCl at pH 4.8 when incubation was at 30 C, the lowest pH tolerance observed in the study. Figure 1 indicates the decimal reduction times for V. parahaemolyticus. The six strains were least sensitive to heat at pH 7.0 and most sensitive at pH 5.0. Differences in lethal effects of heat on the six strains do occur, however, at the same pH. Strain T-3765-1 was the least resistant to heat at pH 5.0, but' was most resistant at pH 7.0. Experimental design may lack the sophistication required to permit emphases on actual numbers; however, V. parahaemolyticus was extremely sensitive to heat treatment at 53 C in a TSB with 3% NaCl medium adjusted to pH 5.0 to 8.0. The organism was least sensitive at pH 7.0. 844 Data presented here were derived by using liquid media, and some caution must be taken if comparisons are to be made with similar pHtemperature-salt conditions in food products or other natural substrates. Strain 0, for example, was reported to survive in shrimp homogenate after heating at 60 or 80 C for 15 min (5). An initial decrease in viable population of strain 0 in whole shrimp, followed by an increase between 4 and 8 days at 7 C, was noted. Kaneko and Colwell (2) reported that 10 C was the minimum temperature for growth for V. parahaemolyticus in a natural environment. Results reported from the conditions observed in this study therefore do not necessarily reflect minimum tolerances to adverse pH and temperature in all systems. However, data may be useful when determining the causative organism in foodborne outbreaks where V. parahaemolyticus is suspect.

v3-fos

2018-04-03T00:30:42.084Z

1973-01-01T00:00:00.000Z

12464285

s2

Lysine-lactose browning products. I. Properties of the reaction products. Fairly typical Maillard-type reaction products were obtained from the lysine-lactose browning system. These substances gave positive ninhy drin (17), orcinol (16) and LOWRY (19) reactions and fraction D1, P1 and P3 showed fairly higher sugar content. Fractions D, and P1 contain 22.4 and 21.8% of sugar, respectively. Fractions D1 and P1 yielded 6.95 and 9.44% of free lysine, respectively, i.e, this corresponded to 25 and 24% of their original total nitrogen levels. A preliminary in vitro test of the fractions (P1 and P3) using M. tuberculosis H37Pv suggests some growth stimulation properties. Recently, activity against micro-organisms has been found to be present in some of these reaction products (4,9,22,23). The products resulting from heating glycine-dextrose inhibit the growth of Phytophthora fragaria (4), and heated skim-milk inhibits the growth of L. bulgaricus-09 (S), while N-D-glucosyl-glycine stimulates the growth of L. gayoni (6). It was recently demonstrated that the product of lysine-xylose browning system has stronger antimicrobial activity for Staphylococcus aureus 2O9 p (23). Reviews on the Bifidus factors (7,8) indicate that milk-whey has nitrogen-containing oligosaccharides which act as a growth factor for certain strains Lactobacillus bifidus and that "Papain hydrolysed casein" also contains similar factors. HABERLAND (9) pointed out that N-(4-carboxy-3 hydroxyphenyl)D-glucosyl-amine has an antitubercular activity. These findings suggest that the amino-carbonyl or the Maillard reaction produce either inhibitors or stimulators of micro-organisms growth. We have used the lysine-lactose browning system as a possible guide to the 2 National College of Food Technology , University of Reading, Weybrige, Surrey, U. K. 251 improvement in purification of anti-mycobacterial factors in milk-whey (10,11). The present paper reports on the properties of melanoidin-like substances result ing from the browning reaction in lysine-lactose systems and their possible bio logical activity in relation to Mycobacterium. Preliminary reports of this work have been published by FUJIWARA and CoULSON (10,11 S1=1-butanol-acetic acid-water (4:1:5, v/v/v upper phase) S2=1-butanol-pyridine-water (4:6: The detection was carried out using aniline hydrogen phthalate for sugars (13) and 0.2 % ninhydrin in acetone for amino acids. Absorption spectra. Absorption spectra were determined using the Hilgar manual and Shimadzu (Beckman type) UV Spectrophotometers, and the Hitachi Infra-red Spectrometer. (15)). Test of activity on microorganisms. The tests for activity on microorganisms were kindly carried out by Dr. J. D. Sleigh of the Bacteriology Department, University of Edinburgh, Scotland, using the Disc method (26). Discs 5mm in diameter used for test were cut from Whatman No. 1 filter paper (such discs were able to absorb about 0.01ml of fluid). The amounts of fractions per disc are given as follows: All solutions were sterilised prior to use with the aid of a Hemmings centrifugal fi lter (Beaumaris Instrument Company, Beaumaris, Anglesey) fitted with "Oxoid" cellulose acetate discs. Infrared absorption spectra The infrared absorption spectra (KBr disk) of P1 and D1 are shown in Fig. 8. Both samples showed characteristic absorption bands representing -OH(3300-3400, 2940cm-1), C-O and C-O-C (1000-1150cm-1) and pyranose ring (875,775cm-1) of glycosidic compounds. Amide (3300-3400, 1640cm-1) and carboxyl groups (1720, 1400cm-1) were detected in both samples. (27) to which was added 10 serum albumin. The inoculum was a 1 in 100 dilution of a 10-day culture of the H37Rv strain of M. tuberculosis in fluid Dubos medium (27). Growth of small rough buff colonies on both P1 and P3 was first observed after only 4 days incuba tion. These colonies were actually, growing on the surface of the disc and when fi lmed they were shown to be rather large and fat acid and alcohol-fast bacilli. Subsequently growth took place all over the slope but there was marked enhancement in the area surrounding the disc. This experiment was repeated and although growth enhancement was again observed it was not nearly so marked-colonies first became visible after some 10 days on this occasion.

v3-fos

2018-04-03T00:31:44.510Z

1973-10-01T00:00:00.000Z

12768670

s2

Fungi that infect cottonseeds before harvest. As a part of an investigation of aflatoxins and other mycotoxins in cottonseeds at harvest, samples of seeds collected from the 1971 crop at locations across the U.S. Cotton Belt were examined to determine the kinds of microorganisms causing internal or seed-coat infection in the field. Aspergillus flavus infection was absent from all seeds examined from most areas but was present in some samples from Arizona, California, and Texas. Fusarium spp., Alternaria sp., and A. niger caused internal infection at many locations; Colletotrichum gossypii and Rhizopus stolonifer were present in seeds from some areas but were generally much less common. Many of the infections with A. niger were in the seed coat. Bacterial infections were fairly frequent. In a series of commerical samples from Arizona. A. flavus infection was found in 61% of seeds, with fiber showing the bright, greenish-yellow (BGY) fluorescence that is diagnostic for A. flavus boll rot. Aflatoxin contamination was also concentration in the same seeds. The above findings agree with previous data showing that aflatoxin contamination of cottonseeds before harvest occurs rarely, if at all, in most parts of the U.S. Cotton Belt and that when such contamination does occur, it tends to be concentrated in seeds with the BGY fluorescence in their fiber and seed fuzz. fore harvest. A. flavus boll rot can be detected by a characteristic bright, greenish-yellow (BGY) fluorescence caused by A. flavus in spots in the fiber (18) and is relatively uncommon. It has been noted particularly in certain western parts of the Cotton Belt (30). Aflatoxin contamination in the seeds at harvest appears to be similarly uncommon but has been found in some samples in the same areas as the boll rot (19). Aflatoxins have been detected most frequently and at highest levels in seeds whose fiber and seed fuzz display the BGY fluorescence (19,20). Aflatoxin contamination of U.S.grown cottonseeds at harvest thus appears from previously available data to be significant only in localized areas. However, because of the present and anticipated future importance of cottonseed products in both animal and human nutrition and the unusual hazards associated with the aflatoxins (15), we considered it desirable to make further observations. Fungi that infect cottonseeds before harvest (3,19). Seed infection with A. flavus has been noted in association with the characteristic BGY fluorescence in the fiber (19). As a whole, however, investigations of preharvest infections of cottonseeds have involved examination of only a relatively small number of seeds from limited cotton-producing areas. Mayne (21) reported that 17 of 41 fungal isolates from 28 samples of cottonseeds from six southern states were A. flavus, but the history of most of her seed samples was not known, and infection may have occurred in damp storage after harvest. Further background information on the cottonseedaflatoxin problem has been detailed and documented with references elsewhere (19). This paper reports results of examinations for fungal infection of cottonseeds grown across the U.S. Cotton Belt under widely variable condi-tions of culture and harvested at known dates. Confirmatory aflatoxin analyses on certain samples are also reported. MATERIALS AND METHODS Bolls from commercial cotton varieties were collected directly from plants on four to eight picking dates at 17 locations across the U.S. Cotton Belt ( Table 1). The maximum weathering period before harvest was not accurately known, but probably it was not in excess of that common with commercial cotton. The bolls were dried at the point of origin and sent to Beltsville. At Beltsville, each boll was ginned individually by hand, and the seeds were delinted in concentrated sulfuric acid, washed first in 1% sodium carbonate solution and then in water, dried, and treated overnight in chlorine gas as described previously to surface disinfect (20). Seeds that were immature or obviously damaged were avoided for the examinations summarized in Table 1. For each picking date, fifty seeds were examined. Five seeds from each boll were planted on 2% water agar in a petri dish and were incubated for 4 days at 30 C in a room with a 12-h light cycle provided by daylight fluorescent lamps, after which they were examined by microscope to detect any fungi or bacteria growing out of them. No distinction was made between A. flavus and A. parasiticus Speare. The 161 commercial seedcotton samples from fields in unspecified parts of Arizona were kindly provided by B. B. Taylor. The samples, as received, weighed in the range of 400 to 2,600 g. Seedcotton consists of seeds with fiber still attached and occurs in locks, one lock for each of the four or five segments of the boll and each lock containing about eight seeds. Analyses for aflatoxin B1 were by a mini-plate screening procedure which detects aflatoxin B1 at levels above 100 parts per billion (ppb), followed by a quantitative assay on 20-by 20-cm Schleicher and Schuell silica gel plates. The mini-plate method, to be detailed elsewhere, consisted essentially of the following. A seed meat sample was shaken with three times its weight of chloroform-acetonitrile (1:2) and onehalf of its weight of 10% aqueous ferric chloride, after which the extract was chromatographed on a 2-by 3-inch (5.08-by 7.62-cm) thin-layer chromatography plate with a 0.75-inch (1.905-cm) band of A120s at the bottom and a 2.25-inch (5.7-cm) band of silica gel on the remainder of the plate. Development was for 8 min with diethyl ether-methanol-water (96:3: 1). For a more quantitative estimate of the aflatoxin level, a sample of the above extract was diluted with benzeneacetonitrile (98:2), and this diluted extract was then spotted on a Schleicher and Schuell plate and developed in an unlined tank with diethyl ether-methanolwater (96:3: 1). Table 1 records data on fungal and bacterial infection of seeds from 17 locations across the Cotton Belt. A. flavus was detected only in the collections from Tulare County, California, and Vernon, Texas, and at low levels at these locations, even though the overall level of microbial infection in most of the samples was First Last slmpick pickB lackville, S.C. 6 8/27 11/29 300 1 0 10 2 17 1 27 0 1 59 College Station, Tex. Stoneville, Miss. 8 In almost all cases, infections of the seeds of Table 1 appeared to involve only a single fungus or bacterium per seed. The infecting fungus generally grew luxuriantly out of the seed at either the chalazal or funicular end. An exception to this was A. niger, which caused both internal (as above) and seed-coat infections. In the latter case, a sparse sporulating outgrowth of the fungus appeared over much or all of the surface of the seed, and hyphae could be seen inside the seed coat but not in the seed meats when the infected seeds were cut open. More than half of the observed infections with A. niger were in the seed coat. RESULTS The general absence of detectable A. flavus infection in seeds from the samples of Table 1 was believed to be real and not a result of any inadequacy in the detection method. The same method was used successfully to detect A. flavus in cottonseeds in previous work (19). Furthermore, it was successfully applied to a series of 161 commercial samples of the 1971 crop in Arizona in the present investigation, with results as shown in Table 2. All locks with BGY-fluorescing fiber were picked out of 25 samples, and 5 seeds from each fluorescent subsample were delinted and examined for fungal infection; of the 125 such seeds, 76 (61%) were infected with A. flavus. For comparison, 25 seeds from locks with non-BGY-fluorescing flavus infections were highly concentrated in the seeds with the BGY-fluorescing fiber. The BGY-fluorescing locks were low in number, constituting only 0 to 1.2% of the weight of the total seed cotton samples. The high general level of fungal infection in the seeds from nonfluorescing locks may have been related to breaks in the seed coat, readily seen in many of them, possibly caused by mechanical harvesting. A. niger was the predominant fungus isolated from these seeds ( Table 2). The fact that A. flavus infections in the 161 Arizona samples were highly concentrated in seeds with BGY-fluorescing fiber suggested that aflatoxins might also be similarly concentrated in these seeds. Aflatoxin analyses were made to check this point. Of the 161 seed cotton samples, 65 had no BGY-fluorescing locks and no detectable aflatoxin B1 by the mini-plate procedure. Of the 96 samples with BGY-fluorescing locks, 51 contained detectable aflatoxin B1 in the seeds from the fluorescing locks and 45 did not. From the same 96 samples, a random selection of non-BGY-fluorescing locks was made for each sample, and only three showed detectable aflatoxins present in their seeds. In each of these three cases, aflatoxins had also been detected in the seeds from the BGY-fluorescing locks in the same samples. Extracts from seeds of the BGY-fluorescing locks were spotted on 20-by 20-cm Schleicher and Schuell thin-layer chromatography plates, and the levels of aflatoxin B1 were estimated ( DISCUSSION The results here reported on A. flavus infections of cottonseeds are in accord with and substantiate previous evidence indicating that A. flavus boll rot and aflatoxin contamination of cottonseeds at harvest involve only a small fraction of the total U.S. cotton crop (19). Data indicate that all of these phenomena are very uncommon in the Southeast and mid-South. All have been obviously present in the Imperial Valley of California and occur at detectable levels in some still-undefined areas of Arizona. A. flavus boll rot has also been detected repeatedly at low to moderate levels in parts of Texas, especially in the region near Brownsville, but also in an area around Dallas. Evidence indicates that A. flavus boll rot is typical of hot, dry climates and that its incidence may be materially increased by insect attack on the bolls (19). Aflatoxin contamination of the seeds at harvest appears to be very uncommon, if present at all, in any growing area where the BGY fluorescence, caused by A. flavus, is not also easily detectable. Such contamination occurs especially in seeds whose fiber or seed fuzz, or both, exhibit the BGY fluorescence. The above observations provide no evidence regarding A. flavus infection and aflatoxin contamination of the seeds during humid transit and storage, matters still very inadequately investigated. The following comments may be made about the several fungi other than A. flavus which were found in the present work to infect cottonseeds before harvest. Alternaria. Cotton may now be added to wheat (4,6,17,22), barley (7,12,17), oats (17,25), rye (17), soybeans (10,24), corn (17), and the very many other plants (23) whose seeds are known to be infected frequently with this fungus in the field. Some isolates under some circumstances produce mycotoxins (5), but no information on such mycotoxins in the cotton crop is known to have been reported. A. niger. This fungus seems to be present in cotton particularly in the western parts of the U.S. Cotton Belt. This conclusion is based more on previously published fiber-infection data (29) than on the seed-infection data here presented. The fungus appears to be more common in field infections of cottonseeds than in field infections of seeds of other plants. C. gossypii. Extensive data on infections of fiber and bolls show this fungus to be geographically localized in areas with moderate to high rainfall (29, M. E. Simpson, P. B. Marsh, and E. C. Filsinger, Plant Dis. Rep., in press). The seed data reported here are less extensive but seem in general accord with this concept. Nigrospora oryzae (Berkley and Broome) Petch. This fungus has been reported by others to be a common cause of cotton boll rot in California (13) but was found rarely in the present study. If we were to select any major infecting fungus as distinguishing the fungal flora of cottonseeds at harvest from that of most other plants, it would be A. niger. We cannot, however, ascribe infection with this fungus to the oil component of cottonseed nor to any other chemical component. The fact that A. niger is a vigorous wound parasite capable of penetrating from wounded into living tissue of the boll wall may be relevant. In the peanut, also an oil-bearing seed, the fungal population before harvest includes numerous species of Penicillium and Aspergillus (14), but the flora in this case are probably influenced strongly by soil contact. The VOL. 26,1973 tendency for A. flavus to occur on small grains, especially under conditions of heating in storage, and the semixerophytic nature of the organism have been noted by Semeniuk (28). Because species of Alternaria and Fusarium cause field infections in the seeds of so many crops that have important food and feed uses, further investigations of mycotoxins produced by these fungi are especially important to U.S. agriculture. Some readers might wonder whether our surface sterilization of cottonseeds in chlorine gas, after delinting in sulfuric acid (20), might kill some fungi found internally in the seeds or whether the chlorine might selectively kill A. flavus. Actually, bolls incubated for several days with A. flavus, as previously described (18), and then examined by this method have shown a high internal seed infection with the fungus; the 61% infection of the seeds from BGY-fluorescing locks recorded in Table 2 also suggests no major killing of the fungus internally in the seeds. Uninfected seeds germinate in high percentage after the chlorine treatment and produce seedlings of normal appearance. Some insight may also be gained by comparing the microbial population of the seeds with that of field-exposed cotton fiber (29) because, in the latter case, no sterilization was used. With the fiber, the microbial population consisted mainly of actinomycetes, Altemaria sp., A. niger, bacteria, C. herbarum, Fusarium spp., and R. stolonifer. In the seeds, all of these were detected except the actinomycetes and C. herbarum. We believe, however, that the absence of these two forms from the detected seed population did not result from their elimination during the sulfuric acid-chlorine treatment. Rather, we think that internal infection of the seeds occurs primarily through the chalazal opening, that the rapidly growing fungi tend to preempt the infection court, and that the slower growing actinomycetes and C. herbarum are eliminated by selective competition. We would agree that a real question about the adequacy of our methods for detection of microorganisms in both cotton fiber and seed may exist in respect to D. gossypina, which probably does not produce recognizable fruiting bodies during the relatively short incubation periods used.

v3-fos

2020-12-10T09:04:16.907Z

1973-06-01T00:00:00.000Z

237233633

s2

Persistence of Aflatoxin During the Fermentation of Soy Sauce Aflatoxin was produced by Aspergillus parasiticus NRRL 2999 but not by A. oryzae during fermentation of soy sauce. Little aflatoxin was degraded within 6 weeks unless Lactobacillus delbrueckii also was present. Sudden outbreaks of "turkey X disease," later considered to be aflatoxicosis, have been attributed to the toxic metabolites of Aspergillus flavus Link ex Fries (1). Strains of Aspergillus, e.g. A. oryzae, are popularly used in Asia in the manufacture of soy sauce as well as other varieties of fermented foods (4). Although a number of strains of A. oryzae have been shown not to produce aflatoxin (5), it is difficult to distinguish A. oryzae from A. flavus or A. parasiticus contaminants in substrates. Because both are widely distributed, either could be involved in an impure fermentation. Another microorganism, Lactobacillus delbrueckii, is commonly used with A. oryzae for producing soy sauce (4). Therefore, the research reported here was undertaken to determine whether an aflatoxin producing A. parasiticus strain could grow with A. oryzae and with L. delbrueckii in soybean koji, whether aflatoxin would be present in the final soy sauce, or whether aflatoxin appeared at some point during preparation, but then was degraded at a later stage in the process. A. parasiticus NRRL 2999, A. oryzae NRRL 1988, and L. delbrueckii NRRL B-445 were obtained from the Northern Utilization Research and Development Div., U.S.D.A., Peoria, Ill.; aflatoxin standards were obtained from the Southern Utilization Research and Development Div., U.S.D.A., New Orleans, La. The procedure for soy sauce production cited by Hesseltine and Wang (6) was adopted, but was modified as indicated in Fig. 1. Fernbach flasks (2.8 liters) were used instead of conventional open "koji" boxes to prevent undesirable contamination, and a clean room was utilized as an incubation chamber. The substrates were inoculated with 8 ml of a I Present address: Bundesanstalt fur Fleischforschung, 8650 Kulmbach, West Germany. culture of A. oryzae NRRL 1988 (1 x 10f spores/ml) and a 5-ml suspension of L. delbrueckii (1 x 108 organisms/ml) into the flasks. Then they were stored at room temperature. To study aflatoxin production during the fermentation of soy sauce, a 2 x 106 spores/ml suspension of A. parasiticus NRRL 2999 was added to the substrate immediately after inoculation with A. oryzae and L. delbrueckii. For the aflatoxin B1 degradation study, an aqueous preparation of aflatoxin B1 (5 gg/kg of substrate) and 0.5 M lactic acid (100 ml/kg of substrate) were introduced immediately after inoculation with A. oryzae and L. delbrueckii. Extraction followed the Ass. of Official Analytical Chemists method suggested by Eppley (3). Soy sauce substrate (50 g) containing wheat, soybeans, and liquid (50 ml) was shaken vigorously with chloroform (50 ml) and decanted. The procedure was twice repeated and A.oryzae and L.delbrueckii Fig. 2 show the amount of aflatoxin in each sample during fermentation and throughout the processing of the koji into soy sauce. The sample containing only A. oryzae and lactic acid bacteria showed no production of aflatoxin during the entire fermentation period (sample I). Aflatoxin (8,500 ag/kg) production by A. parasiticus NRRL 2999 alone peaked after 1 week (sample II). Aflatoxin (7,000 Ag/kg) was produced in the sample inoculated with A. parasiticus NRRL 2999 and L. delbrueckii (sample III). Lower quantities of toxin (2,100 ,g/kg) were detected in sample IV after 1 week, i.e., koji inoculated with A. parasiticus, A. oryzae, and lactic acid bacteria. Persistence of aflatoxin. Observations made with TLC plates indicated that aflatoxin degradation starts immediately after bringing in samples II, III, and IV. The TLC chromatograms of all three samples were nearly identical. No aflatoxin was detected in soy sauce containing only A. oryzae and L. delbrueckii (sample I). Sample II showed only 8% degradation of the aflatoxin in 6 weeks, whereas sample III showed 45% conversion. During this same time interval, there was 55% degradation of the toxin in sample IV. Acid production by L. delbrueckii may have catalyzed the conversion of aflatoxin B, to B2a* Direct addition of aflatoxin B1 and lactic acid (sample V) to the fermentation medium resulted in conversion of aflatoxin B, to derivatives comparable in R. value to aflatoxin B2., a comparatively nontoxic form (2). Lindenfelser and Ciegler (7) found the acid-catalyzed conversion of aflatoxin B, to B2a if a sufficient amount of lactic acid was supplied. The mechanism of the reactions among participating organisms is not clear, but the degradation products from samples III, IV, and V show Rf values lower than those of aflatoxin B1 and B2 on TLC.

v3-fos

2018-04-03T05:39:14.235Z

1973-02-01T00:00:00.000Z

43697623

s2

Lysogeny in lactic streptococci producing and not producing nisin. Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species. Lysogeny has hitherto not been observed in group N streptococci even though bacteriophages have been isolated from these bacteria by many workers (4)(5)(6)(7)(8)(9)(10). The purpose of this work was to determine whether lysogeny occurs in lactic streptococci, including strains producing the antibiotic nisin, and to examine some properties of host-phage systems in these bacteria. MATERIALS AND METHODS Strains. Eighty-seven strains were investigated, including 46 strains of Streptococcus lactis, 24 strains of S. diacetilactis, and 17 of S. cremoris; 12 of the S. lactis strains produced nisin. The strains were kept at 4 C and transferred once a week to a 10% water suspension of defatted powdered milk. The strains came from the Institute of Dairy Industry, Warsaw; Pure Dairy Cultures, Laboratory of Olsztyn; Department of Industrial Microbiology, Technical University of L6dZ. Medium. The medium consisted of the following: beef heart infusion, 1,000 ml; peptone (Gurr), 10 g; yeast extract (Difco), 10 g; NaCl, 5 g; glucose, 10 g; 1 M CaCl2, 1 ml; final pH of the medium, 7.2. The medium was autoclaved at 117 C for 15 min. This medium will be referred to as X. Equipment and chemicals. The equipment and chemicals used were an ultraviolet (UV, bactericidal lamp (Westinghouse G 30 T8), Berkefeld N2 and Seitz EKX filters; and mitomycin C (Nutritional Biochemicals Corp.). Determination of the plaque-forming units. Determination of the plaque-forming units was performed by the method of Horvath and Alffildi (4) or Gratia (see reference 1). Number of viable bacteria. The number of viable bacteria (colony-forming units) was determined by the plate method. The strains were incubated in a water bath or incubator at 30 C. RESULTS AND DISCUSSION Search for lysogens. Search for lysogens was performed by Fisk's method (2) looking for phages in bacterial culture filtrates. In the case of filtrates which hindered the growth of indicator strains, 10to 10-3 dilutions of the filtrates were applied, as well as the undiluted filtrates. This was necessary to distinguish between inhibition of the growth of indicator strains caused by phages and that due to other factors, e.g., nisin, produced by lactic streptococci. This distinction is possible if the filtrates are diluted to 10-3 since at this concentration only the presence of phages, but not the activity of other factors, is detected. The culture filtrates were screened for phages after 3 and 18 h of incubation of the strains and after 3 h of incubation of strains which were treated with UV (usually 260 ergs per mm2) immediately prior to incubation. Each strain was treated as a potential lysogen and as a potential indicator strain. Five lysogenic strains were found among 46 S. lactis strains examined. Four of them (37, 40, 41, 45) liberated phages which could only multiply in one indicator strain, S. lactis 28. A fifth strain, 31, liberated phages which could multiply in several strains belonging to the three examined species. Strains 40, 45, and 28 produced nisin. Phages were obtained sporadically from five other S. lactis strains, but reproducible results were not obtained. Two lysogenic S. diacetilactis strains (84 and 87) were found among the 24 investigated strains. Phages liberated by these strains were able to multiply in a number of strains from the three examined species. No lysogens were found among the 17 tested strains of S. cremoris, in spite of the use of various experimental conditions. Spontaneous liberation of phages. Eighteen-hour cultures were centrifuged and the cells were washed twice with Ringer's solution. The bacteria were then resuspended in a volume of Ringer's solution equal to the volume of the initial culture. The suspensions were diluted 1: 100 with X medium. After taking samples to determine the number of infective centers (CI), the strains were incubated to examine the kinetics of the increase of the number of phages and living bacteria. The controls performed indicated that a maximum of 1 out of 500 observed plaques does not represent CI. Table 1 contains data on the frequency of spontaneous induction which was 1 to 2% and 0.04 to 0.07% for S. lactis and S. diacetilactis strains, respectively. Effectiveness of induction depending on the dose of UV. Bacteria from 18-h cultures were washed twice with Ringer's solution and then resuspended in the same solution; 2.5 ml of this suspension was poured onto each petri dish (5.5 cm in diameter) and irradiated with UV. The number of induced cells and of surviving bacteria were determined immediately. A correlation between the number of induced At the optimal UV dose (260 ergs/mm2), 20 to 37% of the cells were induced in S. lactis and 7 to 10% were induced in S. diacetilactis strains. Only in the case of S. lactis strain 31 was no increase in the number of induced cells after UV treatment found, as compared with spontaneous induction (data not presented in the table). Sometimes, a 90% decrease of the number of phages was observed in this strain after irradiation. In this strain, neither mitomycin C at various concentrations nor a raised temperature was effective in the induction of phages. Investigations now being performed suggest that in this strain the prophage is not integrated into the chromosome. One-step growth of phages. Lysogen suspensions after UV irradiation (260 ergs/mm2-) were diluted 1:100 with X medium and incubated. Samples were taken for determination of the plaque-forming units and the number of living bacteria. One-step growth of the same phages was determined in parallel after infecting sensitive bacteria. The results are presented in Table 2. The course of one-step growth of all S. lactis phages was similar after induction. The latent periods were 80 to 100 min long, and the periods of growth 30 to 60 min; 40 to 46 phages were liberated from each cell. For both lysogens of S. diacetilactis, the latent period was 40 to 60 min, the period of growth 60 to 80 min, and 10 to 25 phages were liberated from each cell. When sensitive bacteria were infected with these phages, the periods of one-step growth of the phages were shorter, but the yields of phages were similar to those obtained after UV induction of lysogens. Lysogenization of sensitive bacteria. To check whether the isolated phages lysogenize indicator strains, suspensions of S. lactis )genic lactic streptococci by using various UV phages (with the exception of phages from strain 31) were applied to S. lactis strain 28, and those from S. diacetilactis) strains of S. diacetilactis 86. All phages were able to lysogenize indicator strains, as most bacterial colonies which grew in the presence of the phages contained phage-resistant cells which liberate phages spontaneously and after UV induction. Delysogenization and relysogenization of nisin-producing strain S. lactis 45. This strain was irradiated on solid medium with a UV dose of 650 ergs/mm2 which inactivated 99.9% of the cells. Among the colonies grown after UV irradiation, five nonlysogenic ones were found which were susceptible to phage 45 and produced nisin. The subclones thus obtained were susceptible to relysogenization with phage 45. Lytic spectrum of the phages. Phages at routine test dilution (RTD) and 1,000 x RTD concentrations were used for lytic spectrum determinations. As is shown in Table 3, phages from strains S. lactis 37, 40, 41, and 45 were able to reproduce in S. lactis strain 28 only. The phages from S. lactis strain 31 and S. diacetilactis strains 84 and 87 had a wide lytic spectrum and were able to multiply in a number of strains (including four nisin producers) belonging to the same and to the two remaining species. The lytic spectrum was slightly more narrow when the phages were used at RTD concentration. Repressor resistance of S. lactic strain 28 after lysogenization by a single phage to the remaining S. lactis phages. Investigations of the lytic spectrum and one-step growth of the isolated phages of S. lactis suggested a great similarity or even identity of these phages. To check whether these phages were identical, the repressor resistance of S. lactis strain 28 (lysogenized with one of the phages) to the remaining phages was determined; it was performed by lysogenization of strain 28 with one of the four phages and by subsequent treatment of this strain with the three remaining phages at RTD concentrations. The results presented in Table 4 suggest that phages 40, 41, and 45 produce a similar type of repressor, because cross-resistance to these phages is observed. It seems that this repressor differs from that produced by phage 37. This indicates that some subtle differences do exist between phage 37 and the remaining phages.

v3-fos

2020-12-10T09:04:17.068Z

1973-03-01T00:00:00.000Z

237230420

s2

Utilization of n-Alkanes by Cladosporium resinae Four different isolates of Cladosporium resinae from Australian soils were tested for their ability to utilize liquid n-alkanes ranging from n-hexane to n-octadecane under standard conditions. The isolates were unable to make use of n-hexane, n-heptane, and n-octane for growth. In fact, these hydrocarbons, particularly n-hexane, exerted an inhibitory effect on spore germination and mycelial growth. All higher n-alkanes from n-nonane to n-octadecane were assimilated by the fungus, although only limited growth occurred on n-nonane and n-decane. The long chain n-alkanes (C14 to C18) supported good growth of all isolates, but there was no obvious correlation between cell yields and chain lengths of these n-alkanes. Variation in growth responses to individual n-alkane among the different isolates was also observed. The cause of this variation is unknown. all isolates, but there was no obvious correlation between cell yields and chain lengths of these n-alkanes. Variation in growth responses to individual n-alkane among the different isolates was also observed. The cause of this variation is unknown. For the past decade the growth of microorganisms in jet fuel, resulting in the formation of biological sludge and the corrosion of fuel tanks, caused serious concern in aviation industry. To date, no completely satisfactory control of microbial contamination has been found (6,7,16). It is now generally believed that a filamentous fungus, Cladosporium resinae, plays the major role in most cases of fuel system fouling (5,8,16). The organism has also been found to grow in hydraulic fluid, diesel fuels, lubricating oil, and other kerosene-type fuels (16). During its growth on jet fuel, C. resinae preferentially utilized the component C9 to C 13 n-alkanes (16). On the other hand, gasoline which contains mostly short-chain alkanes is usually not susceptible to attack by the fungus (10). Recently, Cooney and Proby (3) reported that the growth of C. resinae on all individual n-alkanes, with the exception of n-dodecane, decreased in shake cultures as the chain length increased from n-decane to n-tetradecane. These reports suggest that C. resinae may exhibit a certain degree of substrate specificity with regard to the n-alkanes. At this time, studies on n-alkane utilization by the fungus, which were usually carried out with a single isolate, are either incomplete or qualitative in nature (3,10,16,19). The natural variability of C. resinae (16) is such that results obtained from work done with only one isolate could not be considered representative. Furthermore, differences in cultivation methods and in the amount of hydrocarbon substrate actually available to the organism, which depends partly on the physical state of the hydrocarbon, would undoubtedly complicate the interpretation of observations obtained by different investigators. This communication reports quantitative data on the comparative utilization of individual liquid n-alkanes, ranging from n-hexane to n-octadecane, by several isolates of C. resinae under standard conditions. An attempt was also made to investigate the effect of various n-alkanes on the growth of the organism on glucose. MATERIALS AND METHODS Organism. Isolates 35A, 89A, 95B, and 102B, which were isolated from Australian soils and identified as C. resinae f. avellaneum (14), were obtained from D. G. Parbery, University of Melbourne, Victoria, Australia. They were maintained on Bushnell-Haas glucose-agar slants. Media. Bushnell and Haas (1) mineral salts solution, containing ammonium nitrate as nitrogen source, was used as the aqueous phase in all experiments. Bushnell and Haas agar contained additional 2% agar and 1% glucose. Normal alkanes (.>99% purity) were obtained from Koch-Light Laboratories, Colnbrook, England, and Eastman Kodak Co., New York. Purity of the n-alkanes was checked by both gas-liquid chromatography and thin-layer chromatography; the latter showed that oxygenated impuri-454 ties were absent in all of the n-alkanes used. Jet A-1 commercial fuel was kindly supplied by the Shell Co. of Singapore. Glucose, when used as a carbon source in liquid cultures, was autoclaved separately in concentrated solution and added aseptically to the medium to give a 2% final concentration. Cultivation methods. All liquid culture experiments were carried out in duplicate in standard 250-ml Erlenmeyer flasks. For the preparation of liquid inoculum, the organism was grown on Bushnell-Haas agar slants for one to two weeks at room temperature. A spore suspension was obtained by washing the spores off the agar slants with 10 to 15 ml of sterile distilled water. A 0.3to 0.5-ml sample of this suspension was continuously agitated during inoculation and then pipetted into each flask. The effect of hydrocarbon substrate concentration on growth was determined by adding increasing quantities (1 to 40 ml) of Jet A-1 fuel to a constant volume (40 ml) of the aqueous salt medium. After inoculation, the flasks were incubated statically at 30 C for 35 days. Utilization of individual n-alkanes by the fungus was determined under the following standard conditions. Each flask contained 40 ml of mineral salt medium and 5 ml of the n-alkane being tested; these were added as an overlay on the aqueous surface. After inoculation, all flasks-were incubated statically at 30 C for 35 days. For reference, glucose and Jet A-1 fuel were included as substrates. The effect of individual n-alkanes on spore germination in glucose solution was determined by adding 0.5 ml of the spore suspension to a mixture of 5 ml of the hydrocarbon being tested and 40 ml of the mineral salt medium containing 2% glucose. The flasks were incubated statically at 30 C for 21 days. To investigate the effect of n-alkanes on mycelial growth, flasks containing 2% glucose-mineral salt medium were inoculated with spores and incubated at 30 C for 24 h, by which time tiny white mycelial colonies were clearly visible. A 5-ml sample of the sterile n-alkane being tested was then added aseptically to the mixture, and incubation was continued thereafter for 20 days at 30 C. Gas-liquid chromatography. This was performed with a Varian Aerography model 1200 gas chromatograph equipped with a flame ionization detector. A stainless steel column (6 ft by '/8 inch ca. 81.28 by 0.05 cm]) packed with 80/100 mesh Chromosorb G and coated with 5% SE-30 was used. Temperature was programmed from 40 to 100 C at 2 C/min for the lower n-alkanes and from 100 to 200 C at 4 C/min for the higher boiling n-alkanes. The carrier gas was nitrogen at a flow rate of 25 ml/min. Temperatures of the injection port and the detector were 250 C. Only a single peak was obtained for all of the n-alkanes tested. Dry weight determination. At the end of the incubation period, the cells from each flask were collected by vacuum filtration. They were washed successively with small portions of distilled water and petroleum ether (bp 40 to 60 C) to remove salts and residual hydrocarbon. Finally, the cells were transferred quantitatively to a preweighed dry aluminium boat and dried to constant weight in an oven at 105 C. RESULTS AND DISCUSSION In view of the influence of flask shape and size on the growth of C. resinae (15), uniform batches of 250-ml Erlenmeyer flasks were used in all experiments. Since shaking generally led to lower cell yields, the flasks were incubated statically in agreement with a previous finding (15). The optimum temperature for growth for the four isolates appeared to be at or near 30 C. Hydrocarbon substrate was added as an overlay, since C. resinae is known not to utilize emulsified jet fuel for growth in liquid cultures (2). The organism grew at the hydrocarbonwater interface, forming a partial or complete mycelial mat. The effect of increasing hydrocarbon concentration on the growth of isolate 35A is shown in Fig. 1. It can be readily seem that growth of the organism in the static two-phase liquid medium was dependent upon the concentration of hydrocarbon and that a high cell yield could only be attained when the hydrocarbon concentration exceeded 10% (vol/ vol). Similar phenomenon has been reported previously for a hydrocarbon-utilizing Candida species (17). A likely explanation seems to be the higher oxygen availability to the aqueous medium, with the imposition of a discrete layer of hydrocarbon (4,12). Results of the utilization of individual n-alkanes for growth by C. resinae are shown in Table 1. Although the fungus grew rapidly in 2% glucose and all four isolates gave almost identical large cell mass, the amount of growth on n-alkanes or jet fuel was relatively small. This is, perhaps, a common feature of hydrocarbon-utilizing filamentous fungi, since there have been very few reports of any mold being able to grow rapidly on hydrocarbons (11,18). The three short-chain n-alkanes (n-hexane, n-heptane, and n-octane) did not support any visible growth of the isolates examined. Tanaka et al. (19) had previously reported that n-hexane and n-heptane could not be utilized by a C. resinae isolate, although Leonard & Klemme (10) claimed that their Hormodendrum (later revised as Cladosporium) isolate from jet fuel showed limited growth in n-octane. All of the higher liquid n-alkanes from n-nonane to n-octadecane were assimilated by the fungus, although only limited growth occurred on n-nonane and n-decane. The considerable natural variability of C. resinae (16) decane supported good growth of isolates 35A and 89A; however, it did not support growth of the other two isolates. On the other hand, n-tridecane elicited relatively high growth response in all isolates except isolate 95B. What caused these differences is unknown. In general, the series of n-alkanes, ranging from n-tetradecane to n-octadecane, appeared to be well utilized, but there was no obvious correlation between cell weights and chain length of the n-alkanes. For a given isolate, one particular n-alkane might give a higher growth response than others; the particular n-alkane eliciting that response could be any member of the n-alkane series that supported good growth. Similar behaviour has been described for other filamentous fungi growing on the C,, to C16 n-alkanes (13). Although Cooney & Proby (3) found that the cell yield of C. resinae decreased with increasing chain length of the n-alkanes from n-decane to n-tetradecane, such was not observed in our investigation. The difference could be due to the different cultural conditions used or the fact that only one isolate was tested on a limited number of n-alkanes by these investigators. Both Lowery et al. (11) and Nyns et al. (13) have observed that n-alkanes shorter than n-decane generally supported little or no growth of a large number of hydrocarbon-utilizing filamentous fungi. Some workers have attributed this to the toxicity of the short-chain n-alkanes which, being generally good lipid solvents, could extract certain essential cell lipids from the cell membrane (9,13). In our present study, the inability of the C. resinae isolates to grow on n-hexane, n-heptane, and n-octane suggests either a lack of specific enzymes for transporting and oxidizing these n-alkanes or toxicity due to the short chain n-alkanes themselves, or both. The possible toxicity of n-alkanes was, therefore, tested experimentally by growing C. resinae on glucose together with various n-alkanes added in excess. Results are shown in Table 2. Both n-hexane and n-heptane completely inhibited spore germination in 2% glucose solution, even after prolonged incubation. Only partial inhibition was brought about by n-octane. n-Nonane and all higher n-alkanes tested had no inhibitory effect on spore germination and subsequent growth, the final cell yields being similar to that of the control. The effect of short-chain n-alkanes on mycelial growth appeared to be less marked. Only n-hexane stopped mycelial growth completely, whereas n-octane and higher n-alkanes had no significant inhibitory effect. On removal of the shortchain n-alkanes from the culture flasks under a gentle stream of sterile nitrogen gas, spore germination and resumption of mycelial growth were observed. The final cell yields were similar to those of the control experiments. Thus, the lower n-alkanes, particularly n-hexane, are fungistatic towards C. resinae growing glucose. Preliminary work also indicates that nhexane and n-heptane inhibited spore germination and mycelial growth when Jet A-1 fuel was used as the sole carbon source. This may explain why C. resinae does not grow readily on gasoline, since the latter is rich in shortchain n-alkanes. At present, the inhibitory action of the short-chain n-alkanes on the growth of C. resinae is not well understood, although it is likely to be related to the cell membrane and the higher solubility of these n-alkanes. While this work was in progress, Walker and Cooney (20) reported in a communication that n-hexane apparently altered the cell membrane and decreased the endogenous respiration of C. resinae.

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2020-12-10T09:04:12.757Z

1973-02-01T00:00:00.000Z

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Simplified Gas Chromatographic Procedure for Identification of Bacterial Metabolic Products A rapid and simple procedure is described for analysis of fermentation products from anaerobic bacteria grown in glucose broth. A 1-ml sample of the culture is drained through cation-exchange resin in a Pasteur pipette. The effluent fluid is directly analyzed isothermally in a gas chromatograph for volatile fatty acids (C2 to C6) as well as for lactic, pyruvic, and succinic acids. This procedure is considered to be suitable for routine use in clinical bacteriology. Analysis of fermentation products from glucose was successfully used in identification of anaerobic bacteria (1). The fermentation products were separated isothermally in a gas chromatograph after volatile fatty acids were extracted from the culture broth with ether, and nonvolatile acids were methylated and extracted with chloroform. The time in the chromatograph was about 40 min (1). Rogosa and Love (3) showed that the volatile fatty acids in fermentation media could be separated directly on the porous polymer PAR-1 with programmed heating of the gas chromatograph. Recently, various column materials for analysis of volatile fatty acids in aqueous solution were surveyed, and the porous polymer Chromosorb 101 in a glass column was found to separate volatile fatty acids, C2 to C5, isothermally in about 5 min (2). By using the porous polymer Chromosorb 101, a time-saving procedure has now been worked out for analysis of fermentation products in glucose broth. The fatty acids are analyzed in aqueous solution, and lactic, pyruvic, and succinic acids can be separated together with the volatile fatty acids without any methylation step. MATERIALS AND METHODS Apparatus. The apparatus used was a dualcolumn gas chromatograph (model 5751G, Hewlett-Packard GmbH, WUrtemberg, Germany) equipped with hydrogen flame detectors and a 2-mV span recorder (Servogor, AB Transfers, Stockholm, Sweden). Sensitivity of the electrometer: 1.0 x 10-12 A gave full-scale output on a 1-mV recorder at range 1 and attenuation 1. Coiled glass columns (ca. 1.83 m by 2 mm inner diameter) were used. The columns were packed with a porous polymer (Chromosorb 101, 80/100 mesh; Johns-Manville, Denver, Colo.) with a minimal amount of fine-grade glass wool in both ends. The columns were conditioned overnight at 250 C and repacked from the effluent end of the column, leaving 4 cm of space on the inlet side. The columns were then reconditioned at 250 C for 2 h and then run isothermally at 200 C, with inlet at 200 C and detector at 240 C. A Hamilton syringe of 5-Aliter capacity (75-N) (Hamilton Bonaduz AG, Bonaduz, Switzerland) was used to inject 0.5-Mliter samples. The "dead space" of the syringe was filled with water, and the sample was injected directly into the column immediately above the column packing. Carrier gas flow was 10 ml of nitrogen per min, and the flow rates of hydrogen and air were optimized for acetic acid response. Culture methods. The organisms were grown in prereduced, anaerobically sterilized (PRAS) media as described by Holdeman and Moore (1). Fermentation products from glucose were studied in a broth containing per liter: 10 g of neutralized bacteriological peptone (L34, Oxoid Ltd., London, U.K.); 10 g of yeast extract powder (L21, Oxoid Ltd., London, U.K.); 10 g of glucose; 0.5 g of cysteine HCl H20 and resazurin and salt solution as described by Holdeman and Moore (1). Preparation of sample for gas chromatography. A sample of a culture in the glucose broth was applied on 1 ml of packed cation-exchange resin (AG 50W-X4, 200 to 400 mesh, hydrogen form, washed in water; BioRad Laboratories, Richmond, Calif.) on glass wool in a Pasteur pipette. The sample was allowed to drain through the resin, and the resin was then washed twice with 0.5 ml of water. All fluid from the pipette was collected in a test tube, and this fluid was directly analyzed in the gas chromatograph for volatile fatty acids as well as lactic, pyruvic and succinic acids. For analysis of other nonvolatile acids, 1 ml of the fluid from the previous step was treated with metha-287 nol and sulfuric acid as described by Holdeman and Moore (1). One milliliter of the reaction mixture was then applied on 1 ml of packed anion-exchange resin (AG 1-X8, 200 to 400 mesh, formate form, washed in water; BioRad Laboratories, Richmond, Calif.) on glass wool in a Pasteur pipette. The sample was allowed to drain through the resin, and the resin was then washed twice with 0.5 ml of water. All fluid was collected in a test tube, and the methylated products in the fluid were analyzed in the gas chromatograph. RESULTS AND DISCUSSION The volatile fatty acids, C2 to C,, in mixture with lactic and succinic acids were separated in about 15 min in the gas chromatograph (Fig. 1). The recovery of these fermentations products from a glucose-broth culture was quantitative. In most clinical samples, caproic acid and succinic acid are not expected and the actual time of analysis is usually less than 10 min. The relative retention times of the volatile fatty acids compared to acetic acid (1.0) were: propionic acid, 1.5; isobutyric acid, 2.0; butyric acid, 2.4; isovaleric acid, 3.3; valeric acid, 4.0, and caproic acid 6.5. Pyruvic, lactic, and succinic acids were eluted in asymmetric peaks with some tailing. Compared to the retention time of acetic acid (1.0), these acids started to be eluted at 2.1, 4.9, and 12.9, respectively. Ethanol, propanol, butanol, and pentanol had the following retention times relative to acetic iB acid (1.0): 0.5, 0.9, 1.4, and 2.4, respectively. The methyl esters of the acids had the following retention times compared to acetic acid (1.0): acetate, 0.8; propionate, 1 Pentanol was not separated from butyric acid. However, the acid could be removed from the sample by passing the sample through the anion-exchange resin. If a sample contained a high amount of pyruvic acid, this acid was not separated from butyric acid. By methylating the sample, a separation of these acids was obtained. Lactic acid was usually eluted in only one peak (z, Fig. 2a). However, two other peaks (x and y, Fig. 2a) appeared if the injection of the sample was delayed after the needle of the syringe reached the top of the column packing. To get a quantitative response of lactic acid, the sample had to be in the needle and not in the glass part of the syringe during the injection procedure. The fermentation products of a homofermentative lactobacillus, Lactobacillus acidophilus, are shown in Fig. 2b. The acetic acid in the chromatogram is a component of the glucose broth. Of various peptones and yeast extract powders tested for presence of lactic, pyruvic, succinic, and volatile fatty acids, only Trypticase (BBL, Cockeysville, Md.) was found to contain significant amounts of acetic acid, which makes this peptone unsuitable for use in the fermentation media. The injection of the sample was delayed after the needle of the syringe reached the top of the column packing. Lactic acid gave three peaks: x, y, and z. Conditions of chromatography were the same as in Fig. 1. b, Chromatogram of fermentation products in a glucose-broth culture of Lactobacillus acidophilus (NCDO 929). Conditions of chromatography were the same as in Fig. 1 fermentation products in glucose-broth cultures of Eubacterium limosum and Bifidobac-terium (Actinomyces) eriksonii are shown in Fig. 3. The stability of the porous polymer column was very good. More than 500 samples were separated on the column when the chromatograms presented were run. The separation characteristics of this column were the same as those of newly packed columns. The excellent stability of porous polymers was also noticed by Rogosa and Love (3), when they injected fermentation media directly on such columns. The purification of the sample by cation-exchange chromatography suggested in the present study improved the quality of the gas chromatograms and may also extend the life of the columns. All the fermentation products used in primary identification of anaerobic bacteria (1) could be separated in just one isothermal run in the gas chromatograph. This means that almost any gas chromatograph can be used for this analysis. The simplicity and rapidity of the procedure justify the routine use of this method in clinical bacteriology.

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2018-04-03T02:36:04.197Z

1973-04-01T00:00:00.000Z

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Effects of Patulin and Method of Application on Growth Stages of Wheat When a single, 100-,ug/ml application of patulin, produced by Penicillium urticae Bainier, was applied to growth stages 7, 9, 10, and 10.1 (Feekes scale) of Lee spring wheat (Triticum aestivum L.), decreases in internodal elongation, floret number, seed weight, and seed number were observed. Yields were reduced according to the proximity of application prior to heading. Application of patulin to the soil in crystalline form and dissolved in aqueous solution were also investigated, and the solution method of application was found to be the treatment of choice. A single exposure of growing wheat plants to patulin can produce yield reductions similar to those observed in stubble-mulch farming. Penicillium urticae Bainier, a fungus which produces patulin, has been found in large numbers in the soil in the Great Plains area. In some cases, the P. urticae population comprises 90% of the total fungal population in stubble-mulch wheat farming (J. R. Ellis and T. M. McCalla, Unpublished data). Wheat roots have been found to stimulate the growth of P. urticae over other fungi species (6). Other soil fungi have been reported to produce patulin; however, primarily, P. urticae has been found where plant residues and mulches have been left near the soil surface (11,12,14). The toxicity of patulin has been demonstrated on animals, plants, fungi, and bacteria. In the early 1930's, patulin was used as a broad-spectrum antibiotic but was found to be toxic to humans; consequently, its use was discontinued. In recent years, interest has been centered on its toxicity to plants. It was implicated in problems concerned with apple seedling transplants (3,4). The toxic effect of patulin has been observed on young seedlings, germinating seed, isolated plant tissue, and plants which had continuous applications of patulin until maturity. The effect of patulin on root development, cell division, cell wall development, and enzyme inhibiton has also been reported (10,15). The effect of a single patulin application on wheat plants grown to maturity or the effect of the method of its application has not been demonstrated previously. In the experiments reported here, patulin was applied at specific growth stages to simulate the effect of shortduration P. urticae Banier blooms and subsequent patulin production on wheat plants. Two application methods were used to compare methods being used in research investigations. MATERIALS AND METHODS Patulin used in experiment. The patulin used in this experiment was produced and purified in the laboratory from cultures of P. urticae Bainier isolated from stubble-mulched plots (13). Purity of the antibiotic was verified by using thin-layer chromatography, melting point, and infrared spectroscopy. Soil type. Holdrege silt loam from North Platte, Nebraska, was used in the pot experiment. Waterholding capacity of the soil at one-third bar is 24%. Physical and chemical characteristics of this soil have been described by Norstadt and McCalla (12). Experimental procedure. Thirty-five hundred grams of oven-dried soil was placed in 3.3-liter plastic pots with a layer of 100-mesh nylon cloth covering the drain holes. Optimum fertilizer for wheat production was determined by soil tests and appropriate compounds were mixed with the soil. The following rates of the elements N, P, Mn, Zn, and Fe, respectively, expressed as millimoles per killigram of soil, were added to the soil: 3.57, 0.352, 0.046, 0.061, and 0.082. Lee spring wheat (Triticum aestivum L.) was pregerminated for 3 days, and 7 seedlings were placed in each pot at 2.0-cm depth within a 10-cm circle. Pots were watered to moisture-holding capacity twice weekly with distilled water, Patulin was applied to EFFECTS OF PATULIN ON GROWTH STAGES OF WHEAT Feekes stages 7, 9, and 10 (8) with one aqueous solution application; and Feekes stages 7, 9, 10, and 10.1 with one crystalline application. Three replicates per treatment were used. The solution treatment was applied with a hypodermic syringe and needle in four places around each plant 3.8 cm apart, 2.54 cm from the plant, and 2.54 cm deep. The crystalline application was applied in 0.8-cm holes bored 2.54 cm deep in four equally spaced locations, 3.8 cm apart and 2.54 cm from each plant. After patulin application, the pots were brought to field capacity with distilled water. Pots were placed in an ISCO E-2 growth chamber in three randomized blocks, with one replicate of each treatment in a block. Pots were moved each day so that each treatment was rotated in the block every 8 days. Temperature cycles in the growth chamber were based on Agronomy Farm (Lincoln, Nebraska) averages of bare and bromegrass-covered soil at a depth of 10.16 cm, from April through July 1966 (17). The plants were harvested at maturity (113 days) and observations were made on plant height, internodal elongation, straw weight, chaff weight, grain weight, floret number, and kernal number. The data were analyzed statistically. RESULTS Internodal elongation was markedly affected by the time of patulin application ( statistically significant) between the second and third node, but normal growth occurred thereafter. A decrease was noted in total seed yield (Table 3), indicating a residual effect. In contrast, the crystalline patulin application to stage 7 reduced the growth of the third and fourth internodes (not statistically sigificant) but not the second internode length. A significant reduction in floret number was observed. Solution application to stage 9 wheat plants did not affect second internode growth, but a reduction was noted in the third and fourth internodes and the stem between the last node and head ( Table 2). Stage 9 treatment significantly reduced seed number and total yield. Crystalline treatment reduced fourth to fifth internode growth but not the third to fourth internode or the fifth node to head and also significantly reduced the seed yield. Stage 10 application of solution induced the greatest reduction in seed weight, number, total yield, floret number, and chaff weight. The grain yield (Table 3) was reduced to 0.1 of the control and 0.2 of any of the crystalline applications in this experiment. Stem elongation of the fourth internode and of the upper stem was significantly reduced. The crystalline application to the same stage significantly reduced seed weight, total yield and fourth internode elongation, and elongation between fifth node and head. Table 2. Numbers on the control bar (a) indicate node location. Since stage 10.1 application treatment is made after stem elongation, the crystalline application had no effect on this process. However, the greatest reduction of seed weight and total yield was exhibited by this crystalline application. DISCUSSION The patulin solution application method affected the particular plant growth phase at the time of application, and inhibition was observable for several phases of plant elongation. A residual effect was noted as final wheat yield was reduced with solution treatment applications. Yield reductions increased as patulin in aqueous solution was applied nearer to heading and seed production stages. There was a steady decrease in the seed number, seed weight, floret number, chaff weight, and straw weight. In contrast, the crystalline application was usually not effective on the particular growth stage to which it was applied. The treatment effects were not as great when the crystal treatments were used. The comparison of solution and crystalline applications of patulin showed that patulin must be in solution to produce an effect on the immediate growth phase treated. The crystalline application reduced stem elongation, but the effect, compared to solution application, was delayed. The crystalline form did not reduce the seed weight and number as much as the solution application. The crystalline application had much larger least significant difference (LSD) values, which indicated the greater variability of this type of treatment. The decreased effect of the crystalline treatment could be explained by two factors. The crystals do not dissolve rapidly, thus lowering the patulin concentration in the soil solution. In field treatments, Norstadt, Ellis, and McCalla (Unpublished data) found crystalline patulin in the soil for several weeks after application. Patulin has a half-life in the soil of 24 h (11), and this would also decrease the soil solution concentration. Patulin affects cell division (1, 16), which would cause the reduction in stem elongation noted in this experiment. Cereal crops which suffer damage during these rapidly growing phases often show reduced yields. The action of patulin may take place in several ways. However, a possible mechanism has been shown. Dickens and Jones (5) found that patulin can inactivate S-H groups. They showed that a 1: 1 relationship existed between the number of S-H groups blocked and the number of patulin molecules. This reaction would interfere with enzyme activity and protein synthesis (1, 2, 7), thus explaining the effect of patulin on internode length. The blocking action of important enzymes and interference with cell division processes can reduce plant vigor and yield. This study showed that patulin does have a marked effect on plant development even when plants had only one exposure to patulin. A single exposure to patulin can produce the toxicity problems noted in stubble-mulch farming (9, 11) by reducing plant vigor and final grain yield.

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2018-04-03T01:01:25.892Z

1973-02-01T00:00:00.000Z

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Simplified Gas Chromatographic Procedure for Identification of Bacterial Metabolic Products A rapid and simple procedure is described for analysis of fermentation products from anaerobic bacteria grown in glucose broth. A 1-ml sample of the culture is drained through cation-exchange resin in a Pasteur pipette. The effluent fluid is directly analyzed isothermally in a gas chromatograph for volatile fatty acids (C2 to Cf) as well as for lactic, pyruvic, and succinic acids. This procedure is considered to be suitable for routine use in clinical bacteriology. Analysis of fermentation products from glucose was successfully used in identification of anaerobic bacteria (1). The fermentation products were separated isothermally in a gas chromatograph after volatile fatty acids were extracted from the culture broth with ether, and nonvolatile acids were methylated and extracted with chloroform. The time in the chromatograph was about 40 min (1). Rogosa and Love (3) showed that the volatile fatty acids in fermentation media could be separated directly on the porous polymer PAR-1 with programmed heating of the gas chromatograph. Recently, various column materials for analysis of volatile fatty acids in aqueous solution were surveyed, and the porous polymer Chromosorb 101 in a glass column was found to separate volatile fatty acids, C2 to C5, isothermally in about 5 min (2). By using the porous polymer Chromosorb 101, a time-saving procedure has now been worked out for analysis of fermentation products in glucose broth. The fatty acids are analyzed in aqueous solution, and lactic, pyruvic, and succinic acids can be separated together with the volatile fatty acids without any methylation step. MATERIALS AND METHODS Apparatus. The apparatus used was a dualcolumn gas chromatograph (model 5751G, Hewlett-Packard GmbH, WUrtemberg, Germany) equipped with hydrogen flame detectors and a 2-mV span recorder (Servogor, AB Transfers, Stockholm, Sweden). Sensitivity of the electrometer: 1.0 x 10-12 A gave full-scale output on a 1-mV recorder at range 1 and attenuation 1. Coiled glass columns (ca. 1.83 m by 2 mm inner diameter) were used. The columns were packed with a porous polymer (Chromosorb 101, 80/100 mesh; Johns-Manville, Denver, Colo.) with a minimal amount of fine-grade glass wool in both ends. The columns were conditioned overnight at 250 C and repacked from the effluent end of the column, leaving 4 cm of space on the inlet side. The columns were then reconditioned at 250 C for 2 h and then run isothermally at 200 C, with inlet at 200 C and detector at 240 C. A Hamilton syringe of 5-Aliter capacity (75-N) (Hamilton Bonaduz AG, Bonaduz, Switzerland) was used to inject 0.5-Mliter samples. The "dead space" of the syringe was filled with water, and the sample was injected directly into the column immediately above the column packing. Carrier gas flow was 10 ml of nitrogen per min, and the flow rates of hydrogen and air were optimized for acetic acid response. Culture methods. The organisms were grown in prereduced, anaerobically sterilized (PRAS) media as described by Holdeman and Moore (1). Fermentation products from glucose were studied in a broth containing per liter: 10 g of neutralized bacteriological peptone (L34, Oxoid Ltd., London, U.K.); 10 g of yeast extract powder (L21, Oxoid Ltd., London, U.K.); 10 g of glucose; 0.5 g of cysteine HCl H20 and resazurin and salt solution as described by Holdeman and Moore (1). Preparation of sample for gas chromatography. A sample of a culture in the glucose broth was applied on 1 ml of packed cation-exchange resin (AG 50W-X4, 200 to 400 mesh, hydrogen form, washed in water; BioRad Laboratories, Richmond, Calif.) on glass wool in a Pasteur pipette. The sample was allowed to drain through the resin, and the resin was then washed twice with 0.5 ml of water. All fluid from the pipette was collected in a test tube, and this fluid was directly analyzed in the gas chromatograph for volatile fatty acids as well as lactic, pyruvic and succinic acids. For analysis of other nonvolatile acids, 1 ml of the fluid from the previous step was treated with metha-287 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from APPL. MICROBIOL. nol and sulfuric acid as described by Holdeman and Moore (1). One milliliter of the reaction mixture was then applied on 1 ml of packed anion-exchange resin (AG 1-X8, 200 to 400 mesh, formate form, washed in water; BioRad Laboratories, Richmond, Calif.) on glass wool in a Pasteur pipette. The sample was allowed to drain through the resin, and the resin was then washed twice with 0.5 ml of water. All fluid was collected in a test tube, and the methylated products in the fluid were analyzed in the gas chromatograph. RESULTS AND DISCUSSION The volatile fatty acids, C2 to C,, in mixture with lactic and succinic acids were separated in about 15 min in the gas chromatograph (Fig. 1). The recovery of these fermentations products from a glucose-broth culture was quantitative. In most clinical samples, caproic acid and succinic acid are not expected and the actual time of analysis is usually less than 10 min. The relative retention times of the volatile fatty acids compared to acetic acid (1.0) were: propionic acid, 1.5; isobutyric acid, 2.0; butyric acid, 2.4; isovaleric acid, 3.3; valeric acid, 4.0, and caproic acid 6.5. Pyruvic, lactic, and succinic acids were eluted in asymmetric peaks with some tailing. Compared to the retention time of acetic acid (1.0), these acids started to be eluted at 2.1, 4.9, and 12.9, respectively. Ethanol, propanol, butanol, and pentanol had the following retention times relative to acetic iB acid (1.0): 0.5, 0.9, 1.4, and 2.4, respectively. The methyl esters of the acids had the following retention times compared to acetic acid (1.0): acetate, 0.8; propionate, 1 Pentanol was not separated from butyric acid. However, the acid could be removed from the sample by passing the sample through the anion-exchange resin. If a sample contained a high amount of pyruvic acid, this acid was not separated from butyric acid. By methylating the sample, a separation of these acids was obtained. Lactic acid was usually eluted in only one peak (z, Fig. 2a). However, two other peaks (x and y, Fig. 2a) appeared if the injection of the sample was delayed after the needle of the syringe reached the top of the column packing. To get a quantitative response of lactic acid, the sample had to be in the needle and not in the glass part of the syringe during the injection procedure. The fermentation products of a homofermentative lactobacillus, Lactobacillus acidophilus, are shown in Fig. 2b. The acetic acid in the chromatogram is a component of the glucose broth. Of various peptones and yeast extract powders tested for presence of lactic, pyruvic, succinic, and volatile fatty acids, only Trypticase (BBL, Cockeysville, Md.) was found to contain significant amounts of acetic acid, which makes this peptone unsuitable for use in the fermentation media. The injection of the sample was delayed after the needle of the syringe reached the top of the column packing. Lactic acid gave three peaks: x, y, and z. Conditions of chromatography were the same as in Fig. 1. b, Chromatogram of fermentation products in a glucose-broth culture of Lactobacillus acidophilus (NCDO 929). Conditions of chromatography were the same as in Fig. 1. fermentation products in glucose-broth cultures of Eubacterium limosum and Bifidobac-terium (Actinomyces) eriksonii are shown in Fig. 3. GAS CHROMATOGRAPHY OF METABOLIC PRODUCTS The stability of the porous polymer column was very good. More than 500 samples were separated on the column when the chromatograms presented were run. The separation characteristics of this column were the same as those of newly packed columns. The excellent stability of porous polymers was also noticed by Rogosa and Love (3), when they injected fermentation media directly on such columns. The purification of the sample by cation-exchange chromatography suggested in the present study improved the quality of the gas chromatograms and may also extend the life of the columns. All the fermentation products used in primary identification of anaerobic bacteria (1) could be separated in just one isothermal run in the gas chromatograph. This means that almost any gas chromatograph can be used for this analysis. The simplicity and rapidity of the procedure justify the routine use of this method in clinical bacteriology.

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2020-12-10T09:04:12.697Z

1973-12-01T00:00:00.000Z

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Degradation of Parathion by Bacteria Isolated from Flooded Soil Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite. Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite. Folidol, a commercial formulation of parathion (O, O-diethyl-0-p-nitrophenyl phosphorothioate), is extensively used in India for control of common insect pests of rice. Although parathion is known to be relatively less persistent than chlorinated hydrocarbon insecticides, recently this compound was reported to persist for more than 16 years in a sandy loam soil (13). Parathion undergoes rapid degradation in flooded rice soils either via reduction of the nitro group (12) or via hydrolysis at the P-O-C linkage after repeated applications (10). Degradation of parathion by Flavobacterium sp. isolated from diazinon-amended flooded rice soil ceased at the p-nitrophenol stage (11). This paper reports more extensive degradation of parathion past the p-nitrophenol stage by a single bacterium isolated from flooded alluvial soil and identified as Pseudomonas sp. Formation of nitrite as an end product in the degradation of p-nitrophenol by a species of Bacillus was also investigated. MATERIALS AND METHODS Preparation of parathion-hydrolyzing enrichment culture. One milliliter of aqueous 1,000-ppm solution of parathion was added to 20 g of an alluvial soil from the Institute farm at 2-week intervals. The soils contained in test tubes (25 by 220 mm) were flooded with 24 ml of distilled water. Within 24 to 48 h after the third addition, the standing water over the soils in certain tubes turned yellow, indicating the hydrolysis of parathion to p-nitrophenol (10). This standing water, together with soil suspension exhibiting parathion-hydrolyzing ability, was pooled from several tubes and employed as an enrichment culture. Isolation of bacteria. A dilution of the enrichment culture was mixed with molten modified Wakimoto nutrient agar medium (8) and incubated. Isolates from the agar medium were transferred to a sterile mineral solution [(NH4)2HP04, 0.5 g; MgSO4 7H20, 0.2 g; FeSO4 7H20, 0.001 g; K2HPO4, 0.1 g; Ca(NO3)2, 0.01 g; distilled water, 1,000 ml] containing parathion or p-nitrophenol as the sole carbon source. None of the isolates decomposed parathion, but a species of Bacillus capable of decomposing p-nitrophenol was isolated (10). In another experiment, the enrichment culture was serially diluted, and 1 ml of each dilution was incubated with parathion in a sterile mineral solution following the methods described for diazinon-hydrolyzing Flavobacterium sp. (8). The lowest dilution (10-6) which exhibited parathion-hydrolyzing ability was chosen for further studies. The active parathionhydrolyzing agents in the 106 dilution were multiplied by incubating 1 ml of this dilution with 4 ml of sterile mineral solution containing parathion as the sole carbon source. After 48 h, this solution was streaked on a modified Wakimoto agar medium (8). Individual bacterial isolates developing on the agar medium were transferred to a sterile mineral solution containing parathion. The medium which was incubated with isolate P-6 turned yellow within 24 h, and the yellow color faded within the next 24 h. This isolate was further purified and identified as Pseudomonas sp. Degradation studies. The ability of Pseudomonas sp. to decompose parathion was tested as follows. The mineral solution containing aqueous parathion was passed through a membrane filter (Millipore Corp.; 0.45 Am pore size), and 25-ml samples of this sterile solution (pH 7.1) were distributed in 250-ml Erlenmeyer flasks. The medium was inoculated with 0.1 ml of bacterial suspension in sterile distilled water prepared from 3to 7-day-old cultures. The incubation mixture was incubated at 27 C in a biological oxygen demand (BOD) incubator. Uninoculated media served as control. Methods for extraction (11) and analysis (10) of parathion residues in the incubation mixture have been described earlier. Residues in the medium were extracted three times with 20 ml of chloroform-diethyl ether (1:1), and the solvent fraction was pooled. The residues were evaporated to dryness at room temperature and then dissolved in 2 ml of methanol. The PARATHION DEGRADATION residues spotted on 300-Mm-thick Silica Gel G plates were developed for a distance of 15 cm by employing hexane-chloroform-methanol (7:2:1) as a developing agent. After drying, the authentic compounds of parathion and p-nitrophenol were located by spraying the chromatoplate with 0.5% palladium chloride in 2% HCl followed by 2.5 N NaOH. The silica gel areas of the samples corresponding to parathion were scraped carefully and transferred to a test tube. One milliliter of 2.5 N NaOH was added to each tube, and parathion was converted to p-nitrophenol by alkaline hydrolysis in a water bath for 1 h. After cooling, the volume was made up to 25 ml, silica gel was removed by centrifugation, and the supernatant was read in a Klett-Summerson calorimeter employing a 420-nm blue filter. The amount of parathion in the samples was obtained by multiplying the values for p-nitrophenol by 2.094. p-Nitrophenol in the silica gel areas of the samples opposite to the authentic compound was directly eluted in 0.1 N NaOH. After centrifugation of the silica gel suspension, p-nitrophenol in the supernatant was determined colorimetrically against an appropriate blank as described earlier. In a test to determine whether nitrite was formed during the bacterial decomposition of p-nitrophenol, 20-ml samples of sterile mineral solution without (NH4)2HPO4 and Ca(NO3)2 supplemented with pnitrophenol were inoculated with 0.1 ml of a suspension of Pseudomonas sp. in sterile water. The samples were drawn periodically, and nitrite in the samples was analyzed calorimetrically by using sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (2). Bacillus sp. which decomposed p-nitrophenol as the sole carbon source (10) was tested for its ability to release nitrite from p-nitrophenol as described for Pseudomonas sp. The same bacterium was also tested for its ability to liberate nitrite from intact parathion. RESULTS The bacterial isolate P-6 was gram negative, rod, and aerobic, and was identified as a nonfluorescent species of Pseudomonas. The isolate showed many characteristics of P. multivorans Stanier et al. and appeared to belong or to be closely related to this species. Large inclusions, which were presumably polyhydroxy butyrate, were very clear with the Gram stains. All tests for spores were negative. When Pseudomonas sp. was incubated with parathion, the insecticide was rapidly degraded. The color of the incubation medium turned yellow within 4 h of incubation, indicating the formation of p-nitrophenol. At 20 h, however, the yellow color disappeared, evidently because of the further metabolism of p-nitrophenol. Quantitative analysis of parathion and p-nitrophenol confirmed these findings. At 4 h, about 50 g of added parathion was hydrolyzed and 19 gg of p-nitrophenol was recovered as the major metabolite (Table 1). No other metabolite could be detected in the thin-layer chromatogram of the solvent extract. At 20 h, however, parathion was completely destroyed and no p-nitrophenol could be detected. No appreciable degradation of parathion occurred in the uninoculated control during 20 h of incubation. When the bacterium was grown in a nitrogen-free medium with p-nitrophenol as the sole carbon source at 27 C ± 2 C in a BOD incubator, nitrite nitrogen was released from the organic nitro molecule ( Table 2). The amount of nitrite formed was proportional to the amount of p-nitrophenol degraded. Within 16 h of incubation, 157 ug of p-nitrophenol was decomposed, liberating 51 tig of nitrite. In the uninoculated control, nitrite was not detected. In a preliminary resting-cell experiment, nitrite was formed when living resting cells of Pseudomonas sp. were exposed to parathion in phosphate buffer (pH 7.1). These studies indicated that nitrite was formed from p-nitrophenol. Bacillus sp., isolated from flooded alluvial soil, was reported earlier to utilize p-nitrophenol as a sole carbon source (10). In a test to determine the end product formed during the breakdown of p-nitrophenol by Bacillus sp., the bacterium was incubated with p-nitrophenol in a mineral solution as described for Pseudomonas sp. Within 24 h of incubation, 166 tg of p-nitrophenol was metabolized, releasing 43 Mg of nitrite (Table 3). To test whether Bacillus sp. could release nitrite from intact parathion, the bacterium was incubated with the insecticide for 120 h. p-Nitrophenol and nitrite were not formed. Pseudomonas sp. was subcultured repeatedly on parathionor p-nitrophenol-free modified Wakimoto agar. After the fifth subculture, the bacterium was tested for its ability to degrade parathion or p-nitrophenol as the sole carbon source in a mineral solution as described earlier. The bacterium retained its ability to hydrolyze parathion within 3 h of incubation and then release nitrite from p-nitrophenol within 24 h despite five transfers on parathionand p-nitrophenol-free media. DISCUSSION Parathion metabolism in plant and insect systems via oxidation, reduction of nitro group, or hydrolysis is well established (5), but the stepwise degradation of this insecticide in microorganisms has not been investigated (1). Until recently, the major pathway of parathion metabolism in soils and microorganisms appeared to be the reduction of the nitro group. Now, we have clear evidence that parathion can be hydrolyzed biologically at the nitrophenyl C-O-P bond, both in flooded soils after its repeated additions (10) and in pure culture by Flavobacterium sp. (11,12). Degradation of parathion by Flavobacterium sp., however, ceased Iat the p-nitrophenol stage (11). The results presented in this study clearly established the degradation of parathion past the nitrophenol stage by Pseudomonas sp., leading to the formation of nitrite as an end product. Nitrite formed appeared to persist in pure culture studies with Bacillus sp. and Pseudomonas sp. during the 24-h incubation period. However, in flooded soil nitrite does not accumulate (6), because of its rapid denitrification to molecular nitrogen in -an anaerobic environment. Various nitrophenols are known to be metabolized, liberating nitrite in the process. Corynebacterium simplex formed ni-trite from phenolic substrates containing the nitro group in the para position (3). A species of Arthrobacter released nitrite from a herbicide 3,5-dinitro-o-cresol (4). Similarly, both Bacillus sp. and Pseudomonas sp. used in this study readily released nitrite from p-nitrophenol, utilizing the latter as a sole carbon source ( Table 2, 3). This finding is in agreement with that of Raymond and Alexander (7). Pseudomonas sp., in addition, possessed an enzyme system(s) capable of hydrolyzing parathion. This result is apparently the first report on the degradation of parathion to p-nitrophenol and then to nitrite by the same bacterium. Metabolism of parathion in flooded soil or in pure cultures of microorganisms isolated from flooded soil involves (i) nitro group reduction, leading to the formation of aminoparathion as a major metabolite (9); (ii) hydrolysis to p-nitrophenol as a major metabolite which resists further degradation (11); and/or (iii) hydrolysis to p-nitrophenol followed by the formation of nitrite (this report). The findings reported in this paper show that microorganisms in flooded soils contribute to the rapid breakdown of parathion via the hydrolytic pathway to the end product, nitrite.

v3-fos

2020-12-10T09:04:12.627Z

1973-05-01T00:00:00.000Z

237233315

s2

Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 μg of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken. Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 ,g of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken. Most foods in which Staphylococcus aureus have multiplied and produced enterotoxins and which, when consumed, cause food poisoning are complex colloidal systems. The physical state, independent of the nutrient contribution, may affect the production of enterotoxin. The studies which have been reported previously have been focused on the production of high levels of enterotoxins. Casman and Bennett (2) observed greater enterotoxin A production in semisolid brain heart infusion plates than in liquid; however, gimkovicova and Gilbert (13) did not find large amounts of enterotoxin with this method, although, for one of two strains for which data are reported, there was a slight increase over that produced in broth alone. Neither of these two studies offers a direct comparison of effect of colloidal state itself. Membrane culture methods have been recommended (1,2,7,10) and may exert a surface effect as well as permit dialysis. In foods, the physical state of the substrate may exert an effect upon bacterial growth or enterotoxin production, or both, by changes in aeration, adsorption of metabolic products or nutrients, buffer action, alteration in rate of diffusion, alteration in available water, or changes in the bacterial dispersion and possibly ITechnical paper no. 3530, Oregon Agricultural Experiment Station. related differences in the growth pattern. However, the complex environment is difficult to study. Therefore, in the present study, numbers of colony-forming units and enterotoxin levels were compared in shaken and still cultures which had been varied in physical state by the addition of single colloidal ingredients to basic broth. MATERIALS AND METHODS Strains. The S. aureus S-6 culture, a strain that produces enterotoxins A and B, obtained from M. S. Bergdoll (Food Research Institute, Madison, Wis.), was preserved on porcelain beads (9) for use in the major portion of the study. Enterotoxin B-producing strain 243, enterotoxin A-producing strains 265-1 (from R. W. Bennett, Food and Drug Administration, Washington, D.C.) and 13N-2909 (a high producing mutant strain from S. J. Silverman, Fort Detrick, Frederick, Md.), and enterotoxin C-producing strain 361 (from M. Bergdoll) were used in a follow-up study which compared the NAK broth control with liquid and paste states produced with cornstarch and carrageenan. Colloidal ingredients and dispersions. Liquid, suspension, paste, and gel with each of four different colloids were made with 4% NZ Amine-NAK (Sheffield Chemical, Union, N.J.) supplemented with 50 Ag of thiamine per ml and 1 mg of niacin per ml. In a limited study with two replications, 3% NZ Amine-NAK plus 3% PHP (a pancreatic digest of casein; Mead, Johnson, International, Evansville, Ind.) re-placed the 4% NAK. The colloidal ingredients were cornstarch (Corn Products Refining Co., Argo, Ill.), agar (Noble special agar, Difco), carrageenan (Gelcarin, Marine Colloids, Inc., Springfield, N.J.), and low methoxyl pectin (LMP) (unstandardized product with no compounds added, Sunkist Growers, Corona, Calif.). Proportions used are given in Table 1. The final pH of each was 6.8. (Predetermined amounts of 0.1 N NaOH were added at the time of initial preparation or later, depending upon the colloid.) The pH was checked on random lots during the experiments. Consistencies of the dispersions (Table 1) were determined in standardizing procedures and at intervals during the experimental period. For liquid and paste, the Brookfield viscometer was used; for gels, the percentage sag was measured by taking the height of the gel before and 1 min after removal from the container. Water activity (a,) in each medium was calculated from the value for water potential at 27 C measured in duplicate samples by a thermocouple psychrometer (C-51 Sample Chamber, Wescor, Inc., Logan, Utah), standardized with KCl solutions of predetermined osmotic pressure. Preparation and storage conditions were carefully standardized for all replications. Fifty-milliliter amounts of each medium and of the NAK broth control were put into 300-ml triple-baffled shake flasks (Bellco Glass, Inc., Vineland, N.J.) for shaken samples and into plain 250-ml Erlenmeyer flasks for still samples and covered with gauze-cotton closures secured with clips. Dispersions with starch made by adding weighed quantities of cornstarch to basal medium which had been preheated to 80 C were heated to 90 C with constant stirring. Samples of 50 ml were autoclaved for 15 min at 121 C. For suspensions, 15 medium pearls (approximately 1.8 g) of tapioca (Manhattan Adhesives Corp., Brooklyn, N.Y.) were added to each flask before autoclaving. Agar and carrageenan were dispersed by heating in the basal medium, and were then distributed in 50-ml fractions. After being autoclaved for 15 min, the flasks were held at room temperature until pastes and gels were set (2 h for agar, 1 h for carrageenan). Pastes for both still and shaken samples were then shaken for 2 h. The use of LMP differed from that of the other colloids in that it was necessary to make a slurry of LMP in 95% ethanol before addition to the basal medium. Samples were calculated to be 50 g after adjustment of pH and calcium ion concentration. After 1 min of autoclaving at 121 C, the pH of each was adjusted to 6.8 with precalculated volumes of sterile 0.1 N NaOH. Sterile CaCl2 (5 ml) was added to the paste (0.05 M CaCl,) and gel (0.10 M CaCl2) dispersions. For suspensions thickened with agar, carrageenan, and LMP, 15 6-mm lengths of glass tubing (Exax Raschig Rings, Kimble Products, Toledo, Ohio) were added to each flask before autoclaving. Inoculation. Media, which were 24-h-old and had been held at 37 C a minimum of 15 h either shaken or still, were inoculated with 0.5 ml of a dilution of a 24-h NAK broth culture to give 10' organisms per ml. In the NAK-PHP series, two levels of inoculum, 8 x 103 and 6 x 105 colony-forming units (CFU)/ml, were compared. All flasks, except gels, were swirled to mix; the inoculum was spread over the surface of the gel. After inoculation, triplicate flasks of control NAK broth and duplicate flasks of liquid, suspension, and paste dispersions were incubated at 37 C for both still and shaken (gyratory water bath shaker, New Brunswick Scientific Co., New Brunswick, N.J.; 200 rpm) samples. All gel samples were incubated still. Sampling. Sampling was done at 5, 8, and 24 h. For all of the samples except the gels, 6-ml samples were removed at each of the times. A different gel sample was used at each sampling time. The gels were diluted 2:1 with NAK broth and then mixed by manual shaking except for the use of mechanical blending for cornstarch samples. CFU were determined by direct plating of appropriate dilutions on plate count agar (Difco). The sample was then heat treated at 50 C for 10 min to kill the staphylococci and was cooled, the pH was determined, and the sample was centrifuged for 30 min at 1,200 x g to obtain the supernatant fraction for enterotoxin studies. Starch samples were frozen and thawed twice before centrifugation to facilitate separation of a liquid phase. Enzymatic hydrolysis of agar, starch, and LMP dispersions before centrifugation did not increase enterotoxin recovery or detection in inoculated samples. Enterotoxin assay. Three methods of varying sensitivity were used for the determination of enterotoxin B, and two for enterotoxins A and C. Crowle's micro-slide gel double-diffusion technique as modified by Casman et al. (3) was used to estimate enterotoxin levels prior to Oudin assay and for samples containing too little enterotoxin to quantitate by the single gel-diffusion method. Samples giving negative results were concentrated ninefold with Aquacide (Calbiochem, Los Angeles, Calif.) and retested. The detection limit for the micro double-diffusion technique was 0.2 jg/ml. The reversed passive hemagglutination technique (RPH) adapted to a micro scale by Silverman et al. (12) was used to analyze those concentrated samples which were negative for enterotoxin B with the micro double-diffusion technique. Controls used in all cases included nonsensitized cells and uninoculated suspensions. Since the limit of detection of this method was approximately 0.0007 Ag/ml, a negative RPH sample was considered to be negative for enterotoxin. Development of this technique for enterotoxin A was unsuccessful, nor was the method used for enterotoxin C. The Oudin single gel-diffusion method as described by Hall et al. (6) was used for quantitative assay for the three enterotoxins. The sample which had been dialyzed against 4% NAK broth was placed on top of the prepared agar column, and the tube was incubated in a glass-walled water bath at 30 C. Measurements of the migration of the precipitate band were taken at 24, 48, and 72 h by using a cathetometer with a short-focus telescope. The K value (slope) was then compared to that obtained with known concentrations of enterotoxin (diluted in 4% NAK, pH 7.4). The minimal concentration of enterotoxin for which this method could be used reliably was 4 ug/ml. 826 APPL. MICROBIOL. PRODUCTION OF STAPHYLOCOCCAL ENTEROTOXINS The purified enterotoxins A and B used for these assays were provided by E. J. Schantz (Fort Detrick, Frederick, Md.) and for enterotoxin C, by M. S. Bergdoll. Part of the antisera for enterotoxins A and B and all of that for enterotoxin C were provided through the courtesy of M. S. Bergdoll; much of the antisera for types A and B was produced in the investigators' laboratory. The titers of the antisera from both sources were the same. Statistical analyses. The experiment with strain S-6 was a replicated split-plot design with colloidal ingredients as main plots. One of the four colloids was chosen at random for each day's experimentation in each of two replications. Duplicate samples for each dispersion, and triplicate samples of the NAK broth, were analyzed with respect to CFU, pH, and enterotoxin. Shaken and still samples were analyzed separately at 5, 8, and 24 h. Least significant differences were used to test for differences between each ingredient-dispersion combination. Quantitative measures of enterotoxin were available for 8-and 24-h shaken samples and 24-h still samples. A logarithmic transformation was applied to CFU. Correlations were studied for the 8 and the 24 h data for shaken and the 24-h data for still samples. RESULTS For all of the physical states included, shaking during incubation greatly increased the total amount of enterotoxin at the end of 24 h as has been reported by others (4) and also resulted in earlier detectable enterotoxin. For the samples which were incubated in a thin layer without shaking, the physical state of the inoculated dispersions influenced the yield of enterotoxin. In general, pH and CFU followed trends in enterotoxin levels. Enterotoxin levels are expressed in the text on the basis of the minimal concentrations detectable by the assay methods if below that for which direct quantitation was possible. Data are presented on an unconcentrated basis in Tables 2, 3, and 4. The statistical analysis for the S-6 study is summarized in Table 5. The dispersions varied in viscosity (Table 1) depending upon the concentration of the colloidal ingredient, except for those with LMP in which the viscosity was controlled by the calcium level and carrageenan paste/gel which was controlled by physical treatment after sterilization. The a, was above 0.99 for all; the lower in the range of 0.996 to 0.992 being those made with NAK + PHP. Gels with NAK had an aw of 0.995. Shaken samples, strain S-6: enterotoxin levels. After 5 h of incubation, most shaken samples contained about 0.2 ,g per ml, and all but the carrageenan suspensions had at least (Table 2). Enterotoxin A was present at levels of 0.02 ,g/ml or slightly greater in about half of the samples. At 8 h, enterotoxin B levels did not differ (5% level) for the colloidal ingredients. However, samples containing colloids differed from the controls with differences (1% level) demonstrated among the states of dispersion. Enterotoxin B in the control NAK broth and in the colloidal media averaged 19 Mg/ml with the exception of pastes. Pastes prepared with carrageenan and LMP averaged 3 Mg/ml, whereas those prepared with cornstarch and agar averaged 12 ug/ml. Less efficient aeration may occur but both carrageenan, which gave the thickest paste (Table 1), and LMP, the thinnest, are lower in enterotoxin. A minimum of 0.02 ug/ml of enterotoxin A was present with more than half of the samples having about 0.2 Ag (Table 2). By 24 h the control NAK broth averaged 170 ug of enterotoxin B per ml. Colloidal ingredients and type of dispersion interacted ( Table 5). Concentration of enterotoxin was markedly lower in the paste than in the liquid or suspension except for an opposite trend with starch ( Table 2). Differences in replications were small except for LMP. A minimum of 0.2 ;ig of enterotoxin A per ml was present in all samples except for a few isolated carrageenan samples. Still samples, strain S-6: enterotoxin levels. In still samples, although enterotoxin B was present at 5 h, the amount was generally less than 0.02 ,g/ml (Table 3). After 24 h, the PRODUCTION OF STAPHYLOCOCCAL ENTEROTOXINS colloidal ingredient and type of dispersion each had a significant effect (1% level) ( Table 5). Enterotoxin B was significantly lower in the control NAK broth and in liquid states of colloidal dispersions, except for those with carrageenan. Gels had the highest concentration, from 47 gg/ml with cornstarch to 14 Ag/ml with LMP as compared to an average of 3 for the control broth (Table 3). Enterotoxin A production was also less rapid with still incubation (Tables 2 and 3). Only a few samples from both broth and cornstarch contained as much as 0.02 Mg/ml at 5 h, but the number was similar to that for samples positive for B; at 8 h, scattered samples contained 0.2 Mg/ml or above. After 24 h, nearly all samples had a minimum of 0.2 ug/ml. The effect of colloidal ingredient appeared to be greater than that of type of dispersion. Staphylococcal multiplication and pH changes. When samples were incubated without shaking as compared to shaken, the rate of increase in CFU was less (Tables 2 and 3). The difference was less after 24 h. Differences in pH were not appreciable at 5 h, about 1 pH unit lower at 8 h, and 2 pH units at 24 h, including the gels (Tables 2 and 3). In shaken samples, increase in CFU was rapid from the zero hour count of 106 per ml. The average count in NAK broth was 8.3 x 108 at 5 h and 1.3 x 1010at 8 h. Maximal counts had been reached prior to 24 h. The colloidal ingredient, as compared to the NAK broth, had no significant effect at 5 h but had a slight, although significant (5% level), depressing effect at 8 h. The addition of cornstarch or LMP as the colloidal ingredient had increased the 24-h CFU slightly (significant at the 1% level). The state of dispersion had little effect nor was there a significant interaction between dispersion and colloidal ingredient. Although pH differences were small, effects of the state of dispersion and the interaction with colloids were highly significant statistically. The colloids did not appear to differ until 24 h, when the LMP had the highest average number of CFU (2.8 x 1010 as compared to 7.6 x 109 for the control); pH was the lowest, 7.7. Other strains. Four additional strains were compared after 24 h still incubation in liquid and paste states with cornstarch as the colloid (Table 4). In addition, carrageenan was used for one strain. In samples incubated without shaking, increases in CFU, pH, and the production of enterotoxin were found for the pastes with the exception of CFU of strain 361. In the strain 361 study, the effect of colloid was greater than the effect of type of dispersion. Nutritive effects. Preliminary tests with phosphate buffer dispersions of the colloidal ingredients prior to these studies demonstrated IrA, AND VENN APPL. MICROBIOL. little nutritive effect as indicated by no or slight increases in CFU of inoculated staphylococci except possibly for the higher concentration of carrageenan. There was no enterotoxin production when the colloids had been added to buffer to provide the four types of dispersions. In addition, reducing sugars were determined for the starch dispersions, and then filter-sterilized glucose was added to adjust the control NAK broth and each starch dispersion to the "If quantitation by the single gel-diffusion assay was not possible after concentration of the samples, micrograms per milliliter was estimated from the micro-slide procedure by comparison with enterotoxin dilutions. )RI PRODUCTION OF STAPHYLOCOCCAL ENTEROTOXINS level of the highest concentration found (17 mg/ml in the suspension and gel). Enterotoxin B was low in the still broth and liquid dispersion (average of 0.9 Mg/ml) and higher in the suspension and gel (20 and 13 gg/ml, respectively). A second broth, NAK-PHP, which was higher in protein content was used as the basis for liquid and paste cornstarch dispersions ( Table 6). The trends were little different from those with NAK broth, that with the higher proportion of cornstarch yielded larger quantities of enterotoxin. Limitations. It was more difficult to study the still systems because of the low levels of enterotoxin to be determined. If the concentration reported is below 0.4 Mg/mil, the value was estimated from dilution comparisons and the limits of detection of the methods used. In recovery studies, it was found that enterotoxin A decreased during the concentration procedure and, therefore, reported concentrations below 4 gg/mil are lower than the original. DISCUSSION It is evident that under conditions of forced aeration, there was no increase in enterotoxin production attributable to the addition of the colloidal ingredients. However, under more limited aeration (incubation still), paste and gel dispersions had increased production of enterotoxin. In preliminary investigations, gels surface-inoculated and those inoculated throughout the medium before gelation gave similar levels of enterotoxin. Colony form differs with the consistency of the growth medium; further study may delineate this or other physical factors as being important. Bacterial cells may adhere to colloid particles and thus encounter a different microenvironment (8). Haider et al. (5) in experiments with S. cerevisiae and A. niger observed more efficient utilization of glucose in the presence of montmorillonite. A protective effect has been attributed to several synthetic polymers substituted for part of the serum in media for selected human cell lines (11). Differences in a, appear not to be the cause since all were above 0.99 (14). Since foods represent colloidal systems and have no active aeration during storage, the production of enterotoxin may be favored by the colloidal matrix. However, not all colloids were equally effective.

v3-fos

2018-04-03T03:21:33.147Z

1973-06-01T00:00:00.000Z

2695574

s2

Toxicity of metabolites produced by the "Alternaria". The presence of toxin-producing fungi in foodstuffs and other agricultural commodities is well established (1-3). The Alternaria, Aspergilli, Fusaria and Penicillia have been repeatedly implicated as the principal coinhabitants of products in which toxicity has been demonstrated (4,5). Numerous compounds have been isolated that can explain the toxicity of the Aspergilli, Fusaria, and Penicillia. Among the more important of these are the aflatoxins, patulin, penicillic acid, and sterigmatocystin, because of their carcinogenic potential; the ochratoxins, citrinin, cyclopiazonic acid, and the estrogenic zearalenone because of a variety of high toxicities and their frequent appearance in moldy foodstuffs. By comparison, toxic components of the Alternaria have been studied to only a small extent. The Alternaria are found on wheat, barley, oats, sorghum, corn, and peanuts (4,6,7). Animal feeds and silage that contain these crops, as well as alfalfa and grass hay are also good sources (4). The Alternaria are plant pathogens and thus can contaminate food through field infection as well as through storage. Black spot of Japanese pear, brown spot of tobacco, early blight of tomato and potato,-and seedling chlorosis of citrus are caused by this genus (8). Christensen et al. (4) found Alternaria to be one of the genera most consistently isolated in 943 fungal isolates. Of these Alternaria from foods and animal feeds, 90% were lethal to rats when fed in a sterilized corn-rice mixture. The percentage was greater than that obtained with isolates of Aspergilli, Fusaria, and Penicillia from the same sources. Doupnik and Sobers (9) reported that 31 of 96 Alternaria isolates from tobacco were lethal to chicks. Toxic effects in geese (10) and a hemorrhagic syndrome in poultry (11) have been attributed to Alternaria. Several compounds have been isolated and identified. Most interest has concerned the role of these compounds in plant pathogenesis and their potential use as antibiotics (8,12). An exception is tenuazonic acid, a compound of the tetramic acid class. Interest in its potential for toxicity to animals developed when the compound was shown to have antitumor activity (13,14). When tested, the LD50 doses for sodium tenuazonate administered to mice orally were 81 mg/kg for females and 186 mg/kg for males; in rats the corresponding values were, respectively, 168 and 180 mg/kg (15). Meronuck et al. (16) found that 23 of 34 Alternaria isolates were lethal to rats. Salts of tenuazonic acid were identified in 20 of the 23 lethal isolates, and the compounds were considered to be the major toxins. However, since some isolates were toxic but did not contain tenuazonic acid, other toxins appear to be present. Tenuazonic acid has been found in fieldinfected tobacco (17). Quantitative data on the amounts of tenuazonic acid in toxic extracts are not available, and this information is necessary to better correlate the degree of observed toxicity with the presence of this metabolite. The Alternaria are known to produce compounds of at least two other structural classes. The first are dibenzo-a-pyrones (12,18,19). These are often major components of the crude fungal extracts. The structures of two, alternariol (AOH) and alternariol monomethyl ether (AME), were established in 1953 (12), yet almost no toxicity data are available. Recently, our laboratory isolated and elucidated the structure of two more dibenzo-a-pyrones, altenuene and altenuisol (20,21). We isolated two compounds, which we call altertoxin I and altertoxin II, which now represent a third structural class (unpublished data). Initial investigation of the cytotoxicity of the dibenzo-a-pyrones has indicated a high toxicity (22). For HeLa and lymphoma L5178Y cells, the ID50 values for AOH and AME were 6 and 8 ,ug/ml, respectively. We now present further data on the toxicity of these compounds to bacterial and tissue cell cultures, to female mice, and to fetal mice in utero. The toxicities of crude extracts, pure components, and a combination of two of the components, AOH and AME, are compared. Cytotoxicity HeLa S3 cells were grown in monolayer culture, and the toxicity of purified mycotoxins or crude fungal extracts was evaluated as previously described (23). Bacterial toxicity toward Bacillus mycoides ATCC 6462 was carried out by using the paper-disc agar plate method (24). Toxicity to the Young Adult Mouse Female mice weighing 18-21 g were studied. These included the 4-5 week-old CD-1 mouse (Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts) and 8-10 week-old DBA/2 mouse (Jackson Laboratories, Bar Harbor, Maine). Purified mycotoxins were administered as a single dose intraperitoneally (IP) in 0.1 ml DMSO. Crude fungal extracts were injected IP at a dose of 100 mg/kg/day for 3 consecutive days. The number of deaths that occurred within 14 days was recorded. External signs of toxicity were sought in the animals that survived. After AOH and AME administration, an animal from each test group that survived was sacrificed each month for 10 months. The liver, kidneys, heart, lungs, spleen, and thymus were fixed in 10% neutral buffered formalin for histologic examination. Liver/ body and spleen/body weight ratios were calculated. Fetotoxicity and Teratogenicity DBA/2 mice were used. The day that a vaginal plug was found was considered day 1 of gestation. The test compounds were administered subcutaneously in DMSO or orally in honey:water (1:1) from days 9 to 12 or 13 to 16 of gestation. Controls were untreated or they received the appropriate solvent. The mice were then sacrificed on day 20 of gestation for examination of the young. The number of live, dead, and resorbed fetuses was counted, and the live fetuses were fixed in Bouin's solution for examination of the internal organs by the method of Wilson (25). Some were fixed in alcohol for clearing and staining of the skeleton with alizarin red S (26). Isolation, identification and quantification of the compounds from Alternana isolates (27). The cultures were extracted with acetone-water Environmental Health Perspectives (7:3, v/v) and separated into tetrahydrofuran (THF)-soluble and insoluble fractions. The dibenzo-a-pyrone metabolites AOH, AME, altenuene, and altenuisol were isolated from Alternaria tenuis by silica gel G column chromatography of the THF-soluble extract, an increasing THF-in-benzene elution series being used as reported (20,21). AOH, AME, and altenuene were determined quantitatively as trimethylsilyl derivatives in crude extracts by using the gas-chromatographic method of Pero et al. (28). Concentrations of altenuisol in crude extracts were not determined because an analytical method was not available. However, its presence was determined by thin-layer chromatography (TLC) on 250 p silica gel G after development in 30% THF in benzene (21). Altertoxins I and II were isolated in a similar manner to that above but from Alternaria mali and with an increasing ethyl acetate-in-benzene elution series. The alter-. toxins elute in 30% ethyl acetate in benzene. Altertoxin I has not been crystallized but has the empirical formula C20H1606. Altertoxin II was crystallized from DMSO-water mixtures and has the empirical formula C20 H1 406 Altertoxin I was determined quantitatively by a fluorodensimetric assay. A Zeiss thinlayer chromatogram spectrophotometer with a mercury lamp was used. Altertoxin I was excited by light passing through an M-365 filter and assayed by light emitted at 534 mg. The concentration of altertoxin I was proportional with transmission from 0.1 to 5.0 pg. Altertoxin II gives a black quenching spot under ultraviolet light on the TLC plates. A method for analysis has not been worked out for this compound. (13)1. RultS ,ug/ml. Of the dibenzo-a-pyrones, AOH, AME, and altenuisol were the most toxic. When Bacillus mycoides was the test organism, AOH and altenuisol were the most toxic, but the combination of AOH and AME showed a striking synergistic effect. With a 1:1 mixture of AOH and AME,only 0.25 jig of each was necessary to elicit a zone of inhibition. Similar synergism was not evident with the HeLa cells. aThe lowest concentration of toxin at which there is a measurable zone of inhibition around the assay disc. The crude extracts from Alternaria isolates that had been reported to be lethal to rats showed that most of the cytotoxicity against the bacteria was in the THF-soluble fraction ( Table 2). All of the Alternaria compounds in Table 1 are freely soluble in THF, with the possible exception of the salts of tenuazonic acid. The activities of the crude extracts were much greater than that of the compounds taken singly. Synergism is thus, again suggested. It is notable that both AOH and AME were present in all of the extracts (Table 3). Altertoxin I was also present in most extracts but at much lower concentrations. When administration was to mice, the toxic principles were again found to be in the THF-soluble fraction of the isolates (Table 4). AME produced the least lethality (Table 5). There was no evidence of synergism between AOH and AME as there was in the case of Bacillus mycoides ( Table 5). The mice receiving AOH and AME often were sedated within a few minutes. When recovery occurred it was within 24 hr. The eyes of the mice were dull, and there was occasional stomach spasm and periodic panting. The altertoxins were lethal to the mice at 200 mg/kg. Toxicity with this compound was characterized by inactivity, subendocardial and subarachnoid hemorrhage and blood in the cerebral ventricles. It is seen in Table 3 that altertoxin I occurred in all of the extracts except that from isolate C-2. The effects of AOH and AME on body and organ weight are presented in Table 6. Mice dosed with either AOH or AME weighed slightly less than the controls. Liver/body weight and spleen/body weight ratios were slightly less than in controls, but this was due to reduction of body weight. Histologic aThe dose was equivalent to 300 mg/kg of the initial acetone-water (7:3) extract. bExpressed as number of mice that died in 14 days/number of mice tested. examination of the tissues did not reveal abnormalities. Table 7 shows that the combination of AOH and AME at 25 mg/kg each, administered on days 9-12 of gestation, resulted in an increased percentage of dead and resorbed fetuses/litter and runts/litter. The number of bTwo large spleens were recorded (0.4 10 g and 0.225 g). Environmental Health Perspectives malformed fetuses approached but did not reach significance at the two higher doses. AOH alone, at 100 mg/kg, also increased the percentage of dead + resorbed fetuses/litter and runts/litter. This dosage, however, was shown to have a low degree of toxicity in young adult female mice, as already described. AME, administered on the same days, did not yield effects that reached significance. When AOH was administered at 100 mg/kg on days 13-16 of gestation, the percentage of malformed fetuses increased; AME did not yield effects at the 50 mg/kg dose. Malformations, when seen, were of a variety of types. Most occurred in the controls at lower incidences. The data indicate a fetotoxic effect of AOH at 100 mg/kg and suggest synergism between AOH and AME, since the combination of each at 25 mg/kg yielded significant responses. Conclusion Exposure of humans to acutely toxic levels of Alternaria components through the food supply is unlikely if fungicidal applications and sanitary storage methods are practiced. However, low-level, long-term exposure is an additional hazard. In order to assess the importance of the components of Alternaria as toxic contaminants of our food supply, we must know what toxins are produced in the different species of the genus and how these vary with alterations in their environments. The present study represents a beginning for the determination of the relationship of Alternaria to environmental health problems. We know of at least three classes of toxic metabolites produced by the Alternaria: the dibenzopyrones, the tetramic acids, and the group represented by altertoxins I and II. All have been shown in this work to have some degree of toxicity. A synergistic effect between AOH and AME has also been shown in two of the tests, and both AOH and AME are present in all of the species. Whether or not the compounds discussed represent a hazard to humans remains to be determined. It is the opinion of the authors that further research should be encouraged to establish or negate the importance of these compounds as environmental toxins. Table 7. Fetotoxic and teratogenic effects of alternariol (AOH) and altemariol monomethyl ether (AME) on DBA/2 mice. bAdministered in 0.05 ml DMSO, subcutaneously. Cp I0.05. dp < 0.01. eAdministered in 0.10 ml DMSO, subcutaneously. fAdministered in 0.10 ml honey-water, 1:1.

v3-fos

2020-12-10T09:04:16.707Z

1973-05-01T00:00:00.000Z

237229147

s2

Evaluation of Potential Risk of Botulism from Seafood Cocktails Clostridium botulinum E could not be detected in 35 samples of commercial seafood cocktails, ranging in pH from 4.10 to 4.85. At 30 C, toxinogenesis in homogenates acidified with a citric-acetic acid mixture was prevented at pH 4.86 or lower for crabmeat and at 5.03 or lower for shrimp. Measurements of the rate of acid penetration into the centers of large pieces of flesh indicated that the already small risk of botulism from seafood cocktails could be completely eliminated by using a cocktail sauce at a maximum pH of 3.70 and by cooling the final product to at least 10 C for 24 h. The increasing production of semipreserved convenience-type foods has again focused attention on Clostridium botulinum, even though the overall incidence of this organism in such foods appears to be quite low (for example, see reference 10). Type E has been found in an appreciable proportion of certain raw seafoods: in 13% of whitefish chubs (7), in 10% of vacuumpacked frozen flounder (4), and repeatedly in marine food species off the Pacific Coast of the United States (1, 3). Crab and shrimp provide a good growth medium for C. botulinum (5). When cooked, peeled, and used in the preparation of cocktails in sealed glass jars, these seafoods are not sterilized but are preserved primarily by the bacteriostatic action of acetic acid and secondarily by refrigeration, which, in commercial channels, is not always reliable. In California, the method of preservation has evolved empirically and, while no cases of botulism attributed to such seafood cocktails are known, neither is experimental evidence of their safety available. The purpose of the present investigation was to assess risks of botulism from seafood cocktails. MATERIALS AND METHODS The isolation procedure used for C. botulinum has been described previously (5). Preparation of homogenates. The freshly cooked and peeled meat of Pacific Coast crab (Cancer magister) or the coastal species of shrimp (Pandalus jordani) was homogenized in a blender with an equal weight of water containing 0.5% each of NaCl and sucrose. The final NaCl concentration was 1.25%. A 1:1 mixture of 0.1 N citric and 5% acetic acids was used to adjust pH. Citric acid was selected because it occurs naturally in tomatoes, acetic acid because it is added as vinegar during the commercial preparation of cocktail sauce. The homogenate was then distributed in test tubes, autoclaved, and cooled. The final pH was determined on several tubes of each batch. A Corning Model 10 meter was used for pH determinations. C. botulinum strains and spore production. C. botulinum E strains Beluga and VH, isolated by C. E. Dolman, University of British Columbia; Saratoga, isolated from canned tuna in 1963; and E-8, one of Kushnir's original strains, were used. Because differences in their behavior with respect to pH inhibition were both slight and inconsistent, only Saratoga and VH were used in the later experiments. Spores were produced in the TPG medium of Schmidt et al. (8) modified to contain 0.2% glucose and 0.1% yeast extract (final pH 7.0). Screw-capped jars (8-oz size) filled to within 2 cm of the top were heated for 20 min in boiling water, cooled rapidly, inoculated with 15 ml each of an actively growing culture, and incubated at room temperature (22 to 24 C). The progress of sporulation was observed twice daily by phase-contrast microscopy, and the spores were harvested when about 90% of the cells showed refractile spores. The crop was washed five times and finally suspended in sterile, deionized water. The viable spore count of the stock suspension was made in deep tubes of heart infusion agar (Difco) after heat shocking for 15 min at 60 C. The tubes were overlaid with vaseline and incubated at room temperature (22 to 24 C), and the colonies were counted after 72 and 96 h. Inoculation of crab and shrimp. Tubes of crab meat homogenates were inoculated with C. botulinum E in six runs. Four strains were used in the first two runs and two strains in the last four. Similarly, two strains were used for each of four runs done on shrimp 07 meat. For each run, sets of tubes were prepared containing homogenates at two or three different pH values; each set was divided into either two or four 36-tube subsets, depending on the number of strains used. The tubes were inoculated with approximately 10,000 heat-shocked spores, sealed with vaseline, split into three groups of 12, and incubated at three different temperatures. Tubes at 5.5 C were observed weekly; at 10 C, daily; and at 30 C, twice daily, and were tested for toxin at the first appearance of gas. At the end of each run, tubes of the highest pH step that had remained negative were also tested for toxin. Crab leg muscles were inoculated in the center with about 10,000 spores of C. botulinum E VH; placed in beakers containing sauces of different pH values; stored at 10, 24, and 30 C; and periodically tested for toxin. Detection of toxin. Homogenates were tested by adding to 1 ml of press juice 1 ml of 0.5 N phosphate buffer (pH 6.1) containing 0.2% trypsin (Difco, 1: 250). The pH values of all samples were thus brought to between 6.00 and 6.10, depending on initial acidity. After incubation at 37 C for 75 min, 2 ml of sterile deionized water was added, the mixture was centrifuged, and 0.4 ml of the supernatant fluid was injected intraperitoneally into each of two mice. Type E toxin was confirmed periodically by neutralization with specific antitoxin. The mice were observed for 96 h after inoculation; none died after more than 24 h. Inoculated crab leg muscles were tested by triturating the meat with 1 part of buffer and using the supernatant fluid for trypsinization and injection as just described. Acid penetration in crab leg muscles. Cocktail sauces, minus spices, were prepared from tomato paste, diluted with water to 10 to 11% solids, and adjusted with acetic acid to various pH values. Crab leg muscles measuring about 10 mm across the smallest dimension were placed in beakers of sauce. The ratio of meat to -sauce (40: 60) was that of commercial samples. At intervals, three pieces were removed, rinsed, blotted, and cut transversely. The pH across the section was estimated by means of indicator paper (Macherey, Nagel and Co., Diren, Germany, range 3.8 to 5.8). The calibration of the paper was checked with standard buffers. RESULTS Incidence of C. botulinum E in commercial seafood cocktails. Although the isolation procedure used is sensitive enough to reveal an average contamination of one spore per gram of material, no C. botulinum was detected in any of 35 samples from nine processors. pH of commercial seafood cocktails. The pH of the commercial sauces ranged from 4.10 to 4.85. Of the 35 samples, seven (lobster and halibut) contained pieces of meat up to 10 mm across. The pH at the center of these chunks was always close to that of the sauce, and in all cases below 4.50. Effect of pH and temperature on toxin formation by C. botulinum E in crab and shrimp. Table 1 shows the first appearance of detectable toxin in crab meat homogenates, combining results of tests of all strains. In no case was toxin detected in inactive tubes, whereas tubes showing gas were always toxic. Table 1 does not show the results of incubation at 5.5 C because no growth developed in any of those tubes within 127 days. Because the method of preparation of the homogenates did not allow complete control of the final pH, the differences between some values shown in Table 1 are so small that the corresponding runs should probably be considered replicates. However, it is clear that between pH 4.86 and 5.03 is a fairly narrow grey zone, above which growth (gas formation) and toxinogenesis are apparently unimpeded, and below which both are prevented. Results obtained with shrimp meat homogenates are quite similar and are not tabulated here. In tubes incubated at 30 C, the appearance of toxin took 2 days at pH > 5.25 and 40 days at pH 5.06; at pH < 5.03, no toxin could be detected within 80 days. Incubation at 10 C retarded toxin formation by about 5 days at pH 5.06; at pH < 5.03, the tubes remained negative for 130 days. The crab data, which also apply to the shrimp, indicate that, in order to prevent the formation of detectable toxin by C. botulinum E, the pH of the meat must be brought down to 4.86 or lower. Moreover, this must be done, at the latest, within 20 h at 30 C or 7 days at 10 C. Bringing the pH down rapidly by means of a sauce of the proper acidity would seem to be a simple matter for crab cocktails, in which the meat consists mostly of separate fibers. However, in other cases, such as shrimp and lobster cocktails and seafood cocktails containing halibut, fairly large pieces of meat may be encountered, and the rate of acid penetration becomes a factor. Rate of acid penetration from the sauce into the center of chunks of meat. Crab leg muscles were used in this phase of the work to simulate lobster or halibut chunks as well as shrimp. Table 2 shows combined results from several experiments, in which all three temperatures were not always used, accounting for the many blanks. Room temperature (24 C) was chosen rather than 30 C because it is closer to actual commercial conditions. The target pH was taken as 4.80, rather than 4.86, to allow for the coarser commercial method of measurement. In the series of experiments summarized in Table 1, it was determined that even at pH 7.2 and 30 C toxin could not be detected before 20 h. It would be tempting to assume that, at 24 C, likewise, detectable toxin would not be formed within 20 h, and to conclude from Table 2 that a sauce having an initial pH < 3.91 could lower the pH in the center of a reasonably large piece of meat to a safe level within a safe time However, the difficulty of controlling the rele vant variables under commercial conditions renders this conclusion questionable. A further complication is the presence of tw( competing processes: (i) the elaboration of toxir by C. botulinum and (ii) the concurrent migra tion of acid, which at some point prevent< further toxinogenesis. As temperature lowers the rate of toxin formation slows substantially whereas the rate of acid penetration slows onl1 slightly. Consequently, at 24 C the margin o safety, namely, the difference between the rate of toxin formation and that of acid penetration, is small, a few hours at most; but at 10 C this margin is much greater. Table 3 summarizes the interaction of the two processes. At or above room temperature, sauces of pH > 4.00, at the time of adding to the crab meat, cannot prevent toxinogenesis if C. botulinum E spores are present in large pieces of meat. Although a sauce at pH 3.90 appears to prevent toxin development under the same conditions, the results obtained at the next lower pH step make it prudent to consider pH 3.70 as the maximum. The negative samples were not observed beyond the times indicated because, by then, the center pH was already below 4.80. DISCUSSION The published data on the pH inhibition of C. botulinum E are somewhat conflicting. Segner et al. (9) observed growth of the Beluga strain at pH 5.03 in TPG medium, but Ohye and Christian (6) found that type E could grow at pH 6.0 but not at 5.0 in TYG medium. On the other hand, Dolman and lida (2) reported growth and toxin production by the VH strain in pickled herring (pH 4.0 to 4.2). Little information is available on pH inhibition of C. botulinum E in shellfish meat. Despite differences in growth media and. strains, our data on the acid tolerance of C. botulinum E are in general agreement with those found in the literature. As media for C. botulinum, crab and shrimp meat seem to possess no unusual qualities. The risk presented by products on the market is probably minimal. Although no data are available on lobster and halibut, it has been shown here and elsewhere (5) that the principal ingredients, commercially prepared cooked crab and shrimp meat, appear to be free of C. botulinum spores. Furthermore, the pH of the samples that we examined was low enough to r 25,1973 prevent the growth of the organism. Nevertheless, the known association of C. botulinum E with raw shellfish and its ability to grow at low temperatures make it conceivable that an unusual set of circumstances might result in a hazardous situation. For example, if large pieces of meat heavily contaminated with spores were to be mixed with sauce having too high a pH, and left at room temperature, the spores could grow and produce toxin before the acid penetrated the meat. There are indications that some processors may use a sauce that is not acid enough: When crab or shrimp meat is mixed with cocktail sauce in the usual ratio of 40: 60, the pH of the mixture gradually rises 0.2 to 0.55 units, depending on the original pH of the sauce and the freshness of the meat. During this survey, I encountered sauces with a final pH as high as 4.85; therefore, allowing for a rise in pH of even 0.6, I must conclude that the initial pH of the sauce was somewhere around 4.25 which, as has been shown, is not low enough under certain experimental conditions. Based upon the findings of this investigation, I recommend two key requirements to insure the safety of seafood cocktails. (i) The initial pH of the sauce should be no higher than 3.70, and (ii) as soon as possible after preparation the product should be chilled to at least 10 C and held refrigerated for 24 h. At least with respect to potential botulism, refrigeration need not be maintained beyond this period. These two requirements are easily met and have long been part of the operating procedures of several processors. of conditions. Even in the absence of refrigeration, a sauce having a pH of 3.70 is sufficient to acidify the centers of meat chunks to a safe level before any spores that may be present have had time to grow out and produce toxin. Because the minimum toxinogenesis time of 20 h was actually obtained at 30 C, the true safety factor at room temperature is probably greater than the 7.5 h shown. Nevertheless, the margin of safety is not comfortable enough when one deals with C. botulinum, hence the additional recommendation of rapid initial chilling to retard bacterial growth while allowing acidification to proceed. Under these conditions, any risk of botulism from seafood cocktails should be completely eliminated.

v3-fos

2018-04-03T00:11:02.949Z

1973-11-01T00:00:00.000Z

10487380

s2

Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives' Oleuropein, the bitter glucoside in green olives, and products of its hydrolysis were tested for antibacterial action against certain species of lactic acid bacteria involved in the brine fermentation of olives. Oleuropein was not inhibitory, but two of its hydrolysis products, the aglycone and elenolic acid, inhibited growth of the four species of lactic acid bacteria tested. Another hydrolysis product, fl-3, 4-dihydroxyphenylethyl alcohol, was. not inhibitory. The aglycone of oleuro- pein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCl; 150 ug of either compound per ml prevented growth of Lactobacillus plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 of 17 species of bacteria screened, none of which were lactic acid bacteria. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included four species of lactic acid bacteria and other gram-positive and gram-negative species. Neither crude preparation was inhibitory to growth of the seven species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 C) olives prior to brining renders them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined. pein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCl; 150 ug of either compound per ml prevented growth of Lactobacillus plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 of 17 species of bacteria screened, none of which were lactic acid bacteria. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included four species of lactic acid bacteria and other gram-positive and gram-negative species. Neither crude preparation was inhibitory to growth of the seven species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 C) olives prior to brining renders them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined. Preservation of green olives by brining, according to the Spanish-type process, depends on a lactic acid fermentation in the brine. Failure to develop proper brine acidity may result in various types of spoilage (5,12,13). Etchells et al. (5) found that heating olives prior to brining resulted in a rapid and predictable brine fermentation by pure cultures of lactic acid bacteria, whereas brines of unheated olives failed to develop an acid fermentation and yeasts were the predominant microflora. They suggested the possibility of a heat-sensitive antibacterial compound in the olives. More recently, Borbolla y AlcalA et al. (2) confirmed that heating olives prior to brining encourages an acid fermentation. Fleming and Etchells (6) found that extracts of frozen green olives inhibited lactic acid bacteria. Later studies showed that freezing olives caused the formation of a heat-stable, bitter phenolic compound which was devoid of acid hydrolyzable reducing sugar and inhibited lactic acid bacteria; unfrozen olives did not con-'Paper no. 4109, Journal Series, North Carolina Experimental Station, Raleigh, N. C. tain this compound (7). Formation of the inhibitory compound in frozen olives was accompanied by a decrease in the content of oleuropein, the natural, bitter phenolic glucoside in olives. This decrease, and the fact that the compound from frozen olives was much more inhibitory to lactic acid bacteria than was oleuropein, suggested that the inhibitor might be a degradation product of oleuropein, possibly its aglycone. It was proposed that the improved brine fermentation resulting from heating olives was due to inactivation of an inhibitor-forming system in the olives (7). Results which further support this theory have been presented (H. P. Fleming, J. L. Etchells, T. A. Bell, and W. M. Walter, Jr., Bacteriol. Proc., p. 2,1970). In later studies, Juven et al. (10) found that treating olives with hot alkali before brining enhanced subsequent fermentation. In other work, they reported that oleuropein was inhibitory to several bacteria, including certain lactic acid bacteria (9). Later, however, Juven and Henis (8) found that the aglycone of oleuropein, FLEMING, WALTER, AND ETCHELLS pein to Lactobacillus plantarum. The presence of antimicrobial compounds in olives has been suspected for some time. De-Caro and Ligori (4) found that the water solution remaining after oil was pressed from olives contained a substance which was inhibitory to several bacteria, most of which were gram positive. Recently, it was reported that salts of elenolic acid have antiviral properties (11). This acid is a hydrolysis product of oleuropein (14). The present work was undertaken to determine antimicrobial properties of products resulting from the hydrolysis of oleuropein. A second objective was to determine if unheated, green Manzanillo variety olives would release antimicrobial compounds into the cover brine. MATERIALS (6). Preparation of crude extracts. An ethyl acetate extract of oleuropein was obtained from heated Manzanillo variety olives as described previously (7). A portion of this extract was concentrated in vacuo to remove the ethyl acetate, and the residue was dissolved in 2 N H2SO4. This solution was heated for 1 h at 100 C. The hydrolysate was cooled, adjusted to pH 6 with NaOH, and extracted with ethyl acetate. The inhibitory substance which is formed as a result of freezing olives was obtained as described earlier (7), except that chloroform was used as the extracting solvent instead of ethyl acetate. Dry weights of the three crude extracts were determined. Testing crude extracts and pure compounds for antimicrobial activity. Oleuropein, the aglycone of oleuropein, elenolic acid, fl-3, 4-dihydroxyphenylethyl alcohol, and methyl-o-methyl elenolate were prepared as described previously (14). Solutions of these pure compounds as well as crude extracts from olives were screened for antimicrobial activity by the paper disk bioassay method used previously (6). Appropriate volumes of the solutions were pipetted onto 13-mm diameter paper disks, to give desired dry weight quantities. Solvents were allowed to evaporate before the disks were placed on the seeded agar surface. Control disks, to which only the corresponding solvent was added and evaporated, did not elicit inhibition zones for any of the microorganisms tested. The plating medium was seeded with one drop of a 16-h culture of the test organism grown in cucumber juice broth. Cucumber juice agar (6), pH 5.3, and Trypti-case soy agar (BBL), pH 6.9, were used as assay media. The buffer capacities of the media were determined to be sufficient to prevent drastic changes in pH (less than 0.5 pH units) of the media under the disks due to extracts or pure compounds present on the disks at the levels tested. Oleuropein and the products of its hydrolysis were tested for their effects on L. plantarum cultured in cucumber juice broth with (pH 5.0) and without (pH 5.3) added NaCl. Undiluted cucumber juice broth (2 ml) was placed in 12-by 120-mm tubes. The tubes were capped and autoclaved at 121 C for 10 min. Solutions of the test compounds (1 mg/ml) in 5% (vol/vol) ethyl alcohol were sterilized by filtration through 0.2-,um pore, alpha-8 Metricel membrane filters (Gelman Instrument Co.) and were added aseptically to the tubes of sterile broth. Sterile water and 5% ethyl alcohol were added to appropriate tubes so that the final volume was 4 ml and the concentration of ethyl alcohol was 1% (vol/vol) in all tubes. The pH of the solutions after addition of the test compounds was within 0.2 pH unit of the control broths. The tubes were inoculated with one drop of a 16-h culture of L. plantarum grown in cucumber juice broth and were incubated at 30 C. Growth of L. plantarum in the broth was estimated by determining optical densities at 650 nm with a Lumetron colorimeter. Brining of olives. Whole, green Manzanillo variety olives were washed in cold tap water, and some were subjected to heating and others to freezing treatments. For heating, olives were immersed in 74 C water for 3 min (5) and then cooled in tap water. For freezing, olives were held in plastic bags overnight at -18 C. They were thawed prior to brining. A portion of olives was neither heated nor frozen and served as a control. Because the inhibitory substance is sensitive to alkali (6), the olives were not alkali treated as is normal in the Spanish-type brining process (5). The alkali treatment, in addition to destroying the bitter principle, also probably alters the waxy coating of the fruit which causes greater permeability (10). In the present work the olives were pierced to insure release of nutrients, for microbial growth, from the olives into the brine. After heating or freezing treatments, the olives were pierced by rolling them over a bed of hypodermic needles spaced 10 mm apart and projecting 5 mm above the retaining plate. One-quart (0.946-liter) glass jars were packed with 475 g of olives and 500 ml of cold, sterilized 11.4% NaCl (wt/vol). Olives were held below the surface of the brine by plastic netting (5). The jars were closed with 70-mm diameter, six-lug, "twist-off' caps (White Cap Co., Chicago, Ill.) and held at 3 C for 3 days to allow for equilibration of NaCl with the moisture content of the olives and to permit diffusion of nutrients into the brine. All jars were inoculated with 10 ml of an 18-h culture of L. plantarum that was grown in cucumber juice broth containing 4% NaCl. The jars were loosely capped and incubated at 30 C for 17 days. on June 21, 2020 by guest http://aem.asm.org/ Downloaded from for determining the pH, titratable acidity (calculated as lactic acid), and reducing sugars in brines have been described (5). The antimicrobial compound(s) in olive brines readily partitioned into chloroform or ethyl acetate; oleuropein partitioned only into ethyl acetate. Therefore, for detection of antimicrobial compounds, 10 ml of brine from each jar was extracted with 50 ml of chloroform. Five milliliters of the extract was reserved for determination of ultraviolet absorption at 224 nm with a Cary model 15 spectrophotometer, and the remainder was concentrated in vacuo to 1 ml. This concentrate was bioassayed by the paper disk method by using Leuconostoc mesenteroides 42 as the test organism (6). Another 50 ml of brine was extracted with 50 ml of ethyl acetate. This extract, which contained oleuropein as well as the antimicrobial compounds, was dried over Na2SO4 and then concentrated to 10 ml. The concentrated chloroform and ethyl acetate extracts were analyzed by thin layer chromatography (TLC) by using solvents and procedures described earlier (7). RESULTS Screening of microorganisms for sensitivity to crude extracts. Table 1 shows results of initial screening tests to determine, qualitatively, the sensitivity of selected species of bacteria and yeasts to olive extracts. The oleuropein extract inhibited growth of Bacillus sub- . The remaining bacteria were tested in Trypticase soy agar (TSA, pH 6.9) as well as CJA. A "-" indicates no zone of inhibition; NG indicates that the bacteria did not grow in the medium. The amounts of extracts, dry weight, applied to each 13-mm-diameter disk were: oleuropein, 10 mg; oleuropein hydrolysate, 7.5 mg; extract of frozen olives, 3.5 mg. b The zone of inhibition remained clear for several days, but then growth of the culture began in this region. The hydrolysis extract also inhibited 5 of 11 gram-negative bacteria. The extract from frozen olives was inhibitory to the same bacteria as the oleuropein hydrolysis extract and also inhibited three other species. The seven species of yeasts tested were not inhibited by any of the three extracts. Sensitivity of lactic acid bacteria to oleuropein and products of its hydrolysis. The aglycone of oleuropein and elenolic acid inhibited growth of all four species of lactic acid bacteria tested qualitatively by paper disk bioassay ( Table 2). Zones of inhibition remained clear during extended incubation. Oleuropein, ,3-3,4dihydroxyphenylethyl alcohol, and methyl-omethyl elenolate showed no inhibitory action for any of these bacteria at 1 mg of compound per disk. The above five compounds were tested for their effects on growth of L. plantarum in broth culture without added NaCl (Fig. 1A). Elenolic acid and the aglycone of oleuropein at 100 ,g/ml caused about 11-and 6-h delays, respectively, in the onset of growth; thereafter, the growth rate approached that of the control, even when 200 ,gg/ml levels of these compounds were present. Oleuropein, fl-3, 4-dihydroxyphenylethyl alcohol, and methyl-o-methyl elenolate were not inhibitory to growth at 200 Atg/ml. When 5% NaCl was added to the cucumber juice broth, 100 gg of either the aglycone or elenolic acid per ml delayed growth, as noted by turbidity, of L. plantarum for about 3 days, and the rate was reduced when growth finally began (Fig. 1B). Growth was completely inhibited by 150 gg or more of either of these two compounds per ml when NaCl was present (Table 3). Again, the other three compounds were not inhibitory. Presence of antibacterial activity in the brines of olives. Olives that were heated before brining underwent acid fermentation; 0.85% titratable brine acidity was reached after 17 days (Table 4). A similar level of acidity was reached when the olives were heated prior to freezing. The predominant microbial flora in both cases were rod-shaped bacteria typical of Effect of oleuropein and products of its hydrolysis on growth of L. plantarum. The growth medium was cucumber juice broth, with and without NaCI, and contained 100 pg of the test compounds per ml. Symbols: U, oleuropein; 0, the aglycone; A, elenolic acid; 0, 6-3,4-dihydroxyphenylethyl alcohol; A, methyl-o-methyl elenolate; and 0, the control. Panel A, No NaCI; panel B, 5% NaCl. on June 21, 2020 by guest http://aem.asm.org/ Downloaded from the L. plantarum culture used for inoculation. Inhibitory activity was not detected in extracts of either of these brines. TLC analysis of ethyl acetate extracts of these brines revealed that oleuropein was the major phenolic compound Methyl-o-methyl eleno-_ late aTests were made in cucumber juice broth containing 5% sodium chloride. A " +" indicates inhibition of growth and a "-" indicates no inhibition, as determined by optical density measurements during the 8-day incubation period at 30 C. ND, Not determined. ' Level of compound (,gg/ml). c Growth was delayed for 24 h or less at 50 ug/ml and for 3 to 4 days at 100 jig/ml. present; only traces of other phenolic compounds were detected. Brines of fresh unheated olives, however, did not develop appreciable acidity during incubation (Table 4). The small amount of acidity in these brines probably was due to the natural acids which diffused from the olive tissue. Results were similar with olives that had been frozen, whether or not they were heated after freezing. Chloroform extracts of these brines possessed antibacterial activity and had comparatively high ultraviolet absorption at 224 nm, which is the absorption region of the oleuropein aglycone (14). The level of oleuropein was negligible in ethyl acetate extracts of these brines. Chloroform extracts of the brine contained two phenolic compounds with Ragly values (R. values relative to that of the aglycone) of 0.88 and 1.08. These two compounds were not detected in brines which underwent an acid fermentation. Heating olives at 74 C for 3 min after they had been frozen and thawed did not render them fermentable (Table 4), demonstrating that the antibacterial substance formed by freezing was not significantly destroyed. This fact seems important, as it was suggested earlier (5) that the effect of heat in rendering olives After equilibration of the olives and cover brine, and prior to inoculation, the brines contained approximately 7.4% NaCl, 0.1% acidity, and 0.8% reducing sugar, and were pH 4.5 to 4.9. ' Analyses were made after incubation for 17 days at 30 C. c Determined by paper disk bioassay of a chloroform extract of the brine as described in Materials and Methods. A "+" indicates a zone of inhibition, a indicates no zone. 'Determined by analytical TLC. culture, was not inhibitory to growth of species of lactic acid bacteria involved in the fermentation of brined olives but did inhibit some other species of bacteria. Elenolic acid and the aglycone of oleuropein were inhibitory to growth of lactic acid bacteria, particularly when the growth medium contained 5% NaCl. The aglycone is composed of elenolic acid bound through an ester linkage to fl-3,4-dihydroxyphenylethyl alcohol (14). Since the alcohol was not inhibitory, elenolic acid appears to be the inhibitory moiety of the aglycone. Juven and Henis (8) reported that reducing the amount of yeast extract in their test medium, APT broth, resulted in inhibition of growth of L. plantarum by oleuropein. Oleuropein was not inhibitory when 0.5% yeast extract was present. They suggested that nutrient deficiencies in the medium caused oleuropein to be inhibitory. Test media used in our studies, including olive brines which contained oleuropein, apparently were not nutrient deficient, as this compound did not inhibit lactic acid bacteria. Neither oleuropein nor products of its hydrolysis were inhibitory to the yeast species tested. The tolerance of yeasts to these compounds might explain why yeasts predominated in brines of unheated olives which did not undergo lactic acid fermentation (5). Other reports also indicate that yeasts are more tolerant to olive constituents than bacteria (1,4,9). Since it was first discovered that a mild heat treatment of green olives made brined olives more fermentable by lactic acid bacteria (5), a mechanism has been sought to explain the phenomenon. We theorize, on the basis of present information, that green olives have an enzymatic system which, when the olives are brined, causes the hydrolysis of oleuropein to its aglycone, an antibacterial compound. The aglycone or oleuropein may be degraded to yield elenolic acid, a compound which also is antibacterial. Oleuropein was present in brines of heated olives, whereas the aglycone of oleuropein was not. Lactic acid bacteria readily grew and produced acid in brines of heated olives, further substantiating the noninhibitory property of oleuropein to these bacteria. Walter et al. (14) reported a yield of 0.4% of purified oleuropein isolated from pitted Manzanillo olive8. The actual oleuropein content in the olive tissue was higher than 0.4% because some was lost during purification. A 0.4% concentration of oleuropein in the olives theoretically would yield about 2,700 ,g of aglycone per g of olive flesh. This concentration is much higher than would be needed to inhibit lactic acid bacteria in the brine; only 150 ,ug of the aglycone or elenolic acid per ml was sufficient to completely inhibit growth of L. plantarum when the medium contained 5% NaCl. Cruess and Alsberg (3) suggested that olives contain ,-glucosidase, which hydrolyzed oleuropein when olives were frozen while still on the tree. This enzyme might hydrolyze oleuropein to its aglycone when unheated olives are brined.

v3-fos

2018-04-03T01:37:42.923Z

1973-10-01T00:00:00.000Z

20205844

s2

T-2 toxin as an emetic factor in moldy corn. Extracts of Fusarium poae (NRRL 3287) grown either on sterile corn at 8 C or in Richards solution at room temperature were shown to have emetic activity in pigeons at nonlethal concentration under conditions of oral and intravenous administration. The causative agent was found to be T-2 toxin (3-hydroxy-4,15-diacetoxy-8-[3-methylbutyryloxy]-12,13-epoxy-Delta(9)-trichothecene). Oral and intravenous mean toxic dose values for this compound were found to be 0.72 and 0.15 mg/kg, respectively, as compared with an oral mean lethal dose of 2.75 mg/kg. The fact that T-2 toxin causes emesis at nonlethal concentrations may explain, at least in part, the observance of vomiting as a symptom resulting from ingestion of cereal grains infected with toxic Fusarium species containing T-2 or a similar toxin. Extracts of Fusarium poae (NRRL 3287) grown either on sterile corn at 8 C or in Richards solution at room temperature were shown to have emetic activity in pigeons at nonlethal concentrations under conditions of oral and intravenous administration. The causative agent was found to be T-2 toxin (3-hydroxy-4, 15diacetoxy-8-[3-methylbutyryloxy]-12,13-epoxy-A9-trichothecene). Oral and intravenous mean toxic dose values for this compound were found to be 0.72 and 0.15 mg/kg, respectively, as compared with an oral mean lethal dose of 2.75 mg/kg. The fact that T-2 toxin causes emesis at nonlethal concentrations may explain, at least in part, the observance of vomiting as a symptom resulting from ingestion of cereal grains infected with toxic Fusarium species containing T-2 or a similar toxin. Among the various toxicoses associated with contamination of cereals by Fusarium species (8), there have been intermittent reports of vomiting as a distinct symptom occurring among both animals and humans. Outbreaks of emesis have been reported involving moldy rye (4), wheat (14), rice (11), barley (3), and corn (2). The chemical nature of the causative factor or factors remained unknown. Early attempts to characterize this emetic factor were carried out by Prentice and Dickson, who demonstrated that a water-soluble extract from field-grown corn infected with F. graminearum (synonymous with F. roseum) induced emesis in pigs, dogs, and pigeons. Subsequently they showed that several strains including those of F. graminearum, F. culmorum (synonymous with F. roseum), F. moniliforme, F. nivale and F. poae (synonymous with F. tricinctum, according to the system of Snyder and Hansen [10]) produced an emetic substance when grown on Richards solution for 12 to 40 days (9). In the case of F. moniliforme 111, final purification by preparative thin-layer chromatography (TLC) yielded two substances with emetic activity. The one of higher R, was found to be active at doses of less than 100 ug when given intravenously to pigeons and was not lethal at this concentration. The material of lower R. also produced emesis but killed the pigeon in less than 12 h. These compounds were not characterized except in a preliminary way. MATERIALS AND METHODS In this paper, TD refers to nonlethal toxic dose (i.e., emesis), as opposed to LD (lethal dose), which retains its conventional meaning. The above strains were grown on moist sterile corn at 8 and 25 C and in Richards solution at 25 C in the Department of Plant Pathology, University of Wisconsin, by using previously described conditions (7,9). Pigeons. Healthy adult pigeons (300 to 400 g) were supplied and maintained by the animal care unit, University of Wisconsin. For dosing purposes the pigeons were weighed to the nearest gram. T-2 toxin. Pure toxin for mean TD (TD50) and mean LD (LD5e) studies was obtained in part by preparative isolation from cultures of F. poae (NRRL 3287) as described below and in part from a sample generously supplied by H. R. Burmeister of the NRRL, Peoria, Ill. Oral toxicity. Samples were prepared by dissolving the appropriate amount of material along with commercial corn oil (0.5 ml) in chloroform followed by prolonged evaporation in vacuo at 50 C. Control ethylacetate extracts (0.5 ml) were fed directly. The pigeons could be accurately force-fed by delivering the sample from a plastic 1-ml tuberculin syringe (without needle). In order to achieve relatively uniform stomach content, food (but not water) was withheld for 20 h before testing. After receiving the samples, birds were observed for a period of 1.5 h for emesis and at least 1 week for death. A positive emetic response was defined as a distinct vomiting episode (retching and expulsion of stomach contents) occurring within 35 min, followed by several further episodes over a period of 1 h. For this study, a lethal dose was defined as that which caused death within 40 h. Intravenous toxicity. Samples were prepared by dissolving appropriate amounts of T-2 toxin in 95% ethanol and adding 0.9% saline to give a 27% ethanolsaline solution (0.25 to 0.35 ml). These were conveniently injected via the wing vein of the pigeon by using 1-ml tuberculin syringes fitted with 0.5-in (approximately 1.25 cm) no. 27 needles. The birds were observed 1 h for emesis and at least 1 week for death as above. Isolation of metabolites from F. poae (NRRL 3287). A. Liquid culture. The contents of 14 culture flasks each containing 500 ml of a liquid culture of F. poae (NRRL 3287) were filtered through Whatman no. 2 paper, and the filtrate was adjusted to pH 9.0 with saturated sodium carbonate followed by five extractions with chloroform. The chloroform layers were combined, dried over sodium sulfate, and evaporated in vacuo (40 C) to give an oily residue (861 mg) which was biologically active. Additional active material (86 mg) was obtained by extraction of the mycelial residue with water for 8 h followed by chloroform extraction of the filtrate as before. Thin-layer chromatography (TLC) of the crude material on silica gel (F-254) with ethylacetate as solvent showed a number of spots which were yisualized with ultraviolet light after spraying with 30% sulfuric acid in ethanol and charring at 120 C. Column chromatography of the material recovered from the filtrate (662 mg) on silica gel (Merck E.M., 120 g, 2.7 by 46 cm) was effected with ethylacetate-Skelly B (85:15) as solvent with 1.0-ml (tubes 1 to 232) and 1.7-ml (tubes 233 to 300) fractions being collected. Metabolite fractionation was monitored by the above TLC system and eluate collected into six fractions which were subjected to bioassay. The fraction collected in tubes 200 to 287 was the only active fraction. Preparative TLC of this material (145 mg) on silica gel (Merck E.M. PF-254) with ethylace-tate gave three bands. The upper and lower bands corresponded to inactive material contained in neighboring fractions from the column. The middle, biologically active band was removed and the material was recovered by extraction with 15% methanol in chloroform. The resulting oil (59 mg) proved identical on mixed TLC, proton magnetic resonance, and mass spectrum with an authentic sample of T-2 toxin. B. Corn culture. The moldy corn, supplied as a powder (800 g), was extracted with ethylacetate (4 liters) for 46 h at 25 C with continuous stirring. The mixture was filtered, and the residue was washed with additional ethylacetate (1 liter). Evaporation of the solvent yielded a biologically active yellow oil which was partitioned between water (600 ml) and Skelly B (250 ml). After five extractions with Skelly B, the organic layers were combined and evaporated to give a yellow oil (31 g). The water layer was adjusted to pH 9.0 with saturated sodium carbonate and extracted several times with chloroform. The combined extracts were dried over sodium sulfate and evaporated in vacuo to give an oil (116 mg) which was biologically active. The oil from several work-ups was combined for further fractionation. This oily residue (263 mg) was chromatographed on silica gel (5.5 g, 1.0 by 20.5 cm) with ethylacetate and collected in 0.4-ml fractions. Biological activity was found in a fraction consisting of tubes 27 to 98. This was re-chromatographed on silica gel (5.5 g, 1.0 by 2.05 cm) with Skelly B-ethylacetate (85:15) as eluant in 0.5-ml fractions. Tubes 21 to 23 contained a single compound (4 mg) which was biologically active and which was chromatographically identical with T-2 toxin. Emetic activity was also found in the oil recovered from the Skelly B phase. A portion of this oil (200 mg) was chromatographed on silica gel (5.5 g, 1.0 by 20.5 cm) under linear gradient conditions by using Skelly B (100 g) and ethylacetate-Skelly B (3:2) (100 g) as eluant phases (1.1-ml fractions). Tubes 27 to 50 contained largely corn oil. Biological activity was detected in a combined fraction consisting of tubes 90 to 119. This material (12 mg), although not completely pure, consisted largely of a compound which was identical on mixed TLC with T-2 toxin. RESULTS Pigeon bioassay. The pigeon proved to be a convenient and reliable species for assaying emetic activity and has been used previously in this capacity (1,5). In the case of active fractions, vomiting typically occurred within 35 min and in several cases as early as 10 min. The vomiting response as described earlier was distinct and easy to detect. It has been reported that pigeons injected with emetics typically develop a conditioned response such that subsequent injection of saline controls initiate vomiting (5). This was also observed in this study, and each pigeon was consequently subjected to only one injection. This phenomenon was much less pronounced with oral feeding, and whereas birds were used only one time for the toxicity (9), F. poae was the only strain to exhibit emetic activity when grown in Richards solution at 25 C. Consequently, cultures of F. poae were grown in both media in large scale for further investigation. Examination of F. poae metabolites. When fractions from chromatography of the chloroform extracts from either the corn or the liquid culture were subjected to bioassay, only one component consistently maintained activity. In each case this compound, when purified, was shown to be identical to an authentic sample of T-2 toxin upon comparison of the proton magnetic resonance spectrum, mass spectrum, and behavior on TLC. Further, after determining the emetic potency of T-2 toxin it was possible to attribute all the emetic activity of the crude extract to the T-2 toxin. In the case of the 8 C corn culture, emetic activity was consistently found in the Skelly B fraction as well as in the chloroform extract and was again found to be due to T-2 toxin. The persistent distribution of the toxin in both aqueous and organic phases as well as retention by the mycelium makes quantitation difficult but it is estimated that in Richards solution the toxin is produced to the extent of 14% (by weight) of total metabolites. TLC comparison of the chloroform extracts from both media indicates a very similar pattern of metabolite production. Cursory examination of the remaining nonemetic fractions indicated the presence of at least eight other significant compounds, some of which appear to be trichothecenes. Due to their lack of activity, however, they were not investigated further. Bioactivity of T-2 toxin. With the knowledge that T-2 toxin had emetic activity, it became important to determine whether this activity could be expressed without associated lethality. The results of force-feeding pigeons with increasing doses of pure toxin dissolved in commercial corn oil are shown in Fig. 2. The oral TD,0 and LD5o were found to be 0.72 and 2.75 mg/kg, respectively. The results also show that an emetic dose range exists which is nonlethal. Whereas expulsion of unadsorbed toxin during vomiting might have been expected to reduce the precision of the results, in fact, reasonable, curves were obtained, permitting meaningful comparisons to be made. The corresponding intravenous TD60 was found to be 0.15 mg/kg. Since certain compounds which are antiemetics in humans have been shown to be emetic in pigeons, it was necessary to determine the activity of T-2 toxin in higher species (1). Injection of an intravenous dose of approximately 2.5 mg of toxin into a 10-kg dog resulted in the initiation of vomiting after 35 min in a pattern similar to the pigeon, suggesting but not proving that the response is general. DISCUSSION The fact that four of the five original strains of Fusarium were found to be inactive as emetics after storage makes comparison with the earlier work difficult. Furthermore, only one of two reported emetic substances was detected. Hence, it is likely that the present strains are no longer identical with the original ones. However, although such comparisons are admittedly tenuous, when the same solvent and visualization systems of Prentice and Dickson were used (9), T-2 toxin had an R, which coincided closely with their lethal emetic and suggests that they may in fact be the same compound. It is pertinent that none of the extracts of inactive corn cultures showed the presence of T-2 toxin on TLC and also that extracts of the 25 C corn Dose-response curves for T-2 toxin in pigeons (6). Symbols: 0, emesis resulting from intravenous administration. A total of 26 birds was used. TDi)0 = 0.15 mg/kg; 0, emesis resulting from oral administration. A total of 26 birds was used. TDi0 = 0.72 mg/kg; A, death resulting from oral administration. A total of 47 birds was used. LDi0 = 2.75 mg/kg. cultures of F. poae were inactive and contained no T-2 toxin on TLC. In the case of the liquid cultures, inactive extracts of 4-week cultures of F. culmorum and F. roseum showed some T-2 toxin on TLC, but total metabolite production was negligible. This corroborates the evidence that T-2 toxin is the only significant emetic compound produced by these strains under our conditions. From the intravenous data, T-2 toxin appears to be as potent an emetic in the pigeon as digitoxin (TD., = 0.2 mg/kg) but less potent than alkavervir, a mixture of Veratrum alkaloids (TD99 = 0.03 mg/kg) (1). It can be seen that the TD90 for T-2 toxin corresponds approximately to the LD25 which clearly indicates that a dose range exists where emesis can be expected to be the predominant or exclusive biological activity. The observation of emesis in some cases as early as 10 to 15 min after oral administration, particularly at higher doses, suggests but does not prove that T-2 toxin and not a metabolite is responsible for the emetic activity. This also implies a quite rapid adsorption from the gastrointestinal tract which is consistent with the substantial lipid solubility of the toxin. The recent report (12) that fusarenone-X (3,7, 15-trihydroxy-4-acetoxy-8-oxo-12, 13epoxy-A9-trichothecene), isolated from a strain of F. nivale, is both emetic (subcutaneous minimal effective dose = 0.4 to 0.5 mg/kg) and lethal (subcutaneous LD50 = 2 mg/kg) in 10-day-old Peking ducklings substantiates the fact that certain of the trichothecenes can act as emetics at nonlethal concentrations. Thus, whereas the apparently nonlethal and as yet unidentified emetic of Prentice and Dickson remains elusive, it appears likely that the intermittant but widespread reports of emesis associated with Fusarium-infected cereal grains may be due at least in part to certain trichothecenes, since these are known to widely occur in the genus Fusarium (13).

v3-fos

2018-04-03T01:50:33.088Z

1973-01-01T00:00:00.000Z

31084424

s2

Effect of lysine and lysine plus threonine supplements to rice and wheat protein. The effect of lysine or lysine and threonine as supplements to rice and wheat diets at three different protein levels (5.5, 11.0 and 15.0%) was studied in growing rats with initial body weights of 68 g in experiment I and 55 g in experiment II. The following results were obtained: At the 5.5% protein level addition of lysine alone to the wheat diet had no effect on the growth of the rats, but there was significant effect of lysine plus threonine. At the same level of protein, addition of lysine increased the growth of rats receiving the rice diet, and the effect of threonine with lysine was more significant. At the 15% protein level, addition of lysine had a significant effect on the growth of rats receiving both rice and wheat diets, but no effect of threonine with the wheat+lysine or rice+lysine diet was observed. With 11% protein level, addition of threonine to the lysine-supplemented rice diet had little effect on the growth of rats, whereas it caused a marked improvement in the growth of rats on the wheat diet. It was also observed that the body weight gain or changes in body water correlated well with the lysine intake of rats receiving nonsupplemented as well as those with supplemented rice and wheat diets. The effect of lysine or lysine and threonine as supplements to rice and wheat diets at three different protein levels (5.5, 11.0 and 15.0%) was studied in growing rats with initial body weights of 68g in experiment I and 55g in experiment II. The following results were obtained: At the 5.5% protein level addition of lysine alone to the wheat diet had no effect on the growth of the rats, but there was significant effect of lysine plus threonine. At the same level of protein, addition of lysine increased the growth of rats receiving the rice diet, and the effect of threonine with lysine was more significant. At the 15% protein level, addition of lysine had a significant effect on the growth of rats receiving both rice and wheat diets, but no effect of threonine with the wheat +lysine or rice+lysine diet was observed. With 11% protein level, addition of threonine to the lysine-supplemented rice diet had little effect on the growth of rats, whereas it caused a marked improvement in the growth of rats on the wheat diet. It was also observed that the body weight gain or changes in body water correlated well with the lysine intake of rats receiving nonsupplemented as well as those with supple mented rice and wheat diets. It is well known that lysine and threonine are, reppectively, the first and second limiting amino acids in cereal proteins. Numerous papers on the effect of lysine and threonine supplements to wheat and rice diets have already been reported. Most of these studies have been conducted at protein levels of 10-13% in the wheat diet (1, 2) and of 5-6% in the rice diet (3,4), since the diets were prepared, respectively, from wheat flour or bread backed with wheat flour and rice meal with fat, minerals, and vitamin mixture added. Since the nutritional response of proteins varies depending on the protein level of the diet, the effect of lysine and threonine supplements to the cereal protein would differ with protein levels in the diet. Therefore, protein concentrates were used to investigate the effect of lysine alone and lysine plus threonine as supplements to the rice or wheat proteins at three levels of protein, 5.5, 11.0, and 15.0%, in growing rats, following our pre vious experiment (5) in which three protein levels, 5.5, 11.0, and 19.0%, of the rice or wheat diets were compared. Chocola A: 0.05ml/100g diet were added to supply V.A 1500 I. U. and V. D 150 I. U. Body weight changes and food intake Body weight changes and food intake of rats fed the unsupplemented and supplemented rice and wheat diets at the 5.5, 11.0, and 15.0% protein levels in experiment I are shown in Figs. 1, 2, and 3 Fig. 4. In the rice diet, some effect of lysine supplement on body weight gain was observed at 5.5% (P<0.05), 11.0% (P<0.05 in Expt. I and P<0.005 in Expt. II) and 15.0% (P <0.01) protein levels. In the wheat diet some effect of lysine supplement on the body weight gain was observed at the 11.0% (P<0.01 in Expt. I and P< 0.005 in Expt. II) and 15.0% (P<0.01) protein levels, but not at the 5.5% level. Some effect of threonine added to the rice+lysine diet on body weight gain was observed at the 5.5% protein level (P<0.01 in Expt. I), but there was little effect at the 11.0% protein level (P<0.05 only in Expt. II). On the other hand, supple mental threonine in the wheat+lysine diet affected body weight gain at the 5.5 and 11.0% protein levels (P<0.01 and P<0.01, respectively, in Expt. I, P<0.005 in Expt. II). Protein efficiency ratio The values found in experiment I for protein efficiency ratio (PER) of the rice and wheat diets at all protein levels, and the same diets with lysine or lysine and threonine supplementation are shown in Table 3. PER obtained in experi ment II in the rice and wheat diets are shown in Table 4. From Table 3, im provement of PER in the rice diet with 5.5 % protein by addition of 0.24% lysine- Table 3. PER of rice and wheat diet (Expt. I). Average initial body weight of rats was 68g. Fed for 20 days. EFFECT OF AMINO ACIDS ADDED TO CEREAL 91 HCl (P<0.01) is obvious. Addition of 0.12% threonine to the rice+lysine diet further improved PER (P<0.01). At the 11.0% level of rice protein, supplement of 0.48% lysine-HC1 improved PER (P<0.05 in Expt. I as shown in Table 3 and P<0.005 in Expt. II as shown in Table 4), and an additional supply of 0.24 threonine increased PER further (P<0.01 in Expt. I and P<0.05 in Expt. II). Table 4. PER of rice and wheat diet (Expt. II). Average initial body weight was 55g. Fed for 14 days. On the wheat diet at the 5.5% protein level, the rats failed to grow, and no improvement was observed with the addition of lysine, but PER was significantly improved (P<0.01) with the addition of 0.24% lysine-HCl together with 0.12% threonine. At the 11.0% protein level the lysine supplement had a significant effect on PER (P<0.01 in Expt. I, P<0.005 in Expt. II), and on addition of thre onine to the wheat+lysine diet improvement of PER was also significant (P<0.01 in Expt. I, P<0.005 in Expt. II). Relationship between lysine intake and body weight gain or changes in body water Figure 5 shows the relationship between lysine intake and body weight gain of individual rats for nonsupplemented groups at all protein levels and includes data obtained in the previous experiment(5) at 5.5, 11.0, and 19.0% protein levels. It shows that the body weight gain correlated well with the lysine intake with r=0.962. Correlation between lysine intake and body weight gain of individual rats in experiment I was also observed with r=0.910 for the 5.5 and 11.0% protein levels, including groups with supplemental lysine plus threonine, and 15% protein of rice and wheat diets (except the latter supplemented groups). The relationship between lysine intake and changes in body water for all rats in experiment II and for rats receiving only the lysine plus threonine supplied diets and basal rice and wheat diets was calculated, since changes in body water and body weight gain in the experiment correlated with r=0.922 and are also cited in literature (8,9). As seen in Fig. 6 for individual rats on the rice and wheat diets and the supplement ed diets with lysine plus threonine, correlation between the lysine intake and changes in body water could be observed with r=0.938, which was higher than that calculated for all rats including the supplemented lysine diets (r=0.909). Y. TANAKA, and Y. KAWAGUCHI In our previous studies comparing nutritional qualities of rice and wheat protein(5), we observed that differences in nutritional value of rice and wheat protein are greater than those mentioned in the literature. The present experiments were designed to observe effects of lysine alone or lysine and threonine as supple ments to the rice and wheat proteins at three different protein levels. At the low protein level, 5.5%, the effect of threonine supplement on the growth of rats fed the rice+lysine or wheat+lysine diet was prominent. However , the effect of threonine supplement was also observed on a casein diet with a 5.5% protein level in our previous study (10). Therefore, the effect of threonine on the low protein diet is not only to improve the cereal protein quality, but also generally to promote the growth of rats on low protein diets. On, the other hand , the effect of threonine supplement on growth of rats fed the wheat+lysine diet with 11 .0 protein was statistically significant, this was not the case for the rice+lysine diet at the same protein level. Although the threonine supplement did not improve the growth of rats re ceiving the 11.0% rice diet, it did have an influence on the PER. This effect was greater in experiment I, where food intake in the threonine-lysine group was rela tively low, than in experiment II (P<0.01 and P<0.05, respectively). From the results of the two experiments it may be concluded that the supplement of threonine had a greater effect on the wheat+lysine diet than on the rice+lysine diet when both were fed at the 11% protein level. We thank Shin-shin Shokuryo and Ueda Kagaku for their kind gifts of gluten and the liquid protease-free bacterial a-amylase.

v3-fos

2018-04-03T02:38:28.622Z

1973-06-01T00:00:00.000Z

34464731

s2

Improved Yield of Aflatoxin by Incremental Increases of Temperature Increasing the initial temperature of the rice fermentation of Aspergillus parasiticus NRRL 2999 from 15 to 21 C after 24 h of incubation and then to 28 C after 48 h resulted in about a fourfold increase in total aflatoxin over the usual fermentation which is held constant at 28 C for 6 days. The percentage of aflatoxin B1, the most toxic component, in the total aflatoxin was also increased. Increasing the initial temperature of the rice fermentation of Aspergillus parasiticus NRRL 2999 from 15 to 21 C after 24 h of incubation and then to 28 C after 48 h resulted in about a fourfold increase in total aflatoxin over the usual fermentation which is held constant at 28 C for 6 days. The percentage of aflatoxin B1, the most toxic component, in the total aflatoxin was also increased. The method of Shotwell et al. (7) for the production of aflatoxin offers several advantages, particularly for experiments requiring the continual feeding of aflatoxin to animals. The rice substrate is cheap, the fermentation has no interference in the common analytical methods, the toxic kernels are easily ground to a fine though not dusty powder which mixes easily and thoroughly into a variety of diets, and the yield is good enough so that usually the amount of toxic rice powder added to the diet is too small to alter the nutrient quality. Other advantages are the ease of extraction of the aflatoxin and its purity. Nevertheless, the quantities of aflatoxin consumed during feeding experiments are so large (880 mg during a typical dose response experiment with chickens [9]) that any improvement in yield represents appreciable savings in effort and expense. The present investigation was prompted by the observation of an unusually high yield of aflatoxin from a fermentation exposed accidentally during early incubation to subnormal temperatures as the result of an electric power shortage. A more systematic inquiry into the effect of temperature alterations during the fermentation resulted in a modification which gives about a fourfold increase in the yield of aflatoxin over that obtained with the method of Shotwell et al. (7). Except for the variation in temperature, the rice fermentations were done by the method of Shotwell et al. (7) in the flasks described by Smith and Hamilton (8). The contents of at least 40 flasks were combined for each determination. The results reported are the means of four replicate experiments. The replicate means were subjected to an analysis of variance in which an F-ratio was calculated, and because a significant ratio was obtained, the least significant difference between treatment means was determined (1). The total aflatoxin content of the fermented rice was determined by the method of Nabney and Nesbitt (4) with the modification of Wiseman et al. (10). The flasks were incubated on platform shakers in rooms where the temperature was controlled within 0.5 C of the stated temperature. The percentages of aflatoxin B1, B2, G,, and G2 were determined calorimetrically (4) after separation on thinlayer chromatograms (7). The effect on aflatoxin yield of varying the temperature of the fermentation during the initial 48 h of incubation is shown in Table 1. When the temperature was held at 15 C for 48 h before raising it to the normal 28 C for the remainder of the 6-day incubation period, the yield was increased highly significantly (P < 0.01) to 0.77 mg/g as compared to a yield of 0.46 mg/g when the temperature was maintained at 28 C throughout the fermentation. When the initial temperature of 15 C was increased to 21 C after 24 h and increased to the normal 28 C at the end of 48 h, the yield was increased still further (in a highly significant fashion; P < 0.001) to 1.85 mg/g. When the initial temperature of 15 C was raised to 28 C after 24 h and then to 32 C at 48 h, the yield was decreased highly significantly (P <0.01) from the control value to 0.07 mg/g. The percentages of aflatoxins B1, B2, G1, and G2 in the total aflatoxin are shown in Table 1 for the control fermentation in which the temperature was constant and for the fermentation conditions which gave the highest total yield. The percentages of 71, 9, 16, and 4 for B1, B2, VOL. 25, 1973 G1, and G2, respectively, in the control fermentation agree closely with those obtained by Shotwell et al. (7). When the temperature was raised from an initial 15 C to 21 C after 24 h and then to 28 C after 48 h, the percentages were altered to 88, 2, 9, and 1, respectively. The enhancement of the B: G ratios of the toxins by changing the temperature agrees with earlier observations. Schroeder and Hein (6), Diener and Davis (3), and Schindler et al. (5) found increased production of aflatoxin B in relation to aflatoxin G when the fermentation was done at constant elevated temperatures. Schroeder and Hein (6) obtained evidence that the diminution of aflatoxin G relative to B at elevated temperatures was the result of accelerated catabolism of G at higher temperatures. Our data, in which the final temperature was the same in the two experiments, suggest instead that an enhanced production of aflatoxin B occurred in our experiments. This improved yield of aflatoxin by the use of incremental increases of temperature during fermentation has permitted a considerable savings in the time and effort needed for the production of aflatoxin for feeding trials. An additional benefit has been the enhanced percentage of B1 aflatoxin in the total aflatoxin since aflatoxin B1 is recognized as the most toxic of the components (2). LITERATURE CITED

v3-fos

2018-04-03T06:22:25.169Z

1973-07-01T00:00:00.000Z

24458327

s2

Gamma radiation inactivation of coxsackievirus B-2. The radioresistance of coxsackievirus B-2 was studied when the virus was suspended in Eagle minimal essential medium, distilled water, cooked ground beef, and raw ground beef and irradiated at various temperatures in a cobalt-60 gamma radiation source. The number of surviving viruses at given doses of radiation was determined by a plaque assay system. All destruction curves indicated a first-order reaction. When the virus was irradiated in minimal essential medium at temperatures of -30, -60, and -90 C, D values (in Mrad) were 0.69, 0.59, and 0.64, respectively. When the virus was suspended in water and irradiated at -90 C, the D value was 0.53. Cooked ground beef containing the virus was irradiated at temperatures ranging from 16 to -90 C. The D values were 0.70 (16 C), 0.76 (0.5 C), 0.68 (-30 C), 0.78 (-60 C), and 0.81 (-90 C). Raw ground beef containing the virus was irradiated at -30, -60, and -90 C, and the D values were respectively 0.75, 0.71, and 0.68. The D values indicate that the rate of viral inactivation was dependent on the suspending menstrum. The use of gamma radiation has been advocated as a means of obtaining sterile, organoleptically acceptable raw and cooked foods that require no refrigeration storage and have a long shelf life (10). Safety, enzymatic changes, and microbiological considerations are factors that affect any such food processing system. Present systems utilize low temperature heating followed by irradiation of the frozen (-20 to -40 C) food product. The limited information on inactivation of viruses in foods prompted a study to determine the safety of such foods, and to obtain base line data on viral inactivation. Coxsackievirus B-2 was suspended in Eagle minimal essential medium (MEM) in Hanks balanced salt solution (8,13), distilled water, and ground beef and 14 irradiated at temperatures ranging from 16 to -90 C. The results indicate that the rate of viral inactivation is dependent on the suspending medium. MATERIALS AND METHODS Virus. Coxsackievirus B-2, Ohio 1, VR-29 was passaged in continuous cell cultures (Vero) from African green monkey kidney (Cercopithecus aethiops; reference 32) by using L-15 medium (17) supplemented with 2% fetal bovine serum and 0.07% NaHCO,. Cell monolayers showing advanced cytopathic effects were frozen and thawed three times, and the virus was harvested. The harvest was clarified by centrifugation for 15 min at 1,060 x g at 4 C. The virus titer was determined, and the harvest was dispensed into borosilicate glass ampules; the ampules were flame-sealed and stored at -60 C. This procedure provided a virus pool of known titer for use throughout the investigation. Virus assay. A plaque forming unit (PFU) assay system was used as previously described (30). This system consisted of an agar-medium overlay and monolayer Vero cell sheets (45 cm2) in 6-oz (approximately 0.17 liter) bottles. Radiation soure. A 2,800-C, cobalt-60 gamma radiation source was used. The irradiation chamber consisted of 36 cobalt-60 containing pins immersed in used to position and rotate a cylinder that housed a rig containing the samples of virus materials. Temperatures were maintained during radiations by metering gaseous nitrogen from a liquid nitrogen tank into the irradiation rig. Vertical positioning on the rig of the tubes containing the material being irradiated permitted delivery of eight different doses in a given time. These doses, in Mrad, were 0.43, 0.81, 1.35, 1.88, 2.04, 2.21, 2.67, and 2.82. The Mrad at each tube position in the radiation rig were determined by cobalt glass dosimetry. During dosimetries, cobalt glass strips were placed in tubes containing MEM plus 2% fetal bovine serum, distilled water, cooked ground beef, and raw ground beef. No significant differences were seen in the delivered doses among the menstra. MEM and water irradiation samples. The virus was diluted to approximately 10,000 PFU/ml in MEM (pH 7.0) containing 2% fetal bovine serum, or in distilled water. Next, 1.2 ml of the virus-containing liquid was pipetted into 13 by 53-mm borosilicate glass tubes, which were then flame-sealed. Tubes containing the virus material were equilibrated at the temperature at which they were to be irradiated. Temperatures during irradiation runs were -30, -60, or -90 C. A 1-ml sample from each tube was assayed for viral PFU. Ground beef. The ground beef used in this study was obtained from one animal and was processed, ground, and canned under clean-room conditions at Natick Laboratories, Natick, Mass. The beef was U.S. grade choice, boneless chuck that was ground through a 3A6-in. (approximately 0.46 cm) plate twice; the fat content was 25%. Twenty-six cans containing 400 g each of meat were vacuum sealed and frozen at -30 C. A similar number of cans were prepared with the same batch of meat, sealed, heated to 80 C, and held at this temperature for 15 min. These cans were then stored at -30 C. The unheated meat was designated as raw ground beef, and the heated meat was designated as cooked ground beef. All cans of meat were held at -30 C until used. Meat irradiation samples. One-gram portions of the meat containing approximately 10,000 PFU of coxsackievirus B-2 were placed in individual 13 by 53-mm borosilicate glass tubes, which were then flame-sealed (29). The sealed tubes were held at -60 C until used in an irradiation run. Representative samples were assayed for viral PFU to ensure even distribution of coxsackievirus B-2 among the 1-g portions of the meat prior to irradiation. Meat-virus samples held at a given temperature during irradiation were allowed to equilibrate at this temperature before being lowered into the cobalt-60 well. Four samples were used at each dose in each irradiation run. When an irradiation run was completed, the samples were stored at -60 C until assayed along with positive and negative controls of the same run. All control samples received treatment identical to that received by the irradiated samples, except that the control samples were not irradiated. Viral assay of meat. A method previously shown to give satisfactory virus recovery from ground beef was used (29). This method consisted of making meat slurries by shaking 1-g portions of ground beef in MEM (pH 8.5) and clarifying these slurries by passing the liquid portion through cheese cloth. The filtrate from each 1-g sample was individually titrated by log,0 dilution steps when necessitated by the anticipated PFU number. One-milliliter portions from each dilution were assayed in each of four 6-oz (approximately 0.17 liter) bottles containing Vero cell monolayers. This quadruplicate assay was done for 1-g portions of meat irradiated at lower dose levels; i.e., 1.35 Mrad and lower. At doses of 1.88 Mrad and higher, all of the meat filtrate was assayed. This was done by assaying equal portions of the filtrate in each of four 6-oz (approximately 0.17 liter) prescription bottles containing monolayers of the monkey kidney cells. Calculation of D values. The D value is the dose of gamma radiation that reduces the viral population by 90%. D values were calculated by the following formula. A linear model was assumed, and the parameters P. and 6, were estimated for each run. The model was: Y = F. + P, X + (, where Y = log,. plaque count, P. and , = true but unknown regression coefficients, X = gamma radiation in Mrad, and e = experimental error. This model was used to obtain an estimate for viral radioresistance, and goodness-of-fit tests were performed to confirm the choice of model. The value of one over the estimate of the slope (a,6) and its confidence intervals are the D value in Mrad and its confidence intervals. RESULTS A series of experiments was done to determine the effect of suspending medium and temperature on coxsackievirus B-2 during irradiation. The radioresistance data for coxsackievirus B-2 in MEM plus 2% fetal bovine serum, distilled water, and ground beef are presented in Table 1. All data were linear over the range studied in that more than 90% of the sum of squares was explained by the model. An example of plotted data is presented in Fig. 1. There was little change with temperature among D values of coxsackievirus B-2 irradiated in frozen MEM with 2% fetal bovine serum or in ground beef. The data from the irradiation runs on the virus in cooked and raw ground beef were analyzed to determine if the slopes of the regression lines were equal (21). None of the variance-ratio values, computed to test the hypothesis that the slopes among runs were equal, exceeded the critical value at a = 0.01. These analyses indicated that there were practically no differences among the slopes of the curves for irradiation runs at the given temperatures investigated. VOL. 26,1973 bThese irradiation runs at 0.5 C were previously reported (28). The data are included here for comparison purposes. DISCUSSION Coxsackievirus B-2 was suspended in MEMserum medium, distilled water, cooked ground beef, and raw ground beef and irradiated at different temperatures to determine the effect of suspending medium and temperature on viral inactivation. The limited number of observations in the MEM irradiation runs precluded tight 99% confidence limits. However, computed D values indicated no large difference in the rate of viral inactivation among irradiation runs at -30, -60, and -90 C. D values at the three temperatures were higher than the previously reported D value (0.45 Mrad) computed for the same virus in the same medium at 0.5 C (28). When the virus was suspended in water and irradiated at -90 C, the D value of 0.53 Mrad was significantly greater than the previously reported D value of 0.14 Mrad when the virus was irradiated in water at 0.5 C (28). The higher D values observed for the virus in the frozen material could be due to the inhibition of free-radical formation or to impeding of freeradical travel in the frozen material. The presence of free-radical scavengers, such as fetal bovine serum in the Eagle MEM appears to be related to the higher D value (0.45 Mrad) observed when the virus was irradiated at 0.5 C in this medium as compared to the D value (0.14 Mrad) of the virus irradiated at the same temperature in distilled water (28). No trend in D values with temperature was seen when the virus was suspended in raw ground beef and irradiated at temperatures ranging from -30 to -90 C, nor was any trend in D values with temperature noted in cooked ground beef irradiated at temperatures ranging from 16 to -90 C. Apparently, there is enough free radical scavenging by proteins and other substances in the ground beef to eliminate or reduce the secondary effects of radiation. This is clearly illustrated when the D value of 0.76 Mrad in cooked meat irradiated at 0.5 C is compared with the D value of 0.14 Mrad in water irradiated at the same temperature. If the 12-D concept is used to calculate a food process, the dose required for gamma-radiation sterilization is 12 times the D value. As an example: cooked ground beef containing coxsackievirus B-2 irradiated at -30 C requires 12 times 0.68 or 8.16 Mrad. The D values reported apply only to the menstra, temperatures, and virus investigated. There are variations in the composition of foods and other viral suspending media; however, the reported D values for coxsackievirus B-2 could be utilized as a starting point for viral inactivation studies.

v3-fos

2017-07-29T04:25:02.550Z

1973-04-15T00:00:00.000Z

148027

s2

U. S. D. A.-D. H. I. A. sire summaries in a dairy cattle population undergoing genetic change The dairy cattle populations in the United States and many other countries are undergoing the most rapid genetic improvement for yield in history. This is resulting primarily from the wide use of superior bulls through artificial insemination. As a result of the tremendous effectiveness of present sire summary procedures in identifying bulls of superior genetic merit, some of the assumptions on which these summaries are based are no longer valid. This causes some summaries to be biased. I have described briefly two alternative sire summary procedures that the United States Department of Agricultuve may adopt to eliminate these difficulties and to increase the accuracy of genetic evaluations of dairy bulls. Which procedure we ultimately adopt, depends largely The role of the United States Department of Agriculture (U. S. D. A) in the National Cooperative Dairy Herd Improvement Program (D. H. I. A.) and the National Sire Summ 2 ry and Cow Index Programs is different from the role of similar governmental organizations in many other countries. U. S. D. A. has no direct control over the bulls which are used in artificial insemination. U. S. D. A.'s role is to obtain data from the D. H. I. A. Program, analyse these data, and publish genetic evaluations on all bulls and on registered cows so that individual dairymen, artificial insemination (AI) organizations, breed associations, and others involved in the agri-business complex have accurate and objective information on the transmitting ability of bulls and cows. The artificial insemination industry consists of 2 6 private or cooperative organizations which are in competition with one another in various parts of the country. These organizations breed approximately 50 percent of the cow population. The U. S. D. A.-D. H. I. A. Sire Summaries and Cow Indexes are accepted as the official evaluation of genetic transmitting ability in the United States and as such are the standard reference for genetic merit. The basic goals of artificial insemination in most countries are the same today as they were when AI became generally available to dairymen 20 to 30 years ago. These basic goals are to provide the best germ plasm for yield and other economically important traits to the largest number of dairymen at the lowest possible cost. However, these basic goals have been considerably refined in the past few years in most countries. Dairymen are now demanding more accurate information on yield and on other traits that affect a cows profitability. In the United States this has been due at least in part to the direct relationship which has been shown to exist between a bull's Predicted Difference and the income over feed cost of his daughters as well as other economically important traits (McDeNWr,,D ICKINSON and McDOWLL L ,rg68). The U. S. D. A. started providing genetic evaluations of dairy bulls in the mid-1930 's. From the origin of this program until 19 6 2 variations of the basic daughterdam comparison were used to estimate genetic transmitting ability. In 19 6 2 , the daughter-dam comparison was abandoned in favor of the more accurate herdmate comparison procedure. The present United States Department of Agricultu y e-Dairy Herd Improvement Associntion (U. S. D. A.-D. H. I. A.) Sire Summary Procedures were adopted in 19 6 7 . For approximately five years prior to that time, a rudimentary herdmate comparison had been used based on as few as five daughters, and using an adjusted herdmate comparison based on a five month breedyear-season national average and an average regression for the number of daughters of n + 20 · The following changes were made in ig6! : n 20 i) The number of daughters was accounted for. 2 ) The environmental correlation of half sibs within herds was included by accounting for the distribution of daughters over herds. 3 ) The herdmate average was adjusted for the number of herdmates using the regional-breed-year-season average yield. 4 ) More accurate mature-equivalent age correction factors were adopted. 5 ) Ten daughters were required for a bull to be summarized. Since 19 6 2 , an average adjustment for the genetic level of the herd has been made by crediting the daughter-herdmate deviation with one-tenth of the difference between the herdmate average and the breed average. U. S. D. A.-D. H. I. A. Sire Summary procedures are described in the sire summary book published annually (DrCxirrsorr, McDnrrW!&dquo; NORMAN and KING, rg 7 2). The Predicted Difference formula along with background information and examples has been given by Fo!,!y, BATH, Drcmrrsorr and TUCKER, zg!2). In order to calculate unbiased sire summaries using our present procedures, the following assumptions must be met. I . All sires and dams represented in the summary are random samples of the ' genetic merit of one overall population. 2 . There is no genetic trend in the population. 3 . There is no differential culling among the daughters of the bull versus their herdmates. 4 . The bull's daughters receive no preferential treatment over their herdmates. These assumptions were all quite reasonable when originally adopted. However, assumptions I and 2 have become less and less tenable over the past five years until at the present time, they have become a serious concern. There is evidence of a steadily increasing rate of genetic progress (MCD AN I EL and KING, i 97 2). This causes summaries to decrease as bulls are repeatedly evaluated over a period of several years. To make matters more difficult, the rate of genetic progress does not appear to be constant between regions of the country. This is because : some AI studs have bulls of higher genetic merit than others, and dairymen in some regions make heavier use of genetically superior bulls than in other regions. Assumptions 3 and q. are not serious problems. Assumption 3 can be largely eliminated by better sire summary procedures. Assumption q. will probably always continue to be a problem in a few sire summaries but is becoming less of a problem as time progresses. Approximately two years ago, our U. S. D. A. group embarked on a series of investigations intended to eliminate the necessity of making assumptions I and 2 in the U. S. D. A.-D. H. I. A. Sire Summaries. At about the same time Dr C. R. H EN -DERSON of Cornell University announced the implementation of a new sire summary procedure known as the Direct Comparison Method. He described his procedure at that time as a generalized least-squares method whereby direct comparisons were made among the daughters of AI bulls utilizing their first records. Because of U. S. D. A.'s responsibilities to the entire dairy industry of the United States, the restrictions in Dr HE ND E RSON ' S method at that time of summarizing only AI bulls and using only first records, made it impossible to adopt his metholology for our use. Therefore, U. S. D. A. set out to modify present herdmate procedures in order to eliminate assumptions I and 2 . We did this for several reasons : I ) we realized that improvements were needed in our procedures as soon as possible ; 2 ) we had no idea when Dr H ENDERSON ' S method might be developed to the point where it would be usable under our conditions ; 3 ) the herdmate comparison method was operationally possible on existing computers and we were quite certain that improvements could be made in it which would eliminate current biases. Although we have been cooperating with Dr H!ND!RSOrr on sire summary work for the past several years, the research on these two different methods has been conducted more or less independently. Therefore, it appears at the present time that U. S. D. A. has two alternative procedures which may be developed in the foreseeable future to make improvements in the accuracy of the U. S. D. A.-D. H. I. A. Sire Summaries. DIRECT COMPARISON METHOD OF' SIRE EVALUATION At present, Dr H ENDERSON prefers calling his method Best Linear Unbiased Prediction rather than generalized least-squares. The basis for this method as presently used is the following mathematical model : where : Y lj k l is the age-corrected first lactation yield of the 1-th daughter of sire k in sire-group j made in herd-year -season i. !., hi and g f are fixed effects and Sjk and e ljkl are random effects. Since only one record per cow is used, cow effects cannot be estimated and therefore are not included in the model. As can be seen from the model, differences among sires are estimated in this method by comparing the first lactation yield of the daughters of one sire with that of contemporary daughters of other sires on a within herd-year-season and sire-group basis. Thus, sires may be compared to one another directly, or they may be compared indirectly through comparisons with other sires. This method, as do other methods of sire evaluation, actually gives information on differences among sires rather than absolute estimates of their genetic transmitting ability. In this case, assuming that the mathematical model is truly appropriate for the biological situation and the ratio of variance components is known, this method should give the best unbiased predictions of sire differences from the available data. Basically, the computational procedures are as follows (L ENTZ , MILLER and H!rrD!xsorr, 1971 ). Sires which are to be compared are categorized into sire-groups. Unproven sires should be placed in different groups from the proven sires and the proven sires are best divided into different groups on some logical basis, for example, AI organization. The more sires which are included with large 'numbers of AI daughters in many different herds, the better are the ties among herds and therefore, in general, the better are the estimates of sire differences. The first records on daughters of these sires are sequenced by herd-year-season (HYS). Two fixed seasons are used in each year, December through April and May through November. Since each HYS equation is completed before the next one is begun, each may be absorbed into the sire equations as soon as it is completed; thus, greatly reducing the amount of core storage required of the computer. In addition, the sire-group equations can be obtained later by summing the coefficients for the individual sires in each group and therefore do not need to be dealt with during the initial collection and absorption of the HYS equations. Basically, it is these techniques which make the procedure computationally feasible for large numbers of bulls on existing computers. Up to this point in the calculations the sire within sire-group effects ( Sjk ) have been treated as through they are fixed rather than random as specified by the model. Values for these random variables are estimated by best linear prediction (selection index) methods by adding the variance ratio (62e!62$) to the diagonal element of each sire equation. The variance ratio may be different for each sire group. Adding the ratio breaks the dependency between the group equation and the sire equations within the group. A dependency still exists between the group equations and the absorbed HYS equations and a restriction is needed to obtain a unique solution. Dr H!rrDExsoN has proposed the use of a Lagrange Multiplier equation to eliminate this depencency. This Lagrange Multiplier equation permits one to specify the desired value of any linear function of the solutions, thereby enabling one to maintain a constant base from year to year to compare subsequent evaluations on the same animal. At the present time we anticipate working closely with Dr H E vDERSOrr on further developments to this methodology which will permit summarizing very large numbers of sires, including natural service sires, utilizing all available records on the daughters on each sire and accouting for the intra-herd correlation among each sire's daughters. As of this writing Dr H!rrn!RSOrr has developed a procedure for summarizing natural service bulls and this procedure has been programmed by his coworkers. Work is progressing at present to test out this development. Procedures to include all available lactation records remain to be worked out and U. S. D. A. research geneticists, Dr H. Duane NORMAN and Dr Jeffrey F. K EOWN , will work closely with Dr. H!rrD!xsorr on this. Needless to say, using multiple records per cow will complicate this procedure greatly as well as increasing the amount of computer time significantly. It will require the addition of a cow effects term to the model. Therefore, many of the presently diagonal submatrices will become non-diagonal, resulting in a great increase in the computer time required to process them. U. S. D. A. MODIFIED CONTEMPORARY COMPARISON METHOD Genetic Level of Herdmates and Genetic Trend As indicated previously, our approach to eliminating assumptions i and 2 was to make improvements to the herdmate comparison method which we knew would be operationally feasible when completed. As a result of work by U. S. D. A. research geneticists Dr Ben T. McDANiEL and Dr H. Duane NORMAN it appears that adjusting the herdmate comparison for the genetic level for the herdmates' sires and running repeated iterations of these summaries should largely eliminate biases due to the genetic level of herdmates and genetic trend. These procedures should give a very close approximation to the true least-squares solutions for transmitting ability. The basic modification to the daughter-herdmate deviation formula is as follows. In simple terms, including an adjustment for the genetic level of the herdmates' sires would change this to : (Daughter averageherdmate average !average genetic merit of herdmate sires). Thus, the « average » correction for the genetic level of the herd in the present formula is replaced in the new procedures by a correction for the individual sire of each herdmate. The key to eliminating genetic bias at a given point in time using this method, and also to eliminating genetic bias across time (due to genetic trend) is to obtain improved estimates of the average genetic merit of the herdmate sires through iteration. It has been shown empirically by Dr NORMAN that correcting sire summaries for the genetic level of herdmate sires through iteration should eliminate the major known bias in the present procedures. Therefore, both the correction for the genetic merit of the herdmate sires and repeated iterations of the solutions for transmitting ability are necessary to approach the least-squares solution ; i. e., the unbiased estimate of transmitting abilities of the sires summarized. In addition to this major change in procedures, there are a number of other improvements which could be made to increase the accuracy and completeness of sire summaries utilizing the herdmate comparison procedure. Pedigree Information Pedigree information can be incorporated into these summaries. This would initially include only the bulls' sire and maternal grand sire. The cost of incorporating information from the female side of the pedigree would obviously be much greater than that from the male side. Evidence is accumulating (BUTCHER, 1972 ) that indicates there is little additional information gained from the dam's side of the pedigree if reasonably good estimates of the sire's and maternal grand sire's transmitting ability are available. The procedure utilized in New Zealand has been modified by Dr NORMAN for use in this system. It appears that the most effective use of pedigree information for our purposes is to use it as a basis for grouping. This would mean regressing the daughter-herdmate deviations on the group means using the Repeatability of the sire summary according to the formula : Predicted Difference = Group Mean + R (Daughter-Herdmate Deviation -Group Mean). When little information is available on progeny the pedigree data is quite important relative to the progeny information and should materially increase the accuracy of the first several summaries on a bull. Modified Contemporary Comparison There has been some concern in our country and in other countries as well, that the use of later records ; i. e., records made by cows that have survived culling, would cause bias in sire summaries. It now appears that whatever bias there was from the use of all records arose in large part because of inappropriate age correction factors. The new factors, that will shortly be released, were developed by a procedure devised by Dr P. D. MILLER (MILLER, 1971 ). These factors should decrease the effect of culling bias from later records when they are used in conjunction with first records, and therefore should make the use of all available records a more valuable procedure than before. As a further safety factor against biasing sire summaries from the use of selected records or age factors which are not appropriate for a particular herd, Drs McDANiEi< and NORMAN have developed a modified contemporary comparison procedure which makes the greatest use of the herdmate records which are contemporary to the daughter records. The formulas that would be used to calculate the modified contemporary averages for first records of daughters and for later records of daughter; i. e., records other than first records, are as follows : Modified Contemporary average for daughters' i where N c is the number of first lactation contemporary records, C is the mean of first lactation herdmate records, Wr, is a weighting factor for the number of later lactation herdmate records, and HM L is the mean of the later lactation herdmate records. Modified Contemporary average for daughters' where HM L and C are the same as for the first record formula, N L is the number of later lactation herdmate records and W e is a weighting factor for the number of first lactation herdmate records. Thus, for all daughter records the greatest emphasis would be placed on the daughter -« herdmate » comparisons which in general should be most accurate ; i. e., first records versus first records and later records versus later records. Additional Comments on the Use of Second and Later Lactation Records In spite of the fact that most countries of the world use only first lactation records in sire summaries, we continue to believe that more accurate summaries can be calculated by also using later records. We believe that the evidence for this is considerable and we intend to continue using later records in whatever revisions we make to our sire summary system. The greatest single reason for this decision is the large amount of data which would be lost from the sire summaries under a first record contemporary comparison in our country. McDANIEl&dquo;NORMAN and DiCKrrr-SON ( 1972 ) have shown the following percentage losses of AI bull's daughters in the various breeds : Ayrshire 14 p. 100 ; Guernsey, 9 p. ioo ; Hotstein, 6 p. 100 ; Jevsey, 9 p. ioo ; Brown Swiss, 23 p. 100 . If AI-sired first lactation contemporaries are required, as in the Direct Comparison Method presently used the losses of daughters are even higher : Ayvshive, 22 p. ioo; Guevnsey, 1 8 p. ioo; Holstein, 9 p. 100 ; Jevsey, 20 p. 100 ; Brown Swiss, 34 p. roo. These daughters would be lost from the sire summaries because they would not have first lactation contemporaries available in the same herd-year-season although they would have herdmates. We strongly feel that this loss of data would result in a net decrease in the accuracy of our summaries especially in view of the more accurate age correction factors which will be adopted in the near future and the modified contemporary comparison which would permit differential weighting of different records. Standardizing Records to a Common Age It appears to us that other countries could also gain an increase in accuracy in sire evaluation work if they would start using later records. At any rate, even if they continue to use only first records, there appears to be little justification for doing so without standardizing these to a common age such as average age of first calving. The ages of first calving, which vary generally from 22 or 23 months up to about 34 or 35 months are the ages at which the greatest change in yield takes place as age increases. Therefore, even a few months difference between the average calving age of daughters of one bull and the average calving age of daughters of another bull could cause a serious bias in the genetic evaluation. With relatively small amounts of data the procedures developed by Dr MILLER for calculating age factors could be used in almost any country without difficulty. Therefore, I would make a strong recommendation that all countries give serious consideration to standardizing the lactation records used in sire summaries for age at calving. even if only first records are utilized. With a relatively small geographical area it might be possible to use a single set of factors for an entire country for each breed and trait. In our case the problem is considerably more difficult because of the wide differences in climate and management which are found in different parts of the country. In fact, we will have to use eleven different sets of regional factors to standardize Holstein-Friesen records. We will also adopt separate sets of factors to standardize milk yield, fat yield, and fat percent. We intend to eliminate the mature-equivalent concept and to age-correct all lactation records to the age of average production which in our case is approximately 42 months for each breed. If you are using only first records it probably makes more sense to age-correct to the average age of first calving which in our case would be approximately 27 months of age. Number of Hevdmates and Number of Independent Herdmates On the average, the greater the number of herdmates available the more accurate is the daughter-herdmate deviation. Under our present system the number of herdmates is not taken into account and all daughter-herdmate comparisons are weighted equally. Dr NORMAN has developed a procedure whereby herdmate comparisons based on a larger number of herdmates would receive more weight and also herdmate comparisons from the same herd that were based on different sets of herdmates would receive greater weight than those based on the same herdmates. This will increase the accuracy of the sire summaries because multiple herdmate comparisons from the same herd-year-season very likely contain many of the same herdmates and are therefore all subject to any sampling problems peculiar to that particular group of herdmates. Two other factors which are taken into account are the number of different sires represented among the herdmates and the average Repeatability of the Predicted Differences of these herdmate sires. Both of these factors would affect the accuracy of each daughter-herdmate deviation adjusted for the average genetic merit of the herdmate sires. Adjusting for Residual Herd Effects In most present sire summary procedures, each daughter with the same number of records receives equal weight in the calculation of the daughter-herdmate deviation regardless of the distribution of daughters across herds. This procedure fails to account for the within and between herd variances properly when daughters are unevenly distributed across herds. In fact, the present U. S. D. A.-D. H. I. A. prccedure is one of the few that gives any consideration to the environmental correlation among daughters within a herd. To correct this deficiency, Dr. NoxMaN has devised a procedure utilizing the within and between herd variances which gives greater weighting to daughters which are in herds that have fewer numbers of daughters (NORMAN, McDANiEL and DrcKtNSOx, 1972 ). Our present procedure of weighting every daughter the same regardless of how many there are per herd, leads to a large number of daughters in a single herd domi-nating a sire summary. It takes a very large number of daughters in other herds to counteract the effect of this group of daughters in a single herd. Under the new procedure this deficiency would be eliminated. No matter how many daughters there are in a herd they could not count more than approximately 9 . 5 daughters distributed one per herd. This will also eliminate the present situation whereby the addition of more daughters in a herd that already has a large number of daughters can actually cause the repeatability value to decrease. Preliminary Sire Summaries It is apparent that in the United States, one of the most serious obstacles to more rapid genetic improvement in dairy cattle is that not enough potentially superior bulls are being progeny tested. One way to overcome this deficiency is to obtain sire summaries earlier in bulls' lives so that semen can be saved from the apparently superior bulls and the apparently inferior ones can be eliminated. U. S. D. A. will try to help speed up genetic progress by calculating preliminary sire summaries based on records in progress. We will commence to do this as soon as details can be worked out to acquire the necessary records in progress from the I dairy records processing centers throughout the country. These records in progress will be projected to 3 05 days in milk and the projected records will be weighted according to the length of the record in progress. The preliminary summaries will make it possible to screen bulls between 6 months and a year earlier than is now possible. This will be a screening process only, in that it will be possible to identify those bulls that appear to be either very superior or very inferior. The AI studs can then start banking semen on the superior bulls and can eliminate the infericr ones. It is estimated that the lead time in banking semen from superior bulls would result in approximately 25 ooo additional doses of semen from each of these bulls. These preliminary sire summaries will have two important financial benefits : i) dairymen will have many more highly profitable cows from the additional semen from these seperior bulls, and 2 ) AI studs will derive considerable additional profit from the sales of this semen, thus enabling them to progeny test larger numbers of bulls.

v3-fos

2020-12-10T09:04:17.301Z

1973-02-01T00:00:00.000Z

237229177

s2

Growth of Hansenula holstii on Cadavers Growth of a yeast was observed on prosected cadavers used for demonstration purposes in a medical school. An asporogenous yeast was isolated and identified as an atypical form of Hansenula holstii by analysis of the extracellular polysaccharide. The isolate showed resistance to embalming fluid but was eventually eradicated by addition of picloxidine digluconate to the fluid. purposes at 4 C were never affected. This report concerns the identification of an unusual strain of Hansenula holstii Wickerham as the yeast involved and the methods used for preventing its development on cadavers. MATERIALS AND METHODS Isolation and identification of the yeast. The yeast was isolated on a number of occasions from two of the affected cadavers on 4% malt extract (ME) agar (9), and a representative isolate, NRRL Y-7178, was selected for study. The culture techniques used for identification were described by Wickerham (9,12). Later the composition of the extracellular polysaccharide was determined by the procedures for purification and analysis as described by Slodki et al. (7). The sensitivity of the cadaver yeast to the preservative picloxidine digluconate (Resiguard, Nicholas Laboratories, Ltd., Bucks, England), was determined by inoculating glucose-peptone broth containing this compound at concentrations from 1: 100 to 1: 5,000. All cultures were incubated at 24 C, checked for visual growth, and subcultured after 3 days. RESULTS Colonies of NRRL Y-7178 were white to greyish-white and quite mucoid. Budding was multilateral and the cells measured 2.0 by 2.5 ,gm to 3.5 by 5.0 tim. Neither hyphae nor pseudohyphae were produced on Dalmau plates (9) of yeast morphology agar or corn meal agar. There was no sporulation at 15 or 25 C on ME, yeast-malt (YM), V-8 (9), corn meal, or Gorodkowa agars (8), or on carrot, cucumber, and gypsum blocks. Addition of 2, 5, 10, and 20% glucose or sodium chloride to YM and V-8 agars to increase osmotic pressure did not induce sporulation. Analysis of the extracellular phosphomannan, however, was quite revealing. As shown in Table 1, the polymer formed from glucose (5 g/100 ml) was indistinguishable from phosphomannans elaborated by Hansenula holstii strains, especially the haploid strain NRRL Y-2154 (7). D-Mannose and D-mannose 6-phosphate were the sole products of vigorous acid hydrolysis (2 N HCl, 100 C, 1 hr). Autohydrolysis, i.e., conversion to the phosphomonoester form by heating decationized phosphomannan (pH 2.5, 100 C, 30 min) gave rise to a mixture of phosphorylated oligosaccharides and a monoester phosphorylated mannan fragment. The highand low-molecular-weight components were separated by gel chromatography (M. E. Slodki et al., Proc. 4th Int. Ferment. Symp., in press). The phosphorylated mannan fragment gave a strong precipitin reaction with concanavalin A, whereas the intact phosphomannan gave a weak reaction. As judged by paper electrophoresis in 0.05 M barbital, the oligosaccharide phosphates were a mixture of monosubstituted esters apparently containing 4 to 6 mannose units. The pentasaccharide phosphate ester was the predominant component. All these results are consistent with previous findings on the phosphomannans of H. holstii (4). As with most other phosphomannan-producing yeasts, an extracellular neutral mannan was alternatively formed when orthophosphate was omitted from the fermentation medium (5). The neutral mannan gave the same pattern of enzymatic degradation (M. E. Slodki et al., in press) observed when other H. holstii mannans were incubated with Arthrobacter a-mannosidase. Attempts to mate NRRL Y-7178 with the mating types of H. holstii NRRL Y-2154 and Y-2155 were carried out on ME agar according to Wickerham (10), and on the restricted growth (RG) medium of Herman (2). There was no evidence of conjugation or sporulation on either of these media. The cadaver yeast was significantly more tolerant to the embalming fluid than the other yeasts tested. It grew well in broth containing 2% preservative, a fungistatic effect was seen at 3%, and above 3% concentration the embalming fluid was fungicidal. Four out of the five test yeasts were killed by a concentration of 0.5% preservative and the fifth (C. albicans) by 1%; none showed growth above 0.2% concentration. Picloxidine digluconate was fungicidal to the cadaver yeast at concentrations as low as 1: 5,000. The compound was successfully employed as a preservative of cadavers at a 1: 100 concentration in the presence of 0.5% embalming fluid. DISCUSSION Peterson (3) was apparently the first to report the isolation of a yeast from cadavers. His isolate, extremely tolerant to embalming fluid, was described eventually as the new species Hansenula petersonii Wickerham (11). Both the fermentation and assimilation patterns of this species differ from those of H. holstii (12). A common habitat of H. holstii is the frass of coniferous trees and gums of fruit trees (10,12). Strain Y-7178 differs from the usual isolates not only in habitat, but by its failure to produce hyphae or pseudohyphae and by its inability to assimilate D-galactose, L-sorbose, and soluble starch. It also lacks mating competence but so also do many of the haploid isolates of H. holstii collected from frass of coniferous trees (12). Many isolates of this species also vary in their ability to assimilate carbon compounds and in the degree to which they produce hyphae and pseudohyphae. Nevertheless, Y-7178 could not, with any confidence, be assigned to H. holstii were it not that its extracellular phosphomannan corresponds to that of this species and is, in fact, indistinguishable from the phosphomannan of Y-2154, a sexually reactive haploid strain (4). The specificity of extracellular polysaccharides has clearly been demonstrated (6, 7), but the procedure has seldom been used for purposes of identification. Picloxidine digluconate has previously been shown to be useful in retarding various types of postmortem breakdown when incorporated in preserving solutions (1). The strain of H. holstii isolated from the cadavers was sensitive to concentrations as low as 1: 5,000 but because of other beneficial effects reported (1), the preserving solution was supplemented with picloxidine digluconate at a concentration of 1: 100. Since this recommendation was followed, no trouble with microorganisms attacking prosected specimens has been encountered for 3 years.

v3-fos

2018-04-03T00:16:58.089Z

1973-01-01T00:00:00.000Z

10107045

s2

Cellulolytic bacteria associated with sloughing spoilage of California ripe olives. Sloughing spoilage of California ripe olives during processing is characterized by severe softening, skin rupture, and flesh sloughing. It was assumed that cellulolytic activity was responsible for skin rupture and sloughing of flesh, and so a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing spoilage. A bacterium identified as Cellulomonas flavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose (CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filter paper, enhanced the activity of the Cellulomonas strain when grown in mixed culture, although they did not, in pure culture, have any effect on filter paper. These latter cultures (all degraded olive tissue) represented the genera Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteria belonging to the genera Alcaligenes, Kurthia, and Micrococcus also were used for study of mixed culture fermentation of cellulose by C. flavigena. Cellobiose accumulation at levels of 1.0% (w/v) and above suppressed growth of C. flavigena. In a previous study (11), gram-negative pectinolytic bacteria were reported to be associated with the sloughing spoilage of California ripe olives during processing. However, in spite of rapid softening, rupture of the skin and sloughing of the flesh were not observed. Because skin rupture and flesh sloughing as well as generalized tissue softening are typical symptoms of this spoilage, it was postulated that free-living, cellulase-producing microbes might also be associated with the deterioration. Therefore a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing. This report describes the results of that search and clearly associates cellulolytic bacteria with sloughing spoilage of olives. MATERIALS AND METHODS Isolation and culture media. The basal medium used for growth and isolation of cellulose-decomposing organisms was the one described by Han and Srinivasan (4). The cellulosic substrates were Whatman no. 1 filter paper and carboxymethyl cellulose (CMC) (type 7HF, Hercules Inc.). The latter was used at a concentration of 2.0% as recommended by Goto and Okabe (3). Enrichment, isolation, and purification. The sloughed olives investigated were of commercial origin and represented the Mission, Manzanilla, and Sevillano varieties. The covering liquid was a dilute aqueous solution of lye, or salt, or both. About 1 ml of this solution from the sloughed olives was inoculated into a test tube containing approximately 10 ml of the basal medium and a strip of filter paper so placed that its top portion projected about 3/4 inch (ca. 1.9 cm) above the surface of the liquid. After 5 to 7 days at 30 C on a Rollor drum machine (model TC-5, New Brunswick Scientific Co.), if cellulolytic bacteria were present, a patch of yellow-pigmented material appeared at the air-liquid interface of the paper. Then a small piece of the paper showing slight disintegration at the air-liquid interface was transferred aseptically with sterile forceps to a new tube of the same medium. This process was repeated seven or eight times to enrich the cellulolytic organisms. Then the paper from the last enrichment was removed, macerated in a small amount of sterile physiological saline solution, and streaked onto plates containing nutrient agar, CMC agar (0.5% CMC in basal medium) plus 1.5% agar, and filter paper agar (a plate of nutrient agar covered with a filter paper disc), respectively. Representative colonies which developed on each of these agar media were picked and inoculated into fresh filter paper medium and into CMC gel medium. Cellulolytic cultures were further purified by alternately plating to test purity and enriching in the filter paper medium and CMC gel until pure cultures were obtained. Two filter paper decomposing cultures were obtained. On the basis of morphology and colony characteristics they appeared to be identical, so only one was retained for further study. Four cultures which attacked CMC gel but not filter paper were 62 on March 21, 2020 by guest http://aem.asm.org/ Downloaded from CELLULOLYTIC BACTERIA AND SLOUGHING OF OLIVES also retained for further study. Three noncellulolytic cultures were isolated for mixed-culture fermentation studies. Identification of the bacteria. The cultures retained for further study were identified by conventional methods used for generic and species allocation. General references included Breed et al. (1), Society of American Bacteriologists (9), and Skerman (8). When necessary the original literature was consulted. Preparation of crude cell-free cellulolytic enzyme solutions. The one filter paper decomposing isolate was grown in the basal medium containing 0.25% filter paper (w/v) or 1% CMC (w/v). After incubation for 5 days at 30 C with continuous shaking, cell-free preparations were obtained by centrifugation in a Sorvall, Super Speed R.C.-2, automatic refrigerated centrifuge at 9,500 rev/min for 15 min at 3 to 4 C. The clear supernatant fluid which contained the cellulolytic enzyme(s) was collected. A 1-ml amount of 1% Merthiolate (thimerosal powder, N.F.) was added per 100 ml of solution. The solutions were stored in a refrigerator until used. The four cellulolytic cultures unable to macerate filter paper were grown in the basal medium containing 1% CMC (type 7MF, Hercules Inc.). After incubation for 10 days at 30 C with continuous shaking, cell-free solutions were prepared by centrifugation at 9,500 rev/min for 20 min at 3 to 4 C. The clear supernatant fluid was collected and preserved as described above. Gravimetric determination of cellulolytic activity. The growing 18to 24-hr cultures were inoculated into 250-ml Erlenmeyer flasks containing 100 ml of basal medium and a limited amount of filter paper (110 to 120 mg per flask). After 5 days of incubation at 30 C on a continuous shaker, the flasks were removed, and their contents were filtered, washed, and dried in Gooch crucibles of 30-ml capacity and medium porosity according to the procedure of Lembeck and Colmer (5). Viscosimetric determination of cellulolytic enzyme activity. The activity of the crude enzyme preparations was determined by measurement of the changes in viscosity of the CMC (type 7MF) substrates that were induced by the enzymes with an Ostwald viscosimeter as described by Nortje and Vaughn (7). The volume of crude enzyme solution was always 10 ml. The volume of 1% CMC (T7MF) was always 20 ml. The reaction time was always 30 min. The effect of pH was determined at 30 C. The effect of temperature was studied at pH 6.0 with the enzyme produced in the presence of filter paper and at pH 6.5 with CMC as the substrate. Softening of olives by crude enzyme preparations. The possibility exists that cellulolytic degradation of olive tissue can occur during their storage in salt brine as well as during the final stages of processing when leaching with water is used to remove the lye used to destroy the bitter glucoside oeluropein. It was not possible in the laboratory to exactly duplicate the conditions existing in industry. However, the in vitro tests were designed to duplicate all but the physical pressure produced by 3-to 4-ft depths of olives. Ability of the crude enzyme preparations to soften olive tissue was done with Manzanilla ripe process fruit and with Sevillano variety olives from salt brine storage with preparations adjusted to various pH values (range 4.5 to 8.0) with McIlvaine (6) buffer and preserved with Merthiolate. Controls, regardless of the variety, consisted of olives, buffer solution of the desired pH, and Merthiolate to preserve the mixture. Both of the varieties were submerged in the enzyme solutions for 5 days at 30 C. The ripe process olives were observed daily for signs of softening, skin rupture, and flesh sloughing by sight and feel. After 5 days the Sevillano olives were put through the ripe processes (see Vaughn [101 and Cruess [2] for details). After the lye had penetrated to the pit, the olives were leached with three to four changes of water each day for 5 days. Signs of disintegration were observed daily during the 15 days of processing. The Manzanilla ripe olives were desalted so they would simulate olives during the washing period used to leach the residual lye from the fruits during the final stages of processing prior to canning (see above). It is during the 4to 5-day washing period that sloughing spoilage becomes apparent. The brine storage (7.0% salt, 0.35% total acidity as lactic acid, and pH 4.85) Sevillano fruits during the 15 days required for processing would lose all of the salt and total acidity because of leaching, and the final pH would be in the range of 7.0 to 8.0 during the final washing period. RESULTS Solely on the basis of the tests shown in Tables 1 and 2, the eight isolates described appeared to be in seven different genera and eight species of bacteria. The salient features of each follow. Cellulomonas flavigena. The C. flavigena culture (DS) was the only strongly cellulolytic bacterium isolated. It disintegrated filter paper and CMC readily and caused skin rupture and flesh sloughing of olives. The characteristics are identical to the description of C. flavigena found in Bergey's Manual, 7th ed. (1). The genus Xanthomonas. The two cultures representing the genus Xanthomonas caused liquefaction of CMC gel and disintegrated olive tissue, but did not attack filter paper. The culture DB has been allocated to X. stewartii, although plant pathogenicity was not tested. The other culture (98C) was not allocated to a specific species, although it resembled the descriptions of X. pruni and X. maculifoliigardeniae found in Bergey's Manual (1), the only difference being that culture 98C did not digest milk. The coliform bacteria. The coliform bacteria culture 98A was able to liquefy CMC gel and degrade olive tissue but not filter paper. It had all of the characteristics of Aerobacter cloacae and was so allocated. The other culture (4A) also was cellulolytic to a degree, but was the slowest of the five cellulolytic cultures studied. Even though culture 4A utilized citrate slowly and formed only acid from lactose, it was allocated to the Escherichia intermedia group. An alternative would be to call it Paracolobactrum intermedium on the basis of its anomalous lactose fermentation. Kurthia bessonii. Culture CA was not cellulolytic, but was the only pectolytic organism found in this study. This culture differs only slightly from the descriptions found in Bergey's Manual (1) and given by Skerman (8) for K. bessonii. The main differences are the variability of the Gram stain and the slight amount of acid produced from carbohydrates. The Micrococcus. Culture CW was placed in the genus Micrococcus solely on morphological grounds, but it could not be identified satisfactorily even as to genus because of many variations in morphological and physiological characteristics. These characteristics, however, best match those of the genera Micrococcus or Gaffkya as found in Bergey's Manual (1) or Skerman (8). Alcaligenes faecalis. The other noncellulolytic culture (AW) had all of the characteristics of A. faecalis as found in Bergey's Manual (1) and was so named. It is felt that neither A. faecalis nor the unidentified micrococcus play any important role in mixed fermentation acceleration of cellulolytic activity as will be shown below. All of the isolates involved in this study could grow in the presence of 8% sodium chloride (w/v) in nutrient glucose, tryptone, and yeast extract broth. Cellulolytic activity. The results given in Table 3 compare the attack of C. flavigena on filter paper in pure culture and in mixed culture with the other isolates. The degree of digestion (percent of solubilized cellulose) was 32.35% when C. flavigena was grown alone. Digestion increased about 25 to 50% when the bacterium was grown in mixed culture with the isolates able to use CMC. The noncellulolytic A. faecalis and Micrococcus sp. had little effect on increasing filter paper digestion. Five of the eight cultures involved in this study grew well on CMC-gel and liquefied it at varying rates. However, because of the slowness of their reaction rates, C. flavigena was used to produce crude enzyme to study the effect of pH and temperature on cellulolytic activity as measured by viscosimetry. Data on the effect of pH are shown in Fig. 1; those on the effect of temperature are shown in Fig. 2. faecalis aDegree of digestion of cellulose was measured gravimetrically after the organisms were inoculated into 100 ml of media containing filter paper (Whatman no. 1) as a sole source of carbon. Inoculum was 1.0 ml of cell suspension for single-culture and 0.5 ml of each for mixed-culture fermentation. Incubation was for 5 days at 30 C on a continuous shaker. The cell-free crude-enzyme filter paper preparation was most active at about pH 6.0 and had good activity in the range of pH 5.0 to 7.0, whereas the preparation obtained with CMC as the substrate was most active at about pH 6.5 and had good activity in the range of pH 5.0 to 7.5. As shown, the optimum temperature for activity of the crude enzymes was 50 C regardless of the substrate. Degradation of olive tissue. Crude, cellfree enzymes prepared from the cellulolytic bacteria all softened desalted Manzanilla ripeolive tissue as shown in Table 4. In the range of pH 7.0 to 8.0 the softening was pronounced, and there was skin rupture and flesh sloughing typical of the spoilage as observed under commercial conditions. Nearly identical results were obtained with brine storage Sevillano olives that were treated with crude, cell-free enzymes and then were put through the ripe-pickling process. Figure 3 shows in vitro sloughing spoilage. Softening and skin rupture of olives subjected to the crude enzymes was consistent and involved all of the olives tested to a greater or lesser degree. In contrast, the control olives showed no evidence of softening or sloughing. Cellulolytic activity of pectolytic bacteria previously studied. Seventeen of the 19 cul- microbes might cause the skin rupture and flesh sloughing so characteristic of this spoilage. Some of the pectolytic bacteria previously associated with softening, but not sloughing, were also found to be cellulolytic and also increased cellulose solubilization when grown in mixed culture with C. flavigena. The cultures, pectolytic or not, that degraded CMC all decomposed cellobiose. A concentration of 1.0% cellobiose (w/v) or more was found to inhibit C. flavigena. It is possible that the other bacteria favor the activity of C. flavigena by keeping the cellobiose at a low level. However, noncellulolytic, cellobiose-degrading yeasts grown with C. flavigena did not increase solubilization of the filter paper so this explanation is suspect. Another possibility is that the bacteria unable to decompose filter paper cellulose supply micronutrients to C. flavigena. This latter speculation is suspect, because the two noncellulolytic, cellobiose-negative bacteria used in this study did not materially increase cellulose degradation when grown in mixed culture with C. flavigena. It is also possible that, once C. flavigena has degraded the filter paper to a certain degree of polimerization, the CMC-degrading bacteria can attack the lesser degree of polimerization cellulose and thus increase the total solubilization of the filter paper cellulose. A concise explanation obviously is dependent on further research. In any event, both cellulolytic and pectolytic enzymes produced by various bacteria are involved in sloughing spoilage of olives. C. flavigena and the other cellulolytic cultures also caused marked tissue destruction when grown in sterile olives in brine.

v3-fos

2018-04-03T01:44:18.407Z

1973-02-01T00:00:00.000Z

23577073

s2

Duration of viability and the growth and expiration rates of group E streptococci in soil. In irradiated and nonirradiated feedlot and pasture soils inoculated with group E streptococci, the organism was not recovered 17 days postinoculation from either the irradiated or nonirradiated feedlot soils incubated at 37 C, but survived in the irradiated pasture soils for 24 and 31 days postinoculation. The streptococci survived in irradiated and nonirradiated soils incubated at 4 C for 116 days and in one irradiated feedlot soil for 165 days. The population of streptococci did not increase in either irradiated or nonirradiated soil, and the expiration rate was greater in the soils incubated at 37 and 25 C than at 4 C. With the relatively prolonged duration of viability of group E streptococci in soil at 4 C, it is suggested that soil contaminated with exudate from draining abscesses of infected swine could act as a source of infection during the colder season. In irradiated and nonirradiated feedlot and pasture soils inoculated with group E streptococci, the organism was not recovered 17 days postinoculation from either the irradiated or nonirradiated feedlot soils incubated at 37 C, but survived in the irradiated pasture soils for 24 and 31 days postinoculation. The streptococci survived in irradiated and nonirradiated soils incubated at 4 C for 116 days and in one irradiated feedlot soil for 165 days. The population of streptococci did not increase in either irradiated or nonirradiated soil, and the expiration rate was greater in the soils incubated at 37 and 25 C than at 4 C. With the relatively prolonged duration of' viability of group E streptococci in soil at 4 C, it is suggested that soil contaminated with exudate from draining abscesses of' infected swine could act as a source of infection during the colder season. Recently, it was reported that swine which carry group E streptococci (GES) do exist and possibly are a major factor in the propagation of streptococcic lymphadenitis in swine (SLS) (3, 4; J. A. Schmitz, Ph.D. thesis, Univ. of Missouri, 1971). However, it is also possible that the contamination of soil with exudate from draining abscesses containing GES could constitute a method of infecting a swine herd. This phenomenon could have occurred on a farm where the disease was not eradicated by depopulation of infected swine, disinfection of premises, or the introduction of specific pathogen-free swine (2). It had been observed previously that streptococci, such as Streptococcus agalactiae, S. dcysqualactiae, S. pyogenes, and S. uberis, did survive in damp soil for more than a year (1). This study was designed to determine the duration of viability and the growth and expiration rate of GES in sterilized and unsterilized soil at several temperatures. MATERIALS AND METHODS Experiment 1: Duration of viability of GES in soil. (i) Soils. Irradiated and nonirradiated samples of pasture and feedlot soil from two farms in Missouri designated A and B were used (Table 1). (ii) Sterilization. Soil samples weighing 100 g and air dried were twice irradiated with 170,000 R of gamma radiation per hr for 4 hr from a cobalt 60 'Present address: Department of Veterinary Medicine, Oregon State University, Corvallis, Ore. 97331. cylinder. The interval between irradiation treatments was 72 hr. (iii) Inoculation and incubation. Soil samples, in 500-ml plastic containers without lids, were inoculated with approximately 5 x 109 colony-forming units (CFU) of GES (strain 3X29a) and hydrated to 80% moisture holding capacity (MHC) with sterile isotonic saline solution (ISS). (Strain 3X29a from R. D. Shuman, National Animal Disease Laboratory, Ames, Iowa. Cells were washed twice in isotonic saline after growth for 24 hr at 37 C in Todd-Hewitt broth.) Three irradiated and three nonirradiated samples of each of the four soils were incubated at 4 C and 37 C. Humidity was maintained in the incubator at 37 C by bubbling air entering the chamber at approximately 2 liters/min through a reservoir of water in the chamber. Humidity was maintained in the incubator at 4 C by keeping a pan of water on a lower shelf. (iv) Recovery of GES. Each sample was cultured for GES at various intervals postinoculation (PI) by inoculating each of three tubes containing 30 ml of blood azide-crystal violet (BACV) broth (8) with 2 g of soil ( Table 2). The broth cultures were incubated at 37 C for 24 hr and then centrifuged at 600 x g for 15 min. The sediment was streaked on BACV agar (8) and incubated for 24 to 48 hr at 37 C. One characteristic colony from each plate was used to confirm the serologic grouping of GES (7,9). Experiment 2: Growth and expiration rate of GES in soil. (i) Soils. Irradiated and nonirradiated soils of the Sarpy and Huntington types obtained in Missouri were used (Table 1). (ii) Sterilization. Three 80-g, oven-dried samples of each soil were irradiated twice with 450,000 R of gamma radiation per hr for 2.5 hr at an interval of 96, hr in a cobalt 60 cylinder. (iii) Inoculation and incubation of soils. Soil samples were inoculated with approximately 107 CFU of GES (strain 3X29a) and hydrated to 80% MHC with sterile ISS. Irradiated and nonirradiated specimens of each soil type were incubated at 37 C, 25 C, and 4 C in 100-ml glass containers with lids ajar. (iv) Quantitation of GES. The number of CFU of GES per gram of soil was determined by streak plate colony counts performed on BACV agar. Counts were made on days 0, 1, 2, 4, 8, 16, 23, 30, and 37. Incubation of the plates for 24 to 48 hr at 4 C after the initial incubation at 37 C for 24 to 36 hr enhanced beta-hemolysis and facilitated counting the GES colonies. One characteristic colony from each plate was used to confirm the serologic grouping of GES (7,9). RESULTS Experiment 1. With the feedlot soil incubated at 37 C, GES was detected in the nonirradiated specimens at 4 days PI and in the irradiated specimens at 10 days PI; however, GES could not be isolated from either irradiated or nonirradiated samples at 17 days PI (Table 2). With the pasture soil at 37 C, GES was isolated from nonirradiated samples at 4 days PI but not at 17 days PI. The GES was isolated from all irradiated pasture samples at 24 days PI and from the pasture A sample at 31 days PI. This sample was negative for GES at 52 days PI ( Table 2). The shortest survival time for GES at 4 C was in the soils of feedlot B where the final isolations were made at 31 days PI from the nonirradiated samples and at 59 days PI from the irradiated samples ( Table 2). The GESwas isolated from all of the remaining three soil types, both irradiated and nonirradiated, at 116 days PI. At 165 days PI, the irradiated soil of feedlot A was positive for GES while all other samples were negative. The feedlot A sample was negative for GES at 200 days PI. Experiment 2. The number of CFU of GES per gram of soil was between 106 and 101 in all samples on the day of inoculation. The number of GES in the soil samples incubated at 37 C decreased rapidly, and at 37 days PI only the irradiated Huntington soil contained viable organisms. At 25 C, the expiration rate of GES was slower, with viable organisms present in all but the nonirradiated Huntington soil at 37 days PI. The expiration rate of GES in soil incubated at 4 C was significantly less (P < 0.05, analysis of variance of means) than at 37 or 25 C, with high concentrations of GES in all specimens at 37 days PI (Fig. 1). At each temperature of incubation, there was no significant difference (P > 0.05) in the expiration rates of GES between Sarpy or Huntington soils or between irradiated or nonirradiated soil. DISCUSSION The significant finding in this study was that GES survived longer in soil, specifically at lower temperatures, than these investigators had anticipated. It appeared that there was little or no multiplication of GES in soil, but rather the population of organisms decreased at a rate which was related to temperature. Unquestionably, the technique used in quantitating the GES in experiment 2 resulted in considerable error; however, all specimens were quantitated in the same manner, thus justifying comparisons within the experiment. Irradiation of soil, which was designed to sterilize the soil without greatly altering its physical qualities (5), had less influence on the expiration rate on GES in experiment 2 than was anticipated, although there was an ap-preciable difference in the duration of viability in experiment 1. It is possible that oven-drying the soil samples in experiment 2 reduced the microflora significantly whereas air-drying in experiment 1 did not alter the microflora, thus accounting for the comparable rates of expiration of GES in irradiated and nonirradiated soils in experiment 2 and also for the marked difference in the duration of viability of GES at 37 C between the two experiments. However, even within experiment 1, it appeared that GES survived better in some soils than in others. This variability in the survival time of bacteria in different soils has been observed previously (6). The than pH or organic matter since the survival time of GES was considerably shorter than in the remaining soils of comparable composition. The greater survival time of GES in the pasture soils than in the feedlot soils at 37 C does not agree with a previous report in which survival of enteric bacteria was enhanced by increasing the organic content of soil (6). This disparity may be related to differences in the physiological properties of GES and the enteric bacteria or to the higher incubation temperature. The latter appeared likely considering the prolonged GES survival time of GES in the irradiated soil of feedlot A at 4 C. The isolation of GES from soils incubated at 4 C for 116 and 165 days, in this study, suggests that soil contaminated with exudate from draining abscesses of swine infected with GES could serve as a source of infection for swine during the late fall, winter, and early spring. There is the possibility that the sodium azide and crystal violet in the BACV, which was used as a selective medium, could have had an inhibitory effect on the growth of GES. In a previous study, there was a reduction of approximately 20% in the isolation of GES from BACV as compared to blood agar (unpublished data). However, in the same study, GES was isolated from BACV agar that had been streaked with an inoculum that contained less than fo CFU on blood agar. ACKNOWLEDGMENTS This work was supported by cooperative agreement no.

v3-fos

2020-12-10T09:04:12.852Z

1973-08-01T00:00:00.000Z

237234517

s2

Effects of Various Gases on the Survival of Dried Bacteria During Storage Salmonella newport and Pseudomonas fluorescens were dried together in papain digest broth and sucrose-glutamate, and stored in several gases at various water activities (aw) between 0.00 and 0.40 at 25 C for various periods up to 81 weeks. Both S. newport and P. fluorescens, dried in papain digest broth and stored in air, died rapidly if the conditions were very dry (0.00 aw) or moist (0.40 aw). Storage in carbon dioxide and argon gave greater survival than storage in air but lower survival than did storage in nitrogen or in vacuo. When the organisms were dried in a sucrose-glutamate mixture the differences between the gases were very small, and variations in residual water were less important. Of the inert gases, argon gave the best survival when the organisms were dried in papain digest broth, especially at 0.00 aw; the survival in neon and krypton was lower and in xenon and helium it was much lower. The stability of dried bacteria during storage is dependent on a number of factors, the more important ones being the organism itself, the composition of the suspending fluid in which the organism is dried, the residual moisture, the temperature of storage, and the atmosphere of storage (2). It has been well established by earlier workers that storage in vacuo gives better survival than storage in air (4,(5)(6)(7)(8). However, there is no agreement about the relative merits of other gases and whether or not use of these gases is as good as storage in vacuo. Rogers (6) was one of the first workers to compare storage in vacuo with storage in other gases. He found that survival was highest in cultures stored in vacuo and lowest in those stored in air or oxygen. Atmospheres of nitrogen, hydrogen, and carbon dioxide were intermediate in rates of survival. The observations of Naylor and Smith (4) with Serratia marcescens are in agreement with these findings. Proom and Hemmons (5), working with Neisseria, also found that storage in vacuo gave the best survival, although in this case storage in nitrogen was just as good as storage in vacuo. In this earlier work there had been no control of residual moisture during storage except to dry the particular gas, and a variety of suspending media had been used. In no instance was more than one suspending medium studied in any one experiment. The work of Scott (7) showed that the effect of atmosphere was dependent upon the suspending medium and the moisture level. He found with Salmonella newport that only under the driest conditions and in the absence of sugars was there a marked difference in viability between dried cells stored in air and in vacuo. In the experiments to be described in this paper, the survivals of two organisms, namely, Pseudomonas fluorescens and S. newport, have been determined when they were dried in two suspending fluids and stored in various gases under conditions of controlled moisture levels. MATERIALS AND METHODS Organisms and suspending media. The organisms used were P. fluorescens and S. newport. The two suspending fluids were papain digest broth (7) and a mixture of 0.25 M sucrose and 0.25 M glutamate, Marshall and Scott (3) having shown the protective effects of this combination. Preparation for and freeze-drying of organisms. Both organisms were grown in brain-heart broth medium for 20 h at 30 C with aeration. Equal volumes of each culture were mixed. The resulting suspension was centrifuged and the cells were resuspended to half their original volume in the particular suspending fluid to give approximately 1010 cells of each organism per ml. In this way possible variations in the drying, storage, and rehydration of either oganism were eliminated. EFFECTS OF GASES ON DRIED BACTERIA Replicate ampoules containing 0.2-ml samples of the suspending fluids and organisms were prepared. The ampoules were supported in a rack so that each tube was radially mounted about 200 from horizontal, with its mouth some 2 cm from the surface of the central condenser which was filled with solid CO2 and ethanol. The apparatus accommodated up to 160 ampoules so that all ampoules for a particular experiment were dried together. Cooling was purely evaporative and heating was by radiation from the walls of the steel vacuum chamber. It was found convenient to de-gas the suspensions by evacuation until they had cooled to about 0 to 2 C before the CO2 plus ethanol mixture was added to the condenser. Immediately, the condenser was cooled, the rate of evaporation increased, and the suspending fluids and organisms were promptly cooled to -30 to -35 C. No measurements oi temperature were made during drying which was for 4 h, the time found by Scott (7) sufficient to remove almost all the water from all solutes. The dried cells were prepared for storage immediately after drying. Storage conditions. The ampoules were placed within larger tubes containing phosphorus pentoxide (P20.) for 0.00 a. and 2 ml of the appropriate sulfuric acid solutions (9) for all other a. levels. The larger tubes were first drawn out and then sealed in groups of five on a manifold for individual suspending fluids and water activities. The following conditions for storage were used. (i) In air, the tubes were not vacuated before the final sealing. (ii) In vacuum, the tubes were evacuated in groups of six with a two-stage mechanical pump until the solutions controlling a. boiled, or for at least 60 s before the final sealing. (iii) In nitrogen, the tubes were first evacuated and then flushed to atmospheric pressure with oxygen-free nitrogen (supplied in a cylinder containing alkaline pyrogallol). This was repeated twice before the tubes were finally sealed. (iv) In carbon dioxide, the tubes were treated as for nitrogen, carbon dioxide being flushed into the tubes after the third evacuation and the tubes sealed. (v) For storage in inert gases (argon, helium, neon, krypton, and xenon), the spectrally pure gases were supplied in 1-liter flasks at slightly above one atmosphere pressure (British Oxygen Gases Ltd). A manifold built of capillary tubing was used to conserve the gases for the filling and sealing of groups of six tubes. For each gas, the manifold was evacuated and filled with oxygenfree nitrogen, re-evacuated and filled with the nitrogen, re-evacuated and then filled to one atmosphere with the particular gas, and the tubes sealed. As the pressure at sealing was controlled with a mercury manometer, the possibility of slight contamination by mercury vapor cannot be excluded. Storage and viable counts. Immediately on the completion of sealing, the tubes were stored in the dark in an insulated cabinet at 25 C. Five replicate ampoules for each treatment were stored and after 1, 3, 9, 27, and 81 weeks one ampoule of each was randomly selected for the determination of viable numbers. The contents of each ampoule were rehydrated with 2 ml of saline. Where necessary, decimal dilutions were prepared in saline. At each dilution four plates were poured using brain-heart-infusion agar. One duplicate set of plates was incubated for 17 to 20 h at 37 C for the enumeration of S. newport and the other set was incubated for 6 days at 7.5 C for the enumeration of P. fluorescens. Separate experiments showed that the incubation conditions used completely suppressed colony formation by one organism and permitted full development of the other. S. newport and P. fluorescens were dried together in the two suspending fluids and stored at 25 C in five gases (air, vacuum, nitrogen, carbon dioxide, and argon) at three moisture levels (0.00, 0.20, and 0.40 aw). There were two replicates. In a further experiment (not replicated) storage of both organisms was studied in seven gases (vacuum, nitrogen, helium, neon, argon, krypton, and xenon) at four water activities (0.00, 0.10, 0.20, and 0.30) after drying in papain digest broth. The viable numbers per milliliter in terms of the original undried suspension were expressed as two-figure logarithms derived from the mean counts of duplicate plates. RESULTS P. fluorescens was more sensitive to both the drying and storage treatments. Forty percent of the cells of S. newport were still viable immediately after drying in papain digest broth compared with 100% for sucrose-glutamate. Survival of P. fluorescens after drying in papain digest broth was 25% and 95% in sucrose-glutamate. These survivals form the basis for evaluating changes associated with storage treatments. Figure 1 shows the changes in viability of P. fluorescens for all treatments during 81 weeks of storage. The effects on viability became more evident with time and, except for storage in air, were not apparent until the organisms had been stored for at least 9 weeks. Storage in vacuo and in nitrogen was significantly better than storage in air; carbon dioxide and argon gave intermediate results. The rate of death for storage in any gas and in either suspending fluid was least at 0.20 and generally wes greatest at 0.40 a.. However, the effects of aw on survival were very much greater for cells dried in papain digest than for cells dried in sucrose-glutamate. When P. fluorescens was dried in papain digest, the most significant difference between the gases occurred when the cells were stored at 0.00 a.. In air, storage at 0.00 a. led to death equal to that at 0.40 aw, whereas there were very small differences between 0.00 and 0.20 for vacuum and nitrogen. S. newport was less sensitive than P. fluorescens to all the storage treatments. Table 1 shows levels of survival for S. newport after storage for 81 weeks. The differences between the organisms were more marked when dried in Analysis of variance of the data showed that the average main effects of all factors and all their two-factor interactions were highly significant (at P = 0.001) for both organisms. The three-factor interaction for P. fluorescens was significant at P = 0.01. This was because the means for papain digest and 0.40 aw were very low in relation to those of the other water activities and all the water activities for sucrose-glutamate in all gases. Table 2 shows entries for means over one factor classified in rows and columns for combinations of the remaining two factors in all three possible ways for each organism. The marginal entries for each factor are means over both other factors. The study of means in the two-way tables shows that for P. fluorescens papain digest gave very low viability in air in comparison with sucroseglutamate; viability for air at 0.00 a. was much lower than for 0.20 a. in comparison with the other gases and viability was low for the papain digest-0.00 a. combination; further, the decrease in viability associated with papain digest as compared with sucrose-glutamate was much greater at 0.40 than at 0.20 a.. Similar inconsistencies occurred for S. newport. Comparison of the marginal means for average main effects shows that sucrose-glutamate and 0.20 a. gave the greatest viabilities; as regards gases there was no significant difference between vacuum and nitrogen which were clearly better than the others; these conclusions are true for both organisms. The differences between the seven gases used in the second experiment increased with time to their greatest values at 81 weeks of storage. Table 3 shows the viabilities of both organisms after storage for 81 weeks at all water activities. For P. fluorescens when very dry (0.00 a.) there were no significant differences in storage between vacuum, nitrogen, and argon; xenon was very destructive and the other three gases gave intermediate survival patterns. At 0.30 a. the differences between gases were substantially reduced. Even smaller differences between gases were found after storage at 0.10 and 0.20 6.88 (30) 9.43 a Standard errors of means: n' = 4, ±0.39; n! = 6, ±0.32; n' = 10, ±0.25; n' = 12, ±0.23; n' = 20, +0.18; n' 30, +0.14 on 29 degrees of freedom. The interactions SF x a., SF x gas and a. x gas were significant at 0.1, 0.1 and 5%, respectively. Survival of both organisms was greatest at 0.10 aw and the differences between the gases were substantially suppressed. The only gas giving survival equal to storage in vacuo was argon. DISCUSSION The main aim of these experiments has been to study the changes during prolonged storage and to see whether any gases were different from storage in vacuo. The techniques used ensure that aw is, in fact, controlled during storage and enable valid comparisons to be made between different atmospheres. Because of the small amounts of water hydrating speciments at 0.10 to 0.40 aw, equilibrium would have approached to within 0.01 aw of the appropriate values after storage for 1 week (7). The results emphasize the overriding importance of controlling the level of residual water at about 0.10 aw irrespective of the nature of the atmosphere or suspending fluid. The results of workers where aw is unknown are of limited value. Greiff and Rightsel (1) studied the survival of dried influenza virus when stored in dried gases but the order of stability does not correspond with that reported here for P. fluorescens and S. newport. However, although they used dried gases, the exact values of aw during storage were unknown and indeed may have changed after sealing because of the production of water by the Maillard reaction. Our results do provide, for the first time, unequivocal evidence of differences between gases which are clearly a function of the level of residual water. Because of the interactions between the various factors, greatest survival of dried cells after prolonged storage will only be achieved when careful attention is given to selecting the appropriate level of all conditions concerned. LITERATURE CITED

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2014-10-01T00:00:00.000Z

1973-01-15T00:00:00.000Z

1490449

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Selection in two environments in relation to plateauing in egg production In Random Sample Tests of Putten and Ploufvagan plateauing on a low level is observed in a trait with low heritability i. e. mortality, where selection is directed for low levels and an absolute border is present at o p. 100 . No plateauing is till now observed in a trait with high heritability i. e. egg weight. In a trait with medium heritability, i. e. p. ioo lay plateauing has occurred for different periods but was broken through some times. A selection experiment in two environments, one variable, the other constant showed quite different responses according to trait and environment. Loss of heterosis by mating crossbreds was much more pronounced in a variable environment than in a constant environment. It would be largely recovered by one generation of selection in one sex only. Selection responses for different traits are discussed. INTRODUCTION Poultry breeders will rarely be content with their achievements in improving egg production by breeding and crossbreeding in poultry. Especially when they look at the rapid advances made in breeding for meat production during the last two decades, they may become disappointed in comparing the slow progressif any &mdash; obtained by dogged perseverance in their own branch. This lack of progress under continued selection termed « plateauing u-needs our attention. Cet article a été présenté lors de la reunion du groupe de travail n° 3 (génétique et testage) de la Federation des Branches européennes de la W. P. S. A.,Nouzilly,[7][8] septembre 1971 . Occurrence of Plateauing Before discussing the possible causes of plateauing and the means to avoid these dead ends, it is worth while to investigate to what extent plateauing is really occurring. For this reason data were collected of two Random Sample Tests, viz. the R. S. T. at Putten during the periods 195 6-5 7 through 19 6 1 -6 2 and 19 6 3 -6 4 through 19 6 9 -7 o and those of the R. S. T. at Plouf y agan, Cotes-du-No y d, during the latter period. Egg production data of the year 19 6 2 -6 3 in Putten have net been published because of a fully abnormal production. Progress achieved in three different traits with probably three different levels of heritability will be discussed. Progress in a trait with supposed high he y itability. Egg weight In both R. S. T. stations a steady increase in egg weight was found. Over the period 195 6-70 in Putten a highly significant (P < o.oi) correlation between time (in years) and mean egg weight was found ( y = -!--0 .8 4 6). The regression equation W = 57.93 + 0.241 A shows an annual increase in mean egg weight of a little bit less than one quarter of a gram. Looking at the period of 19 6 3 -70 similar correlations are found both for Putten (-! 0 .8 76 , P < 0 . 01 ) and Cotes-du-Noyd (-!-0 .8 26 , P < 0 . 05 ). The regression coefficients of -+-0 . 4 6 4 and -f-0 . 407 respectively are even higher, indicating that not the slightest sign of plateauing is present. This means that a steady progress in egg weight has been obtained during the last r 5 years, either by improving existing strains and crossbreds or by developing new strains and combinations. The range between the best (max.) and the worst (min.) entry seems to be fairly constant, showing no signs of losses in between entry variance. That the level of egg weight in the Ploufragan test is a little bit lower than in Putten, may have many reasons, like there are different environment, different entries, different numbers of eggs weighed, shorter testing period in Plouf y agan. Summarizing we could state that there are no signs whatsoever for plateauing in egg weight and that eventually when plateauing might occur before long, this might rather be the result of relaxed selection because an optimum has been reached, than of lack of genetic variability. Progress in a tvait with low heritability. Mortality Progress in p. 100 losses per 100 days of the production period has been studied. Expression of p. 100 losses per 100 days has been chosen to reduce the effect of different lengths of testing periods from year to year or between tests. When plateauing might occur in mortality, different causes might be considered : a) because selection is directed towards low mortality (plateauing on a high level needs not to be considered), there is a natural minimum border of p. 100 losses, which can not be surpassed. b) because of a long history of natural and artificial selection for low mortality, the additive portion of heritability will be largely exhausted, causing low heritability and low selection response. As a matter of fact the remaining additive genetic variance will be the calculated regression of effects of genes with non additive gene action rather than a minor residue of purely additive gene effects. The remaining non additive genetic variance should mainly be based on genes showing dominance, overdominance or epistasy for high livability. When frequency of these genes becomes high, the detection of heterozygotes becomes difficult because the majority of recessive genes is carried by heterozygotes. This will inevitably lead to plateauing on a low level of mortality. c) when genetically mortality has reached a minimum level, that even theoretically might be zero, there will still be losses by environmental factors, like diseases and accidents against which no genetic barrier can be present. This will lead towards a phenotypic variability with a skew distribution towards the minimum of zero, which will be responsible for plateauing of mortality on a low level. Definite signs of plateauing were found, though still a rather wide range is present between the best and the worst entry. The minimum mortality is plateauing on a level of about i p. 100 losses per 100 days, though occasionally the border of p. 100 has been reached, which in this case means that no losses have occurred in one entry during the full 344 days of the test. The Putten test shows a nearly significant decrease in average losses during the full period of 195 6 through 197 0 , with r = -0.476 (o.io < P < 0 . 20 ). The regression equation I, = 4 . 7 8 0 -0 . 131 A showing a yearly decrease in mean losses of o.z 3 r p. 100 over the last z 5 years. Both Putten and Ploufragan show a steady, though insignificant decrease in average mortality over the period 19 6 3 through 197 0, (r = -o.3zg and &mdash; 0.464 respectively), with regression coefficients of - 0 . 194 (Putten) ando.126 (Ploufragan). Though the minimum level of mortality is clearly plateauing on a level of p. 100 per 100 days, the total range of entries is still 5 times the minimum level in both tests, indicating that still a lot of work can be done by some breeders in reducing mortality by selective breeding. Progress in egg production Progress in egg production was expressed as p. 100 lay. Calculation has been done on a hen-housed basis. Therefore progress in egg production is partly due to progress in livability. Expression as p. 100 lay has been chosen in order to reduce the effects of variation in registration periods within tests and of differences in testing period between tests. Data of birds housed on litter only have been used. The period of 195 6 through 19 6 2 in Putten shows a slow decline in mean egg production. This decline would have probably been continued in 19 6 2 -6 3 , when no data have been published, and further in 19 6 3 -6 4 . Over the period 195 6 through 19 6 4 the decline is highly significant (r = &mdash; 0 . 922 , P < 0 . 01 ). the regression equation I, = 66-4 6 -1 . 335 A indicating that mean egg production has declined about 4 p. 100 every three years. The causes of this decline are difficult to trace back. The experimental farm being entirely new in 195 6, it is reasonable to suppose that after a few years exogenous conditions have developed, interfering with health conditions in the birds to which some entries were insufficiently resistant. This seems to be confirmed by progress in mortality and the increase in range between best and worst entries both in p. 100 losses and p. 100 egg production on a henhoused basis. In the period 19 6 3 through 197 o a significant increase in egg production has occurred, both in Putten, where the increase was highly significant ( y = + o.g 35 , P < 0 . 01 ) and in Plouf y agan, where the progress was less pronounced (r = + 0 . 7 8 7 , P < 0 . 05 ) but where the starting point was at an I p. 100 higher level. During the last three or four years a striking similarity is present both for highest, mean and lowest levels of entries in the two testfarms. According to the regression formula, Pa = 52-9 9 !--2 .6 93 A for Putten and E = 6 4 . 43 !-0 .65 0 A for Ploufragan, the yearly progress in hen-housed egg production has been somewhat over 2 . 5 p. 100 in Putten and slightly over 0 . 5 p. 100 in Ploufragan. In the two testing stations the ranges between highest and lowest entry seem to have decreased, in Putten from 23 .o p. 100 down to 13 . 1 p. 100 and in Ploufragan from 20 . 5 p. 100 down to 15 . 0 p. ioo. This decrease in range seems largely to be due to an increase of the lower level, by elimination of insufficient entries. Nevertheless there is an indication of improvement in the higher ranges as well. In Putten for a long period, extending from 195 6 through 19 6 5 , the upper limit of entries has been moving between 66.8 and 72 . 0 p. 100 , without any sign of progress. That might indicate that with those flocks under the prevailing circumstances a plateau in egg production of about 6 7 -72 p. 100 has been reached and that no breeder had managed to break this ceiling in performance. The optimal results obtained at Ploufragan in the years 19 6 3 -19 6 5 show similar upper limits between 70 and 73 p. 100 . Starting in 19 6 5 -66 this plateau seems to be raised to a maximum of 7 8 to 7 g p. 100 , both in Putten and in Ploufragan. This indicates that either by selective breeding or by improvement of environment or by both, the plateau which had been present probably for many years, could be raised rather suddenly. The results of Plouf y agan over the last three years show a steady increase in egg production both in the mean and the upper and lower limits, which is quite near to the average increase through the whole period reported. This might indicate that in the Ploufragan results plateauing is not yet occurring. In the Putten results of the last three years however some signs of plateauing seem to be present both in the highest, the lowest and the average level. Taking into account the minimum level of mortality, there is evidence for the supposition that modern layers have reached a plateau of about 8 0 p. 100 survivors production. Future experience only can give the answer whether this is a fixed plateau or not. Possible causes of Plateauing Many causes for plateauing in a quantitative trait can be suggested. Generally spoken, plateauing will occur under selective breeding when the population is in an equilibrium state which can not be changed by selective breeding or when genotype-environment relations occur which counterbalance genetic improvement by environmental deterioration. The latter case however is not very likely to occur. A genetic equilibrium which is resistant to selective breeding requires different propositions : i. In closed flock breeding, the additive part of genotype induced phenotypic variance should be exhausted. This would mean that genes with purely additive action or showing complete or incomplete dominance and favouring the trait selec-ted for, will be completely fixed. For genes showing overdominance either a labile equilibrium should be reached by combined selection and breeding, or the genes should be fixed by continued inbreeding. Similar possibilities are present for genes showing epistasy. Instable equilibria of this kind can always be changed, but not always improved, by reduced or increased selection pressure, by outbreeding or by changing the environment. 2 . In crossbreeding similar but still more instable situations might occur, when the parent populations are partly selected in closed flock breeding. Reciprocal recurrent selection procedures can lead to highly instable and suboptimal performances in crossbred populations. As will be shown later from results obtained by crossbreeding commercial hybrids, dominance and epistasy may have a preponderant influence on heterosis, which can quickly be fixed by selective breeding in one sex only, though further improvement is much more difficult. 3 . As far as environment is concerned, it might well be true that a certain environment is conceiling genetic differences, because phenotypic expression is suppressed by environmental conditions, either because the given environment is prohibitive for phenotypic expression of the genotype or because sources of environmental variation are largely covering up the effects of genetic variation. In the first case either the environment should be radically changed or the selection criterion should be differed ; in the second case, sources of environmental variation should be eliminated. Plateauing of survivors egg production at a level of 8 0 p. 100 will indicate that the average interval between successive eggs in a clutch is about 2 6 hours. With a lighting scheme of 14 hours of light and 10 hours of darkness this will be good for an average clutch length of 4 , giving 8 0 p. 100 production. In order to increase the average clutch length with I egg up to 5 eggs, the ovulation retardation in relation to time should be decreased from 2 to 1 , 5 hours, reducing the average ovulation interval from 2 6 to 25 .5 hours. This change in clutch length will increase egg production from 8 0 p. 100 to 8 3 . 3 p. 100 . It has been shown long ago (VA N AI, B A DA , 1955 ) that selection for clutch length can be a far more effective tool in selection for egg production than selection for egg number itself. Clutch length turned out to be less susceptible to environmental differences than egg number, provided that differences in age, stage of production and lighting pattern do not occur or are largely reduced. From. ducks it is well known that an ovulation interval of 24 hours is good for extremely large clutches and annual egg productions up to 350 eggs for complete breeding flocks. Therefore selection for clutch length is likely to be one of the major tools in overcoming plateauing in egg production. Effect of envi y onmental variation in selection When sources of environmental variation are covering up phenotypic effects of genetic differences, selection effects might be improved by artificially reducing the environmental variation. At the Department of Poultry Husbandry of the Agricultural University of Wageningen an experiment has been started in ig66 for investigation of the occurrence and the importance of genotype-environment interactions. This experiment is also suitable for investigation of the effect of reduction of environmental variation on selection response. Main thought behind the concept of the experiment has been that if environmental and genetic variance are mainly acting additively as mostly is assumed, a radical reduction of environmental variability would result in an increase of both heritability and selection response of quantitative traits. Whenever important interactions between environment and genotype might occur, the elimination of environmental variation might eliminate phenotypic effects of genetic differences between individuals like differences in susceptibility for infections, for fluctuations in daylength, etc. This might lead to reduced selection response and/or environment bound selection effects making the birds less suitable for environments different from the environment they are selected for. Procedure Two polyhybrid populations were produced and divided at random (within maternal half-and full-sibs) over two environments, one « variable », the other « constant ». Selection for a complex production character (estimated total eggshell production) was practised in the two environments seperately, giving rise to two separate lines in each of the two populations, viz. a « constant environment line » and a « variable environment line ». Extensive description of the two environments can be given elsewhere. Here a short description will suffice. The variable environment was created in a conventional layer house with partly open windows and a naturally ventilated ridge, with natural light only, providing daylength fluctuations between 7 . 45 and 1 6. 45 hours all over the year. The birds were housed in pens of 4 X 4 m on litter, with slatted floors underneath the feeders. The constant envivonment was created in an airconditioned battery house at constant temperature (iooc) and humidity (8 0 p. 100 ) with 14 hours of light daily at an intensity between 250 and 65 0 lux. Local differences in light intensity did not show any effect on egg production. Birds were housed in individual cages with 33 cm frontwidth. Birds were housed at 1 8 weeks of age and trapnested daily up to 434 days of age, except for the first year when the birds had been hatched one month later than the following years with hatching from one week in April to one week in May. In that first year trapnesting was stopped at 403 days of age. Course of the experiment 1966: Hatching eggs were obtained from commercial breeders and set for hatching, giving rise to 4 different hybrids : a) a dutch three-way cross in the White Legho y n (A) b) an american hybrid in the White Leghorn (D) c) an Australorp X (RIR X RIR) crossbred from dutch origin (R) d) an Australorp X (RIR X NHs) crossbred from dutch origin (H) All the hybrids were based on different registered strains, except for the Australorp where the same strain was used in both hybrids. The chicks were raised on litter up to 1 8 weeks of age and then divided at random over the two environments and trapnested up to 403 days of age. 1967: Reciprocal crossbreds were made both in the White Leghorn (A X D and D X A) and the heavy breeds (H X R and R X H) by random pen matings in the layer house. Chicks hatched were reared in confinement up to 8 weeks of age and in shelters on range from 8 to 1 8 weeks of age. At 1 8 weeks of age the pullets of each reciprocal mating were divided at random over the two environments and trapnestei up to 6 2 weeks of age. 1968: Pullets housed in the layer house were pen mated at random with randomly chosen males, taking care that all dams were mated with sires of the reciprocal cross (AD male X DA female, etc.). For collecting hatching eggs, those females were used of which large settings could be obtained in order to produce large maternal full-and half-sib families (minimum 6, average about 8 maternal full and half-sibs). So dams used for breeding were selected for (hatching) egg production, but sires were unselected. Each maternal full-and half-sib family was divided at random over the two environments creating the base of the environment-bound lines to be selected. Birds were raised on litter from o to 8 weeks of age and on range from 8 to 1 8 weeks. At 1 8 weeks females were housed either in the layer house (variable environment) or in the battery house (constant environment) and trapnested up to 6 2 weeks of age. 1969: Females hatched 19 6 7 were selected for estimated total egg shell production and used for individual matings with cocks hatched in 19 68 from dams selected for part-time eggshell production. The cocks were selected for part-time eggshell production of their maternal full-and half-sibs. Females previously housed in the layer house were used for reproducing the layer house (variable environment) line, females previously housed on cages were used for reproducing the battery (constant environment) line. Similarly the sires were used on basis of maternal sib performance in either the layer house (for the variable environment line) or the battery (for the constant environment line). Chicks hatched were reared on litter from o to 8 weeks and on range from 8 to 1 8 weeks and then placed in the environment their parents had been selected for. Trapnesting was continued up to 6 2 weeks of age. 1970: A similar selection procedure was followed as in the preceding year and matings were performed similarly. Chicks were reared in the same way as previous years. Pullets from each full sib family of each of the four selected strains were divided at random over the two environments in order to check birds selected in one environment for performance in both the innate and the foreign environment. In every year egg production was recorded 7 days a week. Estimation of total shell production Eggs were weighed individually 3 times with 3 months intervals, 3 eggs in succession. Each time one of the 3 eggs weighed was broken, the shell rinsed with water and weighed in air-dry condition. The average weight of the 3 shells was multiplied with total egg number for calculation of the estimated total shell production. Moreover total and mean egg weight for each group was estimated one day every fortnight. The results of these egg weight data were used in this paper. For birds hatched 19 66 however, because of shortage of labour and equipment, eggs could be weighed only once when the birds were about g months old. Therefore egg weights of birds hatched 19 66 are not strictly comparable with those of the later years. Preliminary results Because many of the data collected have yet to be punched, a complete analysis of data comprising tests of significance has still to be made. Moreover some data have not been calculated in the proper way. For instance data obtained by multiplying, like total egg production in kg. per group have been obtained by multiplying the averages (mean egg number X mean egg weight) instead of multiplying individual data and calculating the mean product afterwards. Egg number of birds hatched ig66 has been corrected for length of period. This however will neither affect the trends in the results or the sign of the differences. Therefore the preliminary results will be presented in the form of graphs without giving the exact figures to the last decimal. Progress in egg number per hen-housed is shown in fig. I Crossbreeding two randomly chosen commercial hybrids, previously bred for combining ability is likely to result in a loss of heterosis in reproductive traits (egg production and livability). A decline in hen-housed egg production is observed in both breeds and in both environments, though much more pronouced in the variable environment in both hybrids. This indicates that heterosis as well as loss of heterosis may be much more pronounced in a variable environment than in a well protected constant environment. Loss of heterosis seems to be more pronounced in the Medium Heavy breed cross than in the White Leghorn probably because of the use of the same sire strain in the two hybrids of the Medium Heavy breed. Selection for egg production in females only has resulted in a complete recovery in the White Leghorn of the losses in the previous year in both environments and a recovery of the major part of loss in egg production in the Medium Heavy breed. This might indicate that the heterosis in the original crossbreds has been largely due to a small number of highly effective autosomal dominant or epistatic genes, easily recovered by selection in the females only. In the variable environment continued selection for total egg shell production in two following years in both sexes has been completely unsuccessfull in the Medium Heavy breed and hardly better in the White Leghorn. The negative result in the Medium Heavy breed in the last year is largely due to an increased mortality as can be seen from fig. I b. In the constant environment a positive response is present, which is more pronounced in the Medium Heavy breed than in the White Leghorn. In both breeds egg production in the variable environment in the last year exceeded slightly the level of the initial commercial hybrids. This may be largely due to environmental circumstances, since the initial hybrids were raised in confinement, whereas later generations have been raised on range from 8 to 1 8 weeks. When kept in the constant environment the lines selected in variable environment show much better results (compare. v with . c c and !-v with -!-c) ; in the White Leghorn these even exceed the original hybrids. Progress in p. 100 losses from diseases and mortality are shown in fig. i b. Losses are lower in the Medium Heavy breeds than in the White Leghorn and also lower in the constant environment than in the variable environment. In the constant environment the increase in losses which had occurred in the second generation in both breeds has been restored, but after two generations of selection mortality is still at the same level as in the initial hybrids. In the variable environment there has been a substantial improvement in the White Leghorrc where mortality was originally very high, but after a continued decrease in the Medium Heavy breed mortality in the last year returned to the original level. Progress in body weight at 1 8 weeks of age is presented in fig. 2 a. Since all birds are kept under the same conditions up to 1 8 weeks of age, no environmental differences are to be expected except foe effects working through selection in the laying period. No important directional changes in body weight at 1 8 weeks seem to have occurred. Progress in body weight at 6 2 weeks of age is presented in fig. 2 b. Except from a tendency for increased body weight in the Medium Heavy breed in the last year, no clear directional changes in body weight have occurred. The same holds true for gain in weight from 1 8 to 6 2 weeks of age, presented in fig. 3 a. Progress in mean egg weight is shown in fig. 3 b. Though heterosis is not expected to be of much importance in egg weight, crossbreeding the commercial hybrids together with suspension of selection results in a severe drop in egg weight in the White Leghorn. After that in the constant environment no important further change in egg weight has occurred, but in the variable environment the decline has continued during the two last generations, notwithstanding the selection for total shell weight. Comparison of results of the same population in variable and constant environment in all cases shows that the environment in itself does not have much influence on egg weight. In the White Leghorn egg weight is about y 2 gram higher in the constant environment in both strains ; in the heavy breed no differences of this kind can be found. The heavy breed shows a steady decline in egg weight in the variable environment but no consistent changes in the constant environment. Progress in mean shell weight is shown in fig. 4 a. In the variable environment shell weight has decreased in both breeds, except for the last generation where a recovery can be observed. Comparable with the trend shown in egg weight, the decline has been much more severe in the White Leghorn than in the Medium Heavy breed. In the constant environment results are quite different.

v3-fos

2020-12-10T09:04:12.672Z

1973-02-01T00:00:00.000Z

237230435

s2

Casein as a Necessary Factor in the Production of Stimulatory Material for Associative Growth of Lactic Streptococci Strains of Streptococcus cremoris KH and HC produced material that was stimulatory for S. cremoris R6 in milk and in the dialyzable fraction of milk, but not in the dialysate fraction of milk, lactic acid whey, or lactose broth. The addition of casein to these latter media permitted the production of this stimulatory material to occur. Tryptone, peptone, and yeast extract could not be substituted for casein in producing the stimulatory material or in initiating associative growth in the lactic acid whey. The minimum concentration of casein required appeared to be from 2.0 to 2.5%. The occurrence of associative or symbiotic activity, or both, among lactic acid starter cultures determines the final quality of the product (1, 5-8, 10, 14). A preliminary study has indicated that increased lactic acid yields can be obtained in milk with Streptococcus cremoris R. when strain KH or HC is also present (12). The stimulatory material produced by these strains appears to be guanine or a guanine-like substance (11). The present study suggests that the constitutents present in milk are involved in the production of the stimulatory material by strains KH or HC. MATERIALS AND METHODS Test organisms. Two strains of S. cremoris, KH and HC, were used for the production of the stimulatory material. A third strain, R., acted as the sensitive organism for estimating this activity. All cultures were grown at 30 C. Titratable acidity was determined by titrating the medium against 0.11 N NaOH and using phenolphthalein and bromothymol blue as indicators in milk and broth, respectively. Sixteen-hour milk cultures were used, except where specified, for preparing the cell-free filtrate as well as for evaluating the stimulatory activity (12). Preparation of milk fractions: (i) casein and lactic acid whey. A 100-ml amount of skim milk was heated to 45 to 50 C in a water bath. To this, 6 times with glass-distilled water. The filtrate, i.e., lactic acid whey, was neutralized with 0.11 N NaOH to pH 7.0 and Seitz-filtered. (ii) Dialyzed and dialysate fraction. Sterilized skimmed milk was dialyzed against distilled water with cellophane at 5 to 8 C for 3 days by using six changes of water. Toluene was the preservative. The dialyzed and dialysate fractions were concentrated to their original volumes under vacuum at 50 C. Media. Milk: Fresh skimmed cow's milk was sterilized at 15 psi for 1 min and steamed the following day for 30 min. RESULTS The lactic acid starter cultures (strains KH, HC, and Rf) were grown individually and in pairs (KH + R6, HC + R,) in milk, in the dialyzed and dialysate fractions of milk, and in lactic acid whey supplemented with peptone, tryptone, yeast extract, or casein. The data indicate that the paired strains produced more acid in milk, in the dialyzed fraction of milk, and in lactic acid whey to which casein had been added; but not in the dialysate fraction of milk, in lactic acid whey alone, or in lactic acid whey supplemented with peptone, tryptone, or yeast extract ( Table 1). The seemingly higher lactic acid production in the whey + casein medium than in the control milk is caused by the longer incubation period (24 versus 8 hr). Cell-free filtrates of strains KH and HC brought about increased production of lactic acid by strain R. only when casein was incorporated in milk or in the dialyzed fraction ( Table 2). This indicates that casein (which remains in the dialyzable fraction) is an important factor in the production of the stimulatory material and the symbiotic activity of strains. To determine whether acid production by strain R. was increased in the presence of stimulatory material in media lacking casein, the cell-free filtrates of strains KH and HC were added at a 1% level to the dialyzed and dialysate fractions of milk, to the dialysate plus casein, to lactic acid whey alone, and to lactic acid whey supplemented with peptone, tryptone, yeast extract, or casein. Acid production by strain R. in the presence of stimulatory material was not increased in media without casein, but was stimulated in both the dialysate and in lactic acid whey when supplemented with casein (Table 3). This would indicate that casein is essential for the proper utilization by strain R, of the stimulatory material produced by strains KH and HC. Since very little acid was produced in media lacking casein, measurements were made after 24 hr whereas, in the other cases, measurements were made after 8 hr. To determine the optimal levels of casein required for associative growth, casein was added at levels of 0.5 to 2.5% to lactic acid whey, and the strains were grown both individually and in pairs. Data indicate that 2.0 to 2.5% casein was required for associative growth ( Table 4). The effect of incorporating casein in lactose broth containing suboptimal levels of nutrients (i.e., half-strength and quarter-strength media) indicates that there was a greater increase in associative growth when casein was added to the quarter-strength medium than to the halfstrength medium (Table 5). DISCUSSION These results suggest that casein is an important factor in the production of stimulatory material by S. cremoris strains KH and HC for symbiotic growth with strain R,. The nature of the components present in casein or casein-rich media that are responsible for this associative growth is still unknown. It has previously been established that lactic acid-producing streptococci are dependent upon an organic nitrogenous source for their supply of essential amino acids needed for growth (9,15). It has also been shown that Streptococcus lactis contains intracellular proteinase, which can function to provide the cell with a mechanism to acquire essential amino acids via the breakdown of milk proteins (2)(3)(4). Presumably, these amino acids are not available in their entirety from simpler nitrogenous materials (tryptone, peptone, etc). Similarly, the proteolytic activity of the endocellular enzymes in S. lactis is greatly reduced if the casein in the medium is replaced by tryptone or peptone (16). The minimal requirements of casein for associative growth of the different strains of S. cremoris appear to be between 2.0 and 2.5% (Table 4). Lower concentrations, although able to support growth, were insufficient to induce any associative effect. When semisynthetic media, i.e., lactose broth (13), were used, the lactic acid-producing culture did not show any symbiosis (Table 5). However, when casein was added to replace part of the tryptone, associated growth occurred. This further strengthens the concept that milk proteins are essential for the symbiotic growth of the lactic acid-producing milk cultures. A similar observation, that milk is essential for the production of stimulatory material, has been noted for Pseudomonas fluorescens in lactic acid starter cultures (10).

v3-fos

2020-12-10T09:04:13.110Z

1973-11-01T00:00:00.000Z

237230632

s2

Growth of Clostridium perfringens in Food Proteins Previously Exposed to Proteolytic Bacilli Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilis-hydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens. Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilishydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens. Clostridium perfringens food-borne illness continues to be a major concern in the food industry (4). Dramatic food processing advances and significant changes in consumer attitudes and marketing approaches have resulted in public acceptance of fabricated synthetic foods utilizing soy protein or casein as the protein base. The production of fabricated foods by using pasteurization processes combined with good sanitation and modern manufacturing practices can result in the removal of common spoilage microorganisms leaving sporeforming bacteria (i.e., Clostridium sp. and Bacillus sp.) without major competition. Earlier studies have shown that small amounts of hydrolyzed proteins in conjunction with soy proteins in synthetic meats were stimulatory to the growth of C. perfringens (15) Effects on C. perfringens growth. To determine any subsequent effect on the growth of C. perfringens, the B. subtilis-cultured sodium caseinate (BSC) and isolated soy protein (BSP) were centrifuged, and the supernatant fluid was added to the growth medium at a concentration equal to 10% of the total sodium caseinate present in the C. perfringens growth medium (i.e., 0.2% BSC added to 1.8% sodium caseinate). The final protein concentration was 2%. Trypticase (BBL), substituted for BSC, served as a reference. Sterile sodium thioglycolate (BBL) (0.6 g/liter), sodium sulfite (Allied Chemical Corp.) (0.2% g/liter), NaCl (Merck & Co.) (2.5 g/liter), and K2HOP4 (J. T. Baker Chemical Co.) (1.5 g/liter) were aseptically added. The volume was adjusted with sterile deionized water to 10 ml for absorbance determinations or 500 ml for colony count determinations. The growth medium was steamed for 20 min before inoculation with C. perfringens. Lack of contamination or B. subtilis growth in the test medium was verified by testing for any viable cells in uninoculated control samples. An 18-h culture of C. perfringens S40 grown in thioglycolate medium without added dextrose (BBL) was centrifuged (4,080 x g for 10 min) and washed in 6.25 x 10-4 M potassium phosphate buffer. The washing procedure was repeated once. The initial inoculum was 108/ml for colony count determinations (1) and 107/ml for absorbance measurements at 650 nm (model 330 spectrophotometer, G. K. Turner Associates, Palo Alto, Calif.). The lag time was estimated from the intercept of the exponential growth slope with the initial inoculation level (10). Lag time estimations were determined from data of three replicates. Colorimetric determination of partial hydrolysis of food proteins by B. subtilis. Partial hydrolysis was measured according to a modification of the method developed by Hull (8). To 4 ml of the protein sample, 4 ml of 0.72 N trichloroacetic acid (Fisher Scientific Co.) was added, and the mixture was filtered. To 1 ml of the filtrate, 3 ml of 7.5% NaCO, and 1 ml of a 1:3 dilution of Folin-Ciocalteau phenol reagent (Fisher Scientific Co.) was added. Color change was measured at 650 nm (Beckman Acta III spectrophotometer, Beckman Instruments, Inc., Fullerton, Calif.) and converted to milligrams of tyrosine per milliliter from a standard curve. Molecular sieving of nontreated and B. subtilistreated food proteins. BSC, BSP, 3% nontreated sodium caseinate, or 3% nontreated isolated soy protein (5-ml sample) was sieved by using gel filtration (Sephadex G-25 fine, Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The method used was a modification of a method by Nekvasilova et al. (14). Size of column was 2.5 by 36 cm, and elution buffer was 0.05 M ammonium bicarbonate (J. T. Baker Chemical Co.) (pH 7.6). The fractions were monitored at 280 nm with a flow-through cell (Beckman Acta III) and collected automatically. The flow rate was 0.5 ml/min. For large volumes of sample, 100 ml of 3% nontreated or B. subtilis-treated sodium caseinate or isolated soy protein was fractionated by using a Sephadex G-25 column (5 by 86 cm) with a flow rate of 2.5 ml/min and collected at 10 ml/tube. Fractionation of active peptide groups on ion exchange gel. BSC, the second peak of gel filtration (Sephadex G-25), 22-h BSC, and untreated sodium caseinate control were fractionated by ion exchange gel. Freeze-dried samples (1 g) were fractionated by gradient elution with a linear pH change according to Carnegie (2). A column (2.5 by 36 cm) was filled with diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) in acetate form and brought to equilibrium with 0.1 M collidine (2,4,6-trimethyl pyridine; Eastman Kodak) acetate buffer (pH 8.55). Elution was affected with collidine acetate buffer at pH 8.55. A pH gradient was obtained by gradual mixing in gradient chambers with 0.1 M acetic acid. Flow rate was 0.75 ml/min. After 1 liter was eluted, 1 M acetic acid was added to the gradient chamber. Approximately 200 fractions consisting of 12 ml/fraction were collected automatically. Detection of protein fractions was made from 1-ml samples from each tube with ninhydrin reagent (Eastman Kodak Co.) by using the Cocking and Yemm modification (3) of Moore and Stein's technique (11). Color change was measured with a Beckman Acta III spectrophotometer at 570 nm and recorded as milligrams of alanine per milliliter by conversion from a standard curve. Amino acid analysis. The amino acid composition of protein fractions was determined by ion exchange chromatography (12). Hydrolysis of the protein samples was carried out in vacuo at 110 C for 24 h under standard conditions described by Moore and Stein (13). RESULTS AND DISCUSSION The effects of added BSC on C. perfringens growth was determined by adding BSC (0.2% final concentration) to the regular sodium caseinate medium. The C. perfringens growth responses were determined by colony count and by turbidity measurements. In Fig. 1, representative growth curves for C. perfringens in a sodium caseinate medium with added BSC treated for 10 to 48 h are shown. BSC from incubations of 12 h or more markedly shortened the lag time. The BSC samples (more than 10 h) produced an effect similar to added Trypticase. Ten-hour or less BSC had little or no influence on C. perfringens lag time (data not presented). Similar results were obtained by the addition of BSP. Incubation periods of 8 h or longer yielded BSP that resulted in a C. perfringens shortened lag time. A shortened lag time also was observed with the addition of the control (7,9,14). The production of toxins has been shown to increase with increasing length of peptide chains while growth remained essentially the same (7) Fig. 2. Isolated soy protein also increased the growth rate of C. perfringens when added to sodium caseinate. This increased rate of growth was consistent with earlier findings (1). Colony count determinations were made to confirm absorbance data (Fig. 3A, B). In this study, 12 and 24 h-treated BSC or 10-and 34-h FIG. 2. Effect of 8to 48-h BSP added to sodium BSP shortened the lag time of C. perfringens. In caseinate medium on the growth of C. perfringens earlier reported data (F. F. Busta, L. B. Smith, (absorbance at 650 nm). One gram of BSP was added and D. J. Schroder, in Spore Research, in press), to 9 g of sodium caseinate per 500 ml. Trypticase was BSC initiated germination and outgrowth of C. substituted for BSP to serve as a reference. perfringens spores (>90% in 8 h) to a greater extent than did the sodium caseinate control. caseinate medium (data not presented). A 260sodium caseinate on the growth of C. perfringens of stativeprosntei.di not (colony count determinations). One gram of BSC was to 280-nm scan ofstimulativeproteindid added to 9 g of sodium casemate per 500 ml. B, Effect reveal the presence of nucleic acids in the of 10-and 34-h BSP added to isolated soy protein on stimulative medium. the growth of C. perfringens (colony count determina-We postulated that B. subtilis incubation tions). One gram of BSP was added to 9 g of isolated hydrolyzed the food proteins and produced soy protein per 500 ml. peptides, especially glycyl-L-asparagine, was demonstrated (9). These and other earlier studies were concerned with toxin production and the concurrent extent of growth, not the rate of growth nor the initiation of growth (lag time). Anticipated responses to specific materials prompted the following study to determine whether the incubation of food proteins with B. subtilis produced specific peptides that decreased the lag time of C. perfringens. T 4. Effect of Trypticase, Casamino Acids, and amino acids added to sodium caseinate on the growth of C. perfringens. One gram of test protein was added to 9 g of sodium caseinate per 500 ml (growth measured by absorbance at 650 nm). Relationship between extent of food protein hydrolysis and C. perfringens growth. Sodium caseinate was supplemented with 0.2% casein hydrolysates (Trypticase and Casamino Acids) or amino acids of similar composition to determine the effect on C. perfringens growth. The addition of Trypticase, Casamino Acids, or amino acids to sodium caseinate (10% of total protein) showed an increased lag time with increased hydrolysis (Fig. 4). This implied that peptides were important initiators of C. perfringens growth in a sodium caseinate medium. This is in agreement with studies by Hauschild (6) which showed the incorporation of "4C from peptides into protein by C. perfringens was greater than incorporation of 4C from amino acids. Fractionation of BSC and BSP. BSC and BSP samples were fractionated to characterize protein hydrolysis resulting from incubation with B. subtilis. Figure 5 presents the Sephadex G-25 gel filtration patterns of BSC and BSP. The change in elution pattern of sodium caseinate from the control to 12 and 24 h of B. subtilis incubation is evident. The extent of protein hydrolysis was determined chemically by using Folin-Ciocalteau phenol reagent. The extent of hydrolysis of sodium caseinate and isolated soy protein during incubation with B. subtilis is shown in Fig. 6. In both protein media, the maximal extent of hydrolysis was reached at approximately 28 h. stimulation was due to peptides of a specific charge. The first fraction (Sephadex fraction 1) of the sodium caseinate control and Sephadex fractions 1 and 2 of 22-h BSC were further fractionated by charge by using a DEAE-Sephadex A-25 column. The DEAE elution patterns for the sodium caseinate control and the BSC fractions appeared similar. The DEAE fractions of the first main peak and the second peak from each sample were collected, evaporated, freezedried, and reincorporated into a sodium caseinate medium at a concentration of 10% of the total sodium caseinate. The effects of these DEAE fractions on the growth of C. perfringens are listed in Table 1. The supplementation of a FIG. 6. Trichloracetic acid-soluble tyrosine residues hydrolyzed from sodium caseinate and isolated soy protein resulting from 0 to 48 h of B. subtilis incubation. Measured with Folin-Ciocalteau phenol reagent (absorbance at 650 nm) and converted to milligrams of tyrosine per milliliter from a standard curve. Trypticase is shown as a reference. The incubation time required for minimal stimulation of C. perfringens (12-h BSC and 10-h BSP) approximated the time of the first detectable protein breakdown shown in Fig. 6. These results can be compared with data on the trichloroacetic acid-soluble fraction of Trypticase resulting in 0.90 mg of tyrosine per ml. They indicate that the shortened lag of C. perfringens was due to partial hydrolysis of the protein. The next step was to determine whether specific peptides or protein fractions were responsible for the shortened lag. BSC and control samples were fractionated on Sephadex G-25. Fractions were isolated, flash-evaporated, freeze-dried, and reincorporated as 10% of the total sodium caseinate in the C. perfringens medium. BSC was used exclusively in this study. The growth curve for each fraction is shown in Fig. 7. All six Sephadex fractions added singly or added to sodium caseinate in combination shortened C. perfringens lag. Each fraction representing a specific size range of hydrolyzed protein exhibited similar effects. Fraction 2 initiated growth in 2.37 h, compared with 2.25 h for Trypticase and 3.75 h for the sodium caseinate control. These data indicated that the stimulative effect of the hydrolyzed protein appeared to be nonspecific with regard to molecular size of the protein fraction. Ion exchange chromatography of Sephadex fractions. The nonspecificity of molecular size of the hydrolyzed protein fractions prompted studies to determine whether this All fractions tested were analyzed for amino acid composition. No composition differences between the sodium caseinate control fractions or the BSC fractions were observed. The decreased lag demonstrated by the BSC fractions, therefore, did not appear to be due to the amino acid composition of the active component(s), because DEAE peak 2 of the control and BSC were of similar composition but differed in their effect on the lag time of C. perfringens. To confirm the lack of specificity of size of the protein molecule or charge of the molecules on shortening the C. perfringens lag, the complete BSC sample was fractionated on the DEAE-Sephadex column. The fractions representing the last DEAE peak were incorporated into a sodium caseinate medium and found to decrease the C. perfringens lag similar to the complete BSC. The fractions representing the last DEAE peak were then separated by gel chromatography on Sephadex A-25. The similarity of the fractions representing the last DEAE peak and the original BSC indicated that the fractions in the last DEAE peak contained all of the original molecular size range (data not presented). These results have demonstrated that a shortened C. perfringens lag resulted from the addition of B. subtilis-treated sodium caseinate and isolated soy protein. The shortened lag was not due to a specific molecular size of protein formed from hydrolysis. The amino acid composition of the BSC peptide fractions that shortened the lag was similar to the composition of the untreated control sodium caseinate fractions that did not shorten the C. perfringens lag time. Therefore, this effect was not due to a particular amino acid composition. Nor was it due to the charge of the protein fragments or peptide molecules, since BSC fractions with a net negative charge (acidic peptide) were stimulatory, whereas samples of the sodium caseinate control with a similar charge were not. There was no apparent difference in the charge pat-tern of the BSC and control sodium caseinate. Peptides of a specific size, charge, or amino acid composition were not solely responsible for the shortened C. perfringens lag period. Therefore, this lag time reduction caused by B. subtilis action was due to hydrolysis of the food protein, apparently making it readily available for utilization by C. perfringens. The inadvertant modification of food proteins by B. subtilis can increase the potential of that protein for supporting growth of C. perfringens. These results indicate the importance of associative growth of proteolytic bacilli with C. perfringens in a food protein medium that is normally inadequate for the growth of C. perfringens. The shortening of the lag time for C. perfringens in a food product has great public health significance. Food processes in which soy proteins are treated with microbial enzymes to improve acceptability, especially through the formation of plastein (5), could result in initiating the growth of C. perfringens in that medium. A food processor fabricating protein foods who uses soy protein or sodium caseinate should be aware of the ramifications of the partial hydrolysis of that protein.

v3-fos

2019-03-20T13:06:17.514Z

1973-10-01T00:00:00.000Z

83537122

s2

COMPARATIVE STUDIES OF THE EFFECTS OF α-TOCOPHERYL NICOTINATE AND THE COMBINATION α-TOCOPHERYL ACETATE AND NICOTINIC ACID By conducting a cross administration of Toc. nic and the combination of Toc. ace and 20% nicotinic acid to cases with insufficient micro-circulation and also by performing the cooling rewarming test during the administration, it was found that Toc. nic is more effective than the combination in reducing the MRT, and that the effectiveness of Toc. nic was not due to the synergic action of tocopherol and nicotinic acid but to the independent action of Toc. nic. By conducting a cross administration of Toc. nic and the combination of Toc. ace and 20% nicotinic acid to cases with insufficient micro circulation and also by performing the cooling rewarming test during the administration, it was found that Toc. nic is more effective than the combination in reducing the MRT, and that the effectiveness of Toc. nic was not due to the synergic action of tocopherol and nicotinic acid but to the independent action of Toc. nic. It EXPERIMENTAL METHODS 1. Drugs and the method of administration. One of the drugs administered was Toe. lie 100 mg and the other, used for controls, was the combination of Toe. ace 100mg and nicotinic acid 20mg. The drugs were put into different capsules having the same outward appearance. Without telling the patients about the differences of the two types of drugs used, 4 capsules a day were administered, 2 in the morning and 2 in the evening when the patients were hungry. One of the drugs was administered for 2-3 weeks, mostly 2 weeks, and 375 the other drug was continuously followed in the same way, and furthermore the drugs was alternately administered for every 2-3 weeks (cross administration of drugs). 2. Subjects in the experiment. As shown in Table 1, the subjects were 10 persons aged 24-60 (2 males and 8 females) and the diagnosed diseases were classified as 3 cases of scleroderma en plaque, 2 of feeling cold, 1 of hyperidorosis localis (palmar and plantar), 1 of acrocyanosis, l of sclerodacteria, l of sclerodermia diffusa, and 1 of microcirculatory insufficiency after congelation. Two of these patients cases (case 5, 6 and 7, 8) were restudied after having discontinued the treat ment once, and 1 patient (case 10, 11) with acrocyanosis was measured with the hand in both horizontal and vertical positions. Although no apparent disturbances were observed in the internal organs of the patients, microcirculation disturbances appeared to develop, and the mean rewarming time (MRT) in the cooling rewarm ing test (CR test) described in the following pages increased and showed Heidel mann's contraction type except for the 27-year-old male with acrocyanosis (case 11) when the fingertip was measured in the horizontal position. 1. The MRT before and after administration and during cross administration of drugs The MRT of each patient before administration, after the initial administ ration, and 2 weeks after changing the drug is shown in Table 1. It can be seen from Table 1 that in most cases the MRT was reduced after the administration of both drugs, and the degree of reduction was more marked when Toc. nic was administered. There were several cases where the MRT continued to reduce after the combination was changed to Toc, nic, and an increase of MRT was observed in not a few cases to which the combination was administered. This tendency was more marked at the fingertip than at the dorsum of the hand. As shown in Table 2, the mean MRT in all the tests was reduced to approximately 60% after the administration of the combination, and to approximately 40% after the administration of Toc. nic when compared with the MRT before ad ministration. In the comparison between the combination and Toc, nic, the MRT in cases to which Toc. nic was administered was more markedly reduced, to about 55% at the fingertip and to 59% at the dorsum of the hand. 2. Correlation between the MRT obtained after the administration of Toc, nic and after the combination The differing cross administration of both drugs provided 37 pairs of readings each for the fingertip and the dorsum of the hand. The relationship of the MRT for each pair of readings is shown in Fig, 1. In the dilatative type (MRT being within 10 min), there were readings where the difference between each pair was slight, but in many pairs of readings, that is 69 out of 74, the MRT was shorter DISCUSSION AND SUMMARY The ester of vitamin E currently used largely for clinical purposes is Toc . ace. In addition, such agents as tocopherol succinate and tocopherol phosphate were developed, but the usefulness of these agents has been limited because of the lack of specific features, hemolytic reaction, and other disadvantages . Under these circumstances, Toc. nic, which has sufficient tocopherol action and no shortcomings such as the flash and short duration of effectiveness observed in nicotinic acid, arrived on the scene and has attracted attention (4,5) . In a previous experiment, the author conducted the cross administration of Toc. nic and Toc. ace in the same way as in the present experiment and showed that with respect to the microcirculatory system Toc. nic had more immediate and higher effectiveness than Toc. ace (1). However, it was not clarified whether such effectiveness was due to the independent effect of Toc. nic or to the synergetic effect of tocopherol and nicotinic acid produced by the hydrolysis of Toc. nic in the body. Accordingly, in order to clarify this point, the author examined the effect of both drugs using the CR test and found a significant difference between the two by cross administering Toc. nic and the combination of Toc. ace and nicotinic acid. Although there are many problems regarding absorption, hydro lysis, dosage, and other aspects, it can be concluded that the effectiveness of Toc . nic is not due to the synergic effect of tocopherol and nicotinic acid but to the independent effect of Toc. nic on the microcirculatory system. The results of the experiment are discussed below. In addition, based on a pair of MRT readings measured after the alternate administration of Toe. nic and the combination, 37 pairs of readings for the as sortment were recorded at the fingertip and also at the dorsum of the hand in order to make comparative studies between the two drugs. In the 19 pairs of readings where the administration of the combination preceded, the MRT after the administration of Toe. nic was shorter in all the paired readings, and in 18 pairs of readings where the administration of Toe. nic preceded, the MRT after the administration of the combination was shorter in only 4 pairs at the dorsum of the hand and 1 at the fingertip. As mentioned above, the findings can be under stood to have been obtained because of the insufficient duration of the administra tion, and cannot be taken as a problem of selecting drugs according to symptoms. Moreover, the values obtained by means of the modulus treatment of these assort ments clearly show the significant difference between the two drugs. Furthermore, the marked superiority of Toe. nic in the extension of MRT implies not only a quantitative difference, but a qualitative difference. Recently, Toe. nic and Toe. ace have not only been separated pharmaceutically as an ester of vitamin E but also have attracted attention due to their different natures. For example, Toe. nic can easily be hydrolyzed in the body, leaving some portion as an ester type for quite a long time, and a difference in the condition of retention in the body be tween the two has been observed (6). Consequently as many more esters join the scene in the future, attention must be paid not only to the question of quantity but also to the question of quality. The observations above describe, our findings that Toe. nic efficiently reduces the MRT in the CR test (thought to be the most accurate measure of the reserve capacity of the microcirculatory system) and that the effect of Toe. nic is not due to the synergetic action of tocopherol and nicotinic acid produced by hydrolysis but to the independent action of Toe. nic.

v3-fos

2020-12-10T09:04:12.214Z

1973-11-01T00:00:00.000Z

237233442

s2

Lactose-Fermenting Salmonella from Dried Milk and Milk-Drying Plants A study of 552 salmonella cultures revealed that 86 (15.6%) of the cultures fermented lactose. These had been isolated from dried milk products and milk-drying plants. Acid and gas were produced in lactose broth. Solid media containing lactose as the key ingredient for the differential reaction were not satisfactory for recognizing salmonella colonies. No problem was encountered in selecting salmonella colonies when bismuth sulfite agar was used. A study of 552 salmonella cultures revealed that 86 (15.6%) of the cultures fermented lactose. These had been isolated from dried milk products and milk-drying plants. Acid and gas were produced in lactose broth. Solid media containing lactose as the key ingredient for the differential reaction were not satisfactory for recognizing salmonella colonies. No problem was encountered in selecting salmonella colonies when bismuth sulfite agar was used. Twort (20) and Kristensen (13) indicated that the environment of salmonella organisms may influence their ability to ferment sugars. The salmonella cultures received at the Veterinary Services Diagnostic Laboratory were isolated from dried milk and milk-drying plants. Such an environment afforded exposure to lactose in the milk. This report concerns a study of these cultures with emphasis on their ability to ferment lactose. MATERIALS AND METHODS Cultures. Cultures (552) were obtained from laboratories of the Agricultural Marketing Service of the U. S. Department of Agriculture. They were received over a period of approximately 3 years and were isolates from dried milk and milk-drying plants. Approximately 175 plants were sampled during a salmonella control program. Media. Brilliant green sulfadiazine (BGS) agar was prepared by a standardized method (1). Bismuth sulfite (BS) agar, triple sugar iron (TSI) agar, and lysine iron agar were prepared by the directions of the manufacturer (Difco). Lactose broth, dulcitol broth, malonate broth, nutrient gelatin, and Jordan tartrate agar were prepared as described by Ewing and Davis (6). Procedures. Plates of BGS and BS were inoculated with each culture. The plates were incubated at 37 C and colonial characteristics were recorded at 24 and 48 h. One colony from each plate was inoculated into lactose broth. When lactose fermentation was indicated on BGS plates (green colonies), one of the colonies was chosen to inoculate the lactose broth. The lactose broth cultures were observed periodically for 30 days before interpreting the test as negative. All cultures that gave no indication of lactose fermentation on the BGS plates were tested for P-D-galactosidase activity (ONPG test) by using the method described by Ewing and Davis (6). Lactose-positive cultures were further examined by inoculating TSI agar, lysine iron agar, tartrate agar, nutrient gelatin, malonate broth, and dulcitol broth. The TSI and lysine iron agar slants were inoculated by stabbing the butt and streaking the slant. Lead acetate strips were placed in the tops of the TSI agar tubes as an additional indicator of hydrogen sulfide formation. All cultures were serotyped by the procedures of Edwards and Ewing (3). RESULTS Eighty-six (15.6%) of the 552 cultures examined were positive in lactose fermentation tests. All lactose-positive cultures produced green colonies on BGS agar, black or brown colonies with sheen on BS agar, and fermented lactose broth within 48 h. ,B-D-Galactosidase 672 activity was not detected in any lactose-negative culture. Reactions produced in TSI agar by the lactose-positive cultures, with one exception, were atypical for Salmonella. Seventy-two cultures produced acid throughout the medium with gas, and 13 cultures produced acid throughout the medium with no gas. Hydrogen sulfide production was indicated by blackening in the medium by 19 cultures and by blackening of the lead acetate strips by all 86 cultures. Reactions in lysine iron agar were typical for Salmonella. All of the lactose-positive cultures gave a positive reaction to the test for lysine decarboxylase. Hydrogen sulfide production in this medium was not recorded. All of the lactose-positive cultures fermented dulcitol with the production of acid and gas and produced acid in Jordan tartrate agar. None utilized sodium malonate and none liquefied gelatin. Thirty-one serotypes were identified from the 552 cultures (Table 1). The lactose-fermenting cultures were limited to three serotypes. S. anatum, S. tennessee, and S. newington. These serotypes were among the five most common types identified in the study. Lactose-fermenting strains of each of these types were found in samples from each of the 3 years. However, the percentage of these types that fermented lactose decreased each year. DISCUSSION The results of this study were compatible with those reported by Twort (20) and Kristensen (13). The lactose-positive strains were serotypes that appeared to be indigenous to the milk plant environment and thus exposed continually to lactose. The three serotypes involved were among the five most commonly found and were isolated during each of the 3 years. It is possible that the ability of these strains to ferment lactose was influenced by their environment. Although the percentage of the cultures fermenting lactose was very high compared to previous statistics, it is not wise to draw conclusions from this concerning their prevalence. These cultures were from a specialized environment which could have influenced the variation. Emphasis is given to the fact that one should continually be alert to variant forms, particularly when examining source materials containing lactose. The observed annual drop in the percentage of lactose-positive variants is interesting. However, information was not sufficient to support any definite conclusions as to the reason for this explanation could be that chronic sources of contamination were detected and eliminated. The observed lack of evidence of hydrogen sulfide production in TSI agar further complicates the process of identification of lactosepositive Salmonella. The use of lead acetate strips, although giving positive results, did not appear to be a completely satisfactory answer to the problem. Lead acetate is the more sensitive indicator of hydrogen sulfide production and, therefore, the results cannot be equated to those in TSI, which is the usual standard and is less sensitive. The results of this study indicate that bismuth sulfite agar and lysine iron agar are especially useful in the isolation and identification of lactose-positive Salmonella. Probably the most important factor is to include a differential plate medium that does not key on lactose fermentation.

v3-fos

2018-04-03T06:12:30.260Z

1973-05-01T00:00:00.000Z

23198061

s2

Evaluation of potential risk of botulism from seafood cocktails. Clostridium botulinum E could not be detected in 35 samples of commercial seafood cocktails, ranging in pH from 4.10 to 4.85. At 30 C, toxinogenesis in homogenates acidified with a citric-acetic acid mixture was prevented at pH 4.86 or lower for crabmeat and at 5.03 or lower for shrimp. Measurements of the rate of acid penetration into the centers of large pieces of flesh indicated that the already small risk of botulism from seafood cocktails could be completely eliminated by using a cocktail sauce at a maximum pH of 3.70 and by cooling the final product to at least 10 C for 24 h. The increasing production of semipreserved convenience-type foods has again focused attention on Clostridium botulinum, even though the overall incidence of this organism in such foods appears to be quite low (for example, see reference 10). Type E has been found in an appreciable proportion of certain raw seafoods: in 13% of whitefish chubs (7), in 10% of vacuumpacked frozen flounder (4), and repeatedly in marine food species off the Pacific Coast of the United States (1, 3). Crab and shrimp provide a good growth medium for C. botulinum (5). When cooked, peeled, and used in the preparation of cocktails in sealed glass jars, these seafoods are not sterilized but are preserved primarily by the bacteriostatic action of acetic acid and secondarily by refrigeration, which, in commercial channels, is not always reliable. In California, the method of preservation has evolved empirically and, while no cases of botulism attributed to such seafood cocktails are known, neither is experimental evidence of their safety available. The purpose of the present investigation was to assess risks of botulism from seafood cocktails. MATERIALS AND METHODS The isolation procedure used for C. botulinum has been described previously (5). Preparation of homogenates. The freshly cooked and peeled meat of Pacific Coast crab (Cancer magister) or the coastal species of shrimp (Pandalus jordani) was homogenized in a blender with an equal weight of water containing 0.5% each of NaCl and sucrose. The final NaCl concentration was 1.25%. A 1:1 mixture of 0.1 N citric and 5% acetic acids was used to adjust pH. Citric acid was selected because it occurs naturally in tomatoes, acetic acid because it is added as vinegar during the commercial preparation of cocktail sauce. The homogenate was then distributed in test tubes, autoclaved, and cooled. The final pH was determined on several tubes of each batch. A Corning Model 10 meter was used for pH determinations. C. botulinum strains and spore production. C. botulinum E strains Beluga and VH, isolated by C. E. Dolman, University of British Columbia; Saratoga, isolated from canned tuna in 1963; and E-8, one of Kushnir's original strains, were used. Because differences in their behavior with respect to pH inhibition were both slight and inconsistent, only Saratoga and VH were used in the later experiments. Spores were produced in the TPG medium of Schmidt et al. (8) modified to contain 0.2% glucose and 0.1% yeast extract (final pH 7.0). Screw-capped jars (8-oz size) filled to within 2 cm of the top were heated for 20 min in boiling water, cooled rapidly, inoculated with 15 ml each of an actively growing culture, and incubated at room temperature (22 to 24 C). The progress of sporulation was observed twice daily by phase-contrast microscopy, and the spores were harvested when about 90% of the cells showed refractile spores. The crop was washed five times and finally suspended in sterile, deionized water. The viable spore count of the stock suspension was made in deep tubes of heart infusion agar (Difco) after heat shocking for 15 min at 60 C. The tubes were overlaid with vaseline and incubated at room temperature (22 to 24 C), and the colonies were counted after 72 and 96 h. Inoculation of crab and shrimp. Tubes of crab meat homogenates were inoculated with C. botulinum E in six runs. Four strains were used in the first two runs and two strains in the last four. Similarly, two strains were used for each of four runs done on shrimp 07 on March 17, 2020 by guest http://aem.asm.org/ Downloaded from meat. For each run, sets of tubes were prepared containing homogenates at two or three different pH values; each set was divided into either two or four 36-tube subsets, depending on the number of strains used. The tubes were inoculated with approximately 10,000 heat-shocked spores, sealed with vaseline, split into three groups of 12, and incubated at three different temperatures. Tubes at 5.5 C were observed weekly; at 10 C, daily; and at 30 C, twice daily, and were tested for toxin at the first appearance of gas. At the end of each run, tubes of the highest pH step that had remained negative were also tested for toxin. Crab leg muscles were inoculated in the center with about 10,000 spores of C. botulinum E VH; placed in beakers containing sauces of different pH values; stored at 10, 24, and 30 C; and periodically tested for toxin. Detection of toxin. Homogenates were tested by adding to 1 ml of press juice 1 ml of 0.5 N phosphate buffer (pH 6.1) containing 0.2% trypsin (Difco, 1: 250). The pH values of all samples were thus brought to between 6.00 and 6.10, depending on initial acidity. After incubation at 37 C for 75 min, 2 ml of sterile deionized water was added, the mixture was centrifuged, and 0.4 ml of the supernatant fluid was injected intraperitoneally into each of two mice. Type E toxin was confirmed periodically by neutralization with specific antitoxin. The mice were observed for 96 h after inoculation; none died after more than 24 h. Inoculated crab leg muscles were tested by triturating the meat with 1 part of buffer and using the supernatant fluid for trypsinization and injection as just described. Acid penetration in crab leg muscles. Cocktail sauces, minus spices, were prepared from tomato paste, diluted with water to 10 to 11% solids, and adjusted with acetic acid to various pH values. Crab leg muscles measuring about 10 mm across the smallest dimension were placed in beakers of sauce. The ratio of meat to -sauce (40: 60) was that of commercial samples. At intervals, three pieces were removed, rinsed, blotted, and cut transversely. The pH across the section was estimated by means of indicator paper (Macherey, Nagel and Co., Diren, Germany, range 3.8 to 5.8). The calibration of the paper was checked with standard buffers. RESULTS Incidence of C. botulinum E in commercial seafood cocktails. Although the isolation procedure used is sensitive enough to reveal an average contamination of one spore per gram of material, no C. botulinum was detected in any of 35 samples from nine processors. pH of commercial seafood cocktails. The pH of the commercial sauces ranged from 4.10 to 4.85. Of the 35 samples, seven (lobster and halibut) contained pieces of meat up to 10 mm across. The pH at the center of these chunks was always close to that of the sauce, and in all cases below 4.50. Effect of pH and temperature on toxin formation by C. botulinum E in crab and shrimp. Table 1 shows the first appearance of detectable toxin in crab meat homogenates, combining results of tests of all strains. In no case was toxin detected in inactive tubes, whereas tubes showing gas were always toxic. Table 1 does not show the results of incubation at 5.5 C because no growth developed in any of those tubes within 127 days. Because the method of preparation of the homogenates did not allow complete control of the final pH, the differences between some values shown in Table 1 are so small that the corresponding runs should probably be considered replicates. However, it is clear that between pH 4.86 and 5.03 is a fairly narrow grey zone, above which growth (gas formation) and toxinogenesis are apparently unimpeded, and below which both are prevented. Results obtained with shrimp meat homogenates are quite similar and are not tabulated here. In tubes incubated at 30 C, the appearance of toxin took 2 days at pH > 5.25 and 40 days at pH 5.06; at pH < 5.03, no toxin could be detected within 80 days. Incubation at 10 C retarded toxin formation by about 5 days at pH 5.06; at pH < 5.03, the tubes remained negative for 130 days. The crab data, which also apply to the shrimp, indicate that, in order to prevent the formation of detectable toxin by C. botulinum E, the pH of the meat must be brought down to 4.86 or lower. Moreover, this must be done, at the latest, within 20 h at 30 C or 7 days at 10 C. Bringing the pH down rapidly by means of a sauce of the proper acidity would seem to be a simple matter for crab cocktails, in which the meat consists mostly of separate fibers. However, in other cases, such as shrimp and lobster cocktails and seafood cocktails containing halibut, fairly large pieces of meat may be encountered, and the rate of acid penetration becomes a factor. Rate of acid penetration from the sauce into the center of chunks of meat. Crab leg muscles were used in this phase of the work to simulate lobster or halibut chunks as well as shrimp. Table 2 shows combined results from several experiments, in which all three temperatures were not always used, accounting for the many blanks. Room temperature (24 C) was chosen rather than 30 C because it is closer to actual commercial conditions. The target pH was taken as 4.80, rather than 4.86, to allow for the coarser commercial method of measurement. In the series of experiments summarized in Table 1, it was determined that even at pH 7 and 30 C toxin could not be detected before 20 h. It would be tempting to assume that, at 24 C, likewise, detectable toxin would not be formed within 20 h, and to conclude from Table 2 that a sauce having an initial pH < 3.91 could lower the pH in the center of a reasonably large piece of meat to a safe level within a safe time However, the difficulty of controlling the rele vant variables under commercial conditions renders this conclusion questionable. A further complication is the presence of tw( competing processes: (i) the elaboration of toxir by C. botulinum and (ii) the concurrent migra tion of acid, which at some point prevent< further toxinogenesis. As temperature lowers the rate of toxin formation slows substantially whereas the rate of acid penetration slows onl1 slightly. Consequently, at 24 C the margin o safety, namely, the difference between the rate of toxin formation and that of acid penetration, is small, a few hours at most; but at 10 C this margin is much greater. Table 3 summarizes the interaction of the two processes. At or above room temperature, sauces of pH > 4.00, at the time of adding to the crab meat, cannot prevent toxinogenesis if C. botulinum E spores are present in large pieces of meat. Although a sauce at pH 3.90 appears to prevent toxin development under the same conditions, the results obtained at the next lower pH step make it prudent to consider pH 3.70 as the maximum. The negative samples were not observed beyond the times indicated because, by then, the center pH was already below 4.80. DISCUSSION The published data on the pH inhibition of C. botulinum E are somewhat conflicting. Segner et al. (9) observed growth of the Beluga strain at pH 5.03 in TPG medium, but Ohye and Christian (6) found that type E could grow at pH 6.0 but not at 5.0 in TYG medium. On the other hand, Dolman and lida (2) reported growth and toxin production by the VH strain in pickled herring (pH 4.0 to 4.2). Little information is available on pH inhibition of C. botulinum E in shellfish meat. Despite differences in growth media and. strains, our data on the acid tolerance of C. botulinum E are in general agreement with those found in the literature. As media for C. botulinum, crab and shrimp meat seem to possess no unusual qualities. The risk presented by products on the market is probably minimal. Although no data are available on lobster and halibut, it has been shown here and elsewhere (5) that the principal ingredients, commercially prepared cooked crab and shrimp meat, appear to be free of C. botulinum spores. Furthermore, the pH of the samples that we examined was low enough to r on March 17, 2020 by guest http://aem.asm.org/ Downloaded from prevent the growth of the organism. Nevertheless, the known association of C. botulinum E with raw shellfish and its ability to grow at low temperatures make it conceivable that an unusual set of circumstances might result in a hazardous situation. For example, if large pieces of meat heavily contaminated with spores were to be mixed with sauce having too high a pH, and left at room temperature, the spores could grow and produce toxin before the acid penetrated the meat. There are indications that some processors may use a sauce that is not acid enough: When crab or shrimp meat is mixed with cocktail sauce in the usual ratio of 40: 60, the pH of the mixture gradually rises 0.2 to 0.55 units, depending on the original pH of the sauce and the freshness of the meat. During this survey, I encountered sauces with a final pH as high as 4.85; therefore, allowing for a rise in pH of even 0.6, I must conclude that the initial pH of the sauce was somewhere around 4.25 which, as has been shown, is not low enough under certain experimental conditions. Based upon the findings of this investigation, I recommend two key requirements to insure the safety of seafood cocktails. (i) The initial pH of the sauce should be no higher than 3.70, and (ii) as soon as possible after preparation the product should be chilled to at least 10 C and held refrigerated for 24 h. At least with respect to potential botulism, refrigeration need not be maintained beyond this period. These two requirements are easily met and have long been part of the operating procedures of several processors. of conditions. Even in the absence of refrigeration, a sauce having a pH of 3.70 is sufficient to acidify the centers of meat chunks to a safe level before any spores that may be present have had time to grow out and produce toxin. Because the minimum toxinogenesis time of 20 h was actually obtained at 30 C, the true safety factor at room temperature is probably greater than the 7.5 h shown. Nevertheless, the margin of safety is not comfortable enough when one deals with C. botulinum, hence the additional recommendation of rapid initial chilling to retard bacterial growth while allowing acidification to proceed. Under these conditions, any risk of botulism from seafood cocktails should be completely eliminated.

v3-fos

2017-06-19T11:47:23.257Z

1973-01-01T00:00:00.000Z

537224

s2

Recent developments in random sample tests for poultry in the united kingdom This paper reports on the six random samples tests conducted by National Poultry Tests Ltd. and discusses the change of emphasis from a Breeder supported test where entry fees were charged and chicks were supplied free to a test which was entirely financed by the Company from its commercial activities and from the sale of a Technical Bulletin called Poultry Testing. The paper reports on the way in which chicks for tes’ting were obtained through third parties without the knowledge of the breeders, and discusses the advantages and disadvantages of obtaining chicks for testing by this method as opposed to the sampling of chicks in hatcheries from several different sources. Also discussed are the difficulties in providing specific’ environments and management conditions for individual strains of bird in accordance with breeders recommendations and the importance of carrying out random sample tests under standard,conditions. ged and chicks were supplied free to a test which was entirely financed by the Company from its commercial activities and from the sale of a Technical Bulletin called Poultry Testing. The paper reports on the way in which chicks for tes'ting were obtained through third parties without the knowledge of the breeders, and discusses the advantages and disadvantages of obtaining chicks for testing by this method as opposed to the sampling of chicks in hatcheries from several different sources. Also discussed are the difficulties in providing specific' environments and management conditions for individual strains of bird in accordance with breeders recommendations and the importance of carrying out random sample tests under standard,conditions. In the seven years since National Poultry Tests was formed six random sample egg production tests have been started. The first four have been completed and the fifth and sixth are currently in production. It is of interest to quickly look at the number of entries and number of different stocks which have been included in each of these tests. Table I lists the number of entries, the number of different commercial stocks in each test and these are divided into white and tinted layers and the brown egg layers. The right hand colum of table i shows the number of pullets housed per entrytogether with the replicate size and number. Entry in the first four tests was on a voluntary basis. The poultry breeders supplied the hatching eggs or day old chicks and these were random sampled by independent officials of the Ministry of Agriculture, advisory service. The breeders in addition paid an entry fee. This entry fee went towards the cost of the testing operation. (') Cet article a été présenté la reunion du grdupe de travail n° 3 (selection et testage) de la Federation des Branches Européennes de la W.P.S.A., Nouzilly-Ploufragan, 6-10 septembre rg 7 r. Opposition to testing amongst the poultry breeders in the United Kingdom rose to such a level in 1970 that it proved impossible to obtain samples of stocks on a voluntary basis for the fifth test. National Poultry Tests therefore decided that it would have to meet the cost of conducting the fifth test from its own income. We realized that we had a duty to the entire industry to provide independent facts and figures on poultry stocks and that this duty had to be met. You well know that the cost of running tests is very high and it puts an enormous burden on a small private company whose only source of income is derived from its farming activities. The fifth test was started by ordering day old chicks of thirty different commercial strains. (a) In five cases the breeders concerned supplied the chicks free and promised donations equivalent to the old entry fees. In addition, they allowed independent officials to random sample the chicks in the hatcheries. (b) We purchased samples of fourteen other commercial strains from the breeders and they also allowed independent officials to random sample the chicks. (c) In the case of three of the commercial strains the breeders concerned were prepared to sell us the chicks but were not prepared to have them random sampled in the hatcheries. (d) A further eight commercial strains were ordered but were found to be unobtainable at the time we required the chicks, for a variety of reasons. This test therefore started with 22 different stocks, twelve white and tinted egg layers and ten brown egg layers which is more than we had had in any of the previous tests. Early in 1971 we decided that one way to raise financial support for our tests was to sell the results we produced. Thus Poultry Testing was launched in April 1971 . This technical bulletin contains month by month test reports, information on various aspects of the tests, individual assessments of various stocks together with articles from leading authorities on technical subjects. Poultry Testing has been extremely well received in all quarters of the industry and although nowhere near large enough, the subscription list is growing at a very encouraging rate. Naturally, what has been written in Poultry Testing has not met with the approval of everybody concerned. This is to be expected when one is dealing with a subject such as the comparative performance of the products of commercial companies. Writing reports about the tests in a monthly bulletin provides an interesting challenge. The success of the tests depends upon the success of Poultry Testing. The readers of Poult y y Testing must find the contents of value otherwise they will not subscribe to it. Thus one is writing the reports extracting information and putting it down in the form which is of most use to the commercial egg producer. One completely new aspect which we have included in Poultry Testing is the individual assessments of stocks. We have been extrem ely well pleased with the interest and assistance we have received from the poultry breeding companies in providing us with sources of information to help us in the work of making sound unbiased assessments of their stocks. We are in many cases dealing with truly international stocks and we make use of information from other countries. This can be from overseas test reports and also from survey data and individual farm results from different countries. It could well be that we should publish much more information on reports from overseas in the interim period before we have a combined summary of European test reports. The sixth series of tests was started in 1971 and a completely new technique was used to obtain the day old chicks. Using a variety of methods we obtained the chicks through third parties without the knowledge of breeders concerned. We can tell you here and now that this method of obtaining stocks is very effective in that we were able to get twenty seven different stocks. Fourteen white and tinted egg layers and thirteen brown egg layers. Our maximum capacity is twenty-eight different stocks and if it had not been for a bad hatch in one instance and the fact that we did not want to wait for an extra two weeks to get replacement chicks we would have had all twenty-eight different stocks in this test. On the other hand making all the arrangements does create a vast amount of work. The ordering and collection of the chicks has to be coordinated so that they all arrive at the test and can be put into the brooder house within a few days of each other. Another draw back to this method is that we were having to pay the full market price for all the chicks and did not obtain any entry fees. It is thus an expensive way of starting a test. It would be obvious to you that tests started in this way can be seen by everybody in the industry to be completely independent. There can be no question of any collusion between the test authorities and the breeders in providing special samples of chicks for the test. However much we know that this does not go on, there are people in the industry who like to use this argument as a criticism of laying tests. It is naive to think that nowadays the poultry breeding companies would go to the trouble of putting specially selected birds into public tests. These companies are selling stock in many countries throughout the world. In some countries the stock is sampled and put into official Governement tests and in others it is entered on a voluntary basis. If the results obtained in the different tests varied too much or if the results in tests were not typical of farm experiences then the credibility of that company would very soon decline. These companies have far too much at stake to take this sort of risk. When purchasing stock on the market there is always the chance of an inferior batch of chicks being bought. The breeder and hatchery organization will know of parent flocks that are not performing well, they will know of poor hatches. In general they will tell their customers that a particular hatch was of poor quality and will offer to supply chicks on an alternative date. The fact that this arises creates another element of chance when purchasing samples for a test. The breeder is much more likely to inform the test authorities that a particular hatch has not gone well if he knows that sampling officials will be calling to select chicks. If just a small batch of chicks are being sold to an individual customer there is a chance that the breeder would not inform him of this. This could add to the variation in results between test samples and is in itself an argument for having more tests and therefore more samples of each strain. This argument is very much in the breeders interests and is why it has been put forward by some of the breeding companies. In the past we have conducted tests where two different layers diets were fed to all stocks. Half the replicates of each entry being fed on one diet and the other half being fed on the second diet. In the last completed we gave the entrants the opportunity to select which of two diets they wished their birds to be fed. We did this despite the fact that we believed that in many cases they were selecting the wrong diet. There are many arguments for and against having variable conditions under which to conduct tests. One can design factorial experiments but this would necessitate very large facilities for testing. One alternative put forward is that each stock should be subjected to the conditions of environment and management that are recommended by the breeder. In effect this means that if one was testing thirty different stocks, one would have to have probably at least 120 separate poultry houses on the one site in order to be able to provide the conditions stipulated by the breeder and also to replicate these. The cost of building a unit of the size and altering its physical conditions before the start of each new test would be far beyond the means of a company of our size. It is possible that location tests could overcome this point and enable a wide range of stocks to be subjected to different conditions. One has to assess that altering the environment and management would change the ranking of the different stocks significantly. One can think of examples where some stocks kept in ten bird cages would produce disastrous results but if kept in three bird cages would perform moderately well. Our main activities have been concerned with the testing of egg laying stocks. We have also conducted tests and experiments of other kinds on commission for individual companies and agencies. We have an open mind on the question of testing of other products. However we would maintain that the testing of stock, laying stock in particular is of greater importance than the testing of feed, equipment or other services. RÉSUMÉ DÉVELOPPEMENTS RÉCENTS DES TESTS SUR ÉCHANTILLONS DE VOLAILLE PRIS AU HASARD DANS LE ROYAUME UNI Cet article est un rapport sur les six tests sur échantillons pris au hasard conduits par National Poultry Tests Ltd. Il discute l'évolution depuis un test supporté par les sélectionneurs, avec paiement de droits d'entrée et fourniture gratuite des poussins, jusqu'à un test entièrement financé par la Compagnie à partir de ses activités commerciales et de la vente d'un bulletin technique appelé Poultry Testing.

v3-fos

2018-04-03T01:00:10.489Z

1973-07-01T00:00:00.000Z

27645929

s2

Microbial penetration through three types of double wrappers for sterile packs. Microbial penetration of sterile packs was studied by using double-wrap (two layers each) muslin, single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC wrappers to wrap 20 gauze sponges (2 by 2 in.). These packs were stored on open shelves of a central sterile supply department of a hospital and processed for sterility at weekly intervals. Microorganisms penetrated the double-wrap muslin as early as 28 days, the single-wrap muslin and single-wrap two-way crepe paper combination in 77 days, and the single-wrap muslin and single-layer BAR-BAC combination in 63 days. A previous study on microbial penetration of sterile packs was conducted to determine, under actual hospital conditions of storage, safe times for sterile storage of four widely used single or double wrappers for packs (1). Viable microorganisms penetrated packs with single wrappers faster than packs with double wrappers, and the time for microbial penetration was less than half as long with open-shelf versus closed-cabinet storage. On the basis of this study, single wrappers were not recommended for sterile packs (1). An extension of that study has been conducted to evaluate the safe time for storage of sterile packs with two additional types of double wrappers to compare them with the most effective wrapper previously studied, double-wrap (four layers) muslin. Because storage on open shelves is common in hospitals, and because such storage resulted in most rapid microbial penetration in the previous study, the comparison of the three types of wrappers was conducted only with open-shelf storage. MATERIALS AND METHODS Standard packs approximately 8 by 10 in. (20.4 by 25.5 cm) consisting of 20 2 by 2 in. (5.1 by 5.1 cm) 12-ply gauze sponges were prepared and sterilized as described by Standard, Mackel, and Mallison (1). These packs were covered with double muslin (each two layers), single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC outer covering. Muslin wrappers used for the standard packs were 140-thread-count material, unbleached, dyed green, laundered, and ironed 1 to 10 times before use. BAR-BAC (Angelica Uniform Co.) wrappers were tightly woven cotton material, dyed green, laundered, and ironed at least one to three times before use. The paper wrappers used were commercially available two-way crepe paper (Dennison Wrap). All wrappers were approximately 24 by 24 in. (61 by 61 cm). The sterile packs were transported in sealed, sterile plastic bags to a hospital central sterile supply department (CSSD). They were placed on open shelves in the same area used for hospital sterile supplies. Test packs were picked up at random in groups of two or four at weekly intervals, placed in sterile plastic bags, and returned to the laboratory for microbiological assay by procedures previously described (1). A total of 252 test packs were assayed. Three series of 14-week evaluations were conducted. On the initial day of each series of evaluations, three packs wrapped in each type of wrapper used in that series were chosen at random, after placement on shelves of the hospital CSSD, and transported back to the laboratory in sterile plastic bags for an initial control assay to confirm that the packs were not contaminated during transportation. In addition, at the time of each weekly pick-up of study packs, two sterile double-muslin-wrapped (4 layers) packs were transported to the hospital and back to the laboratory in sterile plastic bags for assay as weekly transportation controls. A total of 108 initial control packs and weekly transportation control packs were assayed. Temperature and relative humidity were monitored throughout the study by use of 7-day recording hygrothermographs. These instruments were calibrated at weekly intervals with a sling psychrometer. Viable surface contamination settling on the outside of the packs was estimated by using stainlesssteel strips placed open on the storage shelves used for the packs, as described previously (1). RESULTS Microbial contamination was determined for packs with three types of double wrapping: double-wrap (each two layers) muslin, singlewrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC outer covering. Table 1 gives the time in days until the first contamination was found inside packs covered with each type of wrapping material. Contamination occurred as early as 28 days with double-wrap muslin, 63 days with the BAR-BAC and muslin combination, and 77 days with the two-way crepe paper and muslin combination. Tables 2 to 4 show the number of pack weeks of exposure and the number of Temperatures in the CSSD remained between 70 and 80 F (21.1 to 26.7 C) with few exceptions throughout the entire study, and weekly average relative humidities ranged from about 30 to 55%. Table 5 shows the total microbial counts from stainless-steel strips exposed on open shelves to estimate the amount of viable fall-out on sterile packs on the shelves. The microbial counts were calculated on the basis of an area measuring 80 square inches, equal to the exposed surface area of the packs used in the study. About 65% of the settled microorganisms were aerobes grown without heat shocking, about 20% were anaerobes grown without heat shocking, over 5% each were molds and aerobes grown after heat shocking, and less than 5% were anaerobes grown after heat shocking. Only one of the 108 control packs utilized during the study was found to be contaminated. Forty test packs were contaminated during the study. Table 6 shows the type and frequency of organisms isolated from the 41 contaminated packs. The most frequently isolated organisms were Aspergillus spp, Streptomyces spp, and gram-positive sporeforming rods. DISCUSSION The earliest time required for contamination of the packs with double-wrap (four layers) muslin stored on open shelves was 28 days, as in the previous study (1). The amount of microbial contamination collected on stainless-steel strips TAULE 2. Length of storage, number of pack weeks of exposure to contamination, number of positive packs found, and number tested, series 2

v3-fos

2018-04-03T03:41:31.684Z

1973-08-01T00:00:00.000Z

36286634

s2

Induction of Red Color Formation in Cabbage Juice by Lactobacillus brevis and Its Relationship to Pink Sauerkraut. Membrane-filtered cabbage juice, when fermented by Lactobacillus brevis under conditions of controlled pH, frequently produced a water-soluble red pigment. The pigment, presumably responsible for imparting a highly objectionable discoloration to sauerkraut, was formed during the post logarithmic phase of growth. Color development is pH dependent (5.2 to 6.3) and can be suppressed by chemical reductants or anaerobic conditions of growth. The compound responsible for discoloration was purified and partially characterized. The grade of commercial canned sauerkraut is established by a composite value derived from the singular evaluations of character, cut, defect, flavor, and color. Of these factors, color and flavor are given the highest, but equal, considerations (1). The maintenance of a bright, cream to light straw color is necessary for producing a high-quality fermented product. The inability to conform to the highest color standard results in the downgrading of quality; if discoloration is serious, the product may be rejected. One such serious color defect is the formation of red or pink kraut. One type of discoloration is a light pink to deep burgundy coloration, which causes considerable economic losses to processors in Holland (10) and the United States. Since climatic conditions and the varieties of cabbage grown for kraut production vary widely within each locale, it would appear that factors related to the fermentation processes are primarily responsible for inducing the objectionable color formation. Furthermore, the red color develops during the course of the fermentation or immediately prior to processing. The initiation of red color formation in cabbage and sauerkraut apparently proceeds via a series of complex chemical reactions. Recent studies pertaining to the effects of dehydration upon cabbage indicate that ascorbic acid -amino acid interactions are responsible for ' Approved by the Director of the New York State Agricultural Experiment Station as Journal Paper No. 2033, April 24, 1973. producing non-enzymatic discoloration in the dried product (6), whereas in pink kraut the color has been attributed to the formation of a leucoanthocyanidin (4). Since the kraut fermentation arises as a result of a heterogeneous microbial population, it is difficult to assess the role each species contributes to color formation. Although yeasts are known to impart color to kraut (2, 3, 5, 10; E. Steinbuch, Antonie van Leeuwenhoek J. Microbiol. Serol., Yeast Symposium Suppl. no. 35, F39, 1969), the potential induction of red color by pure cultures of lactic acid bacteria associated with the "normal" fermentation of kraut has, to our knowledge, never been reported. Therefore, this paper describes those conditions which contribute to the onset of discoloration, with particular emphasis on the role of Lactobacillus brevis, in inducing a cerise color in filter-sterilized cabbage juice. Growth measurements. Fresh cabbage juice (variety Glory) was prepared as previously described (9). The cabbage sera, sterilized by membrane filtration in a glass assembly, were dispensed aseptically in 10-ml volumes (16-by 150-mm test tubes) for growth and color studies. Each sample was inoculated with one loopful (about 1.8 x 104 cells/ml) of a 24-h culture grown previously in cabbage juice. The viable cell counts were taken at 24-h intervals and were esti-161 STAMER, HRAZDINA, AND STOYLA mated by plating on tryptone-glucose-yeast extractsalts agar medium (8). Sodium hydroxide (0.5 N) or solid calcium carbonate were used for maintaining preselected pH values. Sterile CaCO., when added to cabbage juice at concentrations of 0.25, 0.50, 0.75, 1.0, and 1.5% (wt/vol), provided final pH values of 4.2, 4.4, 5.2, 5.8, and 6.0, respectively, whereas the maintenance of pH by NaOH was controlled by pH-stat. In addition to using smaller volumes (test tubes) for the studies of color and growth development, larger quantities of juice (40 to 80 ml) were fermented in a sterile fermentation assembly equipped with sampling ports, a magnetic stirring bar, and pH electrodes and maintained at 32 or 22 C by a constant temperature water bath. Measurement and purification of colored materials. Since it was desirable to establish the chemical composition of the unknown product(s), larger volumes of juice (5 to 10 liters) were fermented in the hope of obtaining increased quantities of red pigment. It was found, however, that in addition to being difficult to filter-sterilize, the fermented juice was extremely vulnerable to extensive browning, a condition which resulted in excessive losses in the yield of red color. It was observed that juice, when prepared in 150-ml quantities and contained in 250-ml Erlenmeyer flasks was more stable, and therefore was used for subsequent fermentation studies. After fermentation and maximum color production (about 8 days), the juices were centrifuged (20,000 x g) for 20 min. The clear, red juice was extracted four times with 25-ml volumes of diethyl ether. The aqueous phase, containing the red color, was concentrated sixfold under vacuum at 40 C. The red concentrate was treated with acetone (85% saturation) and filtered through paper. The resulting supernatant fraction was evaporated to dryness, dissolved in 10 ml of water, and applied to a polyvinylpyrrolidone (Polyclar AT) column (2.5-by 10-cm) which had been previously equilibrated with water. The column, retaining the adsorbed pigment, was washed with five bed-volumes of water and was subsequently eluted with 900 ml of methanol containing 0.01% hydrochloric acid. After elution, fractions containing the red pigment were concentrated to 0.2 ml, applied as a band on preparative cellulose thin-layer chromatography (TLC) plates, and further purified in the following solvent systems: (i) water-acetic acid-hydrochloric acid (80:25:5); (ii) 2% aqueous acetic acid; and (iii) upper phase of butanol-acetic acid-water (4:1:5). After irrigation, the red band was scraped from the plate, eluted with methanol (50 ml), and concentrated to 10 ml. A sample of the concentrate (4 ml) was subjected to ultraviolet light (UV) and visible spectroscopic analyses. The remainder of the concentrate (5 ml) was evaporated to dryness and the residue was mixed with KBr (0.10 g) and pressed into micropellets for infrared analysis. The sprays used for the qualitative analyses of functional groups included: 2,6-dichloroquinone chlorimide, ferric chloride, and phosphomolybdic-phosphotungstic acid, for phenolic compounds; silver nitrate for reducing materials; and ninhydrin for amino acids. RESULTS AND DISCUSSION While studying the effects of pH upon the growth rates of lactic acid bacteria (9), it was observed that L. brevis, when grown in cabbage juice containing calcium carbonate, imparted a brilliant red color to the fermented extract. Of the five microorganisms commonly associated with the kraut fermentation, L. brevis was the only species which induced color formation in the buffered juice (Table 1). Under these conditions, L. brevis produced a fivefold increase in color (A,,6) within 7 days of incubation at 32 C. In unbuffered juice, the culture produced no apparent changes in color, and the final absorbance values were similar to those displayed by the non-color-forming species. The inability of the cultures, other than L. brevis, to produce a red color in buffered juices cannot be attributed to differences in growth-sustaining properties of the buffered and regular extracts. This was confirmed by plate counts; each juice, when inoculated with about 1.8 x 10' cells per ml, provided final populations of 9 x 108 to 1.2 x 10' cells per ml after 3 days of incubation at 32 C. The rates of color formation as a function of the growth of L. brevis at two temperatures (32 and 22 C) in cabbage are shown in Fig. 1. It may be observed that the growth rates at each respective temperature in both buffered and unbuffered extracts were quite similar. After 5 days of incubation at 32 C, the juice containing no CaCO. yielded a total viable population of 5 x 108 cells per ml. During this time period the initial pH of the medium was abruptly lowered from 6.2 to 3.8, a value which remained constant throughout the 21-day incubation. No red color was formed under these conditions of growth. Although CaCO, had no apparent effect upon growth, it induced most markedly the development of red color. Color production was initiated at about 5 days of incubation at 32 C and reached its maximum intensity 5 days later (Fig. 1). It appeared that color production occurred during the latter stages of postlogarithmic growth and attained its maximum intensity during the stationary growth phase. Incubation beyond 10 days invariably resulted in a marked decrease in absorbance at 558 nm, and after 21 days of incubation the maximum color intensity was reduced more than 60%. The reduction in absorbance at 558 nm was accompanied by a concomitant increase in the 500-nm region suggesting the onset of browning. A comparison of color formation as a function of temperature is also shown in Fig. 1. As might be expected, a 10 degree down-shift in temperature, i.e., from 32 to 22 C, reduced both growth and color development rates by nearly 50%. At 32 C, 6 days were required to achieve maximum cell yields and 8 to 10 days were required for the formation of maximum color intensity, whereas at 22 C, 13 and 19 days were required, respectively, to achieve similar results. Again, as in the case of incubation at 32 C, color production was initiated during the latter phases of growth and occurred only in the juice containing CaCO,. To determine if CaCO, served as a buffering or chelating agent, the effects of various monoand divalent ions upon color production were examined. The addition of the chloride or sulfate salts of calcium, magnesium, manganese, iron, sodium, and potassium, when supplied at a concentration of 0.5%, produced B, viable cell count in juice-containing 1.0% CaCO.; C, red color formation injuice containing 1.0% CaCO,; final pH 5.2; D, color production in regularjuice, final pH 3.8. no enhancement in color response. Therefore, it was concluded that CaCO, played the role of a buffering agent in invoking the color devlopment. Additional evidence that pH was involved, in part, in color formation, was established by growing the culture in cabbage juice with varying concentrations of hydrogen ions. The culture, when maintained at constant pH by sterile NaOH (dispensed by pH-stat) provided color intensities similar to those obtained by the CaCO,-containing systems. As reflected in the increased absorbance values at 558 nm, pH markedly influenced color formation (Fig. 2). Increased quantities of red color were formed as the pH was raised from 4.5 to 6.0. Approximately equal levels of red color were attained in cultures constantly adjusted to pH values of 6.0 and 6.3; higher pH values were not studied. Since the growth rate of L. brevis is suppressed by lowering the pH of cabbage juice (9), the absorbances reported in Fig. 2 represent the maximum values attained as a result of growth at each respective pH. The absorbances observed at pH 5.0 or greater were obtained after 9 days of incubation, whereas those intensities produced at pH 4.5 or less were recorded after 21 days of incubation. These latter prolonged periods of incubation were used to permit the development of maximum color intensities under more acidic conditions. Direct microscopic count showed that each sample reached a minimum population of 9 x 108 cells per ml during the course of the fermentation. The failure of these extended incubations to provide color intensities equal to those observed under the more alkaline conditions show that pH plays a vital role in inducing color formation. This dependence of maximum color formation upon pH, i.e., >5.2, appears to be similar to that reported for the induction of color in dried cabbage by chemical mechanisms (6). However, the route of color generation in the cabbage extracts differs from that of the dehydrated product, in that color development in fermented juices is not only pH dependent, but also requires the presence of L. brevis. Further studies concerning the significance of the bacterium in initiating color response and the inability of sterile sera to undergo spontaneous color changes as a result of chemical or inherent enzymatic reactions likewise were investigated. A 48-h, unbuffered cabbage juice culture, containing 5.5 x 10' cells per ml, showed no evidence of color. However, when 25-ml samples of this culture (pH 3.9) were adjusted to pH 5.5 with NaOH (40%) and 1, color production was initiated absorbances arising as a result of bacterial L (Fig. 3). Maximum color intensity growth under atmospheres comprised of varying Ed after 4 days of incubation; viable air and nitrogen compositions show that aeros remained at about 1.1 x 10' per ml bic conditions were most conducive to color this period. No color development formation (Fig. 4). Although each juice, buf-'ig. 3) when the pH of the juice (3.9) fered at pH 5.7, showed evidence of color, those ared or when the pH of the juice was extracts incubated in the highest air-nitrogen 5.5 and the juice was sterilized by atmospheres (90 to 100% air, respectively), Dfore it was reincubated. provided intensities threefold greater than the ten fermented in 10-ml volumes for 8 corresponding extract grown under nitrogen t 32 C, often possessed a reddish hue only. liquid interphase. This suggested In addition to suppressing color formation, color formation. A comparison of the less aerobic conditions of growth produced lower cell yields in the unbuffered extracts than in the buffered series. As shown in Fig. 4 (8), pH also appears to produce vital interations in limiting growth in cabbage exfects of pH upon red color production by tracts. wn in buffered cabbage juice at 32 C. A, Since discoloration was initiated in part by fermented juices. pH maintained by aerobic conditions, the use of chemical reduc-3or NaOH (0.5 N) dispensed by pH-stat. Effects of pH adjustment upon induction of red color development by L. brevis grown in cabbage juice. Cabbage juice (75 ml) was fermented at 32 C for 48 h (to pH 3.9) and divided into three samples: A, no pH adjustment; B, pH adjusted to 5.5, and then the juice was filter-sterilized; and C, pH adjusted and maintained at 5.5 in a pH-stat. All samples were reincubated at 32 C. 164 reincubated within 16 h was achieve cell number throughout occurred (F was not alte adjusted to filtration be Juice, wh to 10 days a at the airair-induced Fig. 5. (Although S-methyl-cysteine is a nonreducing compound, it is a major sulfur amino acid found in cabbage [11] and its role as potential reductant remains to be clarified.) Of the above compounds examined, ascorbic acid was the more effective color suppressant at lower concentrations (less than 1 mg/ml) than was either cysteine or glutathione, whereas the reducing sulfur materials (cysteine and glutathione) were more effective retardants of color at higher concentrations (2.5 mg/ml). It was also observed that, once the pigment had formed, the color could not be reversed by the addition of the above compounds. Since Smethyl-cysteine was without effect, it appears that this compound provides no color-reducing properties in the kraut fermentation. Attempts to increase the final yield of red pigment by fermenting cabbage juice concentrates consistently resulted in producing a dark brown solution containing little red color. Lyophilized juice, reconstituted to provide 2-to 10-fold increases in soluble solids, or when added to regular juice to provide 2-fold concentrates, resulted in similar failures to produce a bright, red color. This loss in red color also occurred when fermented juices were stored at -50 C for 3 days. Not only is the red color unstable under conditions of rapid freezing and thawing, but it is labile to heat treatment. For example, the color intensities of two fermented juices, pH 3.5 and 5.5, decreased 35% when immersed in boiling water for 30 min. The effects of heat shifted the 558/500 nm ratio from 1.78 to 1.13 and produced browning. This suggests that temperatures used for processing E 0. commercial kraut (75 C) cannot be used advantageously for eliminating red color formation without imparting deleterious discolorations. At room temperature, the pigment is more stable under acidic than alkaline conditions. The adjustment of pH from 4.5 to 1.0 produced no significant change in absorbance at 558 to 568 nm (Fig. 6). At pH 8.5 the extract showed no visible absorption. Upon acidification with hydrochloride (concentrated) the alkaline solution regained its original red color. Although this pH-dependent color response is reversible, the yield, as measured by peak areas, decreased nearly 50%. The inability to extract the red color with ethers (petroleum or diethyl), amyl acetate, FIG. 6. Effect of pH upon the stability of red pigment produced in buffered cabbage juice by L. brevis. Juice (pH 5.2) fermented 8 days at 32 C, centrifuged, and pH adjusted to: A, 4.5 (concentrated hydrochloric acid); B, 1.0 (concentrated hydrochloric acid); C, 8.5 (40% NaOH); D, (C) acidified to pH 1.0 (concentrated hydrochloride) and absorbances were read immediately. chloroform, or n-butanol, but its complete solubility in water-miscible reagents, such as methanol, ethanol, and acetone, show that the pigment is an extremely hydrophilic compound. This pigment, purified by column chromatography and then applied to TLC cellulose plates and irrigated in three solvent systems, provided a visible, singular, red band with the following R, values: water: acetic acid: concentrated hydrochloric acid (80:20:5), 0.09; water: acetic acid (98:2), 0.11; butanol: acetic acid: water (4:1:5), 0.38. The above pigment, when subjected to various spray reagents, failed to show the presence of amino acids, reducing, aromatic, and indole constituents. Spectral analyses of the acidified methanolic elute (Fig. 7) show that the purified pigment vossessed three absorbance peaks, at 558, 272, and 226 nm, respectively. Although these UV absorbances show similarities to the spectral properties assigned to the red constituents of kraut by Gorin and Jans (4), the visible absorbance of the pigment produced by pure culture fermentation occurred at a wavelength considerably higher than they had reported (558 versus 540 nm). Furthermore, the infrared spectrum of the pigment (in KBr) showed absorption bands at the following frequencies: 675 (w); 746 (m); 1,045 (w); 1,075 (m); 1,125 (s); 1,283 (s); 1,387 (s); 1,470 (m); 1,610 (s); 1,737 (s); 2,870 (s); 2,940 (s); 2,960 (s); and 3,500 (m) cm-1. (Abbreviations [absorption intensities]: w, weak; m, medium; s, strong.) A correlation between the above band positions and types of grouping present indicates that the pigment (i) is aliphatic in character (strong signals in the 2,960-2,870 cm-' range), (ii) contains a carbonyl group(s) (1,737 cm-1), (iii) contains a methyl group(s) (1,387 cm-1), (iv) contains a number of hydroxvl groups (3,500 cm 1). Therefore, these data suggest that the red discoloration produced by L. brevis is not a flavonoid, anthocyanin, or anthocyanidin, but rather that the pigment is a saturated aliphatic ester, aldehyde, or diketone, or contains a five-membered ring ketone within its structure. Further determinations of the exact structure of the cabbage pigment were hampered by the difficulties encountered in producing large quantities of the red cabbage juice and by the general lability of the compound responsible for this discoloration.

v3-fos

2018-04-03T01:44:18.407Z

1973-03-01T00:00:00.000Z

30892118

s2

Uptake of Radioactive Strontium and Cesium in Rice Plants (1) Accumulation of Sr and Cs in Rice Grains through Roots* Water-cultured rice plants were exposed to 89Sr or 137Cs through roots for five days at their various growth stages and continued to grow until harvest. The harvested grains were radio chemically analysed and the concentration factors were calculated . The maximum uptake of 137Cs in the grains was found at the booting stage, while that of 89Sr was at the flowering stage . The Cs uptake was 400 times higher at the booting stage, and 30 times higher at the flowering stage than those with Sr. The growth stage dependency of the uptake of Sr and Cs was the most important factor for a selective enrichment of Cs in rice grains. The specific affinity of Cs to cell sap and that of Sr for membrane substances of rice grains probably caused a selective redistribution inside the plant body. * Orally presented at the annual meeting of the Japan Radiation Research Society , 1964. + To be published . INTRODUCTION From standpoint of the environmental contamination with fission products released from nuclear detonation or industrial accidents in nuclear reactor operation, radio-contamination of rice with Sr and Cs is very important problem. The reasons are that rice is a staple food in Japan, and that both radioactive Sr and Cs have long lives and a high availability to crops. During a national radio-contamination survey for 90Sr and 137Cs in soils and rice, which has been conducted in the National Institute of Agricultural Sciences' in co-operation with the Institute of Public Health, it has been pointed out that the content of 137Cs in polished rice is 10 times or more higher than that of 90Sr1-4> For instance 50 pCi of 137Cs per kg and 4 pCi of 90Sr per kg were observed for the polished rice samples collected in 1962, in spite of the ratio of 131Cs to 90Sr being reported as 2.5: 1 in the original fall-out itself'). The mechanisms, governing such selectivity for Cs and Sr in the course of migration of these, elements from fall-out to polished rice, should be studied as one of the most important prcblems of the contamination in food chain. It is well known that there are four pathways in grain contamination: (a) absorption from soil through root (b) plant-base absorption (c) foliar absorption and (d) floral absorption 6)7). The former is an "indirect contamination" and the latter three are "direct contamination". In this paper, root absorption of 89Sr and 137 Cs by water-cultrured rice plant was dealt with. For the sake of comparison, 32P was also applied to the plants, because phosphorus is one of the indispensable elements for plants and its behav iour has been well studied. Table 1. Composition of the nutrient solution Fig. 1. Experimental design of isotope treatments MATERIALS AND METHODS Forty-four days-old rice seedlings were transferred to Wagner pot ('/5000 are) on June 24, 1963, and were grown on water-culture. The composition of the nutrient solution is shown in Table 1. Tap water containing 33 ppm of Ca was used in this water-culture. The culture solution was adjusted to pH 5.5 with HC1 or NaOH, and was renewed every 5 days. The plants were treated with radioisotopes in a green house as schematically shown in Fig. 1. At each stage of the plant growth, 32P was singly applied into a pot, whereas 89Sr and 137Cs were simultaneously added to a pot, with nutrient sol ution in all cases. After isotope treatments the plants were once removed from pot, and the roots were rinsed three times with tap water. And then the plants were transferred to another . pot to be continued to grow under ordinary water culture conditions until harvest. Completely matured rice grains (rough rice, 34g per pot in average) were dried at room temperature for about 10 days and were then husked . The brown rice was polished at the 90% milling rate . Two grams of the polished rice and brown rice were ashed respectively, in an electric muffle furnace by raising the tempera ture gradually up to 450°C and kept at the same temperature for 3 hrs. 89Sr was determined on the basis of the method of Kodaira ,8) and 137 Cs analysis was made by the method recommended in the text book9> (refer to another paper too4)). The procedure is briefly outlined in the appendix. According to this pro cedure, neither cross contamination possibly arising from 89Sr and 137Cs nor inter ference by the constituents of rice grain was observed, as examined respectively in the previous paper10) and in the appendix of this paper . 32P was precipitated as ammonium phosphomolybdate by usual analytical method . (3-radioactivities of 32P and 137Cs were measured by a conventional GM method, whereas for 89Sr a 47r gas flow low background counter was used. Table 2. Concentration factors (C. F.) for 32P in rice grain note: Isotope solution ; 3.1 cpm/ml for treatment P, 6.3 cpm/ml for treatments B, F & M. * See Fig . 1. Table 3. Concentration factors (C. F.) for "'Cs in rice grain note: Isotope solution; 277 cpm/ml. * See Fig . 1. RESULTS The data were expressed in terms of concentration factor (C. F.) in rice grain as defined as follows: Radioactivity (cpm) of the contaminant C. F. = accumulated into 1 g of rice grain R adioactivity (cpm) per 1 g of the initial nutrient solution Table 4. Concentration factors (C. F.) for 89Sr in rice grain note : Isotope solution; 2,631 cpm/ml * See Fig . 1. Fig. 2. Concentration factors in terms of treatments at various growth stages For 32P, as shown in Table 2 and Fig. 2, the concentration factors in both brown rice and polished rice were fairly constant, and kept higher levels than those for 137 Cs and 89Sr, throughout all growth stages. This fact indicates a very high mobility in plant body for phosphorus. For 137Cs, as shown in Table 3 and Fig. 2, the concentration factors obtained from the treatments P, B and F, except for the M, showed almost the similar values, although there was a gentle peak at the treatment B, and kept extremely high contamination levels in comparison with 89Sr. For 89Sr, as shown in Table 4 and Fig. 2, the concentration factors showed a sharp peak at the treatment F. growth stages and continued to grow until harvest. The harvested grains were radio chemically analysed and the concentration factors were calculated . The maximum uptake of 137Cs in the grains was found at the booting stage, while that of 89Sr was at the flowering stage . The Cs uptake was 400 times higher at the booting stage, and 30 times higher at the flowering stage than those with Sr. The growth stage dependency of the uptake of Sr and Cs was the most important factor for a selective enrichment of Cs in rice grains. The specific affinity of Cs to cell sap and that of Sr for membrane substances of rice grains probably caused a selective redistribution inside the plant body. * Orally presented at the annual meeting of the Japan Radiation Research Society , 1964. INTRODUCTION From standpoint of the environmental contamination with fission products released from nuclear detonation or industrial accidents in nuclear reactor operation, radio-contamination of rice with Sr and Cs is very important problem. The reasons are that rice is a staple food in Japan, and that both radioactive Sr and Cs have long lives and a high availability to crops. During a national radio-contamination survey for 90Sr and 137Cs in soils and rice, which has been conducted in the National Institute of Agricultural Sciences' in co-operation with the Institute of Public Health, it has been pointed out that the content of 137Cs in polished rice is 10 times or more higher than that of 90Sr1-4> For instance 50 pCi of 137Cs per kg and 4 pCi of 90Sr per kg were observed for the polished rice samples collected in 1962, in spite of the ratio of 131Cs to 90Sr being reported as 2.5: 1 in the original fall-out itself'). The mechanisms, governing such selectivity for Cs and Sr in the course of migration of these, elements from fall-out to polished rice, should be studied as one of the most important prcblems of the contamination in food chain. It is well known that there are four pathways in grain contamination: (a) absorption from soil through root (b) plant-base absorption (c) foliar absorption and (d) floral absorption 6)7). The former is an "indirect contamination" and the latter three are "direct contamination". In this paper, root absorption of 89Sr and 137 Cs by water-cultrured rice plant was dealt with. For the sake of comparison, 32P was also applied to the plants, because phosphorus is one of the indispensable elements for plants and its behav iour has been well studied. The culture solution was adjusted to pH 5.5 with HC1 or NaOH, and was renewed every 5 days. The plants were treated with radioisotopes in a green house as schematically shown in Fig. 1. At each stage of the plant growth, 32P was singly applied into a pot, whereas 89Sr and 137Cs were simultaneously added to a pot, with nutrient sol ution in all cases. After isotope treatments the plants were once removed from pot, and the roots were rinsed three times with tap water. And then the plants were transferred to another . pot to be continued to grow under ordinary water culture conditions until harvest. Completely matured rice grains (rough rice, 34g per pot in average) were dried at room temperature for about 10 days and were then husked . The brown rice was polished at the 90% milling rate . Two grams of the polished rice and brown rice were ashed respectively, in an electric muffle furnace by raising the tempera ture gradually up to 450°C and kept at the same temperature for 3 hrs. 89Sr was determined on the basis of the method of Kodaira ,8) and 137 Cs analysis was made by the method recommended in the text book9> (refer to another paper too4)). The procedure is briefly outlined in the appendix. According to this pro cedure, neither cross contamination possibly arising from 89Sr and 137Cs nor inter ference by the constituents of rice grain was observed, as examined respectively in the previous paper10) and in the appendix of this paper . 32P was precipitated as ammonium phosphomolybdate by usual analytical method . (3-radioactivities of 32P and 137Cs were measured by a conventional GM method, whereas for 89Sr a 47r gas flow low background counter was used. RESULTS The data were expressed in terms of concentration factor (C . F.) in rice grain as defined as follows: Radioactivity (cpm) of the contaminant C. F. = accumulated into 1 g of rice grain R adioactivity (cpm) per 1 g of the initial nutrient solution For 137Cs, as shown in Table 3 and Fig. 2, the concentration factors obtained from the treatments P, B and F, except for the M, showed almost the similar values, although there was a gentle peak at the treatment B, and kept extremely high contamination levels in comparison with 89Sr. For 89Sr, as shown in Table 4 and Fig. 2, the concentration factors showed a sharp peak at the treatment F. Among the indispensable nutrients, although the discrimination is significant, K is still only one element which has a relatively high similarity to Cs in the behaviour inside the plant body. Numerous studies on the relationship between Sr and Ca in soils and plants have been reported5>s>13>19 zap In most of these studies, a very high similarity of Sr to Ca has been revealed. 2. Growth stage dependency of the uptake of Sr and Cs Generally in plant body, the most metabollically active parts are young organs at each growth stage. Therefore the leaves developed in early growth stage accu mulate various ions very actively. Meanwhile when they become old, other organs emerge and develope to take the place of the most active parts. Conse quently, with the progress of plant growth, the ions accumulated previously in the leaves in early period are redistributed more or less to newly developed organs, finally to grains. Whereas the ions absorbed in late period can be translocated rather directly to grains. According to Takahashi25>, K is mainly accumulated by rice plant in early growth stage, in contrast with the accumulation of Ca which shows a maximum just before flowering stage. In this experiment as shown in Fig. 2, the maximum accumulation of Cs in the grains was resulted from the treatment B (booting stage), while that of Sr was from the treatment F (flowering stage). These patterns of ion uptake were in good accordance with those which was expected from Takahashi's pointing out, taking account of the similarities of Cs to K and Sr to Ca. It seemed that the plant absorbed a large quantity of Cs in early period and then redistributed it into rice grain, but could not absorb it so much in late period due to the decrease in its absorption power. On the contrary the same plant seemed to absorb only a small amount of Sr in early period because of its poor absorption power ; in addition, the redistribution might be difficult by the cause of the affinity of Sr for membrane substances as discussed later. But in late period To compare the degree of accumulation of Cs and Sr in rice grains, the ratio of the concentration factors for these elements was calculated and shown in, Table 5. The ratio of 400: 1 for brown rice was resulted from the treatment B. The ratio of 30: 1 was resulted from the treatment F, by which a maximum accumulation of Sr took place. This experimental fact can be a strong evidence for the explanation of the predominance of 137Cs to 90Sr which has been observed in results of the routine work of radio-contamination survey. Distribution of Sr and Cs in rice grain According to Ozaki26>, polished rice contains 0.13% of P, 0.12% of K and 0.014% of Ca. At present time the value of Ca should be written as 0.006% according to a recent work27>. In any way the content of K is about 10 to 20 times higher than that of Ca in polished rice. If discrimiating function of Cs vs. K and Sr vs. Ca is neglected, in other words, if Cs and Sr behave quite equally to K and Ca respectively, ten to twenty-fold accumulation of Cs against Sr can be expected. This idea also can be one of the interpretations for the predominance of Cs to Sr in polished rice. On the other hand, as shown in Table 6, the concentration factors were com pared in respect of brown rice and polished rice. The ratios were fairly specific to each element (2: 1 for Cs and 3 : 1 for Sr), regardless with the period of treatment. 4. Mechanism of contamination through roots The pathways of 137Cs and 90Sr from soil to rice grain through roots can be outlined in Table 8. Fall-out materials containing about 2.5 of 137Cs and 1 of 90Sr5) are accumulated into soil (refer to Kodaira's previous paper 23)) . Fixation onto soil takes place es pecially significantly for 137CS4)24)28). The ratio of 137Cs to 90Sr present in soil as an exchangeable form (plant available form) becomes different from that of original fall-out materials. In fact Japanese soils collected in 1964 were observed to contain 147 pCi 137Cs/kg and 320 pCi 90Sr/kg, the ratio being 1:2 .** As pointed out by Tensho et all"), if the soil is flooded for the cultivation of lowland rice, a part of 137Cs can be liberated through a replacement action of NH 4+ ion formed under an anaerobic condition. Also as reported by Nishigaki et al 29)30) , the physiological activity of rice root is highly dependent to soil conditions and plant growth, for instance the oxidation-reduction level of the soil, and the distri bution of underground root system in both longitudinal and horizontal directions . In addition to these processes mainly induced by soil conditions , the aforeme ntioned plant nutritional processes accelerate very much a selective enrichment of 137Cs in rice grains . * In this experiment the weight of bran was 109 of that of brown ri ce. The extent of con tamination per unit weight of bran was very high. ** To be published .

v3-fos

2020-12-10T09:04:12.710Z

1973-04-01T00:00:00.000Z

237229161

s2

Effects of Patulin and Method of Application on Growth Stages of Wheat When a single, 100-μg/ml application of patulin, produced by Penicillium urticae Bainier, was applied to growth stages 7, 9, 10, and 10.1 (Feekes scale) of Lee spring wheat (Triticum aestivum L.), decreases in internodal elongation, floret number, seed weight, and seed number were observed. Yields were reduced according to the proximity of application prior to heading. Application of patulin to the soil in crystalline form and dissolved in aqueous solution were also investigated, and the solution method of application was found to be the treatment of choice. A single exposure of growing wheat plants to patulin can produce yield reductions similar to those observed in stubble-mulch farming. Penicillium urticae Bainier, a fungus which produces patulin, has been found in large numbers in the soil in the Great Plains area. In some cases, the P. urticae population comprises 90% of the total fungal population in stubble-mulch wheat farming (J. R. Ellis and T. M. McCalla, Unpublished data). Wheat roots have been found to stimulate the growth of P. urticae over other fungi species (6). Other soil fungi have been reported to produce patulin; however, primarily, P. urticae has been found where plant residues and mulches have been left near the soil surface (11,12,14). The toxicity of patulin has been demonstrated on animals, plants, fungi, and bacteria. In the early 1930's, patulin was used as a broad-spectrum antibiotic but was found to be toxic to humans; consequently, its use was discontinued. In recent years, interest has been centered on its toxicity to plants. It was implicated in problems concerned with apple seedling transplants (3,4). The toxic effect of patulin has been observed on young seedlings, germinating seed, isolated plant tissue, and plants which had continuous applications of patulin until maturity. The effect of patulin on root development, cell division, cell wall development, and enzyme inhibiton has also been reported (10,15). The effect of a single patulin application on wheat plants grown to maturity or the effect of the method of its application has not been demonstrated previously. In the experiments reported here, patulin was applied at specific growth stages to simulate the effect of shortduration P. urticae Banier blooms and subsequent patulin production on wheat plants. Two application methods were used to compare methods being used in research investigations. MATERIALS AND METHODS Patulin used in experiment. The patulin used in this experiment was produced and purified in the laboratory from cultures of P. urticae Bainier isolated from stubble-mulched plots (13). Purity of the antibiotic was verified by using thin-layer chromatography, melting point, and infrared spectroscopy. Soil type. Holdrege silt loam from North Platte, Nebraska, was used in the pot experiment. Waterholding capacity of the soil at one-third bar is 24%. Physical and chemical characteristics of this soil have been described by Norstadt and McCalla (12). Experimental procedure. Thirty-five hundred grams of oven-dried soil was placed in 3.3-liter plastic pots with a layer of 100-mesh nylon cloth covering the drain holes. Optimum fertilizer for wheat production was determined by soil tests and appropriate compounds were mixed with the soil. The following rates of the elements N, P, Mn, Zn, and Fe, respectively, expressed as millimoles per killigram of soil, were added to the soil: 3.57, 0.352, 0.046, 0.061, and 0.082. Lee spring wheat (Triticum aestivum L.) was pregerminated for 3 days, and 7 seedlings were placed in each pot at 2.0-cm depth within a 10-cm circle. Pots were watered to moisture-holding capacity twice weekly with distilled water, Patulin was applied to the soil to give a concentration of 100 ,g/ml (oven-dry basis) (650 gmol/kg). The treatments were: control, Feekes stages 7, 9, and 10 (8) with one aqueous solution application; and Feekes stages 7, 9, 10, and 10.1 with one crystalline application. Three replicates per treatment were used. The solution treatment was applied with a hypodermic syringe and needle in four places around each plant 3.8 cm apart, 2.54 cm from the plant, and 2.54 cm deep. The crystalline application was applied in 0.8-cm holes bored 2.54 cm deep in four equally spaced locations, 3.8 cm apart and 2.54 cm from each plant. After patulin application, the pots were brought to field capacity with distilled water. Pots were placed in an ISCO E-2 growth chamber in three randomized blocks, with one replicate of each treatment in a block. Pots were moved each day so that each treatment was rotated in the block every 8 days. Temperature cycles in the growth chamber were based on Agronomy Farm (Lincoln, Nebraska) averages of bare and bromegrass-covered soil at a depth of 10.16 cm, from April through July 1966 (17). The plants were harvested at maturity (113 days) and observations were made on plant height, internodal elongation, straw weight, chaff weight, grain weight, floret number, and kernal number. The data were analyzed statistically. RESULTS Internodal elongation was markedly affected by the time of patulin application ( statistically significant) between the second and third node, but normal growth occurred thereafter. A decrease was noted in total seed yield (Table 3), indicating a residual effect. In contrast, the crystalline patulin application to stage 7 reduced the growth of the third and fourth internodes (not statistically sigificant) but not the second internode length. A significant reduction in floret number was observed. Solution application to stage 9 wheat plants did not affect second internode growth, but a reduction was noted in the third and fourth internodes and the stem between the last node and head ( Table 2). Stage 9 treatment significantly reduced seed number and total yield. Crystalline treatment reduced fourth to fifth internode growth but not the third to fourth internode or the fifth node to head and also significantly reduced the seed yield. Stage 10 application of solution induced the greatest reduction in seed weight, number, total yield, floret number, and chaff weight. The grain yield (Table 3) was reduced to 0.1 of the control and 0.2 of any of the crystalline applications in this experiment. Stem elongation of the fourth internode and of the upper stem was significantly reduced. The crystalline application to the same stage significantly reduced seed weight, total yield and fourth internode elongation, and elongation between fifth node and head. Table 2. Numbers on the control bar (a) indicate node location. Since stage 10.1 application treatment is made after stem elongation, the crystalline application had no effect on this process. However, the greatest reduction of seed weight and total yield was exhibited by this crystalline application. DISCUSSION The patulin solution application method affected the particular plant growth phase at the time of application, and inhibition was observable for several phases of plant elongation. A residual effect was noted as final wheat yield was reduced with solution treatment applications. Yield reductions increased as patulin in aqueous solution was applied nearer to heading and seed production stages. There was a steady decrease in the seed number, seed weight, floret number, chaff weight, and straw weight. In contrast, the crystalline application was usually not effective on the particular growth stage to which it was applied. The treatment effects were not as great when the crystal treatments were used. The comparison of solution and crystalline applications of patulin showed that patulin must be in solution to produce an effect on the immediate growth phase treated. The crystalline application reduced stem elongation, but the effect, compared to solution application, was delayed. The crystalline form did not reduce the seed weight and number as much as the solution application. The crystalline application had much larger least significant difference (LSD) values, which indicated the greater variability of this type of treatment. The decreased effect of the crystalline treatment could be explained by two factors. The crystals do not dissolve rapidly, thus lowering the patulin concentration in the soil solution. In field treatments, Norstadt, Ellis, and McCalla (Unpublished data) found crystalline patulin in the soil for several weeks after 13 10 13 55 0.42 LSD (P = 0.01) 18 14 18 76 0.52 a LSD, Least significant difference at 5 and 1% probability. One asterisk indicates significance at the 5% level; two asterisks indicates significance at the 1% level. application. Patulin has a half-life in the soil of 24 h (11), and this would also decrease the soil solution concentration. Patulin affects cell division (1, 16), which would cause the reduction in stem elongation noted in this experiment. Cereal crops which suffer damage during these rapidly growing phases often show reduced yields. The action of patulin may take place in several ways. However, a possible mechanism has been shown. Dickens and Jones (5) found that patulin can inactivate S-H groups. They showed that a 1: 1 relationship existed between the number of S-H groups blocked and the number of patulin molecules. This reaction would interfere with enzyme activity and protein synthesis (1, 2, 7), thus explaining the effect of patulin on internode length. The blocking action of important enzymes and interference with cell division processes can reduce plant vigor and yield. This study showed that patulin does have a marked effect on plant development even when plants had only one exposure to patulin. A single exposure to patulin can produce the toxicity problems noted in stubble-mulch farming (9, 11) by reducing plant vigor and final grain yield.

v3-fos

2020-12-10T09:04:17.157Z

1973-07-01T00:00:00.000Z

237235410

s2

Gamma Radiation Inactivation of Coxsackievirus B-2 The radioresistance of coxsackievirus B-2 was studied when the virus was suspended in Eagle minimal essential medium, distilled water, cooked ground beef, and raw ground beef and irradiated at various temperatures in a cobalt-60 gamma radiation source. The number of surviving viruses at given doses of radiation was determined by a plaque assay system. All destruction curves indicated a first-order reaction. When the virus was irradiated in minimal essential medium at temperatures of -30, -60, and -90 C, D values (in Mrad) were 0.69, 0.59, and 0.64, respectively. When the virus was suspended in water and irradiated at -90 C, the D value was 0.53. Cooked ground beef containing the virus was irradiated at temperatures ranging from 16 to -90 C. The D values were 0.70 (16 C), 0.76 (0.5 C), 0.68 (-30 C), 0.78 (-60 C), and 0.81 (-90 C). Raw ground beef containing the virus was irradiated at -30, -60, and -90 C, and the D values were respectively 0.75, 0.71, and 0.68. The D values indicate that the rate of viral inactivation was dependent on the suspending menstrum. The use of gamma radiation has been advocated as a means of obtaining sterile, organoleptically acceptable raw and cooked foods that require no refrigeration storage and have a long shelf life (10). Safety, enzymatic changes, and microbiological considerations are factors that affect any such food processing system. Present systems utilize low temperature heating followed by irradiation of the frozen (-20 to -40 C) food product. The limited information on inactivation of viruses in foods prompted a study to determine the safety of such foods, and to obtain base line data on viral inactivation. Coxsackievirus B-2 was suspended in Eagle minimal essential medium (MEM) in Hanks balanced salt solution (8,13), distilled water, and ground beef and 14 irradiated at temperatures ranging from 16 to -90 C. The results indicate that the rate of viral inactivation is dependent on the suspending medium. MATERIALS AND METHODS Virus. Coxsackievirus B-2, Ohio 1, VR-29 was passaged in continuous cell cultures (Vero) from African green monkey kidney (Cercopithecus aethiops; reference 32) by using L-15 medium (17) supplemented with 2% fetal bovine serum and 0.07% NaHCO,. Cell monolayers showing advanced cytopathic effects were frozen and thawed three times, and the virus was harvested. The harvest was clarified by centrifugation for 15 min at 1,060 x g at 4 C. The virus titer was determined, and the harvest was dispensed into borosilicate glass ampules; the ampules were flame-sealed and stored at -60 C. This procedure provided a virus pool of known titer for use throughout the investigation. Virus assay. A plaque forming unit (PFU) assay system was used as previously described (30). This system consisted of an agar-medium overlay and monolayer Vero cell sheets (45 cm2) in 6-oz (approximately 0.17 liter) bottles. Radiation soure. A 2,800-C, cobalt-60 gamma radiation source was used. The irradiation chamber consisted of 36 cobalt-60 containing pins immersed in 4 meters of deionized water. An overhead device was used to position and rotate a cylinder that housed a rig containing the samples of virus materials. Temperatures were maintained during radiations by metering gaseous nitrogen from a liquid nitrogen tank into the irradiation rig. Vertical positioning on the rig of the tubes containing the material being irradiated permitted delivery of eight different doses in a given time. These doses, in Mrad, were 0.43, 0.81, 1.35, 1.88, 2.04, 2.21, 2.67, and 2.82. The Mrad at each tube position in the radiation rig were determined by cobalt glass dosimetry. During dosimetries, cobalt glass strips were placed in tubes containing MEM plus 2% fetal bovine serum, distilled water, cooked ground beef, and raw ground beef. No significant differences were seen in the delivered doses among the menstra. MEM and water irradiation samples. The virus was diluted to approximately 10,000 PFU/ml in MEM (pH 7.0) containing 2% fetal bovine serum, or in distilled water. Next, 1.2 ml of the virus-containing liquid was pipetted into 13 by 53-mm borosilicate glass tubes, which were then flame-sealed. Tubes containing the virus material were equilibrated at the temperature at which they were to be irradiated. Temperatures during irradiation runs were -30, -60, or -90 C. A 1-ml sample from each tube was assayed for viral PFU. Ground beef. The ground beef used in this study was obtained from one animal and was processed, ground, and canned under clean-room conditions at Natick Laboratories, Natick, Mass. The beef was U.S. grade choice, boneless chuck that was ground through a 3A6-in. (approximately 0.46 cm) plate twice; the fat content was 25%. Twenty-six cans containing 400 g each of meat were vacuum sealed and frozen at -30 C. A similar number of cans were prepared with the same batch of meat, sealed, heated to 80 C, and held at this temperature for 15 min. These cans were then stored at -30 C. The unheated meat was designated as raw ground beef, and the heated meat was designated as cooked ground beef. All cans of meat were held at -30 C until used. Meat irradiation samples. One-gram portions of the meat containing approximately 10,000 PFU of coxsackievirus B-2 were placed in individual 13 by 53-mm borosilicate glass tubes, which were then flame-sealed (29). The sealed tubes were held at -60 C until used in an irradiation run. Representative samples were assayed for viral PFU to ensure even distribution of coxsackievirus B-2 among the 1-g portions of the meat prior to irradiation. Meat-virus samples held at a given temperature during irradiation were allowed to equilibrate at this temperature before being lowered into the cobalt-60 well. Four samples were used at each dose in each irradiation run. When an irradiation run was completed, the samples were stored at -60 C until assayed along with positive and negative controls of the same run. All control samples received treatment identical to that received by the irradiated samples, except that the control samples were not irradiated. Viral assay of meat. A method previously shown to give satisfactory virus recovery from ground beef was used (29). This method consisted of making meat slurries by shaking 1-g portions of ground beef in MEM (pH 8.5) and clarifying these slurries by passing the liquid portion through cheese cloth. The filtrate from each 1-g sample was individually titrated by log,0 dilution steps when necessitated by the anticipated PFU number. One-milliliter portions from each dilution were assayed in each of four 6-oz (approximately 0.17 liter) bottles containing Vero cell monolayers. This quadruplicate assay was done for 1-g portions of meat irradiated at lower dose levels; i.e., 1.35 Mrad and lower. At doses of 1.88 Mrad and higher, all of the meat filtrate was assayed. This was done by assaying equal portions of the filtrate in each of four 6-oz (approximately 0.17 liter) prescription bottles containing monolayers of the monkey kidney cells. Calculation of D values. The D value is the dose of gamma radiation that reduces the viral population by 90%. D values were calculated by the following formula. A linear model was assumed, and the parameters P. and 6, were estimated for each run. The model was: Y = F. + P, X + (, where Y = log,. plaque count, P. and , = true but unknown regression coefficients, X = gamma radiation in Mrad, and e = experimental error. This model was used to obtain an estimate for viral radioresistance, and goodness-of-fit tests were performed to confirm the choice of model. The value of one over the estimate of the slope (a,6) and its confidence intervals are the D value in Mrad and its confidence intervals. RESULTS A series of experiments was done to determine the effect of suspending medium and temperature on coxsackievirus B-2 during irradiation. The radioresistance data for coxsackievirus B-2 in MEM plus 2% fetal bovine serum, distilled water, and ground beef are presented in Table 1. All data were linear over the range studied in that more than 90% of the sum of squares was explained by the model. An example of plotted data is presented in Fig. 1. There was little change with temperature among D values of coxsackievirus B-2 irradiated in frozen MEM with 2% fetal bovine serum or in ground beef. The data from the irradiation runs on the virus in cooked and raw ground beef were analyzed to determine if the slopes of the regression lines were equal (21). None of the variance-ratio values, computed to test the hypothesis that the slopes among runs were equal, exceeded the critical value at a = 0.01. These analyses indicated that there were practically no differences among the slopes of the curves for irradiation runs at the given temperatures investigated. VOL. 26,1973 bThese irradiation runs at 0.5 C were previously reported (28). The data are included here for comparison purposes. DISCUSSION Coxsackievirus B-2 was suspended in MEMserum medium, distilled water, cooked ground beef, and raw ground beef and irradiated at different temperatures to determine the effect of suspending medium and temperature on viral inactivation. The limited number of observations in the MEM irradiation runs precluded tight 99% confidence limits. However, computed D values indicated no large difference in the rate of viral inactivation among irradiation runs at -30, -60, and -90 C. D values at the three temperatures were higher than the previously reported D value (0.45 Mrad) computed for the same virus in the same medium at 0.5 C (28). When the virus was suspended in water and irradiated at -90 C, the D value of 0.53 Mrad was significantly greater than the previously reported D value of 0.14 Mrad when the virus was irradiated in water at 0.5 C (28). The higher D values observed for the virus in the frozen material could be due to the inhibition of free-radical formation or to impeding of freeradical travel in the frozen material. The presence of free-radical scavengers, such as fetal bovine serum in the Eagle MEM appears to be related to the higher D value (0.45 Mrad) observed when the virus was irradiated at 0.5 C in this medium as compared to the D value (0.14 Mrad) of the virus irradiated at the same temperature in distilled water (28). No trend in D values with temperature was seen when the virus was suspended in raw ground beef and irradiated at temperatures ranging from -30 to -90 C, nor was any trend in D values with temperature noted in cooked 16 SULLIVAN ET AL. APPL. MICROBIOL ground beef irradiated at temperatures ranging from 16 to -90 C. Apparently, there is enough free radical scavenging by proteins and other substances in the ground beef to eliminate or reduce the secondary effects of radiation. This is clearly illustrated when the D value of 0.76 Mrad in cooked meat irradiated at 0.5 C is compared with the D value of 0.14 Mrad in water irradiated at the same temperature. If the 12-D concept is used to calculate a food process, the dose required for gamma-radiation sterilization is 12 times the D value. As an example: cooked ground beef containing coxsackievirus B-2 irradiated at -30 C requires 12 times 0.68 or 8.16 Mrad. The D values reported apply only to the menstra, temperatures, and virus investigated. There are variations in the composition of foods and other viral suspending media; however, the reported D values for coxsackievirus B-2 could be utilized as a starting point for viral inactivation studies.

v3-fos

2016-05-04T20:20:58.661Z

1973-04-15T00:00:00.000Z

2457518

s2

A genetic base for estimating the genetic transmitting ability of dairy bulls in populations undergoing genetic change SUMMARY The three major problems encountered in the genetic evaluation of dairy bulls in today's dynamic dairy cattle populations are discussed. These are : a continuing increase in the average genetic merit of the population, an average rate of genetic increase that fluctuates over time, and the formation of genetic sub-populations. Neither a fixed genetic base nor a moving genetic base (presently used by U. S. D. A.) is suitable over a long term basis for calculating genetic evaluations which are unbiased over time and acceptable to people in the field. A new concept, a Stepwise Genetic Base, is described which will enable unbiased comparisons of bulls evaluated at different times and will enable all genetic evaluations to be expressed relative to the popula-tion's average genetic merit at any point in time. U. S. D. A. will adopt this new concept in revised sire summary procedures and most other countries could use the same procedure. INTRODUCTION . Some form of a herdmate comparison is used in virtually all countries that are evaluating the genetic transmitting ability of dairy bulls. Most, if not all, of these procedures are based on the assumption that there is no genetic trend in the dairy cattle population. This assumption was reasonable when adopted, because there had been little genetic progress from sire summary methods used before. However, the success of the herdmate comparison method is apparent to all involved with the genetic improvement of dairy cattle. In fact, the effectiveness of the herdmate comparison has invalidated the basic underlying assumption of nog enetic progress. A major problem at present is how to increase the rate of genetic progress in cattle populations that are becoming continually more difficult to evaluate because of this increase in genetic merit. Practically all countries are faced with essentially the same difficulties inevaluating dairy bulls for genetic merit. Therefore, there should be a general set of principles for overcoming these difficulties. PROBLEMS OP' GENETIC TREND COMMON TO ALL COUNTRIES Probably the most common and most basic problem with which we are faced today is attempting to estimate genetic transmitting abilities in populations in which the average genetic merit is increasing. Of course, the rates of increase may differ considerably from country to country. It is almost certain that all countries having organized programs for genetic improvement of dairy cattle are showing at least some increase in genetic merit at the present time. This problem is compounded even more by the fact that the rate of genetic progress in each country probably changes from time to time because of causes over which most of us have very little control, for example : (i) by chance, several outstanding bulls may be identified in rapid succession followed by a period where fewer outstanding bulls are found ; ( 2 ) the use of outstanding bulls may be tenpered from time to time by the heavy use of bulls with popular names or other popular appeal but which do not have truly superior genotypes, and ( 3 ) errors that are due to sampling variation in the daughters, or from other causes, may result in occasional serious overestimates or underestimates of genetic transmitting ability of individual bulls. To accentuate the problem to an even greater degree most of us no longer deal with genetically uniform and homogenous populations. We are actually dealing with a group of sub-populations that vary in average genetic merit and in the rate of genetic change. For example, in the United States we have many sub-populations that have developed because of the following : 1 . Not all of the artificial insemination (AI) organizations are fortunate enough to have bulls of the same genetic superiority. Therefore, dairymen in certain regions have easier access to the best bulls than do those in others. 2 . Some regions contain higher concentrations of registered or purebred cows than others. On the average, the registered herds probably have higher genetic merit than do the nonregistered herds although the superiority varies from region to region and many nonregistered herds are genetically superior to many registered ones. 4 . Dairymen in some areas of the country are more highly business oriented. This trend generally causes heavier selection for total economic value, meaning primarily yield and a few other traits affecting the economic worth of each animal. 5 . There are vast differences throughout the country in the proportion of herds taking advantage of the dairy record keeping programs (D. H. I. A.). Cows in D. H. I. A. herds outproduce the nontested cows by approximately 4 o p. 100 in milk production. This higher production is certainly due to an important degree to the genetic superiority in these herds. Such superiority results from heavier use of the genetically superior AI bulls and more effective selection and culling practices within the herds. Some of the above influences have probably caused the formation of genetic sub-populations in other countries besides the United States. THE IMPORTANCE OF THE GENETIC BASE These three problems, a continual rise in average genetic merit, fluctuations in the average rate of genetic increase over time, and the formation of sub-populations, make accurate sire summaries, which can be compared over a long time, an extraordinarily difficult problem. This situation demands that we use sophisticated statistical, genetic, and computing techniques. In my previous report I gave a brief overview of two alternative sire summary methods that should aleviate these problems. It appears to those of us at U. S. D. A. that either of the methods -the Direct Comparison Method or the U. S. D. A. Modified Contemporary Comparison Method, should overcome these problems and provide accurate sire summaries on most bulls. However, my previous report intentionally omitted one major pointthe genetic base to which sire summaries are calculated. To make meaningful rankings of bulls over a long period of time, one must be able to compare estimates of the transmitting ability of the same bull summarized at different times, of different bulls summarized at the same time and of different bulls summarized at different times. In other words, one should be able to compare estimates of transmitting ability over a long period of time on the same basis. At the present time, this cannot be done with the U. S. D. A.-D. H. I. A. Sire Summaries and this same deficiency is probably true in many of the countries represented a tthis meeting. However, I would like to suggest a genetic base procedure whereby this can be done. I believe this procedure would be easily understood by people who use sire summaries in the field and who are not highly trained in animal genetics. In the simplest case there are actually only two types of genetic bases, fixed and moving. For reasons explained here, neither of these types of base is adequate under present conditions. FIXED GENETIC BASE The use of a fixed base under the conditions existing in most of our dairy cattle populations would result in the situation that is shown in figure 1 . It is true that, with a fixed base, comparisons can be made among bulls summarized at widely different times. It should probably be mentioned as a side-light that comparisons among bulls evaluated at different times will generally not be as accurate for ranking as will comparisons among bulls evaluated at the same time no matter what genetic base is used. If the sire summaries were to be used strictly by scientists, then the use of a fixed base over a long period of time would be satisfactory. However, most of the people who use this information in the field in all of our countries are not trained as scientists. In the U. S. these people still tend to look on Predicted Differences as a point-estimate of each bull's transmitting ability, rather than considering them as merely a method for determining differences among bulls. When a dairyman is asked what kind of bulls he uses, he seldom replies: « I use only those that rank one, two, three. » More often he will reply : « A bull must have a Predicted Difference for milk of at least + 1 ooo pounds ( 454 kg) » or he will say : « I will use a bull with a Predicted Difference as low as + 8 00 pounds ( 3 6 3 kg) if the bull improves these particular conformation traits in which my herd needs improvement. » The use of a fixed base over a long time interval would cause confusion among such dairymen because bulls that are well above breed average today would be only breed average or even below breed average bulls in the future. Yet their Predicted Differences would still be as high as originally. This can be seen in figure i where a bull with a genetic transmitting ability at level X is above the population average at point A in time, but is below the population average at point B in time. Many dairymen would probably fail to make an appropriate adjustment in the level of PD required for their herd even though bulls of higher genetic merit would continually become available. After an extended period of time, PD's would increase to a very high level and the base would have to be adjusted. It would cause much confusion and loss of confidence if after let's say 10 years or more, a tremendous change was suddenly made in the base. This would mean that evaluations of existing bulls might be lowered as much as 400 kg or more. It would be very dificult for many dairymen to adjust their thinking to such a drastic change in the base after so long a period of time. MOVING GENETIC BASE The use of a moving genetic base would result in the situation shown in figure a. This is actually what the U. S. D. A. uses at the present time. The genetic base is changed every year based on the latest updating of breed averages from the records we have received for use in the sire summaries. This means that sire summaries calculated one year cannot be compared without bias to those calculated in other years. Older bulls have higher evaluations than they should when compared to bulls evaluated more recently in time. This can be seen in figure 2 . One bull whose Predicted Difference is shown by the letter X was progeny-tested at point A in time and another (bull V)-W as tested at point B in time. Both have the same Predicted Difference. However, the bull tested at point B in time (bull Y) is genetically superior to the other bull (bull X) because his transmitting ability is expressed relative to a population of higher genetic merit. This situation causes artificial insemination organizations and dairymen to continue to use older bulls which may actually be genetically inferior to many younger bulls. The moving genetic base also causes repeated sire summaries Annales de G6n6tique animale. -1973 . on a bull to continually decrease or « erode » over time as additional daughters are added. Dairymen tend to lose confidence in the summaries when this happens and those who do not understand the summaries very well continually wonder what is wrong when bulls keep dropping in genetic merit. Also, we believe that it is almost impossible to use a moving genetic base and continually keep all summaries in perspective so that they can be compared over long periods of time. The only way this could be done would be to ; i) adjust all Predicted Differences of herdmate sires every time a sire summary is run, 2 ) resummarize all bulls for which anyone might wish information (i. e., for pedigrees) no matter how old, and, 3 ) discard all previous summaries on all bulls. It would be very costly to carry out these procedures and they would result in a waste of our already limited resources. STEPWISE GENETIC BASE Therefore, it appears that the most realistic approach to this problem, and the one which we intend to adopt as soon as it can be added to our sire summary system, is a combination of fixed and moving genetic' bases such as shown in figure 3 . We have labelled this'a Stepwise Genetic Base. This procedure would permit all summaries to be adjusted to the genetic base presently in use and would permit unbiased comparisons of sire summaries which have been calculated at widely different points in time. The genetic base to which all sire summaries are calculated would be changed periodically, possibly every 3 to 5 years. Changes at 5 year intervals are shown in figure 3 . These changes would be announced in advance and the notification of dairymen and others using the summaries could be carried out before the change so that they would be expecting it and would understand the reasons behind it. When this procedure is adopted, all summaries could be calculated to the present genetic base. Then, summaries for bulls from previous years could be adjusted to bases that would apply to those previous time periods before they are published. The latter procedure would have great public relations benefits in our country. It would eliminate the drastic drop that will likely occur in the Predicted Differences of many of the early AI bulls if their summaries are published relative to the present base. Sire summaries in the years rgy-75 would all be calculated to the genetic base 1970 . Then in 197 6 through ig8o, all summaries would be calculated to the genetic base 1975 . When the genetic base is changed all sire summaries that have been calculated previously could be adjusted to the new genetic base simply by subtracting the amount of change which had occurred between 197 o and 9175 . For example, assuming that the genetic base changed by 400 lbs ( 1 8 1 kg), then, bulls that had been evaluated + i ooo lbs (!-454 kg) at the 1970 base would be adjusted to a PD of + 6 00 lbs (+ 273 kg) at the 1975 base. Summaries for bulls used primarily in previous years could be adjusted to a previous genetic base in a similar manner. In order to make this system workable the present abbreviation for Predicted Difference, « PD o would have to be changed to include the genetic base to which each PD was calculated. Therefore, Predicted Differences calculated to the 1970 base would be labeled « PD, o » and PD's calculated to the 1975 base would be designated « PD, s ». In the same way, PD's for bulls used in previous years could be expressed to bases PD,, or PD... This procedure would have another advantage in the use of sire summaries for pedigree work or for other historical purposes. Most bulls eventually cease to be summarized either because they do not produce any new daughters or because some arbitrary cutoff is designated in the interest of economy of running sire summaries. Under this system when a bull is no longer being summarized he would have a permanent Predicted Difference which would contain the genetic base to which it was calculated. This final PD would go into permanent records and could be used in the future for comparisons with bulls summarized at other times by adjusting the Predicted Difference to whatever common base was being used to compare bulls at that time. This should make pedigree evaluations far more accurate and useful than they are at the present time, since those presently being used may contain serious biases because of genetic trend. As might be expected, the Stepwise Genetic Base will not solve all the problems associated with genetic trend. As with the moving and fixed bases, a single stepwise base may not be entirely appropriate for all genetic sub-populations within a country. There is still the relatively minor problem of genetic trend within the base periods causing some inaccuracies ; however, this will be much less than with our present procedures. In addition, it will be necessary to determine the amount of genetic progress that takes place from one base period to the next so that the genetic base can be changed by a realistic amount. Some additional research input into the sire summary program will be required. In spite of these relatively minor problems, the Stepwise Genetic' Base would result in a major overall increase in accuracy when compared to our present procedures. Needless to say, the adoption of such a revolutionary change would require a massive educational effort by those of us involved in dairy cattle genetics work ; however, I believe that there are many advantages to this system. If a strong educational effort were made before its adoption, then it would be readily accepted by those utilizing sire summaries in our country. In fact, it might be a strong tool to build much greater confidence in sire summaries than we now have among many users in the field. The additional cost in computing time would not be great even under our situation where upwards of 20 00 o bulls are evaluated each year and more than 12 ooo ooo lactation records are used during each sire summary run. It is hoped that adoption of this procedure would eliminate one of the most serious problems in the present U. S. Les trois problèmes majeurs rencontrés pour l'évaluation du niveau génétique des taureaux dans les populations actuelles de bovins laitiers en pleine évolution sont discutés. Il s'agit de l'élévation continuelle du niveau génétique moyen de la population, des fluctuations au cours du temps de cette augmentation, et de la formation de sous-populations génétiques. Un niveau génétique de référence fixé ou variable (utilisé actuellement par l'U. S. D. A.) ne conviennent ni l'un ni l'autre à long terme au calcul d'index qui ne soient pas biaisés dans le temps et qui soient acceptables par les praticiens. Un nouveau concept, le niveau génétique de référence en escalier, est décrit : il permettra la comparaison sans biais de taureaux testés à différentes époques et permettra à toutes les évaluations génétiques d'être exprimées par rapport au niveau génétique moyen de la population, à n'importe quelle époque. L' U. S. D. A. adoptera ce nouveau concept dans ses nouvelles méthodes d'indexation des taureaux : la plupart des autres pays pourrait se servir du même principe. ZUSAMMENFASSUNG EINE GENETISCHE BASIS BEI DER ZUCHTWERTSCH Ä TZUN& FÜR MILCH VON STIEREN IN POPUI,ATIONEN MIT GENETISCHEM TREND Die drei Hauptprobleme, die bei der Zuchtwertschätzung für Milch bei Stieren in den heutigen dynamischen Milchviehpopulationen entstehen, werden diskutiert. Dies sind : Eine kontinuierliche Zunahme des durchschnittlichen genetischen Niveaus der Population, eine durchschnittliche genetische Zuwachsrate, die mit der Zeit schwankt und die Bildung genetischer Sub-Populationen. Weder einer feste noch eine bewegliche (gegenwärtig im U. S. D. A. angewandt) genetische Basis ist über eine lange Zeitdauer geeignet für die Berechnung von Zuchtwerten, die über längere Zeiten frei von systematischen Fehlern und daher für Fachleute annehmbar sind. Ein neues Konzept, eine stufenweise genetische Basis, wird beschrieben, das erlauben wird, ungestörte Vergleiche von Stieren durchzuführen, deren Zuchtwert zu verschiedenen Zeiten geschätzt wird und auch erlauben wird, alle geschätzten Zuchtwerte jederzeit relativ zum genetischen Populationsmittel anzugeben. Das U. S. D. A. wird dieses neue Konzept in den revidiretene Stieren-Prüfungsverfahren annehmen und die meisten andern Länder könnten dasselbe Verfahren verwenden.

v3-fos

2018-04-03T04:42:52.493Z

1973-01-01T00:00:00.000Z

40081298

s2

The relationship between weight gain and free amino acid concentration of plasma and liver in rats fed a diet supplemented with various amounts of lysine. This study was conducted to determine the effect of feeding graded levels of dietary lysine on weight gain and free amino acid concentra-tions in blood plasma and liver of weanling rats. Animals were fed on diets containing 11.6% wheat gluten (equivalent in nitrogen to a 10% casein diet) supplemented with graded levels of L-lysine HCl (0 to 10%) for 14 days. An outline of the results obtained follows: 1) Maximum weight gain was observed with the groups fed the lysine supplement in the 0.64 to 1.8% range. Growth declined with further in-crease of dietary lysine. 2) Blood and liver were sampled after decapitation 6 hr after the final feeding on the 14th day, and amino acid concentrations were determined. Plasma lysine concentrations rose rapidly as dietary lysine increased and reached the maximum level with the 1.8% lysine supplementation. With the large supplement diet, the lysine con-centrations maintained a plateau. Plasma threonine levels were high when dietary lysine was low. 3) Similar responses of lysine and threonine concentrations in liver to the increase of dietary lysine was also observed, but the effect was markedly less than those of plasma. Most other amino acids in plasma and liver were nearly unchanged even when the dietary lysine was varied over a wide range. This study was conducted to determine the effect of feeding graded levels of dietary lysine on weight gain and free amino acid concentra tions in blood plasma and liver of weanling rats. Animals were fed on diets containing 11.6% wheat gluten (equivalent in nitrogen to a 10% casein diet) supplemented with graded levels of L-lysine HCl (0 to 10%) for 14 days. An outline of the results obtained follows: 1) Maximum weight gain was observed with the groups fed the lysine supplement in the 0.64 to 1.8% range. Growth declined with further in crease of dietary lysine. 2) Blood and liver were sampled after decapitation 6 hr after the fi nal feeding on the 14th day, and amino acid concentrations were determined. Plasma lysine concentrations rose rapidly as dietary lysine increased and reached the maximum level with the 1.8% lysine supplementation. With the large supplement diet, the lysine con centrations maintained a plateau. Plasma threonine levels were high when dietary lysine was low. 3) Similar responses of lysine and threonine concentrations in liver to the increase of dietary lysine was also observed, but the effect was markedly less than those of plasma. Most other amino acids in plasma and liver were nearly unchanged even when the dietary lysine was varied over a wide range. the liver was cleared of all blood. The liver was quickly removed, weighed, and then deproteinized in glass homogenizer with a 5-fold amount of 3% sulfosalicylic acid solution. An aliquot of deproteinized filtrate of each group was pooled, and diluted with equal volumes of the pH 2.2 buffer for amino acid analysis. Amino acids were determined by ion-exchange chromatography using a JEOL Model JLC-5AH amino acid analyzer.* A standard amino acid mixture was included in each automatic run of 6 to 12 samples. Serine, glutamine and asparagine were not separated by this chromatography, and therefore this frac tion was reported as serine (5). RESULTS Average weight gain and feed efficiency (gain/feed ratio) of rats fed graded levels of dietary lysine are shown in Table 3. The weight gain increased rapidly served when dietary lysine content was 0.14% (basal lysine-deficient diet) to 0.22 (0.1% lysine HCl addition), then the concentration rose rapidly with increasing level of dietary lysine (2.0% lysine HCl addition), but did not result in any further increase thereafter. On the other hand, the plasma threonine concentra tion was exceptionally high when the dietary lysine was low, but the plasma threonine concentration decreased sharply when the dietary lysine levels were 1.0% or more. Similar responses were also observed for liver free lysine and threonine concentrations, but the ranges of the changes were markedly less than those of plasma. Most other amino acid concentrations of plasma and liver were either unaffected or only slightly changed over the entire range of the dietary lysine levels, except that the serine tended to be lower with the increase of dietary lysine. DISCUSSION In this experiment, the maximum weight gain and gain/feed ratio were observed at a dietary lysine level of 0.64%. This value for lysine requirement is in fair agreement with those reported by MCLAUGHLAN and ILLMAN (7) The results of weight response also indicate that a great growth-depression occurs in rats fed a high-lysine diet, as well as in animals receiving lysine-deficient diets. The shape of the response curve differed from those obtained previously (4) in rats fed by 10% casein diet containing graded levels of methionine and glycine singly. Many studies on animals (1-3, 7, 12-15) and men (16)(17)(18) have demonstrated that plasma amino acid concentrations can be utilized as response criteria for estimating the amino acid requirements. ZIMMERMAN and SCOTT (2) reported that plasma lysine of chicks does not accumulate when the dietary lysine is less than that required for maximum weight gain, at which point the plasma lysine level rises linearly. In this study, however, it was observed that the plasma lysine level at low dietary lysine content remained flat only within the narrow range from 0.14 to 0.22% which level was significantly lower than that of maximum growth. The response curve of plasma lysine in the present experiment showed a sigmoidal shape, and was rather similar to those observed by MORRISON et al. (1) and STOCKLAND et al. (3) when rats were fed graded dietary lysine ad libitum for a long-term period, whereas MCLAUGHLAN and ILLMAN (7) observed that the plasma lysine levels elevated almost linearly with increase of dietary lysine in a short-term study of rats. It may be suggested that the shape of the response curve is appreciably influenced by the feeding method and the experimental period. MORRISON et al. (1) reported that plasma lysine concentration reached a maximum at about the 1.0% dietary lysine level, whereas maximum growth was obtained at the 0.8% dietary lysine level. Our results indicated that the maximum weight gain was obtained at the 0.64% dietary lysine level, much lower than the approximately 1.8% level at maximum plasma lysine concentration. Thus the plateau point of plasma lysine concentration was not necessarily con sistent with the maximum growth point. YOUNG et al. (18) recently indicated that in men plasma lysine levels do not appear to be a response criterion to assess the human maintenance requirement for this amino acid. The present authors' findings on the inverse relationship between plasma lysine and threonine levels confirm those of other investigators (1,19). Threonine has also been shown to increase when tryptophan intake is low in men (16). MORRISON et al. (1) suggested that plasma lysine/threonine ratio may be useful as a very sensitive indicator of lysine nutrition, and that the ratio approaches unity when the dietary lysine level is adequate for growth. As shown in Fig. 1, lysine/threonine ratio plotted against dietary lysine level increased almost linearly as the lysine in the diet was increased, and above approximately 1.8% of dietary lysine maintained a plateau. This bending point corresponded to the upper limit of the maximum gain, like that of the plasma lysine concentration. Threonine concentration in liver of rats receiving lysine-deficient diet also increased. This phenomenon may be related to the increase of nonutilizable threonine for protein synthesis in tissues and the decrease of liver threonine dehydrase of rats fed lysine deficient diet. The data of ZIMMERMAN and SCOTT (2), and OHNO and TASAKI (20) in hens indicate that other amino acid concentrations in plasma can also undergo various alterations in birds fed diets containing graded levels of lysine, whereas the results of this study in rats indicate that other amino acid concentrations in plasma and liver showed no marked and consistent changes, except serine. This difference might be related, in part, to the difference in species and dietary conditions. Even when dietary lysine level was high above 1.8% further elevation of the lysine in plasma and liver did not occur. This may be due to an induction of lysine catabolyzing enzyme in liver, with resulting increased degradation of the excessive lysine intake tending to maintain a constant plasma lysine level. Indeed, with the use of radioactively labeled lysine, BROOKES et al. (8) recently found that the oxidation of lysine does not increase when dietary lysine intake is low but elevates linearly with greater lysine intake. The response of liver free lysine concentration for the graded level of dietary lysine was considerably less than that of the plasma. It seems improbable, there fore, that the liver lysine level can be utilized as a criterion of nutritional adequacy. In this connection, the report of PAWLAK and PION (12), who observed in rats that the free lysine level of muscle was even more sensitive for lysine intake than plasma lysine level, is of interest. We wish to thank Miss Taeko Hashimoto and Mr. S. Takagi for their care of the animals and technical assistance.

v3-fos

2020-12-10T09:04:17.253Z

1973-11-01T00:00:00.000Z

237233554

s2

Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives Oleuropein, the bitter glucoside in green olives, and products of its hydrolysis were tested for antibacterial action against certain species of lactic acid bacteria involved in the brine fermentation of olives. Oleuropein was not inhibitory, but two of its hydrolysis products, the aglycone and elenolic acid, inhibited growth of the four species of lactic acid bacteria tested. Another hydrolysis product, β-3,4-dihydroxyphenylethyl alcohol, was not inhibitory. The aglycone of oleuropein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCl; 150 μg of either compound per ml prevented growth of Lactobacillus plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 of 17 species of bacteria screened, none of which were lactic acid bacteria. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included four species of lactic acid bacteria and other gram-positive and gram-negative species. Neither crude preparation was inhibitory to growth of the seven species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 C) olives prior to brining renders them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined. pein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCl; 150 ug of either compound per ml prevented growth of Lactobacillus plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 of 17 species of bacteria screened, none of which were lactic acid bacteria. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included four species of lactic acid bacteria and other gram-positive and gram-negative species. Neither crude preparation was inhibitory to growth of the seven species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 C) olives prior to brining renders them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined. Preservation of green olives by brining, according to the Spanish-type process, depends on a lactic acid fermentation in the brine. Failure to develop proper brine acidity may result in various types of spoilage (5,12,13). Etchells et al. (5) found that heating olives prior to brining resulted in a rapid and predictable brine fermentation by pure cultures of lactic acid bacteria, whereas brines of unheated olives failed to develop an acid fermentation and yeasts were the predominant microflora. They suggested the possibility of a heat-sensitive antibacterial compound in the olives. More recently, Borbolla y AlcalA et al. (2) confirmed that heating olives prior to brining encourages an acid fermentation. Fleming and Etchells (6) found that extracts of frozen green olives inhibited lactic acid bacteria. Later studies showed that freezing olives caused the formation of a heat-stable, bitter phenolic compound which was devoid of acid hydrolyzable reducing sugar and inhibited lactic acid bacteria; unfrozen olives did not con-'Paper no. 4109, Journal Series, North Carolina Experimental Station, Raleigh, N. C. tain this compound (7). Formation of the inhibitory compound in frozen olives was accompanied by a decrease in the content of oleuropein, the natural, bitter phenolic glucoside in olives. This decrease, and the fact that the compound from frozen olives was much more inhibitory to lactic acid bacteria than was oleuropein, suggested that the inhibitor might be a degradation product of oleuropein, possibly its aglycone. It was proposed that the improved brine fermentation resulting from heating olives was due to inactivation of an inhibitor-forming system in the olives (7). Results which further support this theory have been presented (H. P. Fleming, J. L. Etchells, T. A. Bell, and W. M. Walter, Jr., Bacteriol. Proc., p. 2,1970). In later studies, Juven et al. (10) found that treating olives with hot alkali before brining enhanced subsequent fermentation. In other work, they reported that oleuropein was inhibitory to several bacteria, including certain lactic acid bacteria (9). Later, however, Juven and Henis (8) found that the aglycone of oleuropein, obtained by hydrolysis of the glucoside with fl-glucosidase, was more inhibitory than oleuro-777 pein to Lactobacillus plantarum. The presence of antimicrobial compounds in olives has been suspected for some time. De-Caro and Ligori (4) found that the water solution remaining after oil was pressed from olives contained a substance which was inhibitory to several bacteria, most of which were gram positive. Recently, it was reported that salts of elenolic acid have antiviral properties (11). This acid is a hydrolysis product of oleuropein (14). The present work was undertaken to determine antimicrobial properties of products resulting from the hydrolysis of oleuropein. A second objective was to determine if unheated, green Manzanillo variety olives would release antimicrobial compounds into the cover brine. MATERIALS (6). Preparation of crude extracts. An ethyl acetate extract of oleuropein was obtained from heated Manzanillo variety olives as described previously (7). A portion of this extract was concentrated in vacuo to remove the ethyl acetate, and the residue was dissolved in 2 N H2SO4. This solution was heated for 1 h at 100 C. The hydrolysate was cooled, adjusted to pH 6 with NaOH, and extracted with ethyl acetate. The inhibitory substance which is formed as a result of freezing olives was obtained as described earlier (7), except that chloroform was used as the extracting solvent instead of ethyl acetate. Dry weights of the three crude extracts were determined. Testing crude extracts and pure compounds for antimicrobial activity. Oleuropein, the aglycone of oleuropein, elenolic acid, fl-3, 4-dihydroxyphenylethyl alcohol, and methyl-o-methyl elenolate were prepared as described previously (14). Solutions of these pure compounds as well as crude extracts from olives were screened for antimicrobial activity by the paper disk bioassay method used previously (6). Appropriate volumes of the solutions were pipetted onto 13-mm diameter paper disks, to give desired dry weight quantities. Solvents were allowed to evaporate before the disks were placed on the seeded agar surface. Control disks, to which only the corresponding solvent was added and evaporated, did not elicit inhibition zones for any of the microorganisms tested. The plating medium was seeded with one drop of a 16-h culture of the test organism grown in cucumber juice broth. Cucumber juice agar (6), pH 5.3, and Trypti-case soy agar (BBL), pH 6.9, were used as assay media. The buffer capacities of the media were determined to be sufficient to prevent drastic changes in pH (less than 0.5 pH units) of the media under the disks due to extracts or pure compounds present on the disks at the levels tested. Oleuropein and the products of its hydrolysis were tested for their effects on L. plantarum cultured in cucumber juice broth with (pH 5.0) and without (pH 5.3) added NaCl. Undiluted cucumber juice broth (2 ml) was placed in 12-by 120-mm tubes. The tubes were capped and autoclaved at 121 C for 10 min. Solutions of the test compounds (1 mg/ml) in 5% (vol/vol) ethyl alcohol were sterilized by filtration through 0.2-,um pore, alpha-8 Metricel membrane filters (Gelman Instrument Co.) and were added aseptically to the tubes of sterile broth. Sterile water and 5% ethyl alcohol were added to appropriate tubes so that the final volume was 4 ml and the concentration of ethyl alcohol was 1% (vol/vol) in all tubes. The pH of the solutions after addition of the test compounds was within 0.2 pH unit of the control broths. The tubes were inoculated with one drop of a 16-h culture of L. plantarum grown in cucumber juice broth and were incubated at 30 C. Growth of L. plantarum in the broth was estimated by determining optical densities at 650 nm with a Lumetron colorimeter. Brining of olives. Whole, green Manzanillo variety olives were washed in cold tap water, and some were subjected to heating and others to freezing treatments. For heating, olives were immersed in 74 C water for 3 min (5) and then cooled in tap water. For freezing, olives were held in plastic bags overnight at -18 C. They were thawed prior to brining. A portion of olives was neither heated nor frozen and served as a control. Because the inhibitory substance is sensitive to alkali (6), the olives were not alkali treated as is normal in the Spanish-type brining process (5). The alkali treatment, in addition to destroying the bitter principle, also probably alters the waxy coating of the fruit which causes greater permeability (10). In the present work the olives were pierced to insure release of nutrients, for microbial growth, from the olives into the brine. After heating or freezing treatments, the olives were pierced by rolling them over a bed of hypodermic needles spaced 10 mm apart and projecting 5 mm above the retaining plate. One-quart (0.946-liter) glass jars were packed with 475 g of olives and 500 ml of cold, sterilized 11.4% NaCl (wt/vol). Olives were held below the surface of the brine by plastic netting (5). The jars were closed with 70-mm diameter, six-lug, "twist-off' caps (White Cap Co., Chicago, Ill.) and held at 3 C for 3 days to allow for equilibration of NaCl with the moisture content of the olives and to permit diffusion of nutrients into the brine. All jars were inoculated with 10 ml of an 18-h culture of L. plantarum that was grown in cucumber juice broth containing 4% NaCl. The jars were loosely capped and incubated at 30 C for 17 days. Analyses of fermentation brines. Assay methods for determining the pH, titratable acidity (calculated as lactic acid), and reducing sugars in brines have been described (5). The antimicrobial compound(s) in olive brines readily partitioned into chloroform or ethyl acetate; oleuropein partitioned only into ethyl acetate. Therefore, for detection of antimicrobial compounds, 10 ml of brine from each jar was extracted with 50 ml of chloroform. Five milliliters of the extract was reserved for determination of ultraviolet absorption at 224 nm with a Cary model 15 spectrophotometer, and the remainder was concentrated in vacuo to 1 ml. This concentrate was bioassayed by the paper disk method by using Leuconostoc mesenteroides 42 as the test organism (6). Another 50 ml of brine was extracted with 50 ml of ethyl acetate. This extract, which contained oleuropein as well as the antimicrobial compounds, was dried over Na2SO4 and then concentrated to 10 ml. The concentrated chloroform and ethyl acetate extracts were analyzed by thin layer chromatography (TLC) by using solvents and procedures described earlier (7). RESULTS Screening of microorganisms for sensitivity to crude extracts. Table 1 shows results of initial screening tests to determine, qualitatively, the sensitivity of selected species of bacteria and yeasts to olive extracts. The oleuropein extract inhibited growth of Bacillus sub- . The remaining bacteria were tested in Trypticase soy agar (TSA, pH 6.9) as well as CJA. A "-" indicates no zone of inhibition; NG indicates that the bacteria did not grow in the medium. The amounts of extracts, dry weight, applied to each 13-mm-diameter disk were: oleuropein, 10 mg; oleuropein hydrolysate, 7.5 mg; extract of frozen olives, 3.5 mg. b The zone of inhibition remained clear for several days, but then growth of the culture began in this region. 779 VOL. 26, 1973 tilis, Staphylococcus aureus, and Pseudomonas solanacearum. The extract from acid hydrolysis of oleuropein was inhibitory to growth of all gram-positive bacteria tested, including four species of lactic acid bacteria, B. subtilis, and S. aureus. The hydrolysis extract also inhibited 5 of 11 gram-negative bacteria. The extract from frozen olives was inhibitory to the same bacteria as the oleuropein hydrolysis extract and also inhibited three other species. The seven species of yeasts tested were not inhibited by any of the three extracts. Sensitivity of lactic acid bacteria to oleuropein and products of its hydrolysis. The aglycone of oleuropein and elenolic acid inhibited growth of all four species of lactic acid bacteria tested qualitatively by paper disk bioassay ( Table 2). Zones of inhibition remained clear during extended incubation. Oleuropein, ,3-3,4dihydroxyphenylethyl alcohol, and methyl-omethyl elenolate showed no inhibitory action for any of these bacteria at 1 mg of compound per disk. The above five compounds were tested for their effects on growth of L. plantarum in broth culture without added NaCl (Fig. 1A). Elenolic acid and the aglycone of oleuropein at 100 ,g/ml caused about 11-and 6-h delays, respectively, in the onset of growth; thereafter, the growth rate approached that of the control, even when 200 ,gg/ml levels of these compounds were present. Oleuropein, fl-3, 4-dihydroxyphenylethyl alcohol, and methyl-o-methyl elenolate were not inhibitory to growth at 200 Atg/ml. When 5% NaCl was added to the cucumber juice broth, 100 gg of either the aglycone or elenolic acid per ml delayed growth, as noted by turbidity, of L. plantarum for about 3 days, and the rate was reduced when growth finally began (Fig. 1B). Growth was completely inhibited by 150 gg or more of either of these two compounds per ml when NaCl was present (Table 3). Again, the other three compounds were not inhibitory. Presence of antibacterial activity in the brines of olives. Olives that were heated before brining underwent acid fermentation; 0.85% titratable brine acidity was reached after 17 days (Table 4). A similar level of acidity was reached when the olives were heated prior to freezing. The predominant microbial flora in both cases were rod-shaped bacteria typical of Effect of oleuropein and products of its hydrolysis on growth of L. plantarum. The growth medium was cucumber juice broth, with and without NaCI, and contained 100 pg of the test compounds per ml. Symbols: U, oleuropein; 0, the aglycone; A, elenolic acid; 0, 6-3,4-dihydroxyphenylethyl alcohol; A, methyl-o-methyl elenolate; and 0, the control. Panel A, No NaCI; panel B, 5% NaCl. the L. plantarum culture used for inoculation. Inhibitory activity was not detected in extracts of either of these brines. TLC analysis of ethyl acetate extracts of these brines revealed that oleuropein was the major phenolic compound Methyl-o-methyl eleno-_ late aTests were made in cucumber juice broth containing 5% sodium chloride. A " +" indicates inhibition of growth and a "-" indicates no inhibition, as determined by optical density measurements during the 8-day incubation period at 30 C. ND, Not determined. ' Level of compound (,gg/ml). c Growth was delayed for 24 h or less at 50 ug/ml and for 3 to 4 days at 100 jig/ml. present; only traces of other phenolic compounds were detected. Brines of fresh unheated olives, however, did not develop appreciable acidity during incubation (Table 4). The small amount of acidity in these brines probably was due to the natural acids which diffused from the olive tissue. Results were similar with olives that had been frozen, whether or not they were heated after freezing. Chloroform extracts of these brines possessed antibacterial activity and had comparatively high ultraviolet absorption at 224 nm, which is the absorption region of the oleuropein aglycone (14). The level of oleuropein was negligible in ethyl acetate extracts of these brines. Chloroform extracts of the brine contained two phenolic compounds with Ragly values (R. values relative to that of the aglycone) of 0.88 and 1.08. These two compounds were not detected in brines which underwent an acid fermentation. Heating olives at 74 C for 3 min after they had been frozen and thawed did not render them fermentable (Table 4), demonstrating that the antibacterial substance formed by freezing was not significantly destroyed. This fact seems important, as it was suggested earlier (5) that the effect of heat in rendering olives After equilibration of the olives and cover brine, and prior to inoculation, the brines contained approximately 7.4% NaCl, 0.1% acidity, and 0.8% reducing sugar, and were pH 4.5 to 4.9. ' Analyses were made after incubation for 17 days at 30 C. c Determined by paper disk bioassay of a chloroform extract of the brine as described in Materials and Methods. A "+" indicates a zone of inhibition, a indicates no zone. 'Determined by analytical TLC. more fermentable might be due to destruction of inhibitory compounds. DISCUSSION Oleuropein, at levels up to 200 Ag/ml in broth culture, was not inhibitory to growth of species of lactic acid bacteria involved in the fermentation of brined olives but did inhibit some other species of bacteria. Elenolic acid and the aglycone of oleuropein were inhibitory to growth of lactic acid bacteria, particularly when the growth medium contained 5% NaCl. The aglycone is composed of elenolic acid bound through an ester linkage to fl-3,4-dihydroxyphenylethyl alcohol (14). Since the alcohol was not inhibitory, elenolic acid appears to be the inhibitory moiety of the aglycone. Juven and Henis (8) reported that reducing the amount of yeast extract in their test medium, APT broth, resulted in inhibition of growth of L. plantarum by oleuropein. Oleuropein was not inhibitory when 0.5% yeast extract was present. They suggested that nutrient deficiencies in the medium caused oleuropein to be inhibitory. Test media used in our studies, including olive brines which contained oleuropein, apparently were not nutrient deficient, as this compound did not inhibit lactic acid bacteria. Neither oleuropein nor products of its hydrolysis were inhibitory to the yeast species tested. The tolerance of yeasts to these compounds might explain why yeasts predominated in brines of unheated olives which did not undergo lactic acid fermentation (5). Other reports also indicate that yeasts are more tolerant to olive constituents than bacteria (1,4,9). Since it was first discovered that a mild heat treatment of green olives made brined olives more fermentable by lactic acid bacteria (5), a mechanism has been sought to explain the phenomenon. We theorize, on the basis of present information, that green olives have an enzymatic system which, when the olives are brined, causes the hydrolysis of oleuropein to its aglycone, an antibacterial compound. The aglycone or oleuropein may be degraded to yield elenolic acid, a compound which also is antibacterial. Oleuropein was present in brines of heated olives, whereas the aglycone of oleuropein was not. Lactic acid bacteria readily grew and produced acid in brines of heated olives, further substantiating the noninhibitory property of oleuropein to these bacteria. Walter et al. (14) reported a yield of 0.4% of purified oleuropein isolated from pitted Manzanillo olive8. The actual oleuropein content in the olive tissue was higher than 0.4% because some was lost during purification. A 0.4% concentration of oleuropein in the olives theoretically would yield about 2,700 ,g of aglycone per g of olive flesh. This concentration is much higher than would be needed to inhibit lactic acid bacteria in the brine; only 150 ,ug of the aglycone or elenolic acid per ml was sufficient to completely inhibit growth of L. plantarum when the medium contained 5% NaCl. Cruess and Alsberg (3) suggested that olives contain ,-glucosidase, which hydrolyzed oleuropein when olives were frozen while still on the tree. This enzyme might hydrolyze oleuropein to its aglycone when unheated olives are brined.

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2020-12-10T09:04:16.888Z

1973-02-01T00:00:00.000Z

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Application of the Most-Probable-Number Procedure to Suspensions of Leptospira autumnalis Akiyami A Various statistical tests are presented as evidence that the most-probable-number (MPN) procedure is a reliable method for estimating the density of washed-cell suspensions of Leptospira autumnalis Akiyami A. Colonization of Leptospira was first reported in 1957 by Cox and Larson (2). There is only one known report of the use of this counting procedure for describing growth (4) and there are no reports of its application to survival studies. According to Bodily et al. (1), not all serotypes of Leptospira will colonize, and results between laboratories have not always been reproducible. My attempts at quantitative recovery of pathogenic serotypes on solid media produced, at best, erratic results. Motivated by a desire to study survival of Leptospira at low cell concentrations, and recognizing that semisolid and liquid media have long been used successfully for maintenance and recovery of Leptospira, I undertook an investigation of the applicability of the most-probable-number (MPN) procedure. Estimation of bacterial density by the MPN procedure is based on two assumptions. (i) The distribution of individual cells is assumed to be random in the suspension with complete independence. This precludes use of the method with any microorganism which tends to aggregate in any way. (ii) It is assumed that growth will ensue with the introduction of one or more cells into a tube of medium. Applicability of the procedure is, therefore, essentially dependent on the ability to recover a single cell. MATERIALS transfer every 5 weeks. Antigenic stability was verified periodically by the slide agglutination test with antiserum provided by the Center for Disease Control. Preparation of cells. Cell cultures were grown at 30 C in a medium containing (per liter): Na2HPO4, 1.0 g; KH1PO4, 0.3 g; NaCl, 1.0 g; NH4Cl, 0.25 g; thiamine, 0.005 g; and rabbit serum, 100 ml. After 86 hr of incubation, the cells were recovered by centrifugation for 30 min at 3,000 rev/min, washed twice in buffer (total phosphate, 5.33 mM; pH 7.6), and resuspended in buffer. Cell concentration was standardized by direct count with a Petroff-Hausser chamber and dark-field microscopy. Dilution blanks. Dilutions for MPN determinations were made in 9.0-ml blanks containing Leptospira medium base EMJH (Difco) with 1% rabbit serum. Tubes of recovery medium were inoculated with 0.1 ml of the appropriate dilutions. RESULTS A suspension of washed cells of L. autumnalis was standardized and then serially diluted to 1.0 cell per ml. Three series of 120 tubes each were inoculated with 0.1 ml of the last three decimal dilutions to give a theoretical MPN of 100 cells per ml. Table 1 compares the observed and theoretical ratios of negative tubes to total tubes for the three cell concentrations. The three series of 120 tubes each represent 24 replicate MPN values with 5 tubes per 235 (3); n = s + r = 120; p = 1 -q. dilution and 12 replicate MPN values with 10 tubes per dilution. These MPN values and their 95% confidence intervals are presented in Table 2. Figure 1 shows the 24 replicate MPN values on log-probability paper. A reasonably straight line results, the consequence of a logarithmically normal distribution. The true cell concentration should lie at the 50% probability intersection. Figure 2 is a graphical representation of the 12 replicate MPN values with their 95% confidence intervals. Each confidence interval includes the true cell concentration of 100 per ml based on direct microscopic count and dilution. MPN determinations were completed on a serie f cell suspensions standardized by direct microscopic count and dilution. The results are compared in Table 3. MPN values are expressed as duplicate determinations with 5 tubes per dilution and one determination with 10 tubes per dilution. To compare observed frequencies of MPN codes with theoretically expected frequencies, MPN values from two separate experiments with six different cell suspensions were chosen (Table 4). These MPN tests were completed over time and, therefore, represent different cell concentrations because death was proceeding. This constitutes a stricter test than selecting codes obtained on a single cell suspension. DISCUSSION Each of the statistical tests applied demonstrates that, with supplemented Fletchers medium used for cell recovery, the MPN procedure is a reliable technique for estimating the density of suspensions of L. autumnalis. The observed frequencies shown in Table 1 are within the theoretical frequency plus or minus the expected error for 10 and 1 cells per tube. This is not true for 0.1 cell per tube, but here the problem becomes one of an increasing expected error with a decreasing number of positive tubes. For reliable results, the percentage of positive tubes should be roughly between 35 and 85 (6), which is the case for one cell per tube. The logarithms of MPN values are distributed normally. The mean of the log distribution of MPN values should equal the true cell concentration. The means of 24 five-tube MPN values (106) and 12 ten-tube MPN values (103) agree well with the true cell concentration of 100 per ml. Replicate MPN values from a normal distribution should plot as a straight line on log-probability paper, which is demonstrated in Fig. 1. The mean cell concentration determined graphically is 104, which compares well with the true concentration of 100 cells per ml based on direct microscopic count and dilution. The MPN is not a precise method of measurement; it is only an estimate of the true cell concentration. The 95% confidence interval expresses the imprecision of the method. This interval will include the true cell concentration 95% of the time. Each of the 95% confidence intervals for 12 replicate 10-tube MPN values includes the true concentration of 100 cells per ml. Two of the 24 confidence intervals for replicate five-tube MPN values do not include the true concentration ( Table 2). It is expected that the confidence interval will not include the true concentration 5% of the time or 1.2 times in every 24 tests. In the case of MPN tests completed on 12 different cell suspensions, 2 of milliliter based on direct microscopic count and dilution. (Table 3). This frequency 41.9-208 is slightly greater than the expected 5%. However, the true cell concentrations are based on 12 separate microscopic counts, a method 21. which also has a significant error. The comparison of observed and theoretical frequencies for five-tube MPN codes shows 52.1-258 good agreement (Table 4). An unusually large number of improbable codes would indicate 25. either that the basic assumptions for the MPN procedure do not hold or that something is wrong in the technique. 33.3-165 This procedure was subsequently used to describe exponential death rates for L. autumnalis in defined solutions (5).

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2020-12-10T09:04:12.629Z

1973-03-01T00:00:00.000Z

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Subcutaneous Bacteria in Turkey Carcasses Two methods were employed to quantitate the subcutaneous bacteria in fresh, refrigerated, and frozen turkey carcasses. Relatively few bacteria were detected in the skin-flesh interface and in the flesh as compared with the number of bacteria on the skin surface and in the skin layer. No subcutaneous bacteria were detected in 49% of the skin-flesh interface and flesh samples. The number of bacteria detected in skin samples from carcasses chemically disinfected to kill skin surface bacteria was smaller than that in nondisinfected skin samples. These results indicate that the skin blending method used to quantify microorganisms on poultry carcass skin measures the skin layer flora and that the number of subcutaneous membrane or flesh bacteria measured is not normally large enough to have a significant influence on the results. The flesh of a live animal is essentially sterile; however, during processing, bacteria on the skin surface may contaminate the flesh and skin membrane through severed blood vessels or skin cuts and tears. It has been suggested that most bacterial growth is confined to the skin surface of dressed and eviscerated poultry and that very few bacteria are present in the adjoining flesh (3,6). A review of live human skin structure and its bacterial flora by Price (5) further supports the above assumptions. Several changes occur in skin after death, however, which could affect its function as a barrier to bacterial migration into the inner tissues. The extent of such changes partially depends on the elapsed time after death, the carcass temperature, and the way the carcass was handled. The presence of a significant number of viable bacteria beneath the skin of processed poultry carcasses would be of concern for a number of reasons. It might indicate bacterial migration through the skin enhanced by timetemperature abuse or unsanitary processing. Subcutaneous bacteria in poultry meat might present a potential public health hazard and shorten the shelf-life of the product. Methods employed to reduce the skin surface bacteria level during processing might not necessarily affect subcutaneous bacteria. Analytical methods based on quantifying skin surface bacteria by blending and plating skin samples (1,2) ' Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 1804. 354 might be including a significant number of subcutaneous bacteria in the skin "surface" count. This last possibility was what prompted the study reported herein. It has been reported that a skin sample "blending" method for quantifying the estimated bacterial population on poultry carcass skin yields significantly higher counts than the "cotton swabbing" technique (1,4) or the "carcass rinse" technique (4). However, if a significant number of subcutaneous bacteria were present, the skin "blending" method (2) would reflect both surface and subcutaneous bacteria, whereas the "swab" and "rinse" methods would enumerate primarily surface bacteria. Therefore, it was necessary to determine the relative numbers of subcutaneous bacteria in freshly eviscerated, refrigerated, and frozen-thawed poultry carcasses as compared with the numbers of bacteria in the skin tissue and on the skin surface. MATERIALS AND METHODS Two methods were used to determine subcutaneous bacteria in turkey carcasses. In one method, the right half of the carcass was chemically disinfected by swabbing the skin with 5% phenol for 5 min followed successively by 70% ethyl alcohol, 2.5% sodium hypochlorite, and finally with 70% ethyl alcohol again, each for approximately 1 min. The final application of ethyl alcohol was allowed to evaporate. Skin samples were cut on this half with a sterile brass cutting tool (2.54 cm in diameter), removed from the carcass with sterile forceps (scissors were used to facilitate removal), and homogenized in a Waring blender containing 100 ml of Butterfield's buffered phosphate diluent for 2 min. Three skin samples were removed from the breast and three from the leg. The skin homogenate was plated in plate count agar (Difco). Flesh samples, 1.27 cm in diameter and approximately 1.27 cm deep, were cut below each skin sample that was removed from the disinfected side. These flesh samples were removed, blended, and plated in a similar manner. Carcasses were from three different treatment groups: freshly eviscerated, refrigerated (4 C) for 7 days, or frozen (-29 C) and thawed (8 C for 16 h). Six skin and six flesh samples were removed from the disinfected side from each of duplicate carcasses from each treatment group. Thus, a total of 12 skin and 12 flesh samples were taken from the disinfected side, per carcass group. Six skin samples were removed from the nondisinfected half of each carcass in a similar way. These samples were taken immediately after the samples from the disinfected side of any one carcass had been plated. They were blended and plated as previously described to quantitate the bacteria in the skin tissue. Flesh samples were also removed, blended, and plated as described in the previous paragraph. All plates were incubated at 37 C for 48 h and at 20 to 25 C for 48 h before colonies were counted. The second method used to determine subcutaneous bacteria involved cutting and lifting a portion of carcass skin and swabbing the skin-flesh interface. The skin in the region of the incision was disinfected with 70% ethyl alcohol, which was allowed to evaporate. A V-shaped incision approximately 2.54 cm long was made in the skin with a sterile scalpel. The skin was pulled away from the flesh with sterile forceps. One sterile calcium alginate swab (Calgiswab; Consolidated Laboratories, Inc.) was moistened with one-fourth strength Ringer* solution containing 1% Calgon (Calgon Consumer Products Company, Inc.) and was used to puncture the skin membrane and swab the skin-flesh interface. An area of approximately 1 cm2 was swabbed. The swab was then agitated until dissolved in 10 ml of the modified Ringer solution. All 10 ml of the Ringer solution with the dissolved swab was plated (three plates) in plate count agar (Difco). Plates were incubated at 37 C for 62 h before colonies were counted. A total of 60 samples was taken; 10 samples were removed from each of the duplicate carcasses for each treatment condition. Bacterial counts per square centimeter of skin surface were estimated on each carcass by the same "swab" method. All turkey carcasses used in this study were processed by conventional methods in a laboratory pilot plant. For reasons of availability, male turkey carcasses were used with the first method and female turkey carcasses were used with the second method. RESULTS The ratios of flesh samples yielding any viable bacteria to the number of samples taken by the first sampling method are presented in Table 1, as are the average aerobic plate counts per square centimeter in the flesh samples. Average aerobic plate counts per square centimeter of skin are shown for comparison. Among the 36 flesh samples from the disinfected sides of the turkeys, 27 were positive for bacteria; 24 of 36 from the nondisinfected sides were positive. The replicate averages of the flesh sample aerobic plate counts per square centimeter from fresh, refrigerated, and frozen-thawed carcasses were 10, 36, and 280, respectively, on the nondisinfected side, and 32, 150, and 35, respectively, on the disinfected side. The "disinfected" skin layer of the carcasses yielded replicate average aerobic plate counts per square centimeter of 160, 290, and 110 for fresh, refrigerated, and frozen-thawed carcasses, respectively. The ratios of skin-flesh interface swab samples yielding viable bacteria to the number of samples taken are presented in Table 2, as are the average aerobic plate counts per sample. The average aerobic plate counts per square centimeter of skin surface determined by swabbing are shown for comparison. No bacteria were isolated from 44 of 60 skin-flesh interface swabbings. Each of the three carcass conditions averaged fewer than 10 bacteria per swab sample. The replicate averages of the skin surface aerobic plate counts per square centimeter from fresh, refrigerated, and frozenthawed carcasses were 4,000, 11,000, and 1,100, respectively. DISCUSSION By the skin blending method used (100 ml of blending fluid) and the agar plate count technique, 100 bacteria per square centimeter of skin is the lowest number that can be determined with any accuracy. Aerobic plate counts are only reported to an accuracy of the first two left-hand digits of the calculated average count. Aerobic plate counts greater than 1,000/ square centimeter of skin, determined by blending the skin, would be very slightly, if at all, affected by fewer than 100 subcutaneous bacteria per square centimeter. When one considers the possible sources of contamination of subcutaneous tissue during sampling, the data indicate that there were very few bacteria below the skin tissue in the fresh, refrigerated, or frozen-thawed turkey carcasses tested. There is, however, a possibility that bacteria are firmly imbedded in the rough skin surface or perhaps just beneath the surface layer. Even if this is true, it would be desirable to include them as part of the carcass bacteria count. It appears that subcutaneous bacteria are negligible compared to the level of skin surface bacteria present in fresh, refrigerated, or frozen turkey carcasses. Any significant number of bacteria in the flesh of a turkey carcass would indicate severe time-temperature abuse or unusual contamination through skin cuts and tears, or both. Since turkey carcass skin seems to be a rather effective barrier to bacterial migration into the tissues, an intact eviscerated carcass would be expected to have a longer shelf life than carcass parts or a carcass with large skin cuts or tears. The results of this study indicate that with freshly eviscerated, refrigerated, or frozenthawed turkey carcasses, skin "surface" bacteria counts determined by blending skin samples will not be significantly affected by subcutaneous membrane or flesh bacteria. Skin "surface" bacteria counts are probably only slightly affected, if at all, by subsurface bacteria in the skin layer.

v3-fos

2018-04-03T05:22:35.055Z

1973-04-01T00:00:00.000Z

42643514

s2

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen' Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with 33P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of 33P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with 33P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with 33P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of 33P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with 33P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs. Hungate (19) calculated adenosine triphosphate (ATP) yields from known pathways of ruminal fermentation of carbohydrates. By using a Y ATP = 10 (3,34), he concluded that about 10 g of microbial protein (about 15 g of total cell mass) can be synthesized for each 100 g of carbohydrate fermented. This value was thought to represent an upper limit of the synthetic capacity of anaerobic ruminal fermentation (19). This level of protein synthesis in the rumen is equivalent to 18.3 g of digestible protein per Mcal of digestible energy (36) and is clearly below the requirement of growing ruminants (36). From a husbandry viewpoint, ' this result implied that, for optimal growth or production of ruminants, procedures to allow dietary protein to escape ruminal degradation must be developed. Extensive direct experimental work on quantitative aspects of ruminal microbial protein synthesis indicates, however, that the limits set by Hungate (19) were too low. Thus, growth yield studies with strains of rumen bacteria (11,12,18) and ingesta passage studies with sheep by using purified diets or microbial cell markers (14,15,17,27) indicated that ruminal microbial protein synthesis ranged from 15 to 22 g of microbial protein per 100 g of organic matter fermented. Other recent reports (13,21) have also suggested that the ATP yield per mole of hexose fermented in the rumen is higher than previously anticipated. The experiments described below were initiated to measure absolute bacterial and protozoal protein synthesis rates in whole rumen contents at various times after feeding with short-term, zero time in vitro (6) incubations. Since membrane (phospholipids) synthesis and growth are directly related (31,33) and since growth and protein synthesis are directly related (28) in microorganisms, protein synthesis rates of ruminal microorganisms were assessed by measuring the rate of microbial phospholipid synthesis. MATERIALS AND METHODS Radioactive phosphorus-labeling pattern of cellular phosphorus containing constituents during in vitro incubations of mixed ruminal bacteria: experiment 1. The labeling patterns of cellular phosphorus by 22P-inorganic phosphorus were studied in suspensions of mixed ruminal bacteria in a high and low phosphorus medium in vitro. Rumen contents were collected as outlined by Purser and Moir (37) from sheep fitted with Jarret cannulae (fed ration 1, Table 1) before the morning feeding, placed in a vacuum bottle warmed to 39 C, gassed with oxygen-free C02, and transported quickly to the laboratory. Rumen contents from sheep are quite homogenous, and samples of 100 g or more are representative of whole rumen contents (37). The rumen contents were subsequently squeezed through two layers of cheesecloth. Rumen liquor (500 ml for high PO4 and 800 ml for low PO4 media) was then centrifuged at 500 x g for 15 min at 2 C. The pellet was discarded, and the supernatant was centrifuged at 10,000 x g for 15 min at 2 C. The resulting pellet (bacterial fractions; 300 to 400 mg dry weight for the high PO4 and 150 to 200 mg dry weight for the low PO4 media) was resuspended to the original rumen liquor volume in reduced anaerobic buffer (AB; Table 2) containing either 180 or 35 gg of phosphorus, pH 6.7, per ml. During the centrifugation steps, all tubes and beakers were gassed with 0,-free CO2 to insure anaerobiosis. The ruminal bacteria fractions were then transferred into prewarmed fermentation flasks (1 liter) and incubated at 39 C under 0,-free CO2. Radioactive phosphorus (9P-HP04; ca. 60 x 106 DPM) dissolved in AB was then added to the fermentation flask, and initial subsamples (80 ml) were secured. For the experiments with the high P04 media, substrate (0.66% glucose, 0.5% soluble starch, and 0.15% urea; expressed as percent weight of the final volume of the fermentation system) was added and further subsamples were obtained at 30, 60, 120, 180, and 240 min. For the experiments with the low P04 media, inorganic phosphorus uptake by resting bacteria and actively growing bacteria was studied in the manner outlined by Mitchell and Moyle (31). Thus, substrate (as above) was added after 90 min of incubation. Subsamples (as above) were obtained at 30, 60, 90, 120, 150, 210, 270, and 330 min for the low P04 media fermentations. To stop bacterial activity, subsamples were placed in prechilled beakers which were then placed in a solution of solid CO2 in ethanol. 25,1973 on May 8, 2020 by guest http://aem.asm.org/ Downloaded from When the temperature of the subsample reached 2 to 5 C, the subsamples were centrifuged at 18,000 x g for 15 min at 3 C. The pellet was then resuspended and washed with a small quantity of deionized water and recovered by centrifugation. The pellets were then frozen, lyophilized, and stored for phospholipid, intracellular and total phosphorus, and asp activity analysis. Increases in cellular dry matter and protein (N x 6.25) content were determined on separate subsamples. A sample of cell-free media was secured and frozen for inorganic phosphorus and asP analysis. Radioactive phosphorus incorporation into microbial phospholipids of rumen contents in vitro from sheep: experiment 2. Sheep (fitted with Jarret cannulae) were placed on ration 2 (high protein) or 3 (low protein) ( Table 1, one sheep per ration) for a 3-week period. Rumen contents were collected, as described above, from the sheep before (0 h) and 2 and 4 h after the morning feeding. Upon arrival at the laboratory, 150 g of rumen contents was weighed into a wide-mouth fermentation flask (800 ml) (under an O2-free CO2 atmosphere), and 6 ml of a solution containing 3P-HPO4 (13.2 x 107 DPM) was added. The flask was sealed and then shaken for 30 s, and an initial subsample was removed. Preliminary work showed that 6 ml of 1% crystal violet could be well mixed with 150 g of rumen contents within 30 s under these conditions. Further subsamples were obtained at 30, 60, 120, 180, and 240 min of the in vitro incubation. After each sampling the flask was flushed with 02-free CO2 and resealed. The microorganisms were killed by the addition of 0.05 volume of saturated HgCl,. After washing to remove unincorporated 33P, the samples were frozen and lyophilized. Microbial phospholipid synthesis and microbial protein synthesis in whole rumen contents of sheep: experiment 3. Rumen contents were collected from sheep fed ration 1 (Table 1) before (0 h) and 2, 4, and 9 h after the morning feeding. One sheep served as the donor of rumen contents for two separate experiments. Short-term, zero time method (6,20) in vitro incubations were conducted with rumen contents as described above, except 750 g of rumen contents was placed in the fermentation flasks. After the additions of 33P-H.PO4 (about 44 x 107 DPM), an initial subsample (350 g) was obtained. The final sample was obtained after 60 min. The microorgansims were killed with saturated HgCl2. Each subsample was divided in the following manner. From the well-mixed subsample, a 20-g sample was used to determine 33P incorporation into total microbial phospholipids. The particulate matter was spun down and then resuspended and washed in saline and centrifuged at 18,000 x g three times to remove unincorporated, free, and nonspecifically bound 33P. The residue was then frozen and lyophilized. The remaining rumen contents from the subsamples (305 g) were squeezed through two layers of cheesecloth. The particulate matter was then resuspended in 0.85% NaCl, stirred, and squeezed again through two layers of cheesecloth. Microscopy examination revealed that this procedure removed all except a few small protozoa from the residual plant matter and rumen contents. It was therefore assumed in subsequent calculations that total protozoal protoplasmic mass had been recovered quantitatively from rumen contents by this procedure. A small sample was removed from the rumen liquor for phosphorus and volatile fatty acid (VFA) analysis. The rumen liquor and extract from the second squeeze were then combined. The protozoa and bacteria were separated from the rumen fluid by differential centrifugation as described by Bergen et al. (4). Microscopy examination of the high-speed (bacterial) and 150 x g (protozoal) fractions were done in a separate (i.e., no isotope) study. It was found that the 150 x g (protozoal) and the high-speed (bacterial) fractions were devoid of gross plant material contamination. Previous work had shown that this differential centrifugation system (4) resulted in 150 x g (protozoal) and high-speed (bacterial) fractions which contained only 1.5 and 0.7% (based on dry matter) crude fiber (W. G. Bergen, unpublished data). The 150 x g (protozoal) and high-speed (bacterial) fractions were then frozen and lyophilized. Analytical procedures. Preliminary experiments were conducted to assess the efficacy of various extraction procedures for phospholipids from ruminal microorganisms and the salt wash procedure of Folch et al. (10). During these studies, microbial preparations were extracted and reextracted in organic solvents, and the extracts were then monitored for phospholipids with thin-layer chromatography. Similarly, the salt wash procedure was checked to assure that only inorganic, but not phospholipid, phosphorus was lost during the wash. In a number of trials, upon addition of 33P-HPO4 to chloroformmethanol (2: 1), after the salt wash only 0.3% of the initial radioactivity remained in the solvent. For the experiments described above, microbial phospholipids were extracted from lyophilized rumen contents, high-speed (bacterial), or 150 x g (protozoal) preparations by a modification of the method described by Katz and Keeney (24). Approximately 30 mg of sample was extracted with 5 ml of chloroformmethanol (2:1; vol/vol) in 15 ml of Teflon-lined screw-capped culture tubes. The tubes were rotated for 16 to 20 h at room temperature. The extracts were filtered through a fritted glass Buchner funnel and the nonlipid impurities were removed by the salt wash procedure (10). Sonic treatment of microbial preparations did not improve the yield of total lipid extraction. Phospholipid phosphorus was determined in the washed extract by the ascorbic acid procedure of Chen et al. (7) as modified by Rhee and Dugan (39). Total cellular phosphorus and phosphorus concentration in incubation media were determined by the procedure of Chen et al. (7). Intracellular inorganic phosphorus (IC-P,) was extracted from 30 to (26). Preliminary studies showed that this procedure recovered between 97 and 98% of the phosphorus from standard phosphorus solutions in the presence of 5% PCA. Nitrogen was determined with a micro-Kjeldahl procedure. VFA were determined from media treated with metaphosphoric acid (9) on a Teflon column (198 by 0.05 cm) packed with Chromosorb 101 (60-80 mesh) at 188 C with nitrogen as carrier gas and a hydrogen flame detector. Dry matter of subsamples was determined by drying at 110 C to a constant weight (this took 20-24 h). Suitable samples of all phosphorus-containing extracts (i.e., phospholipids and intracellular phosphorus) were placed in vials containing 10 ml of scintillation fluid (5 g of 2,5diphenyloxazole; 0.05 g 1,4 bis-[2-(4 methyl-5phenyloxazole) ]-benzene, 500 ml of toluene, and 500 ml of Triton X-100) and counted in a Nuclear Chicago 6848 liquid scintillation spectrometer. "3Pphosphorus activity (T 1½2 = 25.3 days) in the counted samples was corrected for counting efficiency and decay, with decay factors derived by Robinson (40). The 33P-phosphorus was obtained from New England Nuclear Corp., Boston, Mass., as H3-'3PO4 in 0.02 N HCl. All glassware used in the experiments was washed with detergent, rinsed in deionized water-12 N HCl (2:1), followed by a final rinse in deionized water. Calculations. To calculate the rate of microbial phospholipid phosphorus synthesis in whole rumen contents (experiment 3), the IC-P1 specific activity (SA) was used as an indicator of precursor pool SA. Since the SA of IC-P, may differ between protozoa and bacteria, these were assessed separately. In this incubation system (experiment 3), the SA of the IC-Pi was measured at the start (SA = 0) and the end of the incubation time. The rate of "3P uptake into cellular phospholipids is dependent on the rate of phospholipid synthesis as well as the rate of change of the SA of the precursor pool (IC-Pi) during the incubation. To properly describe the SA of the IC-P1, more frequent sampling would have been necessary. Thus, an approximation was made to determine the effective SA of the IC-Pi pool during the incubation period. The following formulae describe the calculations: (i) Intracellular phosphorus SA = (final IC-P,-SA + initial IC-P,-SA)/2. (iii) Total phosphorus (micrograms) incorporation into microbial phospholipids = phosphorus incorporation into high-speed pellet (bacterial) phospholipids (net counts/min of 3SP in the PL-PJ/SA of the IC-P1) + phosphorus incorporation into 150 x g pellet (protozoal) phospholipids (net counts/min of 33P in the PL-PJSA of the IC-P,). RESULTS Physical separation of microbial fractions. Sampling of rumen contents and the subsequent fractionation of the rumen contents into microbial fractions is one of the most difficult aspects of microbial investigations in the rumen. Throughout this work rumen samples were withdrawn from the ventral and dorsal sac of the rumen. More importantly, at least 1 liter (from a 4-liter rumen volume of sheep) of contents was removed to insure that a representative sample had been secured. For experiment 1, mixed ruminal bacteria were prepared similar to the procedure outlined by Baldwin and Palmquist (2). It must be recognized that many large bacteria and clumps of bacteria may be lost during the initial low-speed centrifugation step. As Hungate (19) pointed out, suspensions of washed mixed ruminal bacteria contain all kinds of rumen bacteria, but they are not necessarily in the same proportions as found in the total contents. In experiment 3, the 150 x g pellet was designated the protozoal fraction. Although, the protozoa could be nearly quantitatively removed from the rumen fluid, this fraction may also contain sizeable amounts of bacteria (49). These bacteria are large bacteria as well as those harbored within the protozoa. The metabolic activity of these organisms will contribute to the total activity of the 150 x g (protozoal) fraction. In the remainder of the paper, the 150 x g pellet will be called the protozoal fraction, whereas the high-speed pellet will be called a bacterial fraction. Experiment 1. The metabolism of 33P-H3PO, in mixed ruminal bacteria incubated in high or low phosphate medium was studied. Figure 1 depicts the results for the high phosphate medium studies. The SA of inorganic phosphorus medium stayed constant throughout the incubation period. In the presence of substrate there was a linear rise in 33P incorporation into phospholipids and in 33P uptake into IC-Pi and total cellular phosphorus. The increase in dry cell mass and cellular protein (N x 6.25) were significantly correlated (r = 0.99; P < 0.01), and the rate of 33P incorporation into cellular phospholipids and the increase in dry cell mass or cellular protein were significantly correlated (r = 0.91, P < 0.01; r = 0.94, P < 0.01). This high correlation indicates that cellular phospholipid synthesis can be 507 VOL. 25,1973 on used as a marker of total cellular growth. During the 240-min incubation period, the SA of the IC-P, did not equilibrate with the SA of the inorganic phosphorus medium, indicating that, if phospholipid synthesis is to be measured, the SA of the inorganic phosphorus medium will overestimate the SA of the phosphorus precursor pool. Incubation of mixed ruminal bacteria in the low phosphate medium (Fig. 2) in the absence of substrate (0-90 min) resulted in some uptake of 33P into the IC-P1 pool. 33P-phosphorus was not incorporated into the phospholipids during this period. The addition of substrate caused a more rapid uptake of 33P into the IC-P1 pool, the incorporation of 33P into PL-Pi, and an increase (growth) of the bacterial cell mass. The SA of inorganic phosphorus medium showed little change. Again, the SA of the IC-P1 pool did not reach the SA of the phosphorus medium after 240 min of incubation after substrate addition. The rate of 33P incorporation into cellular phospholipids and the increase in cell mass were significantly correlated (r = 0.98; P < 0.01), indicating that cellular phospholipid synthesis can be used as a marker of total cellular growth. Mitchell Since experiment 1 showed that phospholipid synthesis was highly correlated with increases in cellular mass or protein content in mixed ruminal bacteria, experiment 2 was conducted to investigate whether rates of phospholipid synthesis in whole rumen contents (determined in vitro) could be related to dietary protein intakes of sheep. Under these experimental circumstances a significant amount of 33P incorporation into protozoal as well as bacterial phospholipid would be expected. Although protozoal phosphorus metabolism was not studied in detail as for mixed ruminal bacteria, it was assumed that for these organisms phospholipid synthesis would also reflect cellular growth. When a diet containing low levels of protein (N-limiting) is fed to sheep, an increase and then a decline in microbial growth (activity in the rumen) would be expected, whereas for a diet with adequate (or excess) protein a more sustained rate of microbial growth would be expected. Previous work had shown that, when urea is added to a low protein ration, microbial protein synthesis (16) and animal performance (23) are increased. As the results show (Fig. 3), before feeding and at 2 h after feeding the rate of 33P incorporation into microbial phospholipids (determined in vitro) did not differ between the two rations; however, for the high protein ration at 4 h after feeding the rate of 33P incorporation into phospholipids was still high, whereas for the low protein diet the rate of 33P incorporation into phospholipids declined to the prefeeding level. Experiment 3. Experiment 1 showed that phospholipid synthesis and increases in cellular protein were highly correlated and experiment 2 showed that, as expected, the addition of a NH3 source to a low protein diet resulted in more sustained (increased) microbial activity (growth). Thus, these studies indicated that phospholipid synthesis can be used as a marker of microbial growth in whole rumen contents. In this experiment microbial protein synthesis, at various times after feeding, in the rumen of a sheep fed a standard ration was studied. The results were expressed as grams of protein (N x 6.25) synthesized per hour per hypothetical 4-liter rumen. Sheep used in this work showed a rumen volume of 3.5 to 4.0 liters from polyethylene glycol disappearance curves (21). To calculate microbial protein synthesis from phospholipid synthesis, the two parameters had to be related. Walker and Nader (45) used a nitrogen-sulfur ratio to relate 35S incorporation to protein synthesis. Similarly, a nitrogento-phospholipid phosphorus ratio (N/PL-PI) was used to relate inorganic phosphorus incorporation into phospholipids to protein synthesis. Extensive preliminary studies showed that the N/PL-Pi was not constant between various strains of pure culture rumen bacteria or microbial preparations isolated from sheep (Table 3). Thus, the N/PL-P1 ratio had to be determined separately for each in vitro incubation to insure meaningful calculations. 33P-phosphorus and total phosphorus incorporation rates into microbial phospholipids and protein (N x 6.25) synthesis by whole rumen contents during 60-min in vitro incubation periods are given in Table 3. The apparent protein synthesis rate of the 150 x g (protozoal) fraction was equal to the rate noted for bacteria. Although protozoa per se were isolated by the physical separation methods used in this study, the extent of bacterial contamination of the 150 x g fraction was not ascertained. However, it is plausible that one-fifth to one-fourth or more of the cellular mass in the 150 x g fraction was of bacterial origin. Thus, it can be concluded that bacteria contribute 60% or more to overall protein synthesis in the rumen. Recent results by Hungate et al. (21) suggested that the rate of protozoal protein synthesis was nearly equal to bacterial protein synthesis. Overall, the highest rate of protein synthesis was observed at 2 h after feeding, whereas at 9 h after feeding the rate was substantially lower than the rate noted before feeding. There are no apparent reasons for a higher microbial protein synthesis rate at 15 h (0 h in Table 3, Fig. 4) than at 9 h after feeding. Since the 9-h rate was from a single experiment, the rate of protein synthesis may have been underestimated. These results expressed in terms of a 4-liter rumen are depicted in Fig. 4. The relative VFA production rate is plotted on the same graph and is parallel to the changes found for microbial protein synthesis. It is evident that immediately after feeding there was an increase in microbial activity (VFA production) and cellular growth (phospholipid and protein synthesis). Despite differences in rations, these results are in disagreement with the contention of Walker and Nader (46) that microbial protein synthesis in the rumen declines initially after feeding and then increases again to prefeeding rates. DISCUSSION Rumen microbial protein represents a major source of amino acids to the ruminant animal. Quantitative aspects of ruminal microbial protein synthesis have thus been the subject of extensive research efforts. In the past, procedures based on fermentation balances (19), ingesta passage studies with or without microbial cell markers (14), zero time in vitro incubations of rumen contents with 35S or 15N (1,45), single dose or continuous infusion of 15N-urea into the rumen (32,35), and rumen fermentation-turnover models with C, N, and H balance studies (21) have been used to assess microbial protein synthesis. Hungate (19), from known pathways of ruminal VFA production and supposed ATP yields and using Y ATP = 10 (3), calculated that about 10 g of microbial protein can be synthesized for each 100 g of carbohydrate fermented. This value represented an upper limit of the synthetic capacity in the anaerobic ruminal fermentation. This level of protein synthesis in the rumen is equivalent to 18.3 g of digestible protein per Mcal of digestible energy (36). As Purser (36) pointed out, this protein-calorie ratio is below the required protein-calorie ratio to meet the protein requirements of growing ruminants and less than such ratios established from empirical balance studies with growing ruminants. Growth yield studies with pure cultures of rumen bacteria (11,12,18) and ingesta passage studies with sheep, by using nonprotein nitrogen diets or markers for microbial cells (15,17,27), indicated that the microbial protein yield in the rumen was higher than predicted by Hungate (19). Overall, the above studies indicate that 15 to 22 g of microbial protein is formed per 100 g of organic matter fermented. There are a number of alternatives to explain these results. First, the Y ATP value may not 510 APPL. MICROBIOL. on May 8, 2020 by guest http://aem.asm.org/ Downloaded from be universal among bacteria (however, this has been rejected by Payne [40]), or substrate phosphorylation (43) may result in an extra energy yield to the microorganisms. Although the latter process has been implicated in ruminal organisms (44), a more tenable alternative appears to be the recent suggestion that the Y ATP value of 10 is universal but that previously assumed ATP yields per mole of VFA are too low (13,21). To further evaluate the potential of microbial protein synthesis in the rumen, we decided to use the short-term, zero time method. Walker and Nader (45) had used this approach with 35S, but their actual results on protein synthesis rates were quite low. They were able to correct their data by determining a VFA-to-protein synthesis ratio from the in vitro incubation and then multiplying this ratio by an assumed VFA yield. The major problem in the approach of the above authors is that they assumed that the SA of the intracellular precursor pool (in this case 35S-cysteine or 35Smethionine) for protein synthesis equalled the SA of medium 35S-sulfide. Al-Rabbat et al. (1) made similar assumptions in their 15N incorporation studies with ruminal microorganisms. In the present work, it was decided to use phospholipid synthesis as a marker for cellular growth since membrane synthesis is closely related to total cell growth (33,38,50), and since IC-P1 equilibrates readily with phospholipid precursors (5,48) and can be used to determine the SA of the intracellular precursor pool for phospholipid synthesis. The results of experiment 1 showed clearly that, with ruminal bacteria, the SA of inorganic phosphorus medium did not equilibrate with the IC-Pi pool. A very high correlation between cell growth and phospholipid synthesis was noted for mixed ruminal bacteria similar to the observations of Ohki (33) for E. coli. Preliminary work had shown that the N/PL-Pi ratios of the ruminal 150 x g fractions were not similar to the N/PL-Pi of ruminal bacteria. Since the 150 x g fraction is largely composed of protozoa, it was decided that the SA of the IC-Pi must be established for the 150 x g fraction and bacterial fraction separately. For all the calculations below, the rate of protein synthesis by both fractions have been combined to negate the implication ( Table 3) that (apparent) protozoal protein synthesis was equal in magnitude to the bacterial protein synthesis. The results from Table 3 and Fig. 4 were recalculated to estimate daily protein synthesis. The data (Table 4) are expressed as grams of protein produced in a 4-liter rumen per day. To estimate daily protein synthesis, the various measured rates were arbitrarily designated to represent an average rate of various time intervals (summation interval). The estimated daily rate of microbial crude protein (N x 6.25) and true protein (N x 6.25 x 0.85) (42) synthesis in the rumen of a sheep with a 4-liter rumen consuming 158 kcal of digestible energy per 0.75 kg body weight per day was 218 and 185 g, respectively. These values represent a rate of microbial crude and true protein synthesis of 26 and 22 g per 100 g of organic matter digested (fermented; DOM) in the rumen, respectively. These values are higher than the theoretical upper limit proposed by Hungate (19) but similar to the upper values of ruminal microbial protein synthesis per 100 g of DOM in the range of values reported from direct measurements (15)(16)(17)27). The system used in the present work to assess microbial protein synthesis measured absolute but not net rates of microbial protein synthesis which are obtained from ingesta passage studies. The values given above can be recalculated by taking microbial protein turnover into account. Although the extent of turnover of microbial protein and bacterial-protozoal protein interconversions has not been accurately defined, it may be estimated from stud- (42). Estimated rates of microbial crude protein and true protein synthesis per day equals 218.0 and 184.9 g, respectively. Estimated microbial crude protein and true protein synthesis per 100 g of organic matter digested in the rumen [75% digestion (1)1 equal 26.0 and 22.0 g, respectively. ies of bacterial lysis in the rumen (22), from protozoa-bacteria interrelationships in the rumen (8,47), from the recent report on N turnover in ruminants (32), and from microbial turnover data (29) that 25 or more of the total protein synthesized in the rumen may be involved in turnover. On a 25 turnover basis the estimated ruminal protein synthesis rate was 16.1 g of true protein synthesis/100 g of DOM. This value represents a 30-g digestible protein-to-Mcal of digestible energy ratio which is adequate for growing calves and lambs (36).

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2018-04-03T03:38:32.117Z

1973-10-01T00:00:00.000Z

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Sporicidal properties of hydrogen peroxide against food spoilage organisms. The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H(2)O(2) were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with "D" values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H(2)O(2) concentration and the temperature reduced the lag period. Packaging systems that utilize paper or plastic-based packaging materials are used increasingly by the food industry because of the simplicity of operation, the lower raw-material costs, and the easier disposal of the empty container. According to Hsu (4), the most widely accepted of these systems are suitable for aseptic operation, but the nature of the packaging material excludes the use of heat as a sterilizing agent. Among the chemical sterilants available, hydrogen peroxide (H.02) appears to be the most suitable because it does not impart an off-flavor to the product and small residues on the packaging material can be tolerated without adverse effects. The use of H202 for the sterilization of pipes, filters, etc., in the food industry has been reported as early as 1916, viz: Schumb et al., (5). Swartling and Lindgren (6) observed slow sporicidal activity of 11 to 22% H202 at room temperature on spores of Bacillus subtilis and Bacillus cereus var. mycoides which were inoculated on glass and polyethylene surfaces. However, exposure for 15 s to 22% H202 at room temperature, followed by hot-air heating at 125 C for 10 s, substantially reduced the numbers of survivors. Cerf and Hermier (2) reported that 3.5 min of exposure to 15% H202 at 80 C was necessary to achieve four-decimal reductions in populations of the most resistant of 21 Bacillus strains tested. von Bockelmann and von Bockelmann (8) 'Present address: Department of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland. reviewed the literature on the sporicidal properties of H20,. Many of the studies referred to the use of 15 to 20% H202. The absence of data on H202 inactivation of dry spores and the apparent differences in results by using different techniques were discussed. The present study was undertaken to establish the relative resistance of food spoilage organisms to inactivation with H202 and to identify the factors that affect spore inactivation by H202 during aseptic packaging. MATERIALS Preparation of test suspensions. B. subtilis var. globigii and B. coagulans were grown in tryptic soy broth (Difco) and incubated at 37 and 55 C for 4 to 6 days respectively. The spores were harvested by centrifugation, washed three times, suspended in physiological saline, and stored at 4 C until used. B. stearothermophilus and B. subtilis SA 22 were grown on nutrient agar slants (Difco) in 900-ml screw-capped prescription bottles incubated at 55 and 37 C respectively. After 5 days, the spores were recovered from the slants by washing the surface with saline and collecting the suspended cells. S. aureus ATCC 6538 was grown in micrococcus medium (peptone, 5.0 g; yeast extract, 3.0 g; beef extract, 1.5 g; glucose, 1 g; in 1 liter of distilled water, pH 7.4), and incubated at 37 C. Samples from the SPORICIDAL PROPERTIES OF H202 actively growing cell suspension were used in the tests. Clostridium sp. NCA 3679 was grown in thioglycolate broth (Difco), sealed with mineral oil, and incubated at 37 C. Spores were recovered by successive centrifugation and suspension in saline as was previously discussed. Cultures were tested for the extent of sporulation before harvesting by staining smears with malachite green. Counts taken of samples from spore suspensions plated directly from stock and those plated after heating at 80 C for 20 min showed no significant differences, indicating that the stock suspensions were essentially all spores. Enumeration of viable organisms. Standard plate count techniques were used. B. subtilis var. globigii, B. stearothermophilus, S. aureus, and B. coagulans were plated on plate count agar (Difco) and incubated at their optimal growth temperature. B. subtilis spores were plated on tryptone glucose extract agar (Difco). Clostridium sp. 3679 spores were plated on thioglycolate agar and incubated at 35 C in an anaerobic chamber. Initial spore counts were made by plating a sample of the test spore suspension that had been heated for 20 min at 80 C. Spores treated with H202 were not heat shocked so that they were able to retain maximum resistance to H202. H202 treatment: (i) Wet spores at room tempera- immersed in a water bath until the H,02 reached water bath temperature. One ml of spore suspension at room temperature was then introduced to the test tubes rapidly by using a 5-ml plastic disposable syringe fitted with a cannula. After the desired contact time, 1 ml of the mixture was removed by using a sterile syringe and cannula and immediately discharged into the catalase solution, and the numbers of survivors were determined. Average temperatures to which the spores were actually exposed after mixing the cold spore suspension with hot H,02 were determined by recording the temperature change in control test tubes by using H202 and water. (iii) Dry spores. A 1-ml amount of spore suspension was introduced into sterile 25-by 150-mm culture tubes and dried over CaCl2 in a desiccator at 25 C for 72 h. No evidence of spore injury due to drying was found as shown by similar counts obtained from the dried spores and that from the same volume of wet spores. H202 was diluted with distilled water to the same concentration used for the wet-spore treatments after the 4 to 1 ratio mixing and added to the dry spores in the culture tube. A small, sterile, magnetic stirring bar was then introduced, and the mixture was agitated continuously over a magnetic stir plate. At appropriate time intervals, 1-ml samples were removed, and the numbers of survivors were determined, as was previously described for the wet spores. RESULTS AND DISCUSSION Comparative resistance of different microorganisms to H202. The resistance of various microorganisms to 25.8% H202 at 24 C is shown in Fig. 1. As expected, S. aureus was less resistant than any of the spore forms requiring only 1 min of exposure to reduce populations by 593 VOL. 26,1973 six log cycles. Thus, the most rigorous treatment necessary to eliminate spore-forming spoilage organisms should eliminate S. aureus as well. Data reported by Dittmar (3) indicated a very low resistance of this microorganism to HO,, requiring only 10 min in 0.05% H,O, for inactivation. Amin and Olson (1) worked with more resistant strains of S. aureus and reported as much as 171 min of exposure to 0.05% HO, for 99.9% destruction of cells in 250 ml of a 10' suspension per ml. Both groups of investigators used 0.05% H02, a much lower concentration than the 25.8% H02 used in the present investigation. Survival curves for most of the spore-forming organisms showed a lag period after initial exposure where there was a slow reduction in count, followed by a rapid rate of inactivation representative of first-order reaction kinetics. The less the resistance of the organism, the shorter the lag period, and for the two least resistant organisms, the survival curve followed first-order kinetics from the initial exposure. If there was a lag period for these organisms, it was not observed after the initial 30-s interval. The shapes of the survival curves were the same as those reported by Swartling and Lindgren (8) for B. subtilis ATCC 95244 spores in 10% H202. No tailing was observed in the survival curves of the organisms tested. Cerf and Hermier (2) reported a tail in the survival curve of a Bacillus strain isolated from milk when it was exposed to 23% H202 at pH 7.7 and 26 C. According to their data, the tail appeared to be less pronounced at pH 2.9. The present study was conducted by using stabilized H202 (pH 3.8) without adjustment of pH. Schumb (5) points out that HO, has maximal stability at pH 3.5 to 4.0, but becomes increasingly unstable at very low and very high pH values. The presence of the tail in Cerf and Hermier's survival curve could be due to decomposition of H202, resulting in decreased activity at the later stages of exposure. Figure 1 shows that the order of resistance was B. subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus. The "D" values determined from the straight line portion of the inactivation curves were 7.3, 2, 1.8, and 1.5 min, respectively, in 25.8% H02 at 24 C. Clostridium sp. 3679 and S. aureus showed very low resistance to H202. By comparison, Swartling and Lindgren (7) reported a "D" value of 2.3 min for B. subtilis ATCC 95244 in 20% H202, and Cerf and Hermier (2) reported a "D" value of 3.5 min in 23% H,02 at pH 4.6 for the most resistant Bacillus strain isolated from milk. Thus, B. subtilis SA 22 is much more resistant than the organisms previously tested by other investigators. The resistance of B. subtilis var. globigii is comparable to organisms previously tested. Effect of combined heat and HO, treatment on spore survival. Heat shock of spores at 80 C for 20 min prior to H2O2 treatment enhanced their destruction. Because the heatshocked spores were quickly cooled in ice water and immediately treated with H202, the possibility of germinated spores being responsible for the decreased resistance to H202 was eliminated. The inactivation curve for heat-shocked spores of B. subtilis var. globigii (Fig. 2) showed a rapid rate of destruction after 4 min of exposure to H202 compared with untreated spores. This is evidenced by the "D" values of the straight line portion of the curves which were 0.5 and 2 min, respectively. The mild heat treatment alone did not significantly reduce the spore count, but it was sufficient to decrease their resistance to H202. When the unheated spores were exposed to 25.8% H202 for 4 min at 24 C and then, after destroying the H202 by catalase, heated at 80 C in a water bath for 20 min, a six-log cycle reduction in count was observed. The 4 min of treatment with H202 by itself did not reduce the spore count by more than 1 log cycle (Fig. 2), and the heat treatment by itself did not signifi- on March 17, 2020 by guest http://aem.asm.org/ Downloaded from SPORICIDAL PROPERTIES OF H202 cantly alter the count. Yet, the H202 treatment considerably reduced the resistance of spores so that a mild heat treatment inactivated the injured spores. This result is in agreement with data given by Swartling and Lindgren (6) who showed that 11 s of exposure to 22% H202 at room temperature did not inactivate spores of B. subtilis, but that exposure of the spores to hot air at 125 C for 8 to 10 s following the H202 treatment resulted in a 99.2% destruction of spore population. Effect of H202 concentration. Increasing the concentration of H202 increased its sporicidal properties. The survival curves of B. subtilis var. globigii in varying concentrations of H202 at 24 C (Fig. 3) showed that increasing H202 concentrations lowered the time during which the curves showed the initial change in slope, reduced the exposure time, and also decreased the "D" value. Ten minutes of exposure at 10 and 20% was insufficient to start the curve on a exponential decline of surviving spore populations, whereas 6, 4, and 1 min were sufficient when 25.8, 35, and 41% H202, respectively, were applied. "D" values were 2, 1.5, and 0.75 in Fig. 1 through 4 reveals that simple multi-At of temperature. The temperature at plication of the "D" value by the number of log H202 was incorporated has a very marked cycles for the spore inactivation desired is n spore inactivation. Figure 4 shows that insufficient to obtain the required exposure e of inactivation in 25.8% H202 increased time for inactivation because of the initial creasing temperature. persistence of spores, particularly those of B. nination of the inactivation curves shown subtilis SA 22 and B. subtilis var. globigii. However, in most of the curves, the lag does not 10% persist for more than one log cycle of initial A~o * decrease in spore population. Thus, the "D" 0\ \A concept utilized in heat sterilization can also be A.\ \ \\0 utilized for H202 sterilization if, in addition to 20\ \ <% the "D" value, the time required for the first log cycle reduction is incorporated into the calcula-0 tion. In Fig. 5, the "D" value and time required to achieve the first log-cycle reduction in spore Based on the "D" value, the "z" value for B. subtilis var globigii in 25.8% H202 is 40 C. This compares with "z" values of 46 C, 52 C, and 47 C determined from the data reported by Swartling and Lindgren (7) for B. subtilis in 10%, 15%, and 20% H202, respectively. The "z" value is the temperature change required to bring about a 10-fold change in the "D" value. Comparative resistance of wet and dry spores. In a well-mixed system, dry spores are less resistant to H202 (viz. Fig. 6). Spores of B. subtilis SA 22 in 25.8% H202 at 24 C had a first log-cycle inactivation time and a "D" value of 8.5 and 7.3 min respectively when wet, compared to 4.8 and 4.7 min respectively when dry. Spore inactivation started immediately after contact of dry spores with H20., whereas a short induction period was required for wet spores. Because the wet-spore suspension was replaced by water in the H202 solution used for the dry-spore treatment, the actual H202 concen- In an unmixed system (treatment was similar to wet spores at room temperature except that spores were allowed to dry inside the syringe prior to H202 treatment), data scatter was very * pronounced, and in most instances more of the eventually appears in the packaged food and a reasonable margin of safety is allowed in the elimination of pathogenic and food-spoilage microorganisms. Acceptance of H202 has been slow because of uncertainties with regard to its sporicidal properties and the lack of quantitative information necessary to evaluate the sterilizing effectiveness of a specific processing condition within a system. The system described by Hsu (4) is now used primarily on refrigerated or on acid foods. Swartling and Lindgren (7) described microbiological studies on which treatments used in the above system were based. By using 10 to 20% H202, the conditions of exposure to H202 were 0 20 30 40 50 60 TO 80 90 100 sufficient to reduce the population of B. subtilis 95244 (the one species of spore-forming orga-TEMPERATURE 0C nisms studied) by four to six log cycles. Our 5. Effect of temperature of 25.8% H202 on results show that organisms exist that are more :due and time for first log cycle inactivation of resistant than the B. subtilis used by Swartling of B. subtilis var. globigii. and Lindgren (7) sterilizing treatments needed for paper-based packaging materials. The organisms in the present study were able to survive the treatments they recommended. A safer process would result if the most resistant organism in our study, Bacillus subtilis SA 22, is used as a basis for developing requirements for sterilization in H202. Although an anaerobe, Clostridium sp. PA3679 appears to have very low resistance to H202, and the resistance of other anaerobes such as Clostridium botulinum to H202 should be further investigated. The danger from anaerobic organisms in aseptically packaged foods using plastic or paper-based packaging materials is minimized because of the permeability of these materials to oxygen and the minimal vacuum in the container. Our results show that "D" and "z" value concepts used in determining inactivation times in heat sterilization could be applied to H202 if the time required for inactivating the first 90% of the population was used in addition to the "D" value. We have presented data on the "D" values of six microorganisms at 24 C and the "z" value of one organism in 25.8% H202. When more data are compiled on the "D" and "z" values of various microorganisms at different H202 concentrations, it should be possible to evaluate conditions in any aseptic packaging system in which H202 is used for sterilization.

v3-fos

2018-04-03T05:40:50.380Z

1973-08-01T00:00:00.000Z

43757718

s2

Effects of Various Gases on the Survival of Dried Bacteria During Storage Salmonella newport and Pseudomonas fluorescens were dried together in papain digest broth and sucrose-glutamate, and stored in several gases at various water activities (a.) between 0.00 and 0.40 at 25 C for various periods up to 81 weeks. Both S. newport and P. fluorescens, dried in papain digest broth and stored in air, died rapidly if the conditions were very dry (0.00 a.) or moist (0.40 aw). Storage in carbon dioxide and argon gave greater survival than storage in air but lower survival than did storage in nitrogen or in vacuo. When the organisms The stability of dried bacteria during storage is dependent on a number of factors, the more important ones being the organism itself, the composition of the suspending fluid in which the organism is dried, the residual moisture, the temperature of storage, and the atmosphere of storage (2). It has been well established by earlier workers that storage in vacuo gives better survival than storage in air (4,(5)(6)(7)(8). However, there is no agreement about the relative merits of other gases and whether or not use of these gases is as good as storage in vacuo. Rogers (6) was one of the first workers to compare storage in vacuo with storage in other gases. He found that survival was highest in cultures stored in vacuo and lowest in those stored in air or oxygen. Atmospheres of nitrogen, hydrogen, and carbon dioxide were intermediate in rates of survival. The observations of Naylor and Smith (4) with Serratia marcescens are in agreement with these findings. Proom and Hemmons (5), working with Neisseria, also found that storage in vacuo gave the best survival, although in this case storage in nitrogen was just as good as storage in vacuo. In this earlier work there had been no control of residual moisture during storage except to dry the particular gas, and a variety of suspending media had been used. In no instance was more than one suspending medium studied in any one experiment. The work of Scott (7) showed that the effect of atmosphere was dependent upon the suspending medium and the moisture level. He found with Salmonella newport that only under the driest conditions and in the absence of sugars was there a marked difference in viability between dried cells stored in air and in vacuo. In the experiments to be described in this paper, the survivals of two organisms, namely, Pseudomonas fluorescens and S. newport, have been determined when they were dried in two suspending fluids and stored in various gases under conditions of controlled moisture levels. MATERIALS AND METHODS Organisms and suspending media. The organisms used were P. fluorescens and S. newport. The two suspending fluids were papain digest broth (7) and a mixture of 0.25 M sucrose and 0.25 M glutamate, Marshall and Scott (3) having shown the protective effects of this combination. Preparation for and freeze-drying of organisms. Both organisms were grown in brain-heart broth medium for 20 h at 30 C with aeration. Equal volumes of each culture were mixed. The resulting suspension was centrifuged and the cells were resuspended to half their original volume in the particular suspending fluid to give approximately 1010 cells of each organism per ml. In this way possible variations in the drying, storage, and rehydration of either oganism were eliminated. EFFECTS OF GASES ON DRIED BACTERIA Replicate ampoules containing 0.2-ml samples of the suspending fluids and organisms were prepared. The ampoules were supported in a rack so that each tube was radially mounted about 200 from horizontal, with its mouth some 2 cm from the surface of the central condenser which was filled with solid CO2 and ethanol. The apparatus accommodated up to 160 ampoules so that all ampoules for a particular experiment were dried together. Cooling was purely evaporative and heating was by radiation from the walls of the steel vacuum chamber. It was found convenient to de-gas the suspensions by evacuation until they had cooled to about 0 to 2 C before the CO2 plus ethanol mixture was added to the condenser. Immediately, the condenser was cooled, the rate of evaporation increased, and the suspending fluids and organisms were promptly cooled to -30 to -35 C. No measurements oi temperature were made during drying which was for 4 h, the time found by Scott (7) sufficient to remove almost all the water from all solutes. The dried cells were prepared for storage immediately after drying. Storage conditions. The ampoules were placed within larger tubes containing phosphorus pentoxide (P20.) for 0.00 a. and 2 ml of the appropriate sulfuric acid solutions (9) for all other a. levels. The larger tubes were first drawn out and then sealed in groups of five on a manifold for individual suspending fluids and water activities. The following conditions for storage were used. (i) In air, the tubes were not vacuated before the final sealing. (ii) In vacuum, the tubes were evacuated in groups of six with a two-stage mechanical pump until the solutions controlling a. boiled, or for at least 60 s before the final sealing. (iii) In nitrogen, the tubes were first evacuated and then flushed to atmospheric pressure with oxygen-free nitrogen (supplied in a cylinder containing alkaline pyrogallol). This was repeated twice before the tubes were finally sealed. (iv) In carbon dioxide, the tubes were treated as for nitrogen, carbon dioxide being flushed into the tubes after the third evacuation and the tubes sealed. (v) For storage in inert gases (argon, helium, neon, krypton, and xenon), the spectrally pure gases were supplied in 1-liter flasks at slightly above one atmosphere pressure (British Oxygen Gases Ltd). A manifold built of capillary tubing was used to conserve the gases for the filling and sealing of groups of six tubes. For each gas, the manifold was evacuated and filled with oxygenfree nitrogen, re-evacuated and filled with the nitrogen, re-evacuated and then filled to one atmosphere with the particular gas, and the tubes sealed. As the pressure at sealing was controlled with a mercury manometer, the possibility of slight contamination by mercury vapor cannot be excluded. Storage and viable counts. Immediately on the completion of sealing, the tubes were stored in the dark in an insulated cabinet at 25 C. Five replicate ampoules for each treatment were stored and after 1, 3, 9, 27, and 81 weeks one ampoule of each was randomly selected for the determination of viable numbers. The contents of each ampoule were rehydrated with 2 ml of saline. Where necessary, decimal dilutions were prepared in saline. At each dilution four plates were poured using brain-heart-infusion agar. One duplicate set of plates was incubated for 17 to 20 h at 37 C for the enumeration of S. newport and the other set was incubated for 6 days at 7.5 C for the enumeration of P. fluorescens. Separate experiments showed that the incubation conditions used completely suppressed colony formation by one organism and permitted full development of the other. S. newport and P. fluorescens were dried together in the two suspending fluids and stored at 25 C in five gases (air, vacuum, nitrogen, carbon dioxide, and argon) at three moisture levels (0.00, 0.20, and 0.40 aw). There were two replicates. In a further experiment (not replicated) storage of both organisms was studied in seven gases (vacuum, nitrogen, helium, neon, argon, krypton, and xenon) at four water activities (0.00, 0.10, 0.20, and 0.30) after drying in papain digest broth. The viable numbers per milliliter in terms of the original undried suspension were expressed as two-figure logarithms derived from the mean counts of duplicate plates. RESULTS P. fluorescens was more sensitive to both the drying and storage treatments. Forty percent of the cells of S. newport were still viable immediately after drying in papain digest broth compared with 100% for sucrose-glutamate. Survival of P. fluorescens after drying in papain digest broth was 25% and 95% in sucrose-glutamate. These survivals form the basis for evaluating changes associated with storage treatments. Figure 1 shows the changes in viability of P. fluorescens for all treatments during 81 weeks of storage. The effects on viability became more evident with time and, except for storage in air, were not apparent until the organisms had been stored for at least 9 weeks. Storage in vacuo and in nitrogen was significantly better than storage in air; carbon dioxide and argon gave intermediate results. The rate of death for storage in any gas and in either suspending fluid was least at 0.20 and generally wes greatest at 0.40 a.. However, the effects of aw on survival were very much greater for cells dried in papain digest than for cells dried in sucrose-glutamate. When P. fluorescens was dried in papain digest, the most significant difference between the gases occurred when the cells were stored at 0.00 a.. In air, storage at 0.00 a. led to death equal to that at 0.40 aw, whereas there were very small differences between 0.00 and 0.20 for vacuum and nitrogen. S. newport was less sensitive than P. fluorescens to all the storage treatments. Table 1 shows levels of survival for S. newport after storage for 81 weeks. The differences between the organisms were more marked when dried in Analysis of variance of the data showed that the average main effects of all factors and all their two-factor interactions were highly significant (at P = 0.001) for both organisms. The three-factor interaction for P. fluorescens was significant at P = 0.01. This was because the means for papain digest and 0.40 aw were very low in relation to those of the other water activities and all the water activities for sucrose-glutamate in all gases. Table 2 shows entries for means over one factor classified in rows and columns for combinations of the remaining two factors in all three possible ways for each organism. The marginal entries for each factor are means over both other factors. The study of means in the two-way tables shows that for P. fluorescens papain digest gave very low viability in air in comparison with sucroseglutamate; viability for air at 0.00 a. was much lower than for 0.20 a. in comparison with the other gases and viability was low for the papain digest-0.00 a. combination; further, the decrease in viability associated with papain digest as compared with sucrose-glutamate was much greater at 0.40 than at 0.20 a.. Similar inconsistencies occurred for S. newport. Comparison of the marginal means for average main effects shows that sucrose-glutamate and 0.20 a. gave the greatest viabilities; as regards gases there was no significant difference between vacuum and nitrogen which were clearly better than the others; these conclusions are true for both organisms. The differences between the seven gases used in the second experiment increased with time to their greatest values at 81 weeks of storage. Table 3 shows the viabilities of both organisms after storage for 81 weeks at all water activities. For P. fluorescens when very dry (0.00 a.) there were no significant differences in storage between vacuum, nitrogen, and argon; xenon was very destructive and the other three gases gave intermediate survival patterns. At 0.30 a. the differences between gases were substantially reduced. Even smaller differences between gases were found after storage at 0.10 and 0.20 6.88 (30) 9.43 a Standard errors of means: n' = 4, ±0.39; n! = 6, ±0.32; n' = 10, ±0.25; n' = 12, ±0.23; n' = 20, +0.18; n' 30, +0.14 on 29 degrees of freedom. The interactions SF x a., SF x gas and a. x gas were significant at 0.1, 0.1 and 5%, respectively. Survival of both organisms was greatest at 0.10 aw and the differences between the gases were substantially suppressed. The only gas giving survival equal to storage in vacuo was argon. DISCUSSION The main aim of these experiments has been to study the changes during prolonged storage and to see whether any gases were different from storage in vacuo. The techniques used ensure that aw is, in fact, controlled during storage and enable valid comparisons to be made between different atmospheres. Because of the small amounts of water hydrating speciments at 0.10 to 0.40 aw, equilibrium would have approached to within 0.01 aw of the appropriate values after storage for 1 week (7). The results emphasize the overriding importance of controlling the level of residual water at about 0.10 aw irrespective of the nature of the atmosphere or suspending fluid. The results of workers where aw is unknown are of limited value. Greiff and Rightsel (1) studied the survival of dried influenza virus when stored in dried gases but the order of stability does not correspond with that reported here for P. fluorescens and S. newport. However, although they used dried gases, the exact values of aw during storage were unknown and indeed may have changed after sealing because of the production of water by the Maillard reaction. Our results do provide, for the first time, unequivocal evidence of differences between gases which are clearly a function of the level of residual water. Because of the interactions between the various factors, greatest survival of dried cells after prolonged storage will only be achieved when careful attention is given to selecting the appropriate level of all conditions concerned. LITERATURE CITED

v3-fos

2020-12-10T09:04:16.902Z

1973-09-01T00:00:00.000Z

237229138

s2

Staphylococcal Enterotoxins A and B: Solid-Phase Radioimmunoassay in Food An immunoassay employing 125I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less. We recently developed a solid-phase radioimmunoassay test for assaying staphylococcal enterotoxins types A and B in purified form and in culture form (9). The test has also been used to study the antigenic relationships among enterotoxins types A, B, and C (8). The data presented here indicate that the solid-phase radioimmunoassay test is suitable for the detection and quantitation of enterotoxins A and B in several foods. The method is particularly attractive because of high sensitivity and minimal preparation of food extracts for analysis. Current methods for detection of enterotoxins in food either lack sensitivity, require cumbersome extraction and concentration of food extracts, or present problems of nonspecific reactions (1). MATERIALS AND METHODS Purified enterotoxins. Purified staphylococcal enterotoxins A and B were supplied by M. S. Bergdoll, University of Wisconsin, Madison. The purified toxins contained less than 5% impurities (1). The toxins were used for the radiolabeling and spiking of foods. Enterotoxin antisera. Antisera to enterotoxin B were produced in rabbits as previously described (6). Antisera to enterotoxin A were obtained from R. W. Bennett, Food and Drug Administration, Washington, D.C. Pools of anti-A and anti-B contained approximately 1 and 2 mg of antibody protein per ml, respectively, as determined by quantitative precipitation (2). Iodination of enterotoxins. Enterotoxins A and B were radiolabeled with 121I by the chloramine-T procedure (5, 7), modified as previously described (9). Labeled enterotoxin contained approximately 40 XCi activity per Ag of protein. Preparation of antibody-coated polystyrene tubes. Polystyrene tubes (10 by 75 mm) were sensitized with antibody to enterotoxin as previously described (9). Briefly, 1-ml amounts of sodium sul-fate-precipitated antibody in phosphate buffered saline (PBS), pH 7.2 (0.07 M NaCl, 0.07 M phosphate), containing approximately 10 to 20 gg of protein per ml, were added to the polystyrene tubes with a volumetric pipette. After 2 h at room temperature, the antibody was removed and the tubes were washed once with 2.0-ml amounts of 1.0% bovine serum albumin (BSA) in PBS to which sodium azide (0.1% final concentration) had been added as preservative. The tubes were then filled with 1.0% BSA and left overnight at room temperature. The BSA was removed, and the tubes were washed once with PBS and stored inverted at 4 C until used. The preparation of anti-enterotoxin-coated polystyrene tubes is illustrated in Fig. 1. Solid-phase radioimmunoassay. Portions (1 ml) of extracts from foods spiked with or without enterotoxin were added to the antibody-sensitized tubes. The tubes were shaken 10 times and incubated at 37 C for 18 h. The extracts were then removed, and 1 ml of 1% BSA and 0.001 fig of 125I-enterotoxin A or B (in 0.1 ml of 1% BSA) were added to each tube. The tubes were shaken 10 times, incubated at 37 C for 4 h, washed once with 2 ml of PBS, and counted. The inhibition of binding of 1251-enterotoxin was determined by comparing the radioactivity of tubes containing extracts with that of tubes containing only 1% BSA in the 18-h incubation. Counting equipment. Radioactivity was measured with a Packard Auto-Gamma Counter (Model 5320). This system has a counting efficiency of approximately 62% for 1251 in a 1-ml geometry and a background count rate of approximately 50 counts/min. Preparation of food extracts. Various amounts of purified enterotoxin were added to 10-g amounts of ham salad contained in Waring Blenders, followed by the addition of 10 ml of 1% BSA in PBS (with sodium azide to 0.1%). The ham salad was blended at high speed for 3 min at room temperature and then centrifuged for 15 min at 20,000 rpm in an International BD-2 centrifuge at 25 C. The supernatant was collected, and the pH was adjusted to 7.4 to 7.5. The 309 supernatant was recentrifuged at 20,000 rpm for 30 min, removed, passed through a Kimwipe tissue, and stored frozen until used. Cheddar cheese extracts were prepared as described for ham salad, except that 20 ml rather than 10 ml of 1% BSA was added to 10 g of cheese. Extracts of a packaged dry custard mix (Jello) were prepared as described for ham salad, except that the mixture was blended for 1.5 min instead of 3 min. Various amounts of enterotoxin were added to 10-ml samples of condensed milk. The pH was adjusted to 4.5 with 4 N HCl to precipitate the milk proteins. The milk was centrifuged for 30 min at 15,000 rpm at 25 C. The supernatant was removed, and the pH was adjusted to 7.4 to 7.5 with NaOH, followed by recentrifugation at 15,000 rpm for 15 min. The supernatant was collected, passed through a Kimwipe tissue, and stored frozen until used. Extracts from pork and beef salami were prepared as described for cheddar cheese, except that the extract was dialyzed against PBS at 4 C until the dialysate showed no absorption at 215 nm in a Spectronic 600 spectrophotometer. Aromatic substances have a high absorptivity at this wavelength. The salami extracts were not removed from the antibody-coated tubes after 18 h of incubation, as were the other food extracts. Determination of enterotoxin in food extracts. 1251 labeled enterotoxin (0.001 gg/g of food) was added to the various foods and extracted as described above. The ratio of the radioactivity of 1 ml of supernatant extract to the radioactivity of 1 ml of food homogenate before centrifugation was used to determine the concentration of toxin "recovered." RESULTS Ham salad from three sources was studied. For enterotoxin A, recovery varied from 54 to 60% for the different sources of ham salad; for enterotoxin B, it varied from 88 to 97%. Table 1 presents data on the inhibition of binding of 125I labeled enterotoxins A and B to their corresponding antibody-coated tubes by extracts of enterotoxin obtained from one source of ham salad. For the enterotoxin A system, inhibition varied from 23.1 to 55.7%, corresponding to 0.001 to 0.01 tig of enterotoxin A per g of ham salad. Enterotoxin B (0.01 ug/g of ham salad) and ham salad alone gave 7.4 and 14.4% inhibition of the A system, respectively, indicating nonspecific effects. Similar data were obtained for the enterotoxin B system, with 0.001 to 0.01, Ag of B per g of ham salad producing 31.3 to 69.3% inhibition of the binding of labeled enterotoxin B. Inhibitions by extracts of ham salad alone and enterotoxin A in ham salad were 16.7 and 20.4%, respectively. If nonspecific inhibition is taken into consideration, the solid-phase radioimmunoassay test for enterotoxin in ham salad has a lower level of sensitivity of 0.001 to 0.0025 Mg of enterotoxin per g of ham salad. The data obtained for the two other samples of ham salad are not shown but were similar to that presented in Table 1. Cheddar cheese from two sources was studied. In contrast to the 1:1 ratio for ham salad, cheese extraction was performed, using one part cheese to two parts diluent (wt/vol) to reduce the viscosity of the cheese homogenate. Recovery rates ranged from 77 to 98% for enterotoxin A and from 111 to 126% for enterotoxin B. Inhibition data on extracts from one of the cheeses are presented in Table 2. Sensitivity of the assay was approximately 0.0025 Mg/g of cheese. Nonspecific effects, cheese alone and heterologous enterotoxin in cheese, were responsible for about 10 to 15% of the inhibition. Data similar to those presented in Table 2 were obtained with the other cheese source. Inhibition data on custard and enterotoxins A and B are presented in Table 3. The results are similar to those obtained with ham salad in terms of the sensitivity of the assay, and nonspecific inhibitions were approximately 4 to 10%. Recovery values of enterotoxins A and B for the single custard sample examined were 97 and 94%, respectively. Inhibition data on condensed milk are presented in Table 4. Nonspecific and heterologous enterotoxin inhibitions were less than 10%. As little as 0.001 Mg of enterotoxin A or B per ml gave good, specific inhibition (21.4% for A and 24.5% for B). The simplified procedure of extraction by acid precipitation of milk proteins resulted in 68.9 and 74.4% recovery of enterotoxins A and B, respectively. A blind study was conducted for the detection and quantitation of enterotoxins A and B in condensed milk. The unknown samples were prepared by an individual not involved in the assay. Standard curves were constructed, using Table 4 in a manner described previously (9). The results (mean values from triplicate determinations) are presented in Table 5. The enterotoxin type was properly identified for each of 11 samples studied. Furthermore, remarkably good agreement was obtained between the values for the amounts of enterotoxins added to the samples and the amounts determined by radioim-munoassay. Only two determinations showed less than 70% recovery, while one showed 140% recovery. Nine samples showed recoveries that ranged from 75 to 116%. The data establish the solid-phase radioimmunoassay procedure as both a qualitative and quantitative test for enterotoxins A and B in food. The most difficult food encountered thus far in the application of the radioimmunoassay procedure to food has been salami. Extracts from salami, prepared as described for cheddar cheese, have resulted in as much as 50% or greater nonspecific inhibition. We have been able to eliminate most of this nonspecific inhibition by dialyzing the salami extracts against changes of PBS at 4 C until no absorbance has been detected in dialysates at 215 nm in a Spectronic 600 spectrophotometer. Preliminary data on inhibitions with enterotoxin A extracts are presented in Table 6. Nonspecific inhibition was approximately 15%. The lower limit of sensitivity appears to be between 0.005 and 0.01 1g of enterotoxin A/g of salami. The extraction procedure recovered about 52% of the enterotoxin added to the salami. DISCUSSION The data presented here demonstrated that the solid-phase radioimmunoassay procedure is applicable for the sensitive and specific detection of staphylococcal enterotoxins A and B in food. The sensitivity of the assay is in the range of 1 to 10 ng/g of food. It seems that a sufficient number and variety of foods have been examined here to suggest that the procedure is suitable for general application and is not restricted to a few types of foods. The toxin extraction procedures, for example, were quite simple and required minimal manipulation. This is in marked contrast to current techniques for extraction of enterotoxins from food for determination by immunoassay procedures (1). Enterotoxin A is bound by food particles more extensively than is enterotoxin B, as indicated by the extraction studies with 1251 labeled enterotoxins. This phenomenon could possibly be related to the much higher incidence of food poisoning involving enterotoxin A as opposed to (1). It has been shown, for example, that enterotoxin is intimately associated with the bacterial cell wall (3,4). Enterotoxin A receptors in food could possibly react with toxin at the surface of the bacterial cell and thus stimulate the cell to produce more toxin. One would except enterotoxin B to be the most prevalent toxin in staphylococcal enterotoxin food poisoning, since, when compared to enterotoxin A, it is relatively easy to produce in large quantities under laboratory conditions (1). The concept presented above may explain this paradox. It has been shown previously that staphylococcal enterotoxins are antigenically related (8). It has also been demonstrated that this antigenic relatedness is of no consequence from the practical and applied point of view because of the extremely high homologous-to-heterologous inhibition ratios in solid-phase radioimmunoassay. Thus, inhibitions observed with extracts of food treated with 0.01, 0.02, or 0.1 ,ug of heterologous enterotoxin per g of food were no different from those of extracts from foods not treated with enterotoxin. Sensitivity values presented for the assay take into account the slight, nonspecific inhibitory effects, which are usually about 15% or less. Preliminary data (unpublished) indicate that the specificity of the radioimmunoassay is maintained with extracts of food treated with crude enterotoxin preparations or with cultures of nontoxigenic Staphylococcus organisms.

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Book reviews to further promote research interest in rice, particularly the by J.A. GOODE and D. CHADWICH. John Wiley & Sons, application of functional genomics. Many of the resources 605 Third Avenue, New York, NY 10158-0012. 2001. Hardand tools which are developed by rice researchers will be cover, 261 pp., $125.00. ISBN-0-471-49661-8. applicable to the study of other cereals such as wheat and Rice (Oryza sativa L.), is a major food crop worldwide. maize. The quality of, and access to bioinformatics resources The demand for rice will increase with increases in world will play a major role in developing the future potential of population; the demographics of population growth will furrice genomics-based research for meeting the challenges of ther challenge feeding the world’s poor. Genetic enhancement cereal crop production. Bruno et al. present a paper on the through biotechnology is seen as a way to produce more food, state of rice information resources, the needs of the rice comreduce pressure on natural resources, and meet population munity, and proposed bioinformatics activities to support demand. On this premise, Novartis has hosted the Symposium these needs. on Rice Biotechnology: Improving Yield, Stress Tolerance and The next phase of the book is devoted to specific ways to Grain Quality, held at the International Rice Research Institute enhance rice performance. The initial papers in this section (IRRI) at Los Baños, The Philippines, 27–29 Mar. 2000. The deal with the physiology of yield in rice. They look at genetic Novartis Foundation, along with IRRI, invited scientists to promanipulation of rice photosynthesis and of starch synthesis. vide a synopsis of the current state of rice biotechnology. This In the first paper by Ku et al., genes have been introduced in book is a compilation of papers representing the cutting edge the rice plant which encode for C4 photosynthesis. In the of science in March of 2000. In this book, leading researchers second paper, Horton et al. present the response of rice plants from around the world provide on overview of their current to light: Photoinhibiton and photoacclimation under field conresearch in the areas of genome sequencing, bioinformatics, ditions. A third approach to increasing rice yields presented regulation of gene expression, and functionality of genes. Speby Okita et al. involves metabolic engineering of the rice plant cific topics include increasing photosynthesis through gene to improve triose phosphate utilization by enhancing starch manipulation, genetic regulation of disease resistance pathproduction in leaves and developing seeds. ways and drought, salt and freezing tolerance, and use of The next group of papers deal with stress tolerance and biotechnology to improve nutritional characteristics in cereals. include a dissection of defense response pathways in rice by This book underscores the substantial progress that has been Leach, Leung, and Wang; a genetic analysis of plant disease made in rice biotechnology since rice was selected as a model resistance pathways; regulation of systemic acquired resistance cereal crop for molecular genetic research, and provides an by NPPR1 and its partners, and improving plant drought, salt, overview of major advances in rice improvement. The book and freezing tolerance by gene transfer of a single stressalso contains discussions by the participants on future research inducible transcription factor. It is pointed out that increasing areas as well as on public attitudes towards genetically modified stress resistance in the rice plants can play a major role in the crop plants. In the end, John Bennett sums up the symposium increase of yield, especially in marginal situations. with the chapter entitled Summing-up: Cutting-Edge Science The last area the book touches on is breeding for nutritional for Rice Improvement—Breakthroughs and Beneficiaries. The characteristics in cereals. The golden rice story is told, as well papers compiled in this book clearly show the attempt to utilize as a look at developing transgenic grains with improved oils, biotechnology for the improvement of cereal grains, and to proteins, and carbohydrates. develop concepts with practical implications for rice breeding. The purpose of this book is to document an exchange of Rice Biotechnology: Improving yield, stress tolerance and information from researchers who were specifically chosen and grain quality comprises 16 papers, with discussions and comgathered together with the objective to develop research stratements that illustrate the current direction of research by IRRI gies and directions for rice improvement at IRRI during the to improve the yield potential of rice. It is an excellent synopsis next decade. It is a powerful look at the ideas of some of the of rice biotechnology, demonstrating its application towards best minds of our time in the area of rice research. While this increasing rice yield and stability. This would be an excellent book is not for beginners, it is appropriate for those who want textbook supplement for graduate courses in genomics research. phenomena and relationships of the disease are discussed, we are, indeed, rather surprised that he has accomplished his task in so short a time. The work is based upon personal observations, and upon a careful analytical and critical study of all the published cases to the records of which the author could obtain access. When we state that the bibliographical appendix contains two hundred and six separate references,, as well as a long list of theses, all of which have been personally examined by Dr. Monro, and abundantly referred to in the text of the essay, our readers will have some idea of the amount of labour involved in its preparation. The book is one which every physician, called upon to deal with cases of this obscure malady, will find to be of the greatest service to him as a work of reference. It is dedicated to the memory of Maurice Raynaud, and, appropriately enough,, begins with a short biographical sketch of this eminent physician. After introductory chapters on anatomy, statistics,, terminology, and history, the phenomena of Raynaud's disease* under the headings of etiology, local syncope, local asphyxia, and symmetrical gangrene, are exhaustively treated of. The concluding chapters of the work deal with the morbid relationships, the morbid anatomy, the pathology, the diagnosis, and the treatment of Raynaud's disease. After a somewhat careful perusal of Dr. Monro's interesting account, we are obliged to confess that our conceptions of the affection, now commonly associated with Raynaud's name, a-re clinical, rather than pathological or anatomical. While it must be admitted that the group of symptoms, characterised by local syncope, asphyxia, and symmetrical gangrene occurring in paroxysms, constitutes a sufficiently striking clinical picture, it may also be granted that the definite association of these symptoms with a special microscopical or macroscopical lesion in the central nervous system, or elsewhere, has not yet been made clear. This, of course, can only be done when a much larger number of cases presenting Raynaud's phenomena have been subjected to careful investigation after death. Until this has been done, however, we must to some extent remain in doubt whether we are to regard Raynaud's affection as a disease sui generis, or merely as a symptom like albuminuria or dropsy of a number of morbid states. While this is so, there can be no doubt that Dr. Monro has done great service in collating and summarising our present knowledge of the condition,.and so preparing the way for the further investigations which are still necessary to elucidate its true nature. We heartily congratulate the author upon his valuable work, and cordially recommend it to the favourable notice of the profession. 1900. This little work consists of five chapters, of which the first is introductory and new, while the remaining four have already appeared in the Lancet. Chapter I, on the visual memory, gives a lucid explanation of the distinction between visual perception and visual memory, and shows how, on theoretical grounds, investigators have in recent times been led to assume that the visual perceptive centre and the visual memory centre are not the same, but quite distinct from one another. Reviews. In Chapter II the same subject is taken up from the clinical point of view, and illustrated, in the first place, by a case which presented the following features:?Loss of the visual memory of printed and written words and letters, but not of figures; perfect ability to write to dictation, but inability to read what had thus been written; right lateral hemianopsia. The symptoms are attributed to a subcortical lesion in the left occipital lobe, so situated as to destroy the optic radiations passing to the left occipital cortex, and also the fibres passing from both occipital lobes to the left angular gyrus. Dr. Hinshelwood gives another case, where, in addition to right lateral hemianopsia, there was loss of the visual memory for places. As the patient was illiterate, she could not be tested with regard to words. The author points out that memory is not scientifically recognisable as a single faculty, but is simply the sum of a number of individual memories?of vision, hearing, smell, taste, &c. Similarly, if we consider visual memory by itself, we find that this in turn is made up of a series of subordinate visual memories, which are capable of being arranged into two groups?(1) highly specialised visual memories, for the acquirement of which mental concentration and special training are necessary; and (2) less specialised memories, whose acquirement implies no great mental effort on the part of those possessing the sense of vision. The first group of visual memories 4iave to do with words and letters, figures, musical notes; the second group with form and colour, objects and places. It is supposed that one cerebral hemisphere only is, as a rule, educated for the purposes of the first group, while the second group of visual memories are stored in both hemispheres, so that their loss implies a bilateral lesion, and is therefore very uncommon. Chapter III describes an interesting case of partial mindblindness with dyslexia. The patient could read a few words correctly, and then suddenly stopped, owing to complete word-blindness. After a rest, he could read a few words more quite correct^. He was unable to follow his occupation as a tailor, apparently owing to loss of memory of the objects he required to handle, and he had also at times loss of memory for places. Under hospital treatment, including complete abstinence from alcohol, he steadily improved, and ultimately became able to read for any length of time. The disorder is attributed to impaired functional activity of the right and left visual memory centres, or of the fibres on each side connecting the centre with both occipital lobes. It is stated that there was no disturbance of speech, but apparently the presence or absence of agraphia during the alexic period was not determined. Otherwise, in accordance with the teaching of Chapter II. it should have been possible to decide as to a cortical or a subcortical lesion. Chapter IV is on word-blindness without letter-blindness,, and is illustrated by four cases, one of which is original. Such patients can read letters, but not words; can read figures separately, and in any combination; and can write spontaneously, and to dictation, though unable to read the words they have written. The condition is attributed to partial destruction of the visual word centre rather than to a subcortical lesion, since there is no hemianopsia. By partial destruction of the visual word centre is meant destruction of the centre for the visual memory of words, which is regarded as anatomically distinct from, though adjacent to, the centre for the visual memory of letters. Chapter V is on letter-blindness without word-blindness, and includes a very remarkable case which was under the care of Dr. Finlayson two years ago, as well as a case of Sir Wm. Gairdner's, and three cases quoted from other writers. Dr. Hinshelwood is to be congratulated, not only on the importance of his cases, but also on the lucidity of his style, which throws a bright light on a dark subject. practical tendency which will, doubtless, recommend it to many readers. Furthermore, it is profusely illustrated, there Tbeing more than 900 figures, almost entirely drawn from the beautiful collection of Testut, now very familiar in this country. Among the illustrations, a very striking section, contributed l3y Dr. Gerrish, contains a number of photographic reproductions of the surface anatomy of the trunk and limbs, and a series of skiagrams of exceptional merit. In the words of the preface, " the pictorial and diagrammatic illustrations (thanks to the remarkable liberality of the publishers) are phenomenally abundant and of striking artistic excellence," an emphatic statement which cannot be disputed, but which, in its turn, serves to bring into relief the weak part in the composition of the work, namely, the want of due proportion between pencil and pen. The authors have sought, on their own showing, to steer a middle course between the pocket manual and the encyclopaedia, with a bias, frankly admitted, towards the surgical rather than the morphological side of the subject; but everywhere the reader feels that the space devoted to pictorial representation has seriously encroached upon that which should have been allotted to description, which, however concise, should always be accurate and complete. Nearly encyclopaedic in its extent in ohe department, in another the work almost approaches to the miniature. This want of balance is a defect which will be felt by the student rather than the practitioner, who seeks in most cases merely to revive his impressions; for it is only to the trained observer that the dissection or the picture can tell its whole tale, the learner in science must have his attention called to point after point individually before he can acquire the habit of seeing for himself essential details in their true relations. It is abundantly evident all throughout the work that the authors are well acquainted with the subjects they handle, and in spite of what has been said generally, many of the individual sections reach a high mark of descriptive excellence, particularly Dr. Gerrish's chapter on the brain, which, though short, is very clearly written, and presents the main facts to the student from a morphological and developmental standpoint, and the section on the nerves, by Professor Keiller, whose descriptions are brief and pithy. Professor Woolsey's chapter on the joints also calls for special mention, as the work of one accurate and concise in expression, and well versed in the mechanical problems of the skeleton. The chapter to which most exception will probably be taken is that on the muscles, by Dr. Gerrish, for the author, unwisely we think, in the desire to economise space, has so abbreviated his descriptions as to render them little more than mere lists of attachments, positions, and actions. Little or no account of relations is given, and in many cases important actions are unexplained or even neglected altogether. Thus, in the cases of the biceps and triceps of the upper limb, no notice is taken of the action of either muscle upon the shoulderjoint, and in dealing with the tensor vaginae femoris the author neglects to mention the movement communicated to the tibia through the ilio-tibial band. Omissions such as these seem to us to be unjustifiable, even when the necessity for brevity is fully admitted. The chapter in which the minute anatomy of the tissues is described is also disappointingly short, and Dr. Gerrish has not been permitted to do the justice to the subject which its importance demands. Although histology maybe regarded as a common ground on which the anatomist and physiologist meet, the former is not entitled, we think, for that reason to deal with the subject in a half-hearted manner, as claiming but not using to the full the privilege of teaching it, but would be better to relegate it altogether to his neighbour than to treat it with less than its due share of attention. Most of the descriptions in this part of the work are too brief, in our opinion, to be of real value to the student in enabling him to get a comprehensive grasp of the subject. We may cite the account of osseous tissue, in which neither Haversian spaces nor absorption spaces are mentioned, intermediate lamellae being only incidentally noticed, and referred entirely to periosteal origin, and no reference is made to the size of lacunae and corpuscles. What has been said in a general way about histology may all be repeated with even more emphasis with regard to embryology. Professor M/Murrich has been so compelled to condense his descriptions that, while the initiated will follow them with pleasure as a record of the most recent researches, we doubt if the}?' will be intelligently appreciated by any who do not come to the reading with a considerable knowledge of the subject. The compression to which the article has been subjected is also responsible for what cannot but be regarded as a serious defect, namely, the want of that distinction between ascertained fact and conjecture, which should always be drawn in teaching, no matter how probable the conception may appear. In the description of the maturation of the ovum (page 78), the process of the formation of the polar bodies and that of spermatogenesis are spoken of as if they were identical in nature, and thus a speculation, interesting and important, no doubt, and far-reaching in its consequences, may readily be interpreted by the student as something altogether beyond dispute. Here and there Professor M'Murrich commits a slip, common in text-books which treat of human embryology, and, although easily detected by the advanced student, apt to confuse the beginner; on page 86, for instance, the words " upper " and " lower," " behind," &C., are used in the significance in which they are employed in human anatomy ; while, in other cases, as on page 95, words such as " forward" and " above" are made use of in the comparative anatomy sense. The work of publisher and printer has been excellently done; many of the illustrations are most beautifully coloured, and the parts are clearly named, so that in most cases the mastery of the nomenclature is easily attained. We can commend the work very cordially to practitioners and others whose desideratum is a richly illustrated text-book, reliable in its information, and easily handled. The first edition of this work appeared in the spring of 1898, and the fact that a second is required within eighteen months indicates that it has met with a favourable reception. This, no doubt, is largely due to the convenient size of the volume, and to the attempt on the part of the authors to cover the whole field of surgery (even including amputations and excisions) in a book of moderate compass. While we give credit to the authors for their effective condensation, we are afraid the students and practitioners, for whom the book is intended, will often wish they had aimed at greater clearness, even if that involved the addition of a few more lines of letterpress. We notice here, as in most other works emanating from the London schools, a curious jumble of ancient and modern pathology, and a similar admixture of primeval and recent surgical practice. The change brought about by antiseptic theory and practice has had the effect of " leaven," but it has not hospitals the surgeons are still far behind those of the chief provincial ones in the logical carrying out of antiseptic theory in their surgical practice. Further, the Metropolitan surgeons seem to be ignorant of what is being done by their provincial confreres; possibly what was at one time known as the " parochial mind " is now the heritage of the Londoners. The only Glasgow surgeon whose work is recognised in this volume is Professor Macewen, and there is not one of the paragraphs in which his name occurs which gives a fair or accurate account of the operations associated with his name. Under the first head we have the discussion of the treatment of supernumerary digits, webbed-fingers, hammer-toe, metatarsalgia, Dupuytren's contraction, and other affections of the fingers and toes; flat-foot, curved tibia and fibula, genu valgum, varum and recurvatum, coxa vera and congenital dislocation of the hip. Under the second head are included the surgical affections of the skin, nails, lymphatic vessels and glands, fascia, btrsse, muscles, tendons and their sheaths, nerves, veins, and arteries. The discussion of the surgical affections of arteries involves that of the treatment of aneurysm, and this again to the description of the mode of ligaturing the various arteries in their continuity. It will thus be seen that the present volume contains much very important material. We can speak very favourably of the section on deformities, taken as a whole, and are especially pleased with the full and careful description of Lorenz's operative and non-operative treatment of congenital dislocation of the hip. In describing the treatment of the several forms of club-foot, the authors do not get beyond the ideas and practice of a quarter of a century ago, and still pin their faith very largely to the division of tendons and fasciae. In describing talipes equino-varus, they make no reference to the change in the shape of the astragalus, and do not appear to have heard of the removal of that bone as a means of remedying the deformity. In the most severe cases (they say) we have before us four alternatives?(1) the Reviews. forcible restoration of the foot to its proper position bywrenches ; (2) " Phelp's operation," which is simply the operation first described by Dr. George Buchanan, of Glasgow, only performed by the open method instead of subcutaneously; (3) excision of some portion of the tarsus ; and (4) amputation. The authors do not appear to recognise that it is possible in many cases to say positively, from an early date, that the osseous deformity is too great to be remedied by wrenches or the division of tendons, and that in such cases anything short of dealing with the deformed bones is futile. Phelps operation appears to us to be objectionable, not only on the ground that it fails to deal with the osseous deformity, but because the open wound results in cicatricial contraction, which leaves the foot scarcely less deformed than it was at first. In speaking of osteotomy for knock-knee, the description of Macewen's operation is given in his own words, and if this was left to stand without comment it would be quite satisfactory. In expressing a preference, however, for the operation done from the outer side of the femur, the authors ignore the principles laid down by Macewen, and in the use of the splint they recommend for the after-treatment, they still further deviate from the line of treatment he considers essential for complete success. The chapter on the surgical affections of the nerves is fairly satisfactory, and the different methods of restoring the continuity of divided nerves are described with commendable conciseness and clearness. Two and a half pages of small print are devoted to "operations for exposing the main nerve trunks in the upper and lower extremity," but there is no description of operations on the Gasserian ganglion, Meckel's ganglion, or the several branches of the fifth cranial nerve. In a work on surgical treatment, which aims at completeness (as this does), these should certainly not be omitted. The last seventy-five pages treat of the surgical affections of arteries, including aneurysm and the ligature of arteries in their continuity. In this section there is much which challenges criticism, more especially in regard to the pathology of the several conditions described; as, however, the authors do not pretend to give more than the merest outlines of pathology, we may assume that some, at least, of the defects in this department are due to the limitation thus imposed. In speaking of the treatment of aneurysmal varix, they recommend ligature of both artery and vein immediately above and below the communication; this procedure would most likely lead to distal gangrene in persons whose arteries showed atheromatous change, and even in the youngest and healthiest patient would not be entirely devoid of a like risk. The authors have so high an opinion of the transperitoneal method of ligaturing the common or external iliac artery, that they give insufficient attention to the extra-peritoneal operations. It is no doubt true that in cases of aneurysm, the transperitoneal method is easy and fairly safe, but the authors forget that in their work the ligature of arteries will not be O O again described, and that they consequently are treating of ligatures as applied for all conceivable conditions. If it is necessary to ligate either artery for haemorrhage occurring in the course of conditions where asepticism cannot be assured, the transperitoneal road to the artery is not available. In such circumstances the operation devised by Sir Phillip Crampton gives the best access and the greatest security, and we are sorry to observe that it here receives no mention whatever. The woodcuts illustrating the chapter on ligature of arteries are numerous, and are, on the whole, accurate. We must, however, take exception to Fig. 107, which shows the internal carotid artery springing from the common in front of the external carotid, and crossing the latter to reach its outer and posterior aspect, an arrangement we do not remember ever to have seen either in dissecting-room or text-book. We regard this second part of Messrs. Watson-Cheyne and Burghard's important work as marking a distinct advance on the first. The authors appear to have a better grip of their subject, and to speak with more decision and authority. As Mr. Watson-Cheyne is now with our troops in South Africa, we assume there will be some delay in the production of the remaining parts; but we shall await them with interest, tempered by patience, satisfied that when they do appear they will be worthy of our careful perusal. Thus, we are satisfied that, whatever may be the case in Manchester, it is not true of Glasgow that fracture of the humerus is more common than that of the clavicle, and fracture of the shafts of the fore-arm bones more frequent than that of their lower ends (including Colles' fracture). In this respect our experience agrees with the statistics of Stimson, Hamilton, and others, and differs from that of our author. Mr. Piatt was, of course, unable to meet with examples of all possible varieties of fractures and dislocations in the clinique of the Manchester Royal Infirmary, and he has, therefore, added descriptions of the rarer forms of these accidents, drawn from a varietv of sources which he indicates in a bibliography at the end of each chapter. He has thus made the work a fairly complete treatise on the fractures and dislocations of the upper extremity, and we trust he will see his way to follow it up with a corresponding book on those of the lower extremity. Literary style is out of the question in a work of this character, but the descriptions are clear and concise throughout ; and the numerous tables give, in brief, the essential facts as to every case which passed under the author's observation. His study of Colles' fracture is very complete and exhaustive, and he discusses all the points which have exercised the minds of surgeons since the time of Colles, and possibly before that. In regard to the fracture of the styloid process of the ulna as a concomitant of Colles' fracture of the radius?a condition described by Nelaton, Hector C. Cameron, Marcus Beck, and others?he points out that in the 87 cases observed by him this accident was only noted once. This statement is, however, weakened by the admission that " many cases were doubtless overlooked, for it has not been my r.ule to examine specially with regard to this point." Post-mortem specimens, skiagraphs, and the evidence of surgeons who have " examined specially with regard to this point" need not therefore be put aside as fallacious till Mr. Piatt has been able to make further investigations. Some of the cases given in detail are very interesting, and the author mentions in a foot-note one which did not lie strictly within the scope of his enquiry, and was therefore excluded, but would (if we mistake not) be more interesting than any of those he gives. It was a case in which, from a severe blow on the clavicle, the bone, without being fractured, compressed and ruptured the cords of the brachial plexus against the transverse process of the cervical vertebra. This was no mere conjectural diagnosis, but was verified by dissection after death. We must, in conclusion, thank Mr. Piatt for the great pains he has bestowed on the study of this very practical subject, and the interesting volume he has produced. In entering on the production of this work, the first difficulty with the author must have been to define the limits of his subject. On what logical grounds he includes the description of urinary calculus, the surgical affections of the kidney, hydrocele, &c., and excludes salpingitis, endometritis, and the other conditions of the female genital system it would be hard to guess. Certainly the latter are more intimately associated with venereal disease (which is the main theme of the book) than the former, and we can only guess that the reason for their exclusion is the separation in recent years of gynaecological from general surgical practice. By far the most satisfactory sections of the book are those which relate to gonorrhoea and syphilis, and here the author evidently writes from a wide and valuable experience; on the other hand, the description of the surgical affections of the kidney is decidedly feeble and bookish, suggesting a less intimate acquaintance with the subject treated of. The authors experience of venereal disease has not unnaturally led to his entertaining a low opinion of the sexual morality of the average man, but we hope that the extreme opinions he entertains are not justified by actual facts. He quotes with approval the remarkable statements of Noeggerath?" I do not know what the state of matters in other cities is; I did not know how we stood in New York until I questioned the husband of every woman who came under treatment; but I believe we may apply here the dictum of Ricord, that 800 men in 1,000 have had gonorrhoea." And the further conclusion of the same writer, " I believe I do not exaggerate when I say that gonorrhoea in 90 per cent of cases remains uncured. Of every hundred women who have married men formerly affected with gonorrhoea, hardly ten remain well, the others are afflicted by some of the ailments which I have attempted to describe." The sum of this double conclusion is that of all the married women in New York, 72 per cent are suffering from disease of the uterus and its adnexa, due to gonorrhoea in the husband ; surely a monstrous conclusion ! The chapter on the " Diseases of the Sexual Function and Instinct" contains much smart writing; and we are glad to see the necessity for plain-speaking and physiological teaching in regard to sexual matters so forcibly urged. But when Dr. Lydston calmly argues that man is basally a polygamous animal, and woman (his mate) is essentially monogamous, we know not whether to laugh or get angry! Further, we think, he loses his sense of the true proportion of things when he asserts that " Trilby, the fad, did more damage (o the sexual morale of society than all the tabooed obscene books ever written." No doubt the Trilby "fad" was carried in America to an extremity which we in this country have little idea of; but we cannot imagine that the early errors of Du Maurier's fascinating model had any material moral influence on those who read the book or witnessed the play. While fully believing in the germ origin of syphilis, the author is sceptical as to whether the actual bacillus has yet been identified and cultivated. In order to be quite fair, he, however, quotes the description by Lustgarten of his discovery, and the later (though decidedly different) results of the investigations of von Neissen of Wiesbaden. One or two points in the summary of the researches of the latter are worth quoting; thus?" 4. The cause of syphilis is pleo-morpTious bacillus, which is closely related to the more highly organised fungi. 5. The detection of the syphilis germ in the blood is an absolutely sure criterion of the presence of syphilis, and is therefore of the greatest diagnostic value, in doubtful cases, requiring differential diagnosis. 6. Syphilis in all stages is inheritable and communicable. 7. With the therapeutic measures known up to the present time, syphilis is absolutely incurable. Relative healing denotes only a latent state." In regard to the treatment of syphilis the author is clear, definite, and sound. He discusses the value of the newer remedies, especially the vegetable extracts so extensively boomed in America, and the bichromate of potash treatment advocated by Giintz, of Dresden, and arrives at the following eminently safe conclusion :?" While it is undoubtedly best to be liberal with respect to the various new remedies for syphilis, and give different remedies a fair trial, irrespective of their origin, the proportion of cases of syphilis that is curable by the judicious use of mercury and iodine is so large and so gratifying, that the practitioner is hardly warranted in wasting time upon new and strange drugs." The book is for the most part written in excellent English, without those eccentricities of spelling and expression which are usually so prominent in American works. The following sentences, however, are as turgid, inelegant, and unintelligible as anything we have seen, and demonstrate how easy it is even for a careful writer to suffer relapses. " Looming up clearly from the midst of all the confusing clinical facts are the typic, virulent, gonococcic type of urethritis, and the typic, virulent, auto-inoculable, destructive chancroidic ulcer. . As plainly as the clinical specificity of typic gonorrhoea and chancroid, stands out the incontrovertible fact that the local venereal diseases have their origin in genital filth. No matter how it operates, filth is the corner-stone of their development." So proud is Dr. Lydston of these sentences that he prints them in italics ! The get-up of the book is much inferior to what we are accustomed to expect from the Transatlantic medical publishers, and the woodcuts are, many of them, very poor. In Fig. 206, which represents Howard Kelly's method of catheterising the ureters in the female, the illustration is brought into lively contrast with that in Kelly's own work on Clinical Gynecology, a book which represents the high-water mark of medical illustration. Despite the fact that we find much to criticise in Dr. Lvdston's book, we are not blind to its real merits. It is certainly the most complete, sane, outspoken, and honest work on venereal disease in the English language. Surgical Ward Work and Nursing. By Alex. Miles, M.D. Second Edition. London: The Scientific Press, Limited. 1899. This work is directed to supply a want at the foot of the ladder of surgical literature. It is arranged in four sections, dealing respectively with antiseptic surgery, surgical operations, special nursing, the use of rest in surgery, and, lastly, Reviews. bandaging. In the first section are noticed the various lotions, powders, and dressings employed in surgical wards. In the second we have the general preparation of the patient for an operation, followed by notes on the subsequent nursing, with reference to special operations. The third deals with extension and fixation apparatus, and the fourth is devoted to bandaging. We would draw the attention of the author to the following points before issuing a third edition :?On p. 9, volatility is mentioned as one of the advantages of carbolic acid, while on p. 11, non-volatility is looked on as a favourable property of corrosive sublimate, on account of which " it remains antiseptic." We are directed to " antisepticize " catheters (p. 18), and to do likewise to catgut (p. 93). What does this mean ? The plan mentioned on p. 37, of sewing wool into the edge of a mackintosh, does not seem to us to be safe from the point of view of " surgical cleanliness." The author does not consider turpentine as injurious to the skin (p. 38); some surgeons prefer to remove the turpentine by means of methylated spirit to avert its rubefaciant action. So far as we are aware, Higginson's syringe does not give a " constant" stream, and we are surprised that for this purpose no mention is made of the irrigator. The unwinding of soiled bandages after preparing the bed with mackintosh and carbolised towel (p. 40), does not commend itself as in keeping with antiseptic principles. No mention is made of placing a guard on the wound before removing the debris from surrounding parts (p. 41). The author's idea as to the proper method of inserting a safety-pin is a fad (p. 44). " Aseptic " procedure is treated of in a chapter of six pages, in a way that is sure to muddle the understanding of any "junior student" of a logical turn of mind, and of any " nurse-probationer." In a paragraph (p. 61) on preparation of sponge for spongegrafting, after removing calcareous particles ..." the sponge is then antisepticised b}^ 1 to 20 carbolic, and is ready for use." If an antiseptic graft be applied, will it encourage granulation? The section on skin-grafting (p. 121) is incomplete, no mention being made of taking whole thickness of the skin. The text does not tally with the appearances in Fig. 313, in which splints are distinctly figured. While not advocating the use of the perinseal band, we have seen it applied, covered with oiled-silk protective, in order to mitigate the chafing, while elongation of the handkerchief was met by a ratchet and pinion fixed at the lower end of the long splint Fig. 321, and the description of the Staffordshire knot, are sadly wanting. The chapters on special bandages are clear, and the diagrams are good. In spite of slight imperfections, some of which are mentioned above, we think that the book may be commended to those for whom it is intended. It will, undoubtedly, give to them an idea of the rationale of the various procedures which they will see carried out in the wards. Dr. West is to be heartily congratulated on this admirable piece of work. Seldom do we meet with such a happy combination of modesty and freedom from dogmatism on the one hand, with soundness and originality of teaching on the other. The reader does not need to proceed very far before he recognises that these lectures are the outcome of a conscientious and prolonged study of their subject, and he contemplates with satisfaction the addition to his library of such a valuable work of reference. In Part I, granular kidney is considered from the point of view of frequency, age-distribution, morbid anatomy, varieties, etiology, and relation to acute nephritis, arterial disease and gout. Some very important data are given as to the size of granular kidneys, which are found by the author to have a weight above the normal average in 20 per cent of cases. In Part II, we have the signs of granular kidney, viz., high tension, thickened arteries, hypertrophy of the heart, albuminuria, and retinitis. In connection with the fourth of these physical signs, there is a valuable discussion of the subject of physiological albuminuria. Albuminuric retinitis Reviews. is dealt with at considerable length, and the author emphasises the importance of distinguishing the two varieties, viz., (1) the exudative or inflammatory type, which is seen in cases where dropsy predominates, i.e., in parenchymatous nephritis; and (2) the degenerative and hemorrhagic form, which is specially related to granular kidney. Part III deals with symptoms, viz., cardio-vascular and toxemic, and, in connection with the former, the author notes as a new point the relation between aneurysm of large vessels and granular kidney. But there is surely some little slip when we are told that sometimes, in the brain, "a large number of minute haemorrhages are found of that peculiar kind known as miliary aneurysms" (pp. 99, 100). The toxemic symptoms are, of course, varied?digestive, cutaneous, nervous, &c. Parts IV and V are on prognosis and treatment. The author advocates the use of renal extracts, which he has already employed in a tentative manner with encouraging results. There is an appendix of interesting cases, with comments, but illustrative cases are also scattered through the work. An index is provided at the end of the book. 1899. There are now so many excellent works on medical diagnosis that it is with a feeling almost of despair that we take up a newcomer, and we wonder in what it can differ from its predecessors. But the author of the book has considered this objection, and he claims that though he has set before us nothing that is absolutely new, he has brought forward what is old in a. more rational and convincing manner. He says? " Very little that is new will be found in the following pages, but I claim that I have attempted to arrange the old, old phenomena of disease in such a manner as to show more clearly their fundamental meanings and relationships. I have utilised the data of physiology, and the facts of pathological anatomy, as the source from which to draw inferences and deductions, which in turn constitute a critical analysis of clinical symptoms; I have endeavoured by this analysis to lead up to the underlying principles which govern disease as well as health. Once these principles, which are few in number, are recognised,; bedside symptoms become merely illustrations of them, varied, it may be, by local and individual peculiarities, yet ever stamped with such a likeness that the simplest induction will speedily explain the organ of their origin." The above quotation gives, in his own words, the author's position in regard to his subject, his plan of treatment, as well as a brief example of his style of writing. It will thus be understood that the book does not follow the lines of most books on physical diagnosis, nor yet those of a systematic treatise in practice of medicine. It is in a measure a combination of the two. For example, it deals mainly with groups of diseases, pointing out the symptoms and signs common to all, and the symptoms and signs by which the individual diseases may differ. The first two chapters are introductory, and deal with certain pathological phenomena. The third chapter treats of micro-organisms and zymotic diseases. The next five chapters are devoted to the respiratory and circulatory systems, the alimentary tract, the kidneys, to diseases of joints, and to certain affections of the nervous system. The last chapter considers some urgency ? cases, such as haemorrhage, traumatisms, poisonings, &c. In regarding the book as a whole, one cannot but be impressed by its philosophical and logical treatment of the methods of diagnosis; but we doubt if it will appeal to the junior student of medicine. It is too much a general survey of the science of medicine, where certain of the phenomena of disease are cited in illustration of the general theme. But the junior student has not yet become acquainted with these phenomena; and so, from this point of view, we find a good deal that is wanting in the book. For example, in considering the physical signs in the chest, little attempt is made to explain the physics of the sounds obtained by percussion and auscultation. Now, without knowing something of this, the student will in nowise understand the significance of the change in percussion note one observes from day to day in the early stages of pleurisy with effusion. We mean the change from clear to tympanitic, to dull, that we have according to the amount of compression the lung has undergone. Again, in diseases of the nervous system, we would wish a more detailed explanation of the lesions causing the various motor, sensory, and trophic symptoms and signs. For, is it not by referring all these signs to their lesions that we make our diagnosis ? But apart from this, the book *Reviews. is full of interest, and, from its own point of view, quite complete. We have every confidence in recommending it to our readers. It is, we fear, still the custom with many physicians to proceed in the treatment of <iases of dyspepsia without any well-defined views as to the pathological condition of the mucous membrane with which they have to deal, or of the juices which it may secrete. In such a diagnosis we cannot hope for a rational treatment. And so, to all who may come under the above category, we would specially recommend this little book by Dr. Gillespie, for in it will be found information which will greatly aid in the diagnosis of the different forms of dyspepsia. The book begins by describing normal digestion in its various stages. Then we have an account of the methods in use in the physicial and chemical examination of the stomach contents, both in health and disease. The various tests, both qualitative and quantitative, for the different constituents of the gastric juice are fully discussed, and the author gives many useful hints as to the methods best to be employed. A chapter is devoted to a consideration of the mechanical and electrical modes of treating the diseased stomach. Following this is a chapter by Dr. John Thomson on the application of these methods to young children. The volume is clearly written, well illustrated, and we have to congratulate the author on it production. This admirable little book fully attains, in our opinion, the object with which it has been written, viz., " the imparting of accurate knowledge through the students own observation." The author deals with the various substances the examination of which constitutes a laboratory course of physiological chemistry, and a number of extra experiments for advanced students are detailed in small type. In addition, there are inserted here and there blank pages for note-taking. The sections dealing with gastric tests are well worthy of study from the clinical point of view, and the same may be said of the directions for the examination of the urine. The plates at the beginning of the volume, chiefly devoted to urinary sediments, are good. The expression " pustulous," as applied to urine (p. 176), is one which we are not quite -at home with, but we must not carp at parts in a case where the whole is so excellent. The Edinburgh Medical Journal. Edited by G. A. Gibson, M.D., F.RC.P. Ed. New Series?Vol. VI. Edinburgh and London : Young J. Pentland. 1899. If there is nothing very striking in this volume, it at least continues worthily to represent the Edinburgh school of medicine, though it is satisfactory to find that it draws contributions from good men who are not Edinburgh graduates. The summaries of recent advances in medical science strike us as particularly well done.

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Growth of Clostridium perfringens in food proteins previously exposed to proteolytic bacilli. Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilis-hydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens. Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilishydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens. Clostridium perfringens food-borne illness continues to be a major concern in the food industry (4). Dramatic food processing advances and significant changes in consumer attitudes and marketing approaches have resulted in public acceptance of fabricated synthetic foods utilizing soy protein or casein as the protein base. The production of fabricated foods by using pasteurization processes combined with good sanitation and modern manufacturing practices can result in the removal of common spoilage microorganisms leaving sporeforming bacteria (i.e., Clostridium sp. and Bacillus sp.) without major competition. Earlier studies have shown that small amounts of hydrolyzed proteins in conjunction with soy proteins in synthetic meats were stimulatory to the growth of C. perfringens (15) To determine any subsequent effect on the growth of C. perfringens, the B. subtilis-cultured sodium caseinate (BSC) and isolated soy protein (BSP) were centrifuged, and the supernatant fluid was added to the growth medium at a concentration equal to 10% of the total sodium caseinate present in the C. perfringens growth medium (i.e., 0.2% BSC added to 1.8% sodium caseinate). The final protein concentration was 2%. Trypticase (BBL), substituted for BSC, served as a reference. Sterile sodium thioglycolate (BBL) (0.6 g/liter), sodium sulfite (Allied Chemical Corp.) (0.2% g/liter), NaCl (Merck & Co.) (2.5 g/liter), and K2HOP4 (J. T. Baker Chemical Co.) (1.5 g/liter) were aseptically added. The volume was adjusted with sterile deionized water to 10 ml for absorbance determinations or 500 ml for colony count determinations. The growth medium was steamed for 20 min before inoculation with C. perfringens. Lack of contamination or B. subtilis growth in the test medium was verified by testing for any viable cells in uninoculated control samples. An 18-h culture of C. perfringens S40 grown in thioglycolate medium without added dextrose (BBL) was centrifuged (4,080 x g for 10 min) and washed in 6.25 x 10-4 M potassium phosphate buffer. The washing procedure was repeated once. The initial inoculum was 108/ml for colony count determinations (1) and 107/ml for absorbance measurements at 650 nm (model 330 spectrophotometer, G. K. Turner Associates, Palo Alto, Calif.). The lag time was estimated from the intercept of the exponential growth slope with the initial inoculation level (10). Lag time estimations were determined from data of three replicates. Colorimetric determination of partial hydrolysis of food proteins by B. subtilis. Partial hydrolysis was measured according to a modification of the method developed by Hull (8). To 4 ml of the protein sample, 4 ml of 0.72 N trichloroacetic acid (Fisher Scientific Co.) was added, and the mixture was filtered. To 1 ml of the filtrate, 3 ml of 7.5% NaCO, and 1 ml of a 1:3 dilution of Folin-Ciocalteau phenol reagent (Fisher Scientific Co.) was added. Color change was measured at 650 nm (Beckman Acta III spectrophotometer, Beckman Instruments, Inc., Fullerton, Calif.) and converted to milligrams of tyrosine per milliliter from a standard curve. Molecular sieving of nontreated and B. subtilistreated food proteins. BSC, BSP, 3% nontreated sodium caseinate, or 3% nontreated isolated soy protein (5-ml sample) was sieved by using gel filtration (Sephadex G-25 fine, Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The method used was a modification of a method by Nekvasilova et al. (14). Size of column was 2.5 by 36 cm, and elution buffer was 0.05 M ammonium bicarbonate (J. T. Baker Chemical Co.) (pH 7.6). The fractions were monitored at 280 nm with a flow-through cell (Beckman Acta III) and collected automatically. The flow rate was 0.5 ml/min. For large volumes of sample, 100 ml of 3% nontreated or B. subtilis-treated sodium caseinate or isolated soy protein was fractionated by using a Sephadex G-25 column (5 by 86 cm) with a flow rate of 2.5 ml/min and collected at 10 ml/tube. Fractionation of active peptide groups on ion exchange gel. BSC, the second peak of gel filtration (Sephadex G-25), 22-h BSC, and untreated sodium caseinate control were fractionated by ion exchange gel. Freeze-dried samples (1 g) were fractionated by gradient elution with a linear pH change according to Carnegie (2). A column (2.5 by 36 cm) was filled with diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) in acetate form and brought to equilibrium with 0.1 M collidine (2,4,6-trimethyl pyridine; Eastman Kodak) acetate buffer (pH 8.55). Elution was affected with collidine acetate buffer at pH 8.55. A pH gradient was obtained by gradual mixing in gradient chambers with 0.1 M acetic acid. Flow rate was 0.75 ml/min. After 1 liter was eluted, 1 M acetic acid was added to the gradient chamber. Approximately 200 fractions consisting of 12 ml/fraction were collected automatically. Detection of protein fractions was made from 1-ml samples from each tube with ninhydrin reagent (Eastman Kodak Co.) by using the Cocking and Yemm modification (3) of Moore and Stein's technique (11). Color change was measured with a Beckman Acta III spectrophotometer at 570 nm and recorded as milligrams of alanine per milliliter by conversion from a standard curve. Amino acid analysis. The amino acid composition of protein fractions was determined by ion exchange chromatography (12). Hydrolysis of the protein samples was carried out in vacuo at 110 C for 24 h under standard conditions described by Moore and Stein (13). RESULTS AND DISCUSSION The effects of added BSC on C. perfringens growth was determined by adding BSC (0.2% final concentration) to the regular sodium caseinate medium. The C. perfringens growth responses were determined by colony count and by turbidity measurements. In Fig. 1, representative growth curves for C. perfringens in a sodium caseinate medium with added BSC treated for 10 to 48 h are shown. BSC from incubations of 12 h or more markedly shortened the lag time. The BSC samples (more than 10 h) produced an effect similar to added Trypticase. Ten-hour or less BSC had little or no influence on C. perfringens lag time (data not presented). Similar results were obtained by the addition of BSP. Incubation periods of 8 h or longer yielded BSP that resulted in a C. perfringens shortened lag time. A shortened lag time also was observed with the addition of the control (7,9,14). The production of toxins has been shown to increase with increasing length of peptide chains while growth remained essentially the same (7) Fig. 2. Isolated soy protein also increased the growth rate of C. perfringens when added to sodium caseinate. This increased rate of growth was consistent with earlier findings (1). Colony count determinations were made to confirm absorbance data (Fig. 3A, B). In this study, 12 and 24 h-treated BSC or 10-and 34-h FIG. 2. Effect of 8to 48-h BSP added to sodium BSP shortened the lag time of C. perfringens. In caseinate medium on the growth of C. perfringens earlier reported data (F. F. Busta, L. B. Smith, (absorbance at 650 nm). One gram of BSP was added and D. J. Schroder, in Spore Research, in press), to 9 g of sodium caseinate per 500 ml. Trypticase was BSC initiated germination and outgrowth of C. substituted for BSP to serve as a reference. perfringens spores (>90% in 8 h) to a greater extent than did the sodium caseinate control. caseinate medium (data not presented). A 260sodium caseinate on the growth of C. perfringens of stativeprosntei.di not (colony count determinations). One gram of BSC was to 280-nm scan ofstimulativeproteindid added to 9 g of sodium casemate per 500 ml. B, Effect reveal the presence of nucleic acids in the of 10-and 34-h BSP added to isolated soy protein on stimulative medium. the growth of C. perfringens (colony count determina-We postulated that B. subtilis incubation tions). One gram of BSP was added to 9 g of isolated hydrolyzed the food proteins and produced soy protein per 500 ml. VOL. 26, 19873 peptides, especially glycyl-L-asparagine, was demonstrated (9). These and other earlier studies were concerned with toxin production and the concurrent extent of growth, not the rate of growth nor the initiation of growth (lag time). Anticipated responses to specific materials prompted the following study to determine whether the incubation of food proteins with B. subtilis produced specific peptides that decreased the lag time of C. perfringens. T 4. Effect of Trypticase, Casamino Acids, and amino acids added to sodium caseinate on the growth of C. perfringens. One gram of test protein was added to 9 g of sodium caseinate per 500 ml (growth measured by absorbance at 650 nm). Relationship between extent of food protein hydrolysis and C. perfringens growth. Sodium caseinate was supplemented with 0.2% casein hydrolysates (Trypticase and Casamino Acids) or amino acids of similar composition to determine the effect on C. perfringens growth. The addition of Trypticase, Casamino Acids, or amino acids to sodium caseinate (10% of total protein) showed an increased lag time with increased hydrolysis (Fig. 4). This implied that peptides were important initiators of C. perfringens growth in a sodium caseinate medium. This is in agreement with studies by Hauschild (6) which showed the incorporation of "4C from peptides into protein by C. perfringens was greater than incorporation of 4C from amino acids. Fractionation of BSC and BSP. BSC and BSP samples were fractionated to characterize protein hydrolysis resulting from incubation with B. subtilis. Figure 5 presents the Sephadex G-25 gel filtration patterns of BSC and BSP. The change in elution pattern of sodium caseinate from the control to 12 and 24 h of B. subtilis incubation is evident. The extent of protein hydrolysis was determined chemically by using Folin-Ciocalteau phenol reagent. The extent of hydrolysis of sodium caseinate and isolated soy protein during incubation with B. subtilis is shown in Fig. 6. In both protein media, the maximal extent of hydrolysis was reached at approximately 28 h. The first fraction (Sephadex fraction 1) of the sodium caseinate control and Sephadex fractions 1 and 2 of 22-h BSC were further fractionated by charge by using a DEAE-Sephadex A-25 column. The DEAE elution patterns for the sodium caseinate control and the BSC fractions appeared similar. The DEAE fractions of the first main peak and the second peak from each sample were collected, evaporated, freezedried, and reincorporated into a sodium caseinate medium at a concentration of 10% of the total sodium caseinate. The effects of these DEAE fractions on the growth of C. perfringens are listed in Table 1. The supplementation of a FIG. 6. Trichloracetic acid-soluble tyrosine residues hydrolyzed from sodium caseinate and isolated soy protein resulting from 0 to 48 h of B. subtilis incubation. Measured with Folin-Ciocalteau phenol reagent (absorbance at 650 nm) and converted to milligrams of tyrosine per milliliter from a standard curve. Trypticase is shown as a reference. The incubation time required for minimal stimulation of C. perfringens (12-h BSC and 10-h BSP) approximated the time of the first detectable protein breakdown shown in Fig. 6. These results can be compared with data on the trichloroacetic acid-soluble fraction of Trypticase resulting in 0.90 mg of tyrosine per ml. They indicate that the shortened lag of C. perfringens was due to partial hydrolysis of the protein. The next step was to determine whether specific peptides or protein fractions were responsible for the shortened lag. BSC and control samples were fractionated on Sephadex G-25. Fractions were isolated, flash-evaporated, freeze-dried, and reincorporated as 10% of the total sodium caseinate in the C. perfringens medium. BSC was used exclusively in this study. The growth curve for each fraction is shown in Fig. 7. All six Sephadex fractions added singly or added to sodium caseinate in combination shortened C. perfringens lag. Each fraction representing a specific size range of hydrolyzed protein exhibited similar effects. Fraction 2 initiated growth in 2.37 h, compared with 2.25 h for Trypticase and 3.75 h for the sodium caseinate control. These data indicated that the stimulative effect of the hydrolyzed protein appeared to be nonspecific with regard to molecular size of the protein fraction. Ion exchange chromatography of Sephadex fractions. The nonspecificity of molecular size of the hydrolyzed protein fractions prompted studies to determine whether this All fractions tested were analyzed for amino acid composition. No composition differences between the sodium caseinate control fractions or the BSC fractions were observed. The decreased lag demonstrated by the BSC fractions, therefore, did not appear to be due to the amino acid composition of the active component(s), because DEAE peak 2 of the control and BSC were of similar composition but differed in their effect on the lag time of C. perfringens. To confirm the lack of specificity of size of the protein molecule or charge of the molecules on shortening the C. perfringens lag, the complete BSC sample was fractionated on the DEAE-Sephadex column. The fractions representing the last DEAE peak were incorporated into a sodium caseinate medium and found to decrease the C. perfringens lag similar to the complete BSC. The fractions representing the last DEAE peak were then separated by gel chromatography on Sephadex A-25. The similarity of the fractions representing the last DEAE peak and the original BSC indicated that the fractions in the last DEAE peak contained all of the original molecular size range (data not presented). These results have demonstrated that a shortened C. perfringens lag resulted from the addition of B. subtilis-treated sodium caseinate and isolated soy protein. The shortened lag was not due to a specific molecular size of protein formed from hydrolysis. The amino acid composition of the BSC peptide fractions that shortened the lag was similar to the composition of the untreated control sodium caseinate fractions that did not shorten the C. perfringens lag time. Therefore, this effect was not due to a particular amino acid composition. Nor was it due to the charge of the protein fragments or peptide molecules, since BSC fractions with a net negative charge (acidic peptide) were stimulatory, whereas samples of the sodium caseinate control with a similar charge were not. There was no apparent difference in the charge pat-tern of the BSC and control sodium caseinate. Peptides of a specific size, charge, or amino acid composition were not solely responsible for the shortened C. perfringens lag period. Therefore, this lag time reduction caused by B. subtilis action was due to hydrolysis of the food protein, apparently making it readily available for utilization by C. perfringens. The inadvertant modification of food proteins by B. subtilis can increase the potential of that protein for supporting growth of C. perfringens. These results indicate the importance of associative growth of proteolytic bacilli with C. perfringens in a food protein medium that is normally inadequate for the growth of C. perfringens. The shortening of the lag time for C. perfringens in a food product has great public health significance. Food processes in which soy proteins are treated with microbial enzymes to improve acceptability, especially through the formation of plastein (5), could result in initiating the growth of C. perfringens in that medium. A food processor fabricating protein foods who uses soy protein or sodium caseinate should be aware of the ramifications of the partial hydrolysis of that protein.

v3-fos

2020-12-10T09:04:12.199Z

1973-01-01T00:00:00.000Z

237232942

s2

Cellulolytic Bacteria Associated with Sloughing Spoilage of California Ripe Olives Sloughing spoilage of California ripe olives during processing is characterized by severe softening, skin rupture, and flesh sloughing. It was assumed that cellulolytic activity was responsible for skin rupture and sloughing of flesh, and so a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing spoilage. A bacterium identified as Cellulomonas flavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose (CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filter paper, enhanced the activity of the Cellulomonas strain when grown in mixed culture, although they did not, in pure culture, have any effect on filter paper. These latter cultures (all degraded olive tissue) represented the genera Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteria belonging to the genera Alcaligenes, Kurthia, and Micrococcus also were used for study of mixed culture fermentation of cellulose by C. flavigena. Cellobiose accumulation at levels of 1.0% (w/v) and above suppressed growth of C. flavigena. In a previous study (11), gram-negative pectinolytic bacteria were reported to be associated with the sloughing spoilage of California ripe olives during processing. However, in spite of rapid softening, rupture of the skin and sloughing of the flesh were not observed. Because skin rupture and flesh sloughing as well as generalized tissue softening are typical symptoms of this spoilage, it was postulated that free-living, cellulase-producing microbes might also be associated with the deterioration. Therefore a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing. This report describes the results of that search and clearly associates cellulolytic bacteria with sloughing spoilage of olives. MATERIALS AND METHODS Isolation and culture media. The basal medium used for growth and isolation of cellulose-decomposing organisms was the one described by Han and Srinivasan (4). The cellulosic substrates were Whatman no. 1 filter paper and carboxymethyl cellulose (CMC) (type 7HF, Hercules Inc.). The latter was used at a concentration of 2.0% as recommended by Goto and Okabe (3). Enrichment, isolation, and purification. The sloughed olives investigated were of commercial origin and represented the Mission, Manzanilla, and Sevillano varieties. The covering liquid was a dilute aqueous solution of lye, or salt, or both. About 1 ml of this solution from the sloughed olives was inoculated into a test tube containing approximately 10 ml of the basal medium and a strip of filter paper so placed that its top portion projected about 3/4 inch (ca. 1.9 cm) above the surface of the liquid. After 5 to 7 days at 30 C on a Rollor drum machine (model TC-5, New Brunswick Scientific Co.), if cellulolytic bacteria were present, a patch of yellow-pigmented material appeared at the air-liquid interface of the paper. Then a small piece of the paper showing slight disintegration at the air-liquid interface was transferred aseptically with sterile forceps to a new tube of the same medium. This process was repeated seven or eight times to enrich the cellulolytic organisms. Then the paper from the last enrichment was removed, macerated in a small amount of sterile physiological saline solution, and streaked onto plates containing nutrient agar, CMC agar (0.5% CMC in basal medium) plus 1.5% agar, and filter paper agar (a plate of nutrient agar covered with a filter paper disc), respectively. Representative colonies which developed on each of these agar media were picked and inoculated into fresh filter paper medium and into CMC gel medium. Cellulolytic cultures were further purified by alternately plating to test purity and enriching in the filter paper medium and CMC gel until pure cultures were obtained. Two filter paper decomposing cultures were obtained. On the basis of morphology and colony characteristics they appeared to be identical, so only one was retained for further study. Four cultures which attacked CMC gel but not filter paper were also retained for further study. Three noncellulolytic cultures were isolated for mixed-culture fermentation studies. Identification of the bacteria. The cultures retained for further study were identified by conventional methods used for generic and species allocation. General references included Breed et al. (1), Society of American Bacteriologists (9), and Skerman (8). When necessary the original literature was consulted. Preparation of crude cell-free cellulolytic enzyme solutions. The one filter paper decomposing isolate was grown in the basal medium containing 0.25% filter paper (w/v) or 1% CMC (w/v). After incubation for 5 days at 30 C with continuous shaking, cell-free preparations were obtained by centrifugation in a Sorvall, Super Speed R.C.-2, automatic refrigerated centrifuge at 9,500 rev/min for 15 min at 3 to 4 C. The clear supernatant fluid which contained the cellulolytic enzyme(s) was collected. A 1-ml amount of 1% Merthiolate (thimerosal powder, N.F.) was added per 100 ml of solution. The solutions were stored in a refrigerator until used. The four cellulolytic cultures unable to macerate filter paper were grown in the basal medium containing 1% CMC (type 7MF, Hercules Inc.). After incubation for 10 days at 30 C with continuous shaking, cell-free solutions were prepared by centrifugation at 9,500 rev/min for 20 min at 3 to 4 C. The clear supernatant fluid was collected and preserved as described above. Gravimetric determination of cellulolytic activity. The growing 18to 24-hr cultures were inoculated into 250-ml Erlenmeyer flasks containing 100 ml of basal medium and a limited amount of filter paper (110 to 120 mg per flask). After 5 days of incubation at 30 C on a continuous shaker, the flasks were removed, and their contents were filtered, washed, and dried in Gooch crucibles of 30-ml capacity and medium porosity according to the procedure of Lembeck and Colmer (5). Viscosimetric determination of cellulolytic enzyme activity. The activity of the crude enzyme preparations was determined by measurement of the changes in viscosity of the CMC (type 7MF) substrates that were induced by the enzymes with an Ostwald viscosimeter as described by Nortje and Vaughn (7). The volume of crude enzyme solution was always 10 ml. The volume of 1% CMC (T7MF) was always 20 ml. The reaction time was always 30 min. The effect of pH was determined at 30 C. The effect of temperature was studied at pH 6.0 with the enzyme produced in the presence of filter paper and at pH 6.5 with CMC as the substrate. Softening of olives by crude enzyme preparations. The possibility exists that cellulolytic degradation of olive tissue can occur during their storage in salt brine as well as during the final stages of processing when leaching with water is used to remove the lye used to destroy the bitter glucoside oeluropein. It was not possible in the laboratory to exactly duplicate the conditions existing in industry. However, the in vitro tests were designed to duplicate all but the physical pressure produced by 3-to 4-ft depths of olives. Ability of the crude enzyme preparations to soften olive tissue was done with Manzanilla ripe process fruit and with Sevillano variety olives from salt brine storage with preparations adjusted to various pH values (range 4.5 to 8.0) with McIlvaine (6) buffer and preserved with Merthiolate. Controls, regardless of the variety, consisted of olives, buffer solution of the desired pH, and Merthiolate to preserve the mixture. Both of the varieties were submerged in the enzyme solutions for 5 days at 30 C. The ripe process olives were observed daily for signs of softening, skin rupture, and flesh sloughing by sight and feel. After 5 days the Sevillano olives were put through the ripe processes (see Vaughn [101 and Cruess [2] for details). After the lye had penetrated to the pit, the olives were leached with three to four changes of water each day for 5 days. Signs of disintegration were observed daily during the 15 days of processing. The Manzanilla ripe olives were desalted so they would simulate olives during the washing period used to leach the residual lye from the fruits during the final stages of processing prior to canning (see above). It is during the 4to 5-day washing period that sloughing spoilage becomes apparent. The brine storage (7.0% salt, 0.35% total acidity as lactic acid, and pH 4.85) Sevillano fruits during the 15 days required for processing would lose all of the salt and total acidity because of leaching, and the final pH would be in the range of 7.0 to 8.0 during the final washing period. RESULTS Solely on the basis of the tests shown in Tables 1 and 2, the eight isolates described appeared to be in seven different genera and eight species of bacteria. The salient features of each follow. Cellulomonas flavigena. The C. flavigena culture (DS) was the only strongly cellulolytic bacterium isolated. It disintegrated filter paper and CMC readily and caused skin rupture and flesh sloughing of olives. The characteristics are identical to the description of C. flavigena found in Bergey's Manual, 7th ed. (1). The genus Xanthomonas. The two cultures representing the genus Xanthomonas caused liquefaction of CMC gel and disintegrated olive tissue, but did not attack filter paper. The culture DB has been allocated to X. stewartii, although plant pathogenicity was not tested. The other culture (98C) was not allocated to a specific species, although it resembled the descriptions of X. pruni and X. maculifoliigardeniae found in Bergey's Manual (1), the only difference being that culture 98C did not digest milk. The coliform bacteria. The coliform bacteria culture 98A was able to liquefy CMC gel and degrade olive tissue but not filter paper. It had all of the characteristics of Aerobacter cloacae and was so allocated. The other culture (4A) also was cellulolytic to a degree, but was the slowest of the five cellulolytic cultures studied. Even though culture 4A utilized citrate slowly and formed only acid from lactose, it was allocated to the Escherichia intermedia group. An alternative would be to call it Paracolobactrum intermedium on the basis of its anomalous lactose fermentation. Kurthia bessonii. Culture CA was not cellulolytic, but was the only pectolytic organism found in this study. This culture differs only slightly from the descriptions found in Bergey's Manual (1) and given by Skerman (8) for K. bessonii. The main differences are the variability of the Gram stain and the slight amount of acid produced from carbohydrates. The Micrococcus. Culture CW was placed in the genus Micrococcus solely on morphological grounds, but it could not be identified satisfactorily even as to genus because of many variations in morphological and physiological characteristics. These characteristics, however, best match those of the genera Micrococcus or Gaffkya as found in Bergey's Manual (1) or Skerman (8). Alcaligenes faecalis. The other noncellulolytic culture (AW) had all of the characteristics of A. faecalis as found in Bergey's Manual (1) and was so named. It is felt that neither A. faecalis nor the unidentified micrococcus play any important role in mixed fermentation acceleration of cellulolytic activity as will be shown below. All of the isolates involved in this study could grow in the presence of 8% sodium chloride (w/v) in nutrient glucose, tryptone, and yeast extract broth. Cellulolytic activity. The results given in Table 3 compare the attack of C. flavigena on filter paper in pure culture and in mixed culture with the other isolates. The degree of digestion (percent of solubilized cellulose) was 32.35% when C. flavigena was grown alone. Digestion increased about 25 to 50% when the bacterium was grown in mixed culture with the isolates able to use CMC. The noncellulolytic A. faecalis and Micrococcus sp. had little effect on increasing filter paper digestion. Five of the eight cultures involved in this study grew well on CMC-gel and liquefied it at varying rates. However, because of the slowness of their reaction rates, C. flavigena was used to produce crude enzyme to study the effect of pH and temperature on cellulolytic activity as measured by viscosimetry. Data on the effect of pH are shown in Fig. 1; those on the effect of temperature are shown in Fig. 2. faecalis aDegree of digestion of cellulose was measured gravimetrically after the organisms were inoculated into 100 ml of media containing filter paper (Whatman no. 1) as a sole source of carbon. Inoculum was 1.0 ml of cell suspension for single-culture and 0.5 ml of each for mixed-culture fermentation. Incubation was for 5 days at 30 C on a continuous shaker. The cell-free crude-enzyme filter paper preparation was most active at about pH 6.0 and had good activity in the range of pH 5.0 to 7.0, whereas the preparation obtained with CMC as the substrate was most active at about pH 6.5 and had good activity in the range of pH 5.0 to 7.5. As shown, the optimum temperature for activity of the crude enzymes was 50 C regardless of the substrate. Degradation of olive tissue. Crude, cellfree enzymes prepared from the cellulolytic bacteria all softened desalted Manzanilla ripeolive tissue as shown in Table 4. In the range of pH 7.0 to 8.0 the softening was pronounced, and there was skin rupture and flesh sloughing typical of the spoilage as observed under commercial conditions. Nearly identical results were obtained with brine storage Sevillano olives that were treated with crude, cell-free enzymes and then were put through the ripe-pickling process. Figure 3 shows in vitro sloughing spoilage. Softening and skin rupture of olives subjected to the crude enzymes was consistent and involved all of the olives tested to a greater or lesser degree. In contrast, the control olives showed no evidence of softening or sloughing. Cellulolytic activity of pectolytic bacteria previously studied. Seventeen of the 19 cul- microbes might cause the skin rupture and flesh sloughing so characteristic of this spoilage. Some of the pectolytic bacteria previously associated with softening, but not sloughing, were also found to be cellulolytic and also increased cellulose solubilization when grown in mixed culture with C. flavigena. The cultures, pectolytic or not, that degraded CMC all decomposed cellobiose. A concentration of 1.0% cellobiose (w/v) or more was found to inhibit C. flavigena. It is possible that the other bacteria favor the activity of C. flavigena by keeping the cellobiose at a low level. However, noncellulolytic, cellobiose-degrading yeasts grown with C. flavigena did not increase solubilization of the filter paper so this explanation is suspect. Another possibility is that the bacteria unable to decompose filter paper cellulose supply micronutrients to C. flavigena. This latter speculation is suspect, because the two noncellulolytic, cellobiose-negative bacteria used in this study did not materially increase cellulose degradation when grown in mixed culture with C. flavigena. It is also possible that, once C. flavigena has degraded the filter paper to a certain degree of polimerization, the CMC-degrading bacteria can attack the lesser degree of polimerization cellulose and thus increase the total solubilization of the filter paper cellulose. A concise explanation obviously is dependent on further research. In any event, both cellulolytic and pectolytic enzymes produced by various bacteria are involved in sloughing spoilage of olives. C. flavigena and the other cellulolytic cultures also caused marked tissue destruction when grown in sterile olives in brine.

v3-fos

2020-12-10T09:04:16.635Z

1973-01-01T00:00:00.000Z

237229637

s2

Microbiological Testing of Skylab Foods The Skylab manned space flight program presented unique food microbiology problems. This challenge was successfully met by careful evaluation of the total Skylab food system by considering the nature of Skylab foods, their processing and handling, and Skylab food safety requirements. Some of the unique problems encountered with the Skylab foods involved: extended storage times, variations in storage temperatures, no opportunity to resupply or charge foods after launch of the Skylab Workshop, first use of frozen foods in space, first use of a food-warming device in weightlessness, relatively small size of production lots requiring statistically valid sampling plans, and use of the food as an accurately controlled segment of sophisticated life science experiments. Consideration of all of these situations generated the need for definitive microbiological tests and test limits. These tests are described in this paper along with the rationale for their selection. Test results are reported which show successful compliance with the test limits. The unmanned Skylab Orbital Workshop (SOW) is scheduled for launch early in 1973. After attaining orbit, the SOW will be manned by crews who will rendezvous from other space vehicles (Fig. 1). The first Skylab manned phase has a scheduled duration of approximately 28 days. The second and third manned phases are about 56 days each. Each crew will consist of three astronauts. An unmanned phase of approximately 60 days is scheduled between the first and second manned phase, and a 30-day unmanned phase is scheduled between the second and third manned phases. All of the Skylab food will be stored in the SOW at the time of its initial launch. Therefore, there will be no opportunity for resupply or change of foods during flight. The Skylab food system comprises approximately 69 foods which will be stored aboard the SOW according to preselected menus. This food supply must be sufficient for 420 man-days plus a 15% allowance for menu variations. These foods have been produced at the rate of about two foods per week over a period of approximately 9 months. This lengthy production schedule was necessary because of the limited size of the specialized production and test facilities for space foods. This resulted in unavoidably long storage times (Fig. 2). This extended storage had to be taken into consideration in establishing microbiological tests and test limits. In order to identify food production problems and allow for system design improvements, the entire sequence of Skylab foods will be produced two times. The first 9-month production sequence has been completed. These foods were used for crew evaluation, microbiological testing, storage stability studies, systems design studies, engineering systems tests, and nutrient analyses. The second production sequence began 2 months after completion of manufacture of the first. The second sequence will be used for the actual flight, detailed testing, crew training, and nutrient analyses. Skylab foods, other than beverages, are packaged in drawn-aluminum cans with fullpanel pullout lids. The astronaut will assemble these cans into meals in the Skylab foodwarmer tray (Fig. 3). All food (other than beverages) will be consumed directly from the opened cans by using conventional tableware (11). Skylab beverages are packaged in a collapsible, plastic dispenser designed for convenient handling of liquids in zero gravity. The Skylab food-warmer tray provides the first capability to heat foods during space flight. The heaters, located in the walls of the tray cavities, are electrical-resistance wires designed to heat to a maximum of 69.4 C. Higher temperatures have been avoided for boiling must not occur in zero gravity in order to prevent food from being expelled by the boiling action. Boiling will occur near 72.2 C in the SOW, which is approximately one-third atmosphere total pressure. Design requirements were set for the foodwarmer tray so that it would heat frozen food (-23.3 4 5.5 C) to 65 i 3.3 C within 2 hr under zero-gravity conditions. The tray was also required to keep the food at 65 3.3 C until consumed. These design criteria were established to prevent the growth of potential pathogenic microorganisms. The watt density design calculations of the food tray assumed heat transfer only by conduction because convection currents would be minimal in zero gravity (radiant-heat transfer was ignored as being insignificant). Ground-based testing with the complete absence of convection currents in foods could only be approximated. The possibility always remains that food heating in zero gravity will be slower than that indicated by ground-based testing. This would result from poor contact between the food and its container during weightless flight. The Skylab flight menus have been carefully selected prior to flight for nutrient content and will be adjusted in real time as a function of astronaut preference in order to accurately control nutrient intakes during the mission. This nutrient control is a critical part of a sophisticated series of life science experiments designed to elucidate the physiological performance of man during prolonged exposure to weightlessness. The dual application of the food system as both a life support system and a component of complex experiments makes it essential that the food be both accurately defined and safe so that it will not introduce any unknown variable into the experiments. The foregoing brief description of the application of Skylab foods outlines some of the unique situations involved in Skylab food safety. Microbiological test procedures and test limits were selected in order to insure food safety during these flight conditions. These tests have been classified into two categories: those for Skylab foods which are thermostabilized in metal cans and those for all other Skylab foods. This paper describes these microbiological test procedures, the test limits, and the rationale for their selection. The results of the microbiological testing of the first production sequence of Skylab foods are also reported and discussed. MATERIALS AND METHODS Nonthermostabilized food: (i) sampling. Each food package was sequentially serial numbered corresponding to the order of filling. Each microbiological test sample consisted of the first and last packages of each lot plus packages selected by use of a table of 56 APPL. MICROBIOL. Table 1. A representative sample was aseptically removed from each package in this sample. Each sample was about equal in weight and sized so that the composite test sample totaled about 60 g. The exact weight of the complete test sample was recorded to the nearest tenth of a gram, and the sample was aseptically transferred into a sterile blendor cup. A measured quantity of sterile buffered water (ca. 0.066 M PO4, pH 7.0) (1, 2) was added to the composite test sample to produce a 1:10 dilution. This was blended for 2 min. The resultant food slurry (FS) contained the equivalent of 0.1 g of food sample per milliliter of FS. The FS was maintained between 4 and 10 C until promptly used in the test procedures. (ii) Total aerobic count test. A 10-ml amount of FS was transferred into 90 ml of sterile bufferedwater, giving a final dilution of 1:100. One milliliter of the 1: 100 dilution of FS was pipetted into each of five petri dishes, poured with plate count agar (1, 2), incubated at 35 C, and counted after 48 hr. A total of 500 or more colonies on the five plates constituted cause for rejection of the food lot. (iii) Yeast and mold count test. A 1-ml amount of FS was transferred into each of 10 petri dishes and poured with potato dextrose agar which had been acidified with tartaric acid to yield after pouring a pH between 3.3 and 3.7 (9). Plates were incubated at 21 C and counted after 5 days. A total of 10 or more yeast and mold colonies on the 10 plates constituted cause for rejection of the food lot. (iv) Coagulase-positive staphylococci test. A 50-ml sample of FS was transferred into 50 ml of double-strength Trypticase soy broth (TSB) and incubated at 35 C for 2 hr. Then, 100 ml of single-strength TSB containing 20% NaCl was added to yield a final salt concentration of 10% (10). After incubation at 35 C for 24 hr 2 hr, 0.1 ml of the TSB culture was spread on each of two plates of Vogel and Johnson agar and incubated at 35 C. Plates were examined after 24 and 48 hr for the presence of black colonies with yellow zones. Two or more typical representative colonies were transferred to brain heart infusion (BHI) tubes and incubated at 35 C for 24 hr. The remainder of each colony was removed with a loop and emulsified in 0.2 ml of BHL and 0.5 ml of coagulase plasma was added, mixed, and incubated in a 35 C water bath for 4 hr. At the end of 4 hr. negative tubes were noted, and the coagulase test was repeated with the 24-hr culture by using 0.2 ml of the BHI culture and 0.5 ml of coagulase plasma. A single coagulase-positive colony constituted cause for rejection of the food lot. (v) Salmonella test. A 250-ml sample of FS was transferred into 250 ml of double-strength lactose broth and incubated at 35 C for 24 hr. After incubation, 25 ml of lactose broth was transferred to 225 ml of each of Selenite-cystine broth and tetrathionate broth base containing Brilliant Green (1: 100,000) and incubated at 35 C for 18 to 24 hr. One loopful of enrichment culture was streaked on one plate each of three selective media: Brilliant Green sulfadiazine agar, bismuth sulfite agar, and Salmonella-Shigella agar. Brilliant Green sulfadiazine and Salmonella- (vi) Eseherchia coli test. A 1-ml amount of FS was transferred to each of 10 lauryl sulfate tryptose (2) broth tubes and incubated at 35 C for 24 hr. One drop of broth from each positive lauryl sulfate tryptose tube (displaying gas) was transferred with a 1-ml pipette to an EC broth fermentation tube and incubated at 45.5 + 0.2 C for 24 hr. Incubation (not exceeding 24 hr) was carried out in a constant-temperature bath monitored with a certified Bureau of Standards thermometer. Contents of gas-positive EC tubes were streaked on Levines eosine methylene blue agar plates and incubated at 35 C for 24 + 2 hr. Two typical colonies were picked from each eosine methylene blue plate and transferred to a plate count agar slant and incubated at 35 C for 24 hr. Growth on plate count agar slants was confirmed for E. coli types through the establishment of the IMViC pattern according to standard procedures. IMViC patterns of + + --or -+ -were considered confirmed E. coli types I and II. Presence of a single confirmed E. coli type I or H constituted cause for rejection of the food lot. (viM) Clostridium perfringens test. A 0.1-ml amount of FS was spread onto each of 10 petri plates containing Shahidi-Ferguson perfringens (SFP) agar (12) and overlayed with 10 ml of SFP overlay agar (egg yolk emulsion omitted). Plates were placed into Gaspak anaerobic jars (BBL) (or equivalent) and incubated at 35 C for 24 hr. Black colonies surrounded by a zone of precipitate were counted. Typical colonies were confirmed in lactose motility agar. Lactose motility agar was boiled for 10 min and cooled immediately prior to use. Inoculated tubes were incubated at 35 C for 24 hr. Nonmotile lactosepositive cultures in lactose motility agar were considered to be Clostridium perfringens. A total of more than 10 confirmed colonies on the 10 plates constituted cause for rejection of the food lot. Thermostabilized food: (i) incubation test. The size of the sample for incubation testing is indicated in Table 1 as sample size for each given lot size. When necessary, the sample size was increased so that each retort batch in the lot was represented by an equal number of cans. The cans selected from each retort batch included at least one can from the bottom, one can from the center, and one can from the top of the batch of cans as physically located in the retort. Two separate comparable samples were drawn and incubated for 14 days. One sample was incubated at 32 2 C and another at 55 i 2 C. After incubation, the cans were manually examined for evidence of gas production. The presence of one can that became a flipper, springer, soft swell, or hard swell (8) after incubation at either temperature constituted cause for rejection of the food lot. (ii) Microbiological tests. A subsample (Table 1) of cans was randomly selected from the cans after their incubation. Each can in the subsample was microbiologically tested with all incubations performed at the same temperature at which the can had been incubated. About 2 g of food from each can in the subsample was aseptically transferred, in duplicate, into (i) freshly prepared fluid Thioglycollate medium and (ii) TSB with 0.1% yeast extract. Both media were prepared in 25-by 150-mm culture tubes filled to about one-half depth. The fluid Thioglycollate was incubated anaerobically by using anaerobic jars. After 9 days of incubation, the tubes were examined for indication of microbial growth. All doubtful tubes were confirmed by streaking 0.1 ml of the incubated broth on a Trypticase soy agar plate and incubating the plates for 24 hr under the same conditions (atmospheric and temperature) under which the original tubes were incubated. The presence of microbial growth in any of the microbiological test culture tubes or plates constituted cause for rejection of the food lot. RESULTS AND DISCUSSION The Skylab food microbiological test regimen was selected after evaluation of the benefit-risk ratio of each solution to problems judged in the context of the total food system (3). Furthermore, microbiological testing itself was considered as only one segment of a total food safety system. Equally essential elements of this safety system included the test procedures and test criteria for raw materials, storage, processing and shipping environment controls, personnel monitoring, intentional and unintentional additives, and examination for in-storage degradation (browning pigment accumulation, increase in hydroperoxides, and decrease in protein digestibility). It was recognized that no test regimen, per se, could assure 100% safety of Skylab food. For example, regardless of testing, food itself remains a microbiological growth media, and the safety hazard is determined by initial microbial contamination as well as by subsequent factors which impact elements of the total system. The selection of the Skylab microbiological test sample was given careful consideration. The sample examined in food microbiological testing usually represents an extremely small segment of the production lot. The statistical validity of such sampling is frequently further degraded by pooling of selected subsamples. The sampling plan for Skylab foods was influenced greatly by the unique size of the production lots. Production sequence sizes for Skylab foods ranged from only 500 to 10,000 individual food packages. The S-4 level of sampling defined in Military Standard 105D (7) was judged to be most appropriate for sampling these lots because of the previous history of low microbiological counts in space foods produced in the Skylab-type clean-room space food environments (5,6,10,13). Each Skylab food that was not preserved by thermostabilization was studied from the viewpoint of published reports of its epidemiological association with food-borne disease and its potential for supporting microbial growth. This study included processing procedures, microbiological data from similar flight foods, recommended limits established by public health organizations, and proposed crew handling procedures (including in-flight heating and refrigeration). On the basis of these studies, the requirement for testing each nonthermostabilized food for one or more of the following groups of organisms was determined: total aerobic organisms, E. coli, Salmonella, coagulase-positive staphylococci, C. perfringens, and yeast and mold. The test limits applied to each nonthermostabilized Skylab food are equivalent to those shown in Table 2. The results of the microbiological testing of the first Skylab food production sequence are shown in Table 3. The total aerobic count was imposed primarily as a monitor of overall sanitation of food production procedures. All of the food items were found to be within the requirement of less than 10,000 aerobic organisms per gram. The yeast and mold requirements were included to assist the quantitation of and limitation of spacecraft contamination as well as for food safety considerations. With the exception of strawberries and cream style corn, all of the foods examined possessed relatively low counts. The requirements for E. coli, coagulase-positive staphylococci, and Salmonella were selected in order to limit known pathogens rather than to test for "indicator" organisms. Thirteen of the foods were presumptively positive for E. coli. However, these foods were negative when examined in EC broth. None of the foods were positive for coagulase-positive staphylococci or Salmonella. C. perfringens tests were performed on the 15 foods which required warming prior to consumption and in which it was judged that C. perfringens might be present (see Table 3 for food items tested). The requirement of not greater than 100 per g was based upon the assumptions that (i) a total dose of 106 viable organisms are required to produce detectable signs or symptoms; (ii) there would be no lag phase; (iii) a generation time is 20 min; and (iv) any one meal would not contain more than 100 g of contaminated food (foods involved are either entrees which appear only once per meal or rehydratable foods which contain about 30 g per serving, dry weight). Based on these assumptions, it would require any one meal to be held for approximately 2 hr in the temperature range for growth of C. perfringens before it could cause a clinical episode. SFP agar was the method selected for enumeration of C. perfringens. SFP agar is less selective than sulfite-polymyxin-sulfadiazine or tryptone-sulfite-neomycin agar (4). This nonselectivity somewhat increases the conservativeness of the test limit. Fifteen of the food items which required warming prior to consumption were examined for the presence of C. perfringens, and all were essentially negative. The adequacy of the thermostabilization process was verified by two test procedures. First, incubation tests determined whether or not gas was produced in the sealed cans during 32 and 55 C incubation. Subsequent to incubation, the cans were examined microbiologically to detect any microbial growth which may have occurred without gas production. The extended Skylab storage time and variations in storage temperature necessitated a more rigorous microbiological testing plan than normally applied for thermostabilized foods. None of the 12 Skylab thermostabilized foods (ham salad sandwich, butterscotch pudding, tuna salad sandwich spread, lemon pudding, chili with meat, pineapple, turkey and gravy, applesauce, hot dogs with tomato sauce, peaches, pears, stewed tomatoes) produced gas or microbial growth when subjected to this test regimen. Three food items, catsup, peanut butter, and fruit jam, could be considered to be preserved by thermostabilization. However, these foods were tested via the methods outlined for nonthermostabilized foods due to the relatively low temperatures involved in the thermostabilization of these products. The results of the microbiological examination of the first production sequence of Skylab food demonstrated that all of the established microbiological safety requirements were satisfied. In evaluating these results, it should be emphasized that these products are not off-theshelf items. All products were produced following strict specifications. Compliance with requirements was verified at numerous inspection points during the production. The foods involved were processed by a wide variety of methods including: freeze-dehydration, spray drying, intermediate moisture, thermostabilization, and freezing. These microbiological test results reveal the types and numbers of microorganisms important to public health which are associated with this wide variety of foods produced under ideal conditions. As such, these test data contribute to information needed to establish realistic food safety standards. The Skylab food safety standards represent standards designed to provide maximal practical consumer protection derived from realistic appraisal of the benefit risk ratios involved in the total system.

v3-fos

2019-04-11T13:12:01.472Z

1973-12-01T00:00:00.000Z

222383455

s2

Influence of Temperature and Humidity on Microbial, Enzymatic, and Physical Changes of Stored, Pickling Cucumbers Pickling cucumbers stored at five temperatures and four relative humidities were examined for growth of eight microbial groups, the activities of two enzyme systems (pectinolytic and cellulolytic), and weight loss. Twenty-four storage tests for 6 days each were conducted during the 2-year study. In general, microbial populations of the eight groups increased rapidly at the higher temperature (>21 C) and humidity (>70%) treatments. Moisture loss of the cucumbers was rapid with combinations of high temperatures and low humidities. The results suggest that the best environmental conditions for storage or transport of cucumbers would be a combination of low temperature (10 C) with high relative humidity (about 95%). These conditions minimized undesirable microbial, enzymatic, and physical changes of stored, pickling cucumbers. microbial populations of the eight groups increased rapidly at the higher temperature (>21 C) and humidity (>70%) treatments. Moisture loss of the cucumbers was rapid with combinations of high temperatures and low humidities. The results suggest that the best environmental conditions for storage or transport of cucumbers would be a combination of low temperature (10 C) with high relative humidity (about 95%). These conditions minimized undesirable microbial, enzymatic, and physical changes of stored, pickling cucumbers. In recent years, pickle processors have had to extend the holding time of their harvested crop, often for several days, because of the extended times necessary for transit from growing areas to the manufacturing plant. Today, transportation distances by truck equal to that between cucumber production areas in Mexico and processors in the Toronto, Ontario (Canada), area are not uncommon. Also, due to year-round customer demand for pickle products made from raw cucumbers (freshly packed or pasteurized pickles; refrigerated overnight dills), an estimated 3 to 4 million bushels (75,000 to 100,000 tons [about 80,000 metric tons]) of pickling varieties are transported, under refrigerated conditions, from southern growing areas of the country to pickle processors in northern states, prior to their harvest season. Removal of field heat and respiration heat of freshly harvested cucumbers, either by forcedair cooling (18) or by hydro-cooling (4,20), for preventing or delaying spoilage losses has been investigated. The effects of cucumber storage temperature and holding time on the quality of pasteurized pickles also have been reported (5,16). However, physiological or chill damage, characterized by water-soaked spots, pitting, or tissue collapse, was observed (1,8,21) when cucumbers were precooled and stored below 10 C. Fellers and Pflug (17), however, recommended a storage temperature as low as 34 F (1.1 C). General recommendations for storage and transit conditions of pickling and "produce" or "slicer-type" cucumber varieties have commonly been 10 + 2 C, at a relative humidity (RH) of 80 to 85% or higher (5,19,23 its retained blossom, and two of their hydrolytic enzyme systems, pectinase and cellulase, have been studied (12,22). These enzymes, particularly pectinase, have been implicated directly as causing softening of commercially brined cucumbers (12). Based on this background (12,14,22), the present study was designed to follow population changes of the above-named eight microbial groups, and activities of the two enzyme systems, on pickling cucumbers from the time of harvest through 6 days of storage at various temperatures and RHs. MATERIALS AND METHODS Environmental control storage rooms and cucumber samples. The three walk-in rooms, used for the storage tests, were located in the Department of Biological and Agricultural Engineering, and were equipped with temperature (-1 C) and humidity (±+3% RH) controls. After calibration and adjustment, the desired environmental conditions were pre-set for each room. Four runs in each of the three rooms were made in each of 2 years for a total of 24 tests ( wire-screen trays fitted with wooden frames. During the second season, the trays were suspended on scales for recording daily changes in cucumber weight. For analysis, each sample consisted of 20 to 25 cucumbers. These were collected aseptically in sterile paper bags and examined promptly for their microbial populations and enzymatic development. Microbiological procedures. Populations were estimated for the eight groups of microorganisms occurring on the fruit at the start of each storage test and on a 1-or 2-day sampling schedule for 6 days. The procedures for estimating populations for the various microbial groups were described by Etchells et al. (11,12,13), but included certain improvements by Borg et al. (3) and Costilow et al. (7), directed primarily to determining the acid-forming bacteria. The eight groups were: (i) total aerobes, (ii) aerobic spores, (iii) total anaerobes, (iv) anaerobic spores, (v) coliform bacteria, (vi) acid-forming bacteria; (vii) yeasts, and (viii) molds (higher fungi). Measuring enzyme activity. Pectinolytic and cellulolytic activities were measured on each sample prepared for microbiological examination. The activities of enzymes were determined by viscosity methods as reported by Bell et al. (2). The percentage of loss in viscosity was converted to enzymatic activity units from a standard curve for each enzyme assay. RESULTS AND DISCUSSION An experiment, conducted during the latter part of the first year's study, showed that cucumbers that were blended in a saline (0.85% NaCl) diluent had higher microbial plate counts than platings made from cucumber washings in saline. During the second year's study, microbial counts and enzyme determinations were made on both washings and blended extracts to determine the extent of this difference. Based on 53 cucumber samples, blending increased the average counts of various microbial groups from 1.3 to 2.6 times ( Table 2). Blending increased enzymatic activity even more: pectinolytic and cellulolytic assay units, on the average, increased by six and three times, respectively. Initial microbial population estimates on cucumbers. The weights of freshly harvested, pickling cucumber samples examined during the 2-year study and initial populations of the eight groups of microorganisms occurring on these samples are presented in Table 3. The numbers of microorganisms on an individual cucumber can be estimated by multiplying the counts by 20, the average weight for a single fruit. If allowances are made for season, size, and other variables, the populations of the various microbial groups found initially on the fresh fruit were reasonably comparable to those reported by Etchells et al. (14). However, it is evident from the present study that the time of harvest had a strong influence on initial populations; in most instances the numbers increased as the season progressed from the middle of June to early July. A similar progressive, initial increase during the harvest in the numbers of higher fungi (molds) on cucumber blossoms was noted earlier (12). The initial increase in mold populations as the season progresses probably relates directly to the reduced quality of the late-season fruit often received by pickle plants, especially under the 944 APPL. MICROBIOL. weather conditions usually prevailing in the southeast. High humidity, high temperatures, and the absence of refrigeration during this period reduce the quality of cucumber fruit. STORAGE OF PICKLING CUCUMBERS Such conditions favor rapid production of pectinolytic and cellulolytic enzymes, elaborated by many species of molds known to be present on the pickling cucumber (12,22 North Carolina shippers, and those further south, of greenstock pickling cucumbers report that more spoilage claims arise from late-than from early-season shipments. Population changes of microbial groups. Temperature and humidity influenced the development of eight groups of microorganisms on cucumber fruit. The relative rate and extent of development of the two most numerous groups -aerobes and coliforms-and of the molds during six days of storage at five temperatures and at four humidities are illustrated in Fig. 1. To simplify and facilitate comparisons of population growths with initial counts, the estimated population at each sampling interval was divided by the initial population and the log of this ratio was plotted. At the highest temperature tested (27 C), populations of the three microbial groups increased at all four relative humidities. In most instances, storage at 10, 16, or 18 C prevented any marked increase (less than 10 times) in microbial populations. After 6 days at 90 to 95% RH, however, the numbers aerobic bacteria and molds increased markedly. Molds rapidly at 21 C as at 27 C. Although there was a definite lag in the growth of the total aerobes and coliforms at 21 C, as compared to 27 C, growth at 80 to 85% RH was comparable at both temperatures. The four humidity treatments were about equally effective as to control of microbial growth at the lower temperatures. Microbial growth was less at 10 and 16 C than at 18 C and above. The maximal populations of the eight microbial groups on the cucumbers under some of the different environmental storage conditions are presented graphically in Fig. 2. Aerobes and coliform bacteria predominated; population counts remained high during storage and each increased by 10-fold or more at 27 C. At 10 C and 70 to 75% or lower RH, the populations of these two microbial groups remained fairly constant; at higher humidities counts increased. Populations of anaerobic bacteria and anaerobic spores increased somewhat at 27 C, but changed only slightly at 10 C. Even though cucumbers at the two lower humidity treatments were not examined for these two groups, there was evidence that the higher humidity treatments encouraged growth. In the past, acid-forming bacteria have been exceedingly difficult to separate from the very high populations of other microbial groups present on the cucumber. An earlier study (14) reported a few thousand per gram of cucumber in comparison to over a thousand times this number for other bacterial groups. The population ratios of the different microbial groups were very similar in this study, and acid-forming bacteria were present in small but significant numbers. The acid-formers are the most important microbial group present on the cucumbers because certain species of lactobacilli and of pediococci are the predominant acid-forming microorganisms in the natural lactic fermentation of cucumbers for salt-stock pickles (6,15). The acid they produce has a preserving effect on the cucumber in the brining operation. The acid-forming bacteria were present in populations of nearly 10,000/g on the initial cucumber samples (Fig. 2). In storage at 27 C, this count increased to over 100,000 in the direct order of increasing humidity conditions of storage. At 16 C, there was little change in the population of the acid-forming bacteria, except for a small increase at the 90 to 95% humidity treatment. Before storage, yeasts and molds (Fig. 2) were present on the cucumbers in substantially lower numbers than most of the bacteria. The initial mold counts were 3,000 or 4,000/g of cucumber, about twice the yeast counts. We have studied these two microbial groups, molds and yeasts, extensively (9-12, 14, 22) because they cause certain spoilage problems, e.g., softening spoilage by molds, bloating (hollow cucumbers) by fermentative yeast species, and surface-growth of oxidative, film-forming yeast species on the brine of salt-stock tanks. Thus, these two groups are highly undesirable organisms in pickle brines and in the pickle plant. The growth and elaboration of enzymes (particularly softening enzymes by molds) on or in the retained blossom of the cucumber under the environmental conditions of this study are most important. At 10 C, the mold population remained fairly constant at the lower humidities tested-70 to 75% and below; population increased slightly at 80 to 85% and still more at 90 to 95%. This indicates that high moisture conditions encourage growth of some mold species even at the lowest temperature used (10 C). However, at 27 C (optimum for most species) the molds increased rapidly from initial counts of about 5 x 103 to nearly 106/g, with the highest humidity treatments (80 to 95%) showing the largest populations. Molds are, at best, a most difficult group for which to obtain a true population count; however, coupled with visual observations, cucumbers having high mold populations likewise showed large amounts of fungal growth and decay. Yeast populations (Fig. 2) increased more than 10-fold on the cucumbers stored at 27 C and were not influenced greatly by humidity. At 10 C, the yeasts were somewhat restricted at the lower humidity treatments. Overall (Fig. 2) maximal populations of the various groups obtained from the cucumbers VOL. 26, 1973 947 did not indicate competitive or inhibitory properties of one group of organisms for another. The populations of each group remained on the cucumber at about the same or higher populations during exposure to the various environmental treatments, with one or two exceptions. Increase in storage temperature was a greater factor in microbial population increase than was humidity. At all temperatures studied, increased humidity generally brought on increased microbial growth. Enzyme development. Increased levels of pectinolytic and cellulolytic enzymes on the stored cucumbers ( Fig. 3 and 4) correlated closely with the development of mold growth. At 10 C there was little or no enzyme activity at humidities up to 70 to 75%, and development of the two enzyme systems appeared to be delayed more than 2 days even at high humidities. At 27 C, both enzymes increased in activity very rapidly on cucumbers stored at 80% RH and above; there was about a 2-day lag before noticeable increases in enzyme levels were observed in fruit stored at 70% humidity and below. The pectinolytic and cellulolytic enzyme activities from the first year's study (Fig. 3) were measured on samples of fruit washings and from the second year's on blended samples (Fig. 4). Activities of pectinolytic and cellulolytic enzymes averaged six and three times higher, respectively, in blended samples than in washings (Table 1) STORAGE OF PICKLING CUCUMBERS data, the activity levels of the two enzymes for the two seasons, on fresh and stored cucumbers at 80 to 85% RH, appear more comparable. Physical changes. Cucumbers have a moisture content of about 95% and are very susceptible to rapid weight loss accompanied by visible shriveling. Table 4 shows the average weight loss per day at various temperature and humidity treatments. These data may be slightly higher than those from commercial practice because our cucumbers were spread over a wire mesh screen about two to three cucumbers in depth and not stored in bulk containers. Weight loss was most rapid at 55 to 60% RH at 27 C and averaged 25% per day, with severe shriveling. In general, cucumbers stored at 90 to 95% RH lost less than half as much weight daily as those stored at 55 to 60% RH, regardless of temperature. Thus, the lowest rate of cucumber weight loss was at 90 to 95% humidity with the lowest storage temperature used, 10 C (Fig. 5). The rate of weight loss of pickling cucumbers in storage reported herein agrees with a study by Wells (24) who reported that other fruits such as apples, lemons, oranges, peaches, grapefruit, and avocados in storage lost weight as the humidity decreased and temperature increased. ACKNOWLEDGMENTS This investigation was supported by a research grant from Pickle Packers International, Inc., St. Charles, Ill.; and our special thanks are given to W. R. Moore, Jr., Executive Vice President, for his support of this study. We also acknowledge the generous support of John N.

v3-fos

2020-12-10T09:04:16.591Z

1973-10-01T00:00:00.000Z

237233715

s2

Bacillus sp. ATCC 27380: a Spore with Extreme Resistance to Dry Heat An unusual mesophilic Bacillus sp. was isolated from heated soil, and a cleaned spore preparation showed extraordinary resistance to dry heat (D125C = 139 h) and relative sensitivity to moist heat (D80C = 61 min). Biochemical tests and morphology fit no described species. For the past several years, we have been involved in the study of dry-heat inactivation kinetics of bacterial spores as related to terminal sterilization of spacecraft destined to detect extraterrestrial life. These investigations have included spores in their naturally occurring environments (soils and dusts) as well as environmental isolates cultured with various types of media (2,3). Prior to 1960, relatively little was known about inactivation by dry heat at temperatures below 160 C, and workers initially involved in the problem of sterilizing a spacecraft at approximately 125 C began using spores of Bacillus subtilis var. niger to study changes in dry-heat resistance levels due to pressure, gases, moisture, and combinations of these and other physical parameters. The spores of this organism, now commonly used as a biological indicator for dry-heat cycles, came to be considered as a representative of "average" resistance (4). In earlier studies of naturally occurring spore populations in 25 soil samples collected in various parts of the United States, we observed D125 c values (length of time necessary to effect 90% kill at 125 C) ranging from 16 to 126 min (2). Pure Portions (0.1 ml) of the suspension were applied to stainless steel strips (0.5 inch by 0.5 inch), and the dry heat resistance level was determined by a method reported earlier (2) using Trypticase soy agar (TSA; BBL) supplemented with soluble starch and yeast extract (3) as the recovery medium. Figure 1 shows the survivor curve when four strips were heated at each interval and the D125C value of 24 h was calculated from a best-fit regression line of the mean data points (ignoring N., the unheated controls) by using a least squares method. During an end-point determination with this naturally occurring spore population, in which survivors at the 48-h interval were recovered in broth (3), an unusual, slow-growing sporeformer was isolated after 1 month of incubation at 32 C. Positive broth tubes were recognized only when vigorously agitated, thereby showing a clear, compact, mucoid sediment with no turbidity in the supernatant broth. When subcultured on supplemented TSA at 32 C, growth and sporulation were also slow, requiring 10 ical to slightly oval, central to subterminal spores having rough, stainable walls. After leaving the swollen sporangium, the spores appeared to increase in size from 1.2 gm to approximately 1.7 gm while assuming a more oval shape. Vegetative cells became pleomorphic in older cultures, and the highly sculptured spore surfaces were discernible using light microscopy; the less dense forms showing surface texture were most likely spore wall debris left after germination. Figure 2 is a scanning electron photomicrograph of a single spore showing the surface morphology in detail with suggestion of exosporium remnants. The honeycomb pattern of polygonal depressions surrounded by straight ridges is similar to that seen in electron photomicrographs of "B. megaterium 350" described by Robinow (10) and the freeze-etched preparations of B. polymyxa and B. fastidiosus shown by Holt and Leadbetter (6). Attempts at identification of this isolate by standard biochemical methods (12; R. E. Gordon, W. C. Haynes and C. H-N. Pang, The Genus Bacillus, U.S.D.A., Agricultural Handbook no. 427, in press) have shown that it fits no described pattern. Positive tests were obtained with (i) growth in 2, 4, and 10% NaCl broth, and (ii) production of catalase. Negative tests were (i) motility, (ii) utilization of citrate, (iii) hydrolysis of starch, gelatin, or casein, (iv) production of acetylmethylcarbinol or indole, (v) reduction of nitrate or methylene blue, (vi) growth at 45 C (growth was poor at 37 C), and (vii) utilization of carbohydrates (arabinose, glucose, lactose, sorbitol, rhamnose, mannitol, or xylose). Growth on supplemented TSA in a Brewer Anaerobic Jar (BBL) was negative in 14 days at 32 C. The Bacillus sp. isolate was submitted to the American Type Culture Collection and was given an accession number of 27380. An actively growing culture was streaked on thickly poured 100-by 15-mm plates of AK #2 sporulation agar (Difco) supplemented with 20 ,gg of magnesium sulfate per ml and 80 Aig of calcium chloride per ml. After incubation for 20 days at 32 C, growth was harvested and cleaned by a method described previously (2), and spores were resuspended in phosphate-buffered distilled water (BDW; reference 1) held at 4 C. Survival of the spores when exposed to dry heat at 125 C was determined by the stainless steel strip assay method (3) modified by insonating for 60 s with a Biosonic III Ultrasonic Probe (2) at 60% maximum intensity prior to plating the rinse solution from each strip. The moist heat D value was determined by placing inoculated stainless steel strips in tubes containing 10 ml of BDW and heating the tubes at 80 C for appropriate intervals in an oil bath (Blue M Electric Co., Blue Island, Ill., Mod. MW1115A). Assay subsequent to cooling of the tubes was identical to the dry-heat determination. All heating and assay manipulations were conducted in a vertical laminar flow clean room (3). Plates were incubated at 32 C and counted at 2-day intervals for 2 weeks, and Fig. 3 shows the results when five test units were heated at each interval. The Survival of spores of Bacillus sp. ATCC 27380 exposed to 80 C moist heat or 125 C dry heat. concave downward (8) shapes of both survivor curves indicate that Bacillus sp. ATCC 27380 spores require either dryor moist-heat activation (5); it is emphasized that the maximum level of germination in the dry-heat system was reached only after 24 h at 125 C. The gentle slope of the dry-heat curve (D = 139 h) indicates that 125 C is barely the threshold temperature of lethality. The extreme magnitude of the dry-heat resistance (5, 7-9, 11) and also the difference in resistance levels between dry and moist heat may make this organism a valuable tool in the elucidation of spore germination mechanisms, the biophysical nature of spores, mechanisms of heat inactivation, and possibly as a more stringent biological indicator for dry-heat sterilization cycles. Services were provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration under Contract W-13,062. We are indebted to Ruth Gordon of Rutgers University for her helpful comments and assistance with identification schemes for Bacillus spp. Sincere appreciation is also expressed to H. Orin Halvorson and to personnel of the Applied Science Division (Div. 5252), Sandia Laboratories, Albuquerque, New Mexico, for their cooperation.

v3-fos

2022-02-23T16:14:54.676Z

1973-04-02T00:00:00.000Z

247038719

s2

Climatic factors influencing agriculture III the low rainfall tract of Bellary in Mysore State .\ I'-;TRAUI'. Th e W'nt'ml climatic feat ures of Bellary bused mainly 011 the 1111.\11 recorded at tl.le Suil CUll il£'r~'ll ­ l io.llc.muoMch "'Arlll for rh e pcrlod IlJ)n ·6!l nre prt'JlSl'llhod. The short pcrilHlraiufall lilltll of the farm I~ compan-d with IIM.year rain fall rec ord IJC t ile Bellary Observatory rl'gMding amounts, d lstrtbution und mont hly probabilinee of oc currence of ra in of specific amount. Introduction Bellary in ) Iysore Stat e lies in th e low rainfall zone receiving less than 750 mm of rainfall. Though th e Bellary area is situatcd within th e influence of hoth monsoons, most of the annu al rainfa ll is contribu ted by the northeast monsoon. The frequent failu re of rains render it n problem area. Bellary is reporte d to have experienced severe famine s in 1 82~, 1833, 18M, IH66-67, 1876-78,1891-92,1 896·97,1921and 1923and Rubramania m (1964 has observed a minimu m of four drought years of v arying inte nsit ies in a normal decade. An attempt is made in thi s paper to discuss th e characteristics end var iat ions in rainfall with reference to the sowing of crops in the tract. Climatic features Average val ues of various me teorologica l parameters like tempe rature, low level (10 ft) wind, sunshine, evaporation, maximum daily rainfall and maximum hourlv int ensity, based on t he data recorded at th e ,;,eteorological observatory at the Soil Conservation Resear ch F arm, Sreedharagadda (9 Ion nort h of Bcllary) for the period 1956-59, are gi"en in Table 1. Short period data of many meteorological parameters can be ave raged to represent cli mate of a place; however, for rainfall, because of large inter and intra-annu al variations, a long series dat a is required for meaningful studies, Therefore, for th e est imate of variability, th e longer series data of t he Bellary~let eorologi cal Observatory ha s been used, The percentage of vari ability est imated after th e method of Logan (1957) using t he semi-intraquartile deviation from th e media n expressed as a percentage, is given in Table 1. 163 Comparison of f arm and obserm lory rainfaU series It is generally recognised th at a month is too long a period to analyse, study and understand th e feutures of distribut ion of rainfa ll, especially fur agricultural purposes under rainfed cond itions. Weekly rainfall data was readily available for the farm rainfall series. Before analys ing this, it was decided to compare th e same with the long series observatory data regarding fluctuations in annual rainfall, distri bution and probabilities of occurrence of given amount of rainfall in a month, The following procedure was adopted. (n) Fluauations in annual rainf all -The two usual methods of obtaining th e central tendency of a series of rainfall recordings a re : (i) arithmetic mean , and (ii) median. Church eI al, (19Il ) after critically going through both th e methods; recommended t he median to represent th e tru e central tendency, while Foster (IW9) suggestro the usc of mean as more desira ble for statistical soundness. The comparative figures of mean and median rainfall hoth for th e fann data and th e Bclls rv Observatory da ta, adopti ng th e meth od given by Elh ance (1958) are presented in Tabl e 2. Thougll th e farm data do not show much difference bet ween th e mean and th e median, th e IOO-year record for Bellary has a difference of 78· 5 mm between t he mean and th e median being 507· 9 and 429·4 Il1Ill respectively. Obviously, th e wet and dr y spells d uring the short period of observations at tbe research farm has affected th e annual mean and median values as compar ed to the long period of records for th e observatory station. The extreme fluctuati ons of the long series ra infall data for th e l 00-year period (1870-1969) abou t t he mean and the median are illustra ted in Fig. 1.

v3-fos

2018-04-03T01:40:46.997Z

1973-11-01T00:00:00.000Z

19307048

s2

Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives. More reliable and rapid test procedures for Salmonella detection would be beneficial to the food industry. The cultural procedure outlined in the Bacteriological Analytical Manual (BAM) specifies a 4to 6-day time period to test food material for the presence of salmonellae (13). The occurrence of salmonellae contamination in various products from processing plants, however, is generally low (14). The use of rapid sensitive screening procedures are justified as long as the procedure permits proper testing of raw materials, in-process samples, and finished products to assure that Salmonella contamination has not occurred. Several procedures for the rapid detection of Salmonella have been reported. The rapid procedures utilize enrichment broths of one type or another and proceed to a poly "H" agglutination or a fluorescent-antibody (FA) reaction. One procedure for the rapid determination of Salmonellae was reported by Banwart and Kreitzer (1). This procedure requires 24 to 49 h of testing time and involves the use of a specialized glass apparatus in which carbohydrate fermentation and motility characteristics are used as the basis for Salmonellae detection. Another accelerated (50 h) detection procedure, referred to as "enrichment serology" (ES), has been reported by Sperber and Deibel (17). This procedure was compared to the cultural procedure outlined in the Bacteriological Analytical Manual by Fantasia et al. (4). This method was as accurate and sensitive as the BAM method as well as being more rapid. However, Sperber and Deibel also reported that three species were not detectable by the suggested pooled "H" antisera. These are S. agona (group B), S. quinhon (group x), and S. pullorum gallinarum (group D). Because the ES procedure depends upon the flagellar antigen which is produced almost exclusively by motile Salmonella, it is most likely that a nonmotile variant will not be detectable by utilizing the ES procedure alone (17). The FA technique has also been proposed in many screening procedures for detecting Salmonella organisms in meat (7,8), eggs (16), nonfat dry milk (14,15), and other foods (9,10). The FA procedure has undergone evalutions whereby its inherent problems have been disclosed (6,8,9). Thomason reported that, when a direct FA procedure was applied to 304 environmental, food, and feed samples, a level of 3.1% false negatives occurred (18). An improved FA system utilizing a Zeiss immunofluorescent microscope with a quartyiodine (Halogen) light source combined with a "Fluoro-kit" (Clinical Sciences, Ind., Whippany, N.J.) has been made commercially available. This study was designed to compare the ES and FA techniques for detection of salmonellae with the cultural method and to evaluate the combination of ES and FA results as a potential means for eliminating the weaknesses inherent in each of the systems when used alone. MATERIALS AND METHODS Subsamples (126) representing 22 different naturally contaminated condiments, food products, food by-products, and animal feeds were used in this investigation. The naturally contaminated samples were obtained from R. H. Deibel, University of Wisconsin, Madison, Wis., and from N. F. Insalata, General Foods Corp., Post Division Research, Battle Creek, Mich. All samples were stored in separate containers at 4 to 5 C to help maintain any existing populations of salmonellae. The selection of the pre-enrichment broth media was dependent on the type of product or food prototype to be analyzed. All milk products and milk by-products were pre-enriched in sterile distilled water containing 0.002% brilliant green dye and 0.6% tergitol anionic 7 (3,12,13). Baker's yeast samples were pre-enriched in nutrient broth (BBL). All other samples were pre-enriched in lactose broth (BBL) (3,13). A 10% (wt/vol) ratio of sample material to pre-enrichment broth was maintained, and all preenrichment media were tempered to 40 C prior to use. All samples undergoing pre-enrichment were incubated for 18 to 24 h at 35 C. The pH levels of all pre-enriched cultures after 18 to 24 h of incubation were between pH 4.5 and 6.4. Samples (1 ml) of each pre-enrichment culture were inoculated into separate tubes containing 9.0 ml of 1.1 strength selenite cystine (BBL) and tetrathionate broths (BBL) (17) tempered at 40 C. The selectivity of the tetrathionate broth was enhanced with a final concentration of 0.002% brilliant green dye (12,17). All samples of pre-enriched cultures inoculated into selective enrichment media were incubated at 35 C for 22 to 26 h. FA procedure. The "CSI, Fluoro-kit for Salmonella screening" (Clinical Sciences, Inc.) was utilized. Salmonella "OH" somatic antisera conjugate A through S (prepared by CSI, Inc.) and FA Salmonella poly antisera conjugate A through S, including 0 factors 1 through 25, 27, 28, 30, 34 through 41, 45, and Vi, (Difco), were tested on formalinized (0.15% final concentration of formalinized saline) 0.001-ml samples of each selective enrichment broth culture. Formalin was used as a safety factor for protection of laboratory personnel. Slides were prepared, fixed, rinsed, stained, rinsed, washed twice in phosphate buffer, rinsed, dried, and mounted with a cover slip in complete compliance with the instructions supplied by the manufacturer (Clinical Sciences Inc., Whippany, N.J.). All wells of the Clinislide were examined with the use of a Zeiss immunofluorescent microscope equipped with a 12-V, 100-W halogen lamp, x 40 and x 100 oil immersion Planapo and Plan iris diaphragm objectives, respectively, a 490-nm interference FITC filter, a KG1 heat absorption filter, a BG 38 red suppressing filter, a Zeiss no. 53 barrier filter, a dark-field ultracondenser, and x8 narrowangle eyepieces. ES procedure. Tubes containing 10 ml of M broth (Difco) were inoculated with 0.05-ml samples of each selective enrichment broth culture. The tubes containing M broth were tempered at 40 C prior to use. All inoculated M broths were incubated at 35 C for 7 h. After 7 h of incubation, H-agglutination tests were performed on all M broth cultures by the procedure outlined by Sperber and Deibel (17). Nonspecific agglutination tests (ES controls) were also performed on all M broth cultures by using 0.1 ml of physiological saline in place of the 0.1 ml of poly H antisera for the H-agglutination procedure. Cultural procedure. The salmonellae cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual was utilized with minor modifications (13). Differential selective plates of brilliant green agar (BBL), bismuth sulfite agar (BBL) aged 3 days at 4 to 5 C, and salmonella-shigella agar with additions of 1% sucrose and 0.65% agar (17) were streak-inoculated with one loopful (0.01 ml) of each selective enrichment culture. The streaked differential selective agar plates were incubated at 35 C for 24 to 48 h. After 24 h of incubation elapsed, the plates were examined for suspect salmonellae colonies. Negative plates were reincubated at 35 C for an additional 24 h and subsequently re-examined for suspect salmonellae colonies. Several colonies which were considered suspect on any given plate after 24 and 48 h of incubation were picked and inoculated into and onto triple sugar iron (TSI) agar slants and lysine iron agar (LIA) slants (2,5). Inoculated slants of TSI and LIA were incubated at 35 C for 18 to 24 h. Cultures which exhibited typical Salmonella-positive reactions with TSI or LIA were transferred to brain-heart infusion (BHI) broth (BBL) and incubated at 35 C for 6 to 8 h. Flagellar (H) antigen tests were performed by using the poly H antisera pool reported by Sperber and Deibel (17) for the ES test, plus Z44 which was added to detect S. quinhon. Analyses were performed on the same sample by using the FA, ES, and combination FA and ES procedures. Applying the BAM procedure as a refer-ence standard, FA positives and negatives, ES positives and negatives, as well as FA and ES combination test positives and negatives were determined. Various statistical tests were applied to the data to ascertain the reliability of the FA and ES procedures as compared to BAM procdure for detecting salmonellae. RESULTS AND DISCUSSION Responses to the various detection procedures for a total of 126 samples, representing 22 types of finished product, are shown in Table 1 (18). These investigators reported that the problem of the FA method lies in its tendency to indicate the presence of salmonellae in sample which are cultural negatives. The ES procedure detected 64 of the 66 positive samples after interacting the results from both selective enrichment broths and considering any positive response to be Salmonella. ES from selenite cystine broth gave higher numbers of false negatives than ES from tetrathionate broth. If it were not for one egg noodle product in which the tetrathionate gave 8 out of 10 false negatives, the decision to drop the use of selenite completely might have been made. The apparent anomalous result demonstrates the need to recommend the use of both selenite and tetrathionate and to read the ES test as positive if either broth yields a positive response. The two salmonellae-positive samples missed by the ES procedure in this investigation were both lactalbumin. In both samples the salmonellae appeared to be outgrown by a competing organism in the natural microflora of the lactalbumin. The remaining 60 of the 126 samples which were analyzed for the presence of salmonellae were negative by the BAM procedure. The FA fluoro-kit with CSI FA Salmonella conjugate sera yielded 37 negatives and 23 presumptive positives corresponding to the 60 BAM negatives. When Difco FA Salmonella poly sera was used in place of the CSI conjugate with the FA fluoro-kit procedure, 46 negatives and 14 presumptive positives which were cultural negatives were detected. One explanation for this result could be that the FA procedure is more sensitive than the BAM procedure and consequently detects positives that the cultural procedure has not detected. This would appear as false alarms in the test data, since the BAM procedure was chosen as the standard and VOL. 26,1973 positives not detected by this procedure are classified as false alarms. When results of the ES procedure without nonspecific agglutination controls were compared with the 60 BAM negatives, there were 54 samples in agreement and 6 presumptive positives. When the nonspecific agglutination controls were included, the response improved to 59 negatives and only 1 presumptive positive in comparison to the 60 cultural negatives. A summary of the data obtained from the materials analyzed for salmonellae by the BAM, ES, and FA procedures is shown in Table 2. The decision strategy applied to the ES and FA reaction columns (R.) shown in Table 2 incorporated results obtained from both selenite cystine selective enrichments and tetrathionate selective enrichments. A positive response which occurred in any one of the selective enrichments was read as an overall positive reaction. For a negative reaction, both the selective enrichments must show negative responses. A control reaction was applied to the serological procedures of the ES test to negate positive nonspecific agglutination reactions. If the serological test of the ES procedure yielded a positive response and the serological control did not show nonspecific agglutination, this was recorded as a positive serological response for salmonellae. Applying the statistical chi-square test to the data indicates that the 44-h FA and 50-h ES test procedures are adequate in detecting BAM positives. All BAM-positive samples were FA positive. The BAM and ES results were not identical, but, with 95% confidence, there was no significant difference in their ability to detect Salmonella. The ES test procedure without nonspecific agglutination control reactions was significantly different at the 95% confidence level from the BAM procedure in detecting negatives. However, no significant differences were found (at the 95% confidence level) between the ES test procedure with nonspecific agglutination control reactions and the BAM procedure in detecting negatives. Running the ES test procedure with nonspecific agglutination control reactions materially reduced the ES false-alarm probability without reducing the probability of detecting BAM positives. There was a significant difference (at a 95% confidence level) between the BAM procedure and the FA procedure using either CSI or Difco FA Salmonella sera conjugate in detecting negatives. No significant differences were found (at a 95% confidence level) between CSI or Difco FA Salmonella sera conjugate in detecting negatives. The combination of FA and ES procedural results for the detection of salmonellae was also compared to results obtained by utilizing the Salmonella cultural procedure outlined in the U. S. Food and Drug Administration's Bacteriological Analytical Manual. The FA procedure using CSI sera combined with the ES test with and without nonspecific agglutination controls (FA,-ES-1 and FAk-ES-2, respectively), and the FA procedure using Difco sera combined with the ES test with and without nonspecific agglutination controls (FAd-ES-1 and FAd-ES-2, respectively) represent the possible combinations observed in this investigation. A summary of the data obtained by combining the FA and ES procedures is shown in Table 3. The Fk-ES-1, Fk-ES-2, FAd-ES-i, and FAd-ES-2 combinations each exhibited two missed detections. However, the differences werenot statistically significant. Specifically, no significant difference was found (at a 95% confidence level) between FA-ES combinations and the BAM procedure in detecting positives. This investigation revealed that, whenever presumptive positive results were obtained with the FA and ES procedures, the BAM procedure also revealed positive confirmation for the presence of salmonellae. Incorporating positive FA and ES results in a combination FA-ES technique revealed that the FA-ES positives were equivalent to BAM positives. The FAk-ES-1 combination exhibited one false alarm. No significant difference was found (at a 95% confidence level) between FA&-ES-1 and the BAM procedure in detecting negatives. The FAd-ES-1 combination exhibited two false alarms. The differences were not statistically significant. No significant difference was found (at a 95% confidence level) between the FAC-ES-1 combination or the FAd-ES-2 combination and traditional cultural procedure in detecting negatives. Combining FA and ES results yielded greater accuracy than when either of the two rapid screening procedures was used alone. The suggested analytical scheme is shown in Fig. 1. In use, the 44-h FA results would be obtained first. If FA readings are negative, the sample is negative at this point and no further testing would be required. If the FA readings are positive, completion of the 50-h ES test along with nonspecific agglutination controls is necessary. If the ES test also yields a positive reaction and a negative nonspecific agglutination control reaction, the sample is deemed positive and no further testing would be required. If the ES test yields a negative reaction and the control remains negative, or if the ES test yields a positive reaction and the control indicates nonspecific agglutination (a nonspecific agglutination negates the ES test), then, with at least 92.7% probability (at the 95% confidence level), the sample may be considered negative. Since there is a high probability of the sample being negative, it is suggested that product from a manufacturing operation be released but held under recall control while the traditional cultural procedure (BAM) is being completed. The factors which may cause the apparent negative when the sample is truly positive would be the presence of nonmotile salmonellae or suppression of salmonella growth by competitive microorganisms, both of which situations would interfere with or preclude a positive ES response.

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2020-12-10T09:04:12.215Z

1973-09-01T00:00:00.000Z

237230958

s2

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar. During the past decade, Hawaii has reported the highest incidence of human salmonellosis to the Center for Disease Control in Atlanta, Ga. (4). Two surveys, conducted in 1960 to 1962 and 1967, on the epidemiological aspects of salmonellosis in Hawaii established that food products played a significant role as the source and in the transmission of salmonellae (2,3,5). A Salmonella surveillance project supported by the U.S. Department of Health, Education, and Welfare from January 1970 to April 1971 investigated the sources and transmission of salmonellae in food products, animal feeds, market equipment, and abattoir environments in Hawaii. A total of 15,071 specimens was examined during this period. Of this number, 11,226 were food samples. Food and food products examined included powdered food prod. ucts, eggs and egg products, and carcasses and viscera of beef, poultry, and pork of intrastate, interstate, and foreign origin. In conjunction with this project, the effectiveness and selectivity of Brillian Green (BG) and Hektoen enteric (HE) agar media for isolating salmonellae from food products were compared. MATERIALS AND METHODS Sampling procedures. The cotton swab method was used in obtaining carcasses and viscera samplings of beef, pork, and poultry. All the meat samplings were obtained by swabbing an area 4 by 4 inches (10.16 by 10.16 cm [2]). In instances where the surfaces of the meat products were semidry, the swabs were moistened with sterile normal saline prior to sampling. A sampling thus obtained was placed directly into a test tube containing 8 ml of Tetrathionate Brilliant Green (TBG) enrichment broth. The inoculated specimen was immediately delivered to the laboratory, assigned a number, and placed in a 37 C incubator. Laboratory methods. Isolation procedures for foods, feeds, etc., recommended by the Center for Disease Control were followed (1). After 18 to 24 h of incubation, inoculated TBG broth was streaked onto BG and HE agar plates. The BG and HE agar plates were examined after overnight incubation for the presence of Salmonella-like colonies. Three colonies were picked from each suspicious plate and inoculated into triple sugar iron agar slants and incubated at 37 C. Cultures exhibiting Salmonella-like reactions (mainly alkaline slant, acid, and gas butt, with or without H1S formation) were checked for urease activity by the rapid urea test (a 1: 20 dilution of urea and buffer solution). Urease-negative cultures were inoculated into a series of biochemical test media, including 1% sucrose, mannitol, lactose, and salicin broths, tryptone broth, 10% lactose agar slant, Simmons citrate agar slant, and motility test medium. Cultures showing biochemical reactions characteristic of the genus Salmonella were checked with Salmonella polyvalent somatic and grouping sera and submitted to the State's Regional Salmonella Typing Center for definitive identification. Various brands of dried milk, instant breakfasts, and powdered haupia (coconut pudding) were examined for Salmonella contamination. For each sampling, 100 g were reconstituted in a 2-liter flask with 1,000 ml of sterile distilled water containing 20 ml of a 0.1% aqueous BG solution (a 1: 50,000 concentration). The 288 inoculated broth was incubated at 37 C. After 24 h of incubation, 10 ml of the primary broth was transferred to a 250-ml flask containing 100 ml of TBG broth and reincubated. Loopfuls of the primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. A dozen eggs was considered as a single specimen. Each dozen eggs was cracked manually (by using a sterile glove for each specimen) into a 2-liter flask containing 1,000 ml of nutrient broth. The inoculated broth was incubated at 37 C. After 24 h, 10 ml of the primary broth was transferred to a 250-ml flask containing 100 ml of TBG broth and reincubated. Loopfuls of the primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. Each 30 g of egg noodle specimen was inoculated into a 250-ml flask containing 100 ml of nutrient broth. After 24 h of incubation, 1 ml of the primary broth was inoculated into a test tube containing 9 ml of TBG broth and reincubated. The primary and secondary broths were streaked onto BG and HE agar plates after 24 and 48 h. RESULTS Effectiveness of BG agar and HE agar media. A comparative study of BG and HE agar media was undertaken from January 1970 to April 1971 to determine their effectiveness for isolating salmonellae from food products. Of 11,226 food samplings, 1,662 (or 14.9%) were positive for salmonellae on one or both isolation media (Table 1). Although BG agar detected significantly more specimens positive for salmonella (1,475 of 11,226) than did HE agar (1,315 of 11,226), and the difference is significant (XI = 10.47, p < 0.005), there were 187 positives that would not have been detected if HE agar were not used as the second plating media and 347 if BG had not been used. Table 2 summarizes the incidence of Salmonella in the food products examined during the study. Eggs and egg products. Of the 200 dozen (from 7 farms) local cracked eggs, 4 (or 2.0%) were positive. Salmonella infantis was isolated from three of the four dozen eggs which were positive. From 236 dozen local whole eggs (from 9 brands), 3 (or 1.3%) were positive. S. cerro was isolated from the two dozens of eggs which were positive. From the 103 mainland whole eggs (from 2 brands) examined for Salmonella, no positives were isolated. No positives were isolated from the 14 local egg noodle samplings. Powdered food products. A total of 101 powdered products was examined for Salmonella, with negative results. Samples included 74 (from 7 brands) dried milk, 25 (from 3 brands) instant breakfasts, and 2 (from 1 brand) haupias (coconut puddings). I --L Bovine carcasses and viscera. From a total of 860 beef samplings, only 1 (0.5%) positive was isolated from 211 foreign beef carcasses. S. infantis was isolated on HE agar medium. No salmonellae were isolated from the local and mainland beef samplings, which consisted of 370 (354 carcasses and 16 tripe) and 279 (253 carcasses and 26 tripe) specimens, respectively. The beef samplings were obtained from two meat companies, one sausage factory, and four supermarkets. Pisces carcasses. Approximately 77 island "reef' fishes were examined for salmonella contamination. BG and HE agar media failed to detect the presence of salmonella on fish carcasses. The fish samplings were obtained from one open market and a supermarket. Poultry carcasses and viscera. Of 3,713 chicken carcasses and viscera, 3,526 were of local and 187 were of mainland origin. Out of 2,927 local chicken carcasses, 65 (or 2.2%) were positive. BG agar isolated 59 (90.7%) positives from the carcasses and HE agar yielded 16 (24.6%) positives. From 599 local chicken viscera, 8 (or 1.3%) were positive. BG agar isolated five (62.5%) positives from the viscera and HE agar isolated four (50%) positives. The predominant Salmonella serotypes isolated were S. heidelberg, S. typhimurium, and S. saint paul. The chicken specimens were obtained from four local abattoirs and two supermarkets. No positives were found in the 62 mainland chicken carcass samplings. Of 125 mainland chicken viscera, 2 (or 1.6%) were positive. The two serotypes isolated were S. typhimurium and S. derby. They were isolated from BG agar medium. The mainland samples were obtained from one sausage factory, three supermarkets, and one drive-in restaurant. Porcine carcasses and viscera. A total of 5,922 pork carcasses and viscera was examined for salmonellae. Salmonellae were isolated from 234 (or 9.2%) of the 2,543 local pork carcasses and 1,30Y7 (or 56.9%) of the 2,296 local hog viscera. A 3-month viscera pasteurization study was carried out in the hog slaughterhouse from September to December, 1970, by utilizing methods described by Paul Yoder (personal communication). Prior to the study, the contamination percentage ranged from 70 to over 90. Due to the pasteurization treatment of all hog viscera before marketing, the degree of contamination fell considerably. A total of 170 hog viscera was subjected to pasteurization, of which 9 (5.3%) yielded salmonella. S. derby and S. anatum were the two serotypes that survived a few pasteurization treatments. The predominant serotypes isolated were S. derby, S. ana-tum, and S. typhimurium. The local pork carcass samplings were obtained from 1 abattoir, 3 processing pork plants, and 16 supermarkets. The hog viscera were sampled from one local abattoir. From 891 mainland pork carcasses, 38 (or 4.3%) were positive. S. typhimurium and S. derby were the predominant serotypes isolated. No salmonellae were recovered from 192 mainland viscera samplings. The mainland pork samplings were obtained from three supermarkets and one restaurant. Table 3 summarizes the distribution of Salmonella serotypes isolated from the various food products. A total of 28 serotypes was isolated. Of the 15 serotypes frequently isolated, only 1 (S. worthington) was isolated more frequently on HE agar medium, but the difference was not statistically significant. Selectiveness of BG agar and HE agar. To compare the selectiveness of BG and HE agar media, a 2-week study was conducted from 1-15 April 1971. A total of 410 food specimens was examined. Of this number, three specimens were positive for Salmonella on BG and four on HE agar media. There was a remarkable difference in the number of nonlactose-fermenting (Proteus, Pseudomonas, Paracolons) subcultures made from BG agar medium (92 subcultures) and HE agar medium (11 subcultures) ( Table 4). This study demonstrated that HE agar medium is far more selective than BG agar medium. DISCUSSION An ideal selective medium for the isolation of Salmonella from various kinds of specimens should be inhibitory against the rapid lactosefermenting coliforms and other nonlactose-fermenting bacteria such as Proteus, Pseudomonas, Citrobacter, etc., and expedite the identification of Salmonella by eliminating the bacteriological examination of these extraneous colonies. On HE agar medium, Salmonella colonies appear as transparent blue-green colonies, with or without black centers (H,S production). The size of the colonies ranged from 0.5 to 2 mm. HE agar medium inhibited most of the coliforms and other nonlactose-fermenting bacteria, thereby facilitating the identification of Salmonella from food products. BG agar medium supported the growth of most of the nonlactose as well as the rapid lactose-fermenting bacteria. On BG agar medium the Salmonella colonies ranged from 0.5 to 1 mm in size. The other nonlactose fermenters as well as the Salmonella colonies appear as transparent red colonies, with or without H2S formation. Therefore, more suspicious BG agaf plates than HE agar plates were selected out for picking, and this may account for the higher number of Salmonella isolates from BG agar. In conclusion, BG agar merits consideration as an isolation medium for food surveys because of its effectiveness in isolating salmonellae. HE agar, although not as effective, was far more selective, and its use would require less effort and supplies to eliminate Salmonella-like isolants. The epidemiological implications of survey results are presented and discussed in a companion paper.

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2016-05-12T22:15:10.714Z

1973-12-01T00:00:00.000Z

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The evaluation of chemical mutagenicity data in relation to population risk: the need for better methods of extrapolation. In the fall of 1966 the Food and Drug Administration established an Advisory Committee on Protocols for Safety Evaluation. The first problem to be reviewed was the requirement for reproduction studies in the safety evaluation of pesticides and food additives , and the adequacy of the current methods involved. Reproduction studies are designed to give information on: (1) effects on male and female fertility, (2) effects during gestation on the mother and the fetus, (3) effects appearing after parturition on the mother (for example, lactation) and the offspring (for example, growth, development, sexual maturation), and (4) mutagenic effects , particularly those which might not appear in the first generation. The report of panel dealing with this problem was written in November 1968 and published later (1). During the time the panel was considering the problem, a report prepared by the Genetic Study Section under the chairmanship of Dr. James F. Crow (2), became available to us (C. C. Cockerham, personal communication). The emphasis in this particular report of the Genetic Study Section was that there is reason to be concerned about chemicals as a mut-agenic risk equivalent to radiation, possibly even more serious. The concern, of course, is with the welfare of future generations as well as with the health of contemporary populations. It is useful to emphasize again that *Division of Toxicology, Food and Drug Administration , Washington, D. C. 20204. methods to detect mutagenic effects that are useful for the evaluation of mutagenic risks must deal with the potential for genetic changes that are transmissible to future generations. Without this clearly in mind we can easily become confused regarding the import of our laboratory findings. I believe it is of interest to this group to cite certain portions of the Reproduction Panel Report (1). 1. "There should be a repository of information on the mutagenic potential of chemicals. Such information should include chemical nature, quantitative information on mutagenicity, the particular test systems on which this information is based, and other biological effects including carcinogenesis." 2. "The task of demonstrating that a chemical constitutes no genetic hazard is somewhat different and inherently much more difficult than carrying out and evaluating other toxicity tests including reproduction tests. The 'permissible level' takes on new meaning when multiplied over future generations. MIutagens often act at very low concentration. Definitive tests of their effects in mammals are tedious and expensive. Generally , the … methods to detect mutagenic effects that are useful for the evaluation of mutagenic risks must deal with the potential for genetic changes that are transmissible to future generations. Without this clearly in mind we can easily become confused regarding the import of our laboratory findings. I believe it is of interest to this group to cite certain portions of the Reproduction Panel Report (1). 1. "There should be a repository of information on the mutagenic potential of chemicals. Such information should include chemical nature, quantitative information on mutagenicity, the particular test systems on which this information is based, and other biological effects including carcinogenesis." 2. "The task of demonstrating that a chemical constitutes no genetic hazard is somewhat different and inherently much more difficult than carrying out and evaluating other toxicity tests including reproduction tests. The 'permissible level' takes on new meaning when multiplied over future generations. MIutagens often act at very low concentration. Definitive tests of their effects in mammals are tedious and expensive. Generally, the more sensitive and feasible the screening test is for chemical mutagens, the further the test organism is removed from man. Little information is available about the applicability of non-mammalian assay systems to mammals." 3. "Because there is already evidence that some chemicals are mutagenic in some test systems, all new chemicals to which human December 1973 populations are to be exposed should be tested in some way for mutagenic effects. The most definitive tests (specific locus tests and backcross or genetic load tests) for chemical mutagenesis in mammals are clearly impractical because they require approximately one-half million animals, or more, depending on test circumstances, to detect a doubling of the mutation rate. Feasible is the dominant lethal test in mice and/or rats as outlined by Bateman" (3). "It is likely that occasionally a chemical, which does not constitute a hazard to human health under the actual conditions of use, might be shown to be mutagenic by simpler test systems. For example, a chemical when added to the culture media of microorganisms might produce a detectable increase in mutation frequency, but be innocuous to man because of degradation in the gut, detoxification, or other reasons. The same comments are appropriate for cytological tests with cultivated cells. Further tests would be in order for a chemical which is suspect as might be indicated by positive results of simple screening tests or by existing information such as known mutagenicity of related compounds already recorded in the registry." "Test procedures in simple systems are also open to the criticism that negative results do not prove that a chemical will not be mutagenic to man. Among possible reasons for this are the high degree of specificity of the tests and production of active metabolites. Consequently, the proper evaluation of the non-mutagenicity of chemicals may be accomplished best through a series of tests, first being direct tests on microorganisms for specific effects and on cultivated mammalian cells for chromosomal aberrations. In the event that results of direct tests are negative (which still leaves the possibility of mutagenic metabolites being formed in the animal) or that the potential value of the substance warrants the effort, tests in mammalian systems should be undertaken. 4. "The simple bacterial tests are for back mutations, and only if mutagens produce both forward and backward mutations with roughly equal frequencies are these tests ade-ouate. Since results of different systems do not always agree, at least two bacterial systems should be used to test for specific effects." "Of the tests currently available (November 1968) using mammalian systems, three types are suggested: (1) dominant lethal tests in mice (possibly rats); (2) cytological tests of cells removed from treated animals, and (3) mammalian host-mediated microbial assays. If results of a dominant lethal test and of one other test in a mammalian system as well as results of the 'direct tests' are negative, the chemical may be judged to be nonmutagenic. When the results of 'direct tests' are positive, additional data comparing the metabolism of the compound in the animals used in the mammalian tests to its metabolism in man should be available before the hazard of mutagenesis in humans can be evaluated." Since this report was written there has been very rapid development in mutagenesis assay systems and their application to a great variety of substances. The specific concern over the contributions of environmental agents to the problem of genetic disease was thus widely recognized and we are now at an important crossroad as we recognize the need to better equip ourselves for the task of extrapolating from the results of our laboratory studies to the potential risk to human populations. The Mrak Commission Report (4) contained a chapter on mutagenicity of pesticides. The various methods for mutagenicity testing were fully reviewed by the Mutagenicity Panel and a recommended program for mutagenicity testing was set forth. The recommended protocol included the test of each compound in three mammalian systems, the dominant-lethal, host-mediated, and in vivo cytogenetic, by appropriate methods of administration reflecting human exposure and also parenterally and at high dose levels such as maximal tolerated doses. Also, each compound should be tested in ancillary microbial systems, preferably those detecting both single nucleotide changes and effects involving more than one gene. Environmental Health Perspectives The precision of testing such systems would be such that doubling of the control level of mutation would be statistically significent at the 5%7 level. If one or more of the three mammalian tests shows a significant effect, the test is regarded as positive. A pesticide is regarded as negative if none of the tests is significantly different from its control. If only the microbial test is positive, more detailed mammalian tests are indicated. These recommendations and others not quoted indicated, on the one hand, a degree of confidence in the precision and interpretability of results from these methods, and on the other, a degree of uncertainty which accurately reflected the lack of experience of all concerned with the problems of methodology and with the interpretation of results from mutagenesis testing. There seems to be agreement among two separate panels that the dominant lethal tests on rodents, somatic and/or germinal cell cytogenetics and the newly developed host-mediated assay were the methods of choice in any program of safety evaluation that was to include specific attention to the problem of mutagenesis. Therefore, when the Food and Drug Administration, in response to the directive of the White House Conference on Nutrition, undertook to re-examine the substances used in food that had been listed as generally recognized as safe -(GRAS), it was logical that the question of mutagenesis should be seriously considered. All of these substances had had a considerable history of human use but had not been studied as extensively as any new food additive. There were gaps in knowledge, therefore, especially in regard to newer questions such as mutagenicity and teratogenicity, and these could be dealt with only by application of appropriate laboratory methods. Therefore, about a year or two ago, the FDA undertook mutagenicity testing of GRAS compounds on a relatively large scale. From the beginning it was recognized that not only were substances being tested but that new laboratories which had contracted to undertake the task were acquiring experi-ence in a new area, and that the test systems themselves were being scrutinized. The Mrak Commission Panel had stated that "the testing procedures recommended above must be constantly updated and improved to reflect new techniques and new data." This large-scale undertaking by FDA would provide new experience and data,-and would inevitably have an impact'on our -understanding of the methods used and our confidence in results and our ability to interpret these results in terms of the risk to human populations. I believe we can all agree that essential to any procedure used for safety evaluation is an acceptable degree of reproducibility. Equally important is that the test procedure relate to a health-hazard to 'which man 'is-known to be susceptible. For instance, we-' know that people are poisone&c-in-the same way that our laboratory animals -are whenwe determine lethal doses. We know that man is subject to cardiovas'cular effects, carcinogenicity, and teratogenicity -and other chronic and acute health -problems', so that studies of these specific problems -in animal models are easily translatable, -at least conceptually. The situation with mutagenicity testing has been very-different than that where biological endpoints have been traditionally used. Here the endpoints are not always clearly related to the structural or functional effects which are of known concern to man. Indeed, the relationships seem sometimes on tangential and based on purely mechanistic consideration. It is assumed, for example, that if the compound is found to induce a sex-linked recessive lethal mutation, it would also induce an increased frequency of galactosemia, phenylketonuria, or any number of other recessive conditions in which the specific gene product has been rendered functionless by mut-ation.-This concept is supported better by theory than by observation or direct demonstration, but it points out an essential fact. A great deal more is known about mutagenesis in terms of mechanism than is true formost areas of toxicology. In mutagenesis, the chemical nature of the target substance is known and the molecular mechanisms are at least as well understood as any event in biology. Thus, mutagenesis may occupy a special place in toxicology wherein test system endpoints and human health effects may possibly not be as stringently related as appears necessary in other areas. On the other hand, we must be certain that our experimental model is relevant to the problem, that we are comparing apples to apples, and not to oranges. We must, therefore, identify the genetic events which are of concern to man and focus our efforts on these events by selecting test systems which are suitable for such purposes. Presumably, we know a good deal about the types of mutations which are of concern in man from the specific protein products generated by mutation. For instance, it is known from studies of the globin molecule that gene mutations in man include point mutations. These have either been shown or presumed to include base substitution or base addition or deletion. Small chromosomal deletions either of a terminal or interstitial nature are also known to be responsible for gene mutations in mammals and as such deserve our concern. While there are a number of systems capable of measuring any one of these effects, we need to select the one or ones most closely relevant to man. We further need to be mindful of the need to assess the effects and consequences of pharmacokinetic factors upon the compound in question. Ultimately, we must derive quantitative information in sufficiently precise terms to define mutagenic risk. In addition to gene mutations, it is obvious that man is subject to a significant cytogenetic disease burden as exemplified by Down's, Klinefelter's and Turner's syndromes and a variety of other conditions known to be of cytogenetic origin. These heritable conditions are predicated on certain types of cytogenetic anomalies but it is not known whether these anomalies are induced by the same chemicals which are capable of inducing gene mutations and if so, whether such alterations can accumulate within the germinal stem line and be transmitted to future generations. Such information is necessary if we are to achieve proper understanding of the problem leading towards effective safety or risk evaluations. Indeed it is appropriate to cite Brent, who emphasized: Do not adopt a testing protocol whose results are uninterpretable for human situation (5). Clearly, we must address ourselves to this central question and objectively examine and evaluate all mutagenicity tests in terms of whether they can or will provide the kind of information which is applicable to human safety evaluation. This is the important charge for this Workshop. Specifically, we must ask the following question of any current or proposed mutagenicity test system: (1) Are the events measured heritable and to what extent would they accumulate either in germinal stem cells or within the human population? (2) Does the assay properly account for all essential pharmacokinetic factors relevant to human exposures? (3) Are the genetic events measured of concern to human health? (4) Is the assay applicable to the types of exposures which are of greatest presumed hazard to man, i.e., what is the probability of a given dose reaching a given number of people? (5) To what extent can the assay yield quantitative information useful in ranking chemical mutagens in terms of mutagenic potency? If this Workshop Study Group agrees that these questions are essential ones to ask, the suggestion is made that they serve as a basis for the panel discussions.

v3-fos

2018-04-03T05:29:04.229Z

1973-03-01T00:00:00.000Z

20846273

s2

Subcutaneous Bacteria in Turkey Carcasses' Two methods were employed to quantitate the subcutaneous bacteria in fresh, refrigerated, and frozen turkey carcasses. Relatively few bacteria were detected in the skin-flesh interface and in the flesh as compared with the number of bacteria on the skin surface and in the skin layer. No subcutaneous bacteria were detected in 49% of the skin-flesh interface and flesh samples. The number of bacteria detected in skin samples from carcasses chemically disinfected to kill skin surface bacteria was smaller than that in nondisinfected skin samples. These results indicate that the skin blending method used to quantify microorganisms on poultry carcass skin measures the skin layer flora and that the number of subcutaneous membrane or flesh bacteria measured is not normally large enough to have a significant influence on the results. Two methods were employed to quantitate the subcutaneous bacteria in fresh, refrigerated, and frozen turkey carcasses. Relatively few bacteria were detected in the skin-flesh interface and in the flesh as compared with the number of bacteria on the skin surface and in the skin layer. No subcutaneous bacteria were detected in 49% of the skin-flesh interface and flesh samples. The number of bacteria detected in skin samples from carcasses chemically disinfected to kill skin surface bacteria was smaller than that in nondisinfected skin samples. These results indicate that the skin blending method used to quantify microorganisms on poultry carcass skin measures the skin layer flora and that the number of subcutaneous membrane or flesh bacteria measured is not normally large enough to have a significant influence on the results. The flesh of a live animal is essentially sterile; however, during processing, bacteria on the skin surface may contaminate the flesh and skin membrane through severed blood vessels or skin cuts and tears. It has been suggested that most bacterial growth is confined to the skin surface of dressed and eviscerated poultry and that very few bacteria are present in the adjoining flesh (3,6). A review of live human skin structure and its bacterial flora by Price (5) further supports the above assumptions. Several changes occur in skin after death, however, which could affect its function as a barrier to bacterial migration into the inner tissues. The extent of such changes partially depends on the elapsed time after death, the carcass temperature, and the way the carcass was handled. The presence of a significant number of viable bacteria beneath the skin of processed poultry carcasses would be of concern for a number of reasons. It might indicate bacterial migration through the skin enhanced by timetemperature abuse or unsanitary processing. Subcutaneous bacteria in poultry meat might present a potential public health hazard and shorten the shelf-life of the product. Methods employed to reduce the skin surface bacteria level during processing might not necessarily affect subcutaneous bacteria. Analytical methods based on quantifying skin surface bacteria by blending and plating skin samples (1,2) ' Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 1804. 354 might be including a significant number of subcutaneous bacteria in the skin "surface" count. This last possibility was what prompted the study reported herein. It has been reported that a skin sample "blending" method for quantifying the estimated bacterial population on poultry carcass skin yields significantly higher counts than the "cotton swabbing" technique (1,4) or the "carcass rinse" technique (4). However, if a significant number of subcutaneous bacteria were present, the skin "blending" method (2) would reflect both surface and subcutaneous bacteria, whereas the "swab" and "rinse" methods would enumerate primarily surface bacteria. Therefore, it was necessary to determine the relative numbers of subcutaneous bacteria in freshly eviscerated, refrigerated, and frozen-thawed poultry carcasses as compared with the numbers of bacteria in the skin tissue and on the skin surface. MATERIALS AND METHODS Two methods were used to determine subcutaneous bacteria in turkey carcasses. In one method, the right half of the carcass was chemically disinfected by swabbing the skin with 5% phenol for 5 min followed successively by 70% ethyl alcohol, 2.5% sodium hypochlorite, and finally with 70% ethyl alcohol again, each for approximately 1 min. The final application of ethyl alcohol was allowed to evaporate. Skin samples were cut on this half with a sterile brass cutting tool (2.54 cm in diameter), removed from the carcass with sterile forceps (scissors were used to facilitate removal), and homogenized in a Waring blender containing 100 ml of BACTERIA IN TURKEY CARCASSES Butterfield's buffered phosphate diluent for 2 min. Three skin samples were removed from the breast and three from the leg. The skin homogenate was plated in plate count agar (Difco). Flesh samples, 1.27 cm in diameter and approximately 1.27 cm deep, were cut below each skin sample that was removed from the disinfected side. These flesh samples were removed, blended, and plated in a similar manner. Carcasses were from three different treatment groups: freshly eviscerated, refrigerated (4 C) for 7 days, or frozen (-29 C) and thawed (8 C for 16 h). Six skin and six flesh samples were removed from the disinfected side from each of duplicate carcasses from each treatment group. Thus, a total of 12 skin and 12 flesh samples were taken from the disinfected side, per carcass group. Six skin samples were removed from the nondisinfected half of each carcass in a similar way. These samples were taken immediately after the samples from the disinfected side of any one carcass had been plated. They were blended and plated as previously described to quantitate the bacteria in the skin tissue. Flesh samples were also removed, blended, and plated as described in the previous paragraph. All plates were incubated at 37 C for 48 h and at 20 to 25 C for 48 h before colonies were counted. The second method used to determine subcutaneous bacteria involved cutting and lifting a portion of carcass skin and swabbing the skin-flesh interface. The skin in the region of the incision was disinfected with 70% ethyl alcohol, which was allowed to evaporate. A V-shaped incision approximately 2.54 cm long was made in the skin with a sterile scalpel. The skin was pulled away from the flesh with sterile forceps. One sterile calcium alginate swab (Calgiswab; Consolidated Laboratories, Inc.) was moistened with one-fourth strength Ringer* solution containing 1% Calgon (Calgon Consumer Products Company, Inc.) and was used to puncture the skin membrane and swab the skin-flesh interface. An area of approximately 1 cm2 was swabbed. The swab was then agitated until dissolved in 10 ml of the modified Ringer solution. All 10 ml of the Ringer solution with the dissolved swab was plated (three plates) in plate count agar (Difco). Plates were incubated at 37 C for 62 h before colonies were counted. A total of 60 samples was taken; 10 samples were removed from each of the duplicate carcasses for each treatment condition. Bacterial counts per square centimeter of skin surface were estimated on each carcass by the same "swab" method. All turkey carcasses used in this study were processed by conventional methods in a laboratory pilot plant. For reasons of availability, male turkey carcasses were used with the first method and female turkey carcasses were used with the second method. RESULTS The ratios of flesh samples yielding any viable bacteria to the number of samples taken by the first sampling method are presented in Table 1, as are the average aerobic plate counts per square centimeter in the flesh samples. Average aerobic plate counts per square centimeter of skin are shown for comparison. Among the 36 flesh samples from the disinfected sides of the turkeys, 27 were positive for bacteria; 24 of 36 from the nondisinfected sides were positive. The replicate averages of the flesh sample aerobic plate counts per square centimeter from fresh, refrigerated, and frozen-thawed carcasses were 10, 36, and 280, respectively, on the nondisinfected side, and 32, 150, and 35, respectively, on the disinfected side. The "disinfected" skin layer of the carcasses yielded replicate average aerobic plate counts per square centimeter of 160, 290, and 110 for fresh, refrigerated, and frozen-thawed carcasses, respectively. The ratios of skin-flesh interface swab samples yielding viable bacteria to the number of samples taken are presented in Table 2, as are the average aerobic plate counts per sample. The average aerobic plate counts per square centimeter of skin surface determined by swabbing are shown for comparison. No bacteria were isolated from 44 of 60 skin-flesh interface swabbings. Each of the three carcass conditions averaged fewer than 10 bacteria per swab sample. The replicate averages of the skin surface aerobic plate counts per square centimeter from fresh, refrigerated, and frozenthawed carcasses were 4,000, 11,000, and 1,100, respectively. DISCUSSION By the skin blending method used (100 ml of blending fluid) and the agar plate count technique, 100 bacteria per square centimeter of skin is the lowest number that can be determined with any accuracy. Aerobic plate counts are only reported to an accuracy of the first two left-hand digits of the calculated average count. Aerobic plate counts greater than 1,000/ square centimeter of skin, determined by blending the skin, would be very slightly, if at all, affected by fewer than 100 subcutaneous bacteria per square centimeter. When one considers the possible sources of contamination of subcutaneous tissue during sampling, the data indicate that there were very few bacteria below the skin tissue in the fresh, refrigerated, or frozen-thawed turkey carcasses tested. There is, however, a possibility that bacteria are firmly imbedded in the rough skin surface or perhaps just beneath the surface layer. Even if this is true, it would be desirable to include them as part of the carcass bacteria count. It appears that subcutaneous bacteria are negligible compared to the level of skin surface bacteria present in fresh, refrigerated, or frozen turkey carcasses. Any significant number of bacteria in the flesh of a turkey carcass would indicate severe time-temperature abuse or unusual contamination through skin cuts and tears, or both. Since turkey carcass skin seems to be a rather effective barrier to bacterial migration into the tissues, an intact eviscerated carcass would be expected to have a longer shelf life than carcass parts or a carcass with large skin cuts or tears. The results of this study indicate that with freshly eviscerated, refrigerated, or frozenthawed turkey carcasses, skin "surface" bacteria counts determined by blending skin samples will not be significantly affected by subcutaneous membrane or flesh bacteria. Skin "surface" bacteria counts are probably only slightly affected, if at all, by subsurface bacteria in the skin layer.

v3-fos

2018-04-03T05:17:51.344Z

1973-12-01T00:00:00.000Z

42296993

s2

Microbiological Profiles of Four Apollo Spacecraft Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (104 per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch2. A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft. Microbiological profiles of automated and manned spacecraft are being determined on a continuous basis due to national and international agreements which stipulate that microorganisms which have a potential of being transported in a viable state to the surface of the moon be enumerated and identified, and an inventory of the levels of contamination at each landing site be maintained (13). In addition, because the existence of life on other planets is possible, scientific investigations for determining the possibility of extraterrestrial life forms must not be jeopardized. Contamination of Mars and other planets of biological interest with terrestrial microorganisms will be controlled to the extent that the probability of contamination will be 1 in 1,000 (10-3) (5,10,11,15). Dry-heat sterilization of the spacecraft will be employed to achieve these objectives. The assessment of microbial contamination levels, especially bacterial spores on space hardware, will be one of the essential controlling parameters in the sterilization of interplanetary spacecraft because the number of bacterial spores present on the spacecraft, as determined by microbiological assays, will determine the extent and character of the dry-heat sterilization cycle (2,3,10,11). The objective of this study was to determine and compare the levels and types of microorganisms on various components of four Apollo spacecraft. MATERIALS AND METHODS Microbiological assays were conducted on Apollo spacecraft during assembly and testing, and sampling locations were selected on the interior and exterior surfaces of various spacecraft components. A prerequisite for sites was that they be representative surfaces of the entire spacecraft and be accessible throughout the sampling periods. The Command Module (CM), Lunar Module ascent stage, Instrument Unit (IU), Saturn *S-4B stage (S-4B), and Spacecraft Lunar Module Adapter (SLA) were interior surfaces studied. Exterior surfaces included the ascent and descent stages of the Lunar Module, and, for Apollo 15, the first Lunar Roving Vehicle (LRV). The various spacecraft components were studied at three periods during assembly and testing. The CM was sampled at 14 days, 7 days, and 24 h, and other spacecraft components were sampled at 14 days, 7 days, and 57 h, respectively, before launch. At each interval, 15 immediately to the laboratory, agitated on a Vortex mixer for 5 to 10 s, placed in an ultrasonic bath (tank LTH60-3; generator, A-300; Branson Instruments, Inc., Stamford, Conn.) containing a 0.3% (vol/vol) Tween 80, and insonated for 2 min at 25 kHz (14,18,19). Randomly, sterile swabs were moistened in sterile distilled water and then returned to sterile screw-cap tubes containing sterile buffered rinse solution with 0.02% (vol/vol) solution of Tween 80. These swabs were then assayed as described above and served as controls. After insonation, replicate portions from each tube were plated with Trypticase soy agar (TSA; BBL). For Apollo 12, portions also were spread over the surface of blood agar (TSA plus 5% defibrinated sheep blood), MacConkey agar (BBL), and Mycophil agar (BBL). Spore assays were performed by heat shocking the remaining rinse fluid in each tube at 80 C for 15 min and plating with TSA. Brewer jars for anaerobic incubation were flushed three times with a gas mixture of nitrogen (80%), carbon dioxide (10%), and hydrogen (10%), filled a fourth time with the gas mixture, and connected to an electrical source for 45 min for catalytic removal of oxygen. The CM interior surfaces of Apollo 15 were sampled at approximately 9 h (preflight) prior to launch and also after the mission (postflight) when the CM was taken on board the recovery vessel. The sampling procedures were similar to the above, except that each cotton swab was placed into 10 ml of sterile veal infusion broth. The postflight samples were kept at 4 C, transported to the Planetary Quarantine Laboratory at Cape Kennedy, Fla., and assayed within 30 h after being taken. In addition to plating on TSA, portions were spread over the surfaces of blood agar and blood agar enriched with vitamin K and hemin. All media except TSA were incubated at 37 C under aerobic, anaerobic, and CO2 conditions. The TSA culture plates were incubated at 32 C under aerobic conditions. All laboratory procedures were performed in a horizontal laminar flow clean bench (7) to eliminate background contamination. Other details of the sampling procedure have been described previously (14). Plates were incubated at 32 C and colony counts were performed after 48 and 72 h. For each Apollo mission, 1,000 to 2,000 colonies were picked randomly from culture plates, gram stained, and identified. All isolates were subsequently lyophilized and stored for future reference. Micrococcaceae were classified by the scheme of Baird-Parker (1), aerobic sporeformers (Bacillus spp.) were classified by the method of Smith et al. (24), Enterobacteriaceae were classified by the schemes of Edwards and Ewing (4), and the Pseudomonas-Achromobacter-Flavobacterium group and related gram-negative bacteria were classified by the method described by Shewan et al. (23). Bergey's Manual (7th ed.) was used for classifying other groups of bacteria. RESULTS AND DISCUSSION Comparison of the levels of microbial contamination detected on the four Apollo spacecraft is shown in Unidentified ascomycete 1 microorganisms per square foot of surface for each of the component parts were relatively consistent for the four Apollo spacecraft. Although the levels of total microorganisms were similar for all CM, IU, and S-4B, the concentrations of bacterial spores and molds on the latter two components were higher than on the CM, which was consistent with what was found on previous Apollo spacecraft (21). The highest percentage of bacterial spores was detected on the surfaces of the SLA, although this component had the lowest number of microorganisms. The constant flushing of the SLA with high volumes of filtered air might have reduced the vegetative microbial population due to physical removal or desiccation, resulting in a relatively Table 2 shows the types of aerobic mesophilic microorganisms isolated from each of the Apollo spacecraft by using TSA. The distribution by types of microorganisms on the four Apollo spacecraft was remarkably similar. Vegetative microorganisms of human origin such as Staphylococcus spp., Micrococcus spp., and the Corynebacterium-Brevibacterium group accounted for the vast majority of microbial contamination detected. This pattern is consistent with previous Apollo spacecraft (17,20,21,22). The percentage of these microbial types (i.e., indigenous to humans) as detected on the various components of the Apollo spacecraft is shown in Table 3. The highest percentages were found on the interior surfaces of the Command and Lunar Modules. The levels of microorganisms associated with soil and dust (bacterial sporeformers, molds, and actinomycetes) have increased with each Apollo spacecraft. Normally, these types of microorganisms reflect the degree of environmental and personnel controls employed, and when environmental controls are relaxed there is a marked increase in the types of microorganisms originating from soil and dust. Nineteen genera of molds were isolated from Apollo spacecraft (Table 4), with Aspergillus, Bipolaris, Curvularia, and Penicillium being the predominant. The elimination of the 21-day back-contamination quarantine period of the Apollo 15 mission made possible the first opportunity to take postflight microbiological samples on the interior surfaces of the CM. In addition to the 14-day, 7-day, and 24-h sampling of the CM, a 9-h (preflight) sample was taken by a member of the astronaut back-up crew. The postflight samplin'g was conducted by the flight surgeon on board the recovery vessel. Samples were Table 5. The levels of microorganisms increased in some areas by 3 logs per square inch. A total of 1,682 microorganisms were isolated and identified from the Apollo 15 spacecraft. Of these isolates, 183 and 327 were obtained from the interior surfaces of the CM at preflight and postflight sampling periods, respectively. Tables 6 and 7 list the types of microorganisms detected in the CM from pre-and postflight samples employing various media and incubation methods. These microorganisms were isolated from TSA, blood agar, and enriched blood agar. All media except TSA were incubated at 37 C under aerobic, anaerobic, and CO2 conditions as requested by the Manned Space Center (MSC). Three types of microorganisms (Streptococcus-Viridans group, Peptostreptococcus spp., and Lactobacillus spp.) were detected only on postflight samples. For the identification data to be meaningful to MSC, colonies resulting from postflight samples were selected by the MSC protocol, i.e., every different colonial type on every culture plate was picked. The standard method used in the Planetary Quarantine Laboratory employs a template with randomly selected points for picking colonies only from aerobic TSA plates. With the exception of the three types of microorganisms detected from postflight sample plates incubated under special conditions (CO2 or anaerobic), all other types were detected on aerobic TSA plates. Nine types of microorganisms were detected on TSA which were not detected on the other Analysis of the types of microorganisms isolated from the various sites on the surfaces of the CM of Apollo 15 from pre-and postflight samples revealed that no apparent recovery pattern existed. Any microorganism which was isolated was equally likely to be found on any given site. It is evident from the results obtained that the levels and types of microorganisms on surfaces of Apollo spacecraft remain relatively constant among spacecraft (20-22) but greater than some of the automated (6,9,16) spacecraft. This was to be expected since Apollo spacecraft are assembled and tested in environmental areas (20) which do not have the same degrees of environmental and personnel controls exerted in those areas used for automated spacecraft (25). If the total number of microorganisms was used as a criterion for evaluating spacecraft microbial cleanliness, the Apollo 14 spacecraft was clearly the most contaminated Apollo spacecraft flown to date. However, if numbers of microorganisms indigenous to soil were used as the standard, the Apollo 15 would be the most contaminated. It has been shown that man is the chief source of microbial contamination to which spacecraft are exposed when assembled and tested in rigidly controlled environments such as high-quality conventional clean rooms (8,12) and, to a greater extent, in laminar flow clean rooms. When environmental constraints are non-existent or nominal, the percentage of soil microorganisms in the total microbial population increases. This might explain the increase of these microorganisms on the Apollo spacecraft with each mission.

v3-fos

2018-04-03T01:33:56.859Z

1973-12-01T00:00:00.000Z

30007356

s2

Supplemental effect of lysine on growth depression of rats fed wheat gluten diets containing excessive amount of some individual amino acids. The effects of lysine supplementation on the growth depression causes by the excessive addition of some individual amino acids to a wheat gluten diet in which lysine is the first-limiting amino acid were studied. Male weanling rats were fed 11.6% wheat gluten (equivalent to the N content of 10% casein) diets containing 5.0% of single amino acids, L-arginine-HCI, L-tryptophan, L-phenylalanine, L-methionine and L-tyrosine, with or without 0.6% of L-lysine ⋅HCl for 3 weeks. The growth retardation produced by the excess arginine was reversed by the sup-plement of lysine, but the adverse effects caused by the excessive addition of the other amino acids were not alleviated. The moderate elevations of arginine and lysine levels in blood plasma produced by excess arginine diet did not altered by the supplemental lysine. In contrast, the plasma levels of phenylalanine, tyrosine, tryptophan and methionine elevated by the excessive feeding of corresponding amino acid were decreased fairly by the supplement of lysine. The activities of liver arginase did not changed appreciably by the addition of excess arginine and the supplemental lysine. It is suggested that the decrease in growth when excess arginine is added to lysine-deficient diet causes the reduction of lysine utilization and increases need for lysine. The severity of the growth depression occurs in rats fed a low protein diet containing excessive amount of single L-amino acids is dependent not only upon the kind of amino acid supplemented (1-3) and the protein content in diet (2)(3)(4) but also upon the source of dietary protein (2,5). Previous study (5) showed that a) the growth-depressing effect of individual amino acid added to a wheat gluten diet is generally severer than those added to casein and egg albumin diets; b) regardless of the protein source, excess methionine, phenylalanine and tyrosine are more toxic than those of other amino acids; and in addition c) when the wheat gluten was used, tryptophan and arginine also cause severe growth depression, although arginine only causes a slight growth depression when casein or egg albumin was supplied as the protein source. Why does excess arginine depress the growth of rats when added to a wheat gluten diet? What effect does lysine, which is the first limiting amino acid in the wheat gluten, have on the adverse effect of excess amino acids? To elucidate these problems, the present study was undertaken to investigate the effect of lysine supplement on the growth depression which produced in rats fed wheat gluten diets containing excessive amount of single L-arginine, L-trypto phan, L-phenylalanine, L-tyrosine and L-methionine. EXPERIMENTAL Male weanling rats (55 to 58g) of Donryu strain were separated into twelve groups of 4 animals each. The animals were housed individually in a tempera ture and light-controlled room and were allowed food and water ad libitum for 3 weeks. The basal diet contained 11.6% wheat gluten2 (equivalent to a nitrogen content of 10% casein); 5.0% soybean oil; 5.0% salt mixture3; 1.0% vitamin mixture3; 0.1% choline chloride; 2.0% cellulose powder and a-potato starch to make 100%. Vitamin A (2,000 IU) and D2 (200 IU) were also added.4 Additions of 5.0% of single L-arginine• HCI, L-tryptophan, L-phenylalanine, L-methionine and L-tyrosine, with or without 0.6% of L-lysine• HCL were made at the expense of a-starch. This supplemental level of lysine to the basal diet was adequate for the requirement of lysine (6). When the experimental diet containing 5.0 arginine• HCl was prepared, 2.0% of sodium bicarbonate was included at the replacement of cellulose powder. Body weights were recorded daily and total dry food consumptions were measured. At the end of the experimental period, animals were killed by decapitation 2 hr after the final feeding. The blood was centrifuged in heparinized tubes, and equal volumes of plasma were pooled from each rat. Plasma was deproteinized with equal volumes of 3% sulfosalicylic acid, and the free amino acid concentrations were determined as described pre viously (5) using an automatic amino acid analyzer.5 The decapitated animals were perfused in situ with ice-cold 0.9% saline, then the livers were excised, weighed and homogenized in two volumes of ice-cold 0.14M KCI. Protein, RNA and DNA contents of the liver homogenates were determined by the procedure used earlier (3). Liver arginase activity in the homo genates was assayed by the same manner as reported previously (7), and the activity was expressed as µmole of produced urea per g of wet liver per hour. RESULTS The growth response curves of rats fed the diets containing excessive amount of some individual amino acids, with or without lysine supplement are shown in Although rats fed the basal (11.6% wheat gluten) diet grew very slowly, the supplement of 0.6% lysine HCl to the diet produced a significant improvement in weight gain. As might be expected, the addition of 5.0% of single arginine, tryptophan, phenylalanine, methionine, and tyrosine to the basal diet caused growth depression to varying degrees. The rats receiving excess arginine diet resulted in growth retardation and maintained the initial body weight at the end of the feeding period, whereas animals fed excess methionine diet caused most significant reduction in weight where the remarkable weight loss occurred during the first few days of the feeding period after which rats remained the weight. The growth retardations caused by excess tryptophan, phenylalanine, and tyrosine were graded between those caused by arginine and methionine. These data agree closely with the results of previous report (3). When rats were fed the excess arginine diet supplemented with lysine, the growth rate was considerably increased, and the weight gain at the end of the feeding period reached 80% of the basal diet supplemented with lysine. The difference between both groups was not significant (see Table 1). In contrast, the supplement of lysine to the excess tryptophan, phenylalanine, methionine and tyrosine diets failed to improve the growth rate, though the lysine addition to the excess phenylalanine diet showed a tendency to somewhat alleviate the growth depression, but did not statistical significances. In spite of whether lysine was supplemented to the excess tyrosine diet or not, the animals developed external pathological lesions, and all rats fed the diet supplemented with lysine died within 8 to 15 days, while all animals fed excess tyrosine diet died within 15 to 20 days. Thus, the supplement of lysine to the excess tyrosine diet rather shortened the survival time of animals. In earlier experiment (5), when rats were fed excess tyrosine diet, two out of seven animals survived during the 3-week period. This discrepancy is probably due to the fact that the rats used weighed somewhat less than those of the previous experiment. The data of food intake, liver weight and some liver compositions are presented in Table 1. Food intake of rats fed the excess arginine diet supplemented with lysine increased significantly more than that of the animals fed the excess arginine diet without lysine, whereas in rats with the supplement of lysine to the excess tryptophan, phenylalanine and methionine diets, the food intake did not increase over the values of diets without lysine, respectively. Thus, a close relationship between growth depression and decrease of food intake was indicated. Liver weight, liver total DNA, protein and RNA contents, protein/DNA and RNA/DNA ratios of rats fed excess arginine diet supplemented with lysine were significantly higher than those of animals receiving excess arginine diet, but when rats were fed the excess tryptophan, phenylalanine and methionine diets, these values were unaffected by the supplement of lysine to the diets, though the total DNA and protein contents tended to be higher. Fig. 2. Plasma amino acid patterns in rats fed the basal (11.6% wheat gluten), basal plus 5% arginine• HCI, and basal plus 5% arginine• HCl plus 0.6% lysine• HCl diets, re spectively. Analysis was performed on a pooled sample from four rats. The free amino acid patterns of blood plasma are shown in Fig. 2. The amino acid pattern of rats fed the excess arginine diet resembled the pattern of animals fed the excess arginine diet supplemented with 0.6% lysine• HCI, except the threonine concentration. In agreement with the results reported previously (5), the addition of excess arginine to the wheat gluten diet produced the elevation of arginine and lysine levels in plasma. Furthermore, the supplement of lysine to the excess arginine diet tended to result in increases of arginine and lysine levels in plasma. The decline of plasma threonine concentration in animals fed diets supplemented with lysine was consistent with the results of previous observa tions (6,8 The effects of dietary arginine and lysine on the activity of liver arginase are shown in Table 2. The arginase activity of rats fed a diet containing excess argi nine showed a tendency to increase as compared to the control, but the difference did not show any statistical significance. This result is essentially consistent with other reports (7,(9)(10)(11). The adequate supplement of lysine to the basal and the excess-arginine diet did not result in any appreciable effect on arginase activity. DISCUSSION The present studies demonstrate that the growth-depressing effect caused by excessive dietary arginine could be prevented by the supplementation of dietary lysine which is the first-limiting amino acid in a wheat gluten diet, but the adverse effects caused by the excessive addition of the other amino acids, such as trypto phan, phenylalanine, methionine or tyrosine, could not be reversed by supplemental lysine. Accordingly, it is clear that a) the adverse effects cause by excessive individual amino acids are not necessarily alleviated with the supply of the first limiting amino acid, and b) there exist a certain relationship between dietary argi nine and lysine. In relation to the situation of a), it is of interest that the extreme elevation of the corresponding plasma amino acid caused by the excess feeding of single amino acids were decreased by supplemental lysine, the most limiting amino acid, regard less of the fact that growth depressions were not counteracted. In contrast, little information has been accumulated about the effect of lysine supplement on excessive dietary arginine. O'DELL and SAVAGE (14) reported that the addition of 1.2% arginine to a sesame diet with limited lysine caused growth depression in chicks, but not when the diet was adequate in lysine. HILL et al. (20,21) also noted in chicks a similar effect from excess arginine added to a diet first limited in lysine. The mechanism by which excess lysine increases need for arginine in the chick has been extensively studied, and some investigators have suggested that the induction of kidney arginase by excess lysine is responsible in part for the possible mechanism (15,16,(22)(23)(24). The present data in rats about the plasma arginine and lysine levels and liver arginase activity concerned with the mechanism did not furnished direct infor mation as to the cause, that is, in the rat the supplementation of lysine to the excess arginine diet failed to produce appreciable effects on plasma lysine and arginine levels, and liver arginase activity. In contrast to this, in the chick, it was shown that when arginine is added to a excess-lysine diet, lysine level in plasma is markedly reduced (22,23,25), and results in increased arginase activity of kidney (15,16,23). In any case, it is suggested that the addition of excess arginine to the diets in which lysine is the first-limiting amino acid in rats causes a reduction of lysine utilization and increases the need of lysine. Experiments are in progress to eluci date the cause of the arginine and lysine relationship in the rat. We are grateful to Mrs. Taeko Aito for assistance with these experiments.

v3-fos

2018-04-03T05:52:42.569Z

1973-01-01T00:00:00.000Z

44772382

s2

Protein isolates from Chlorella algae, Torula yeasts, and hydrocarbon-assimilating microorganisms. Preparation of protein isolates from the cells of Chlorella, Torula yeasts, and hydrocarbon-assimilating microorganisms is described. Simple pretreatment of the cells with alkali, acid, or some organic solvents enhanced the protein extraction efficiency and made the following purification procedures easier. Bleached cells of Chlorella obtained by growing the algae in a culture medium with high C/N ratio at high temperature were found to release protein more easily than do the normal cells. Structural changes in the cell wall region detected under the electron microscope may be responsible for this. The extracted protein was further purified. Amino acid composition of the protein isolates was determined, and their nutritional values were calculated. Among the essential amino acids the sulfur-containing amino acids were found to be the first limiting amino acid. Supplementing the isolate with methionine resulted in a significant increase in its nutritional value (PER) which became comparable to those of egg albumin and milk casein. The digestibility of the protein isolate from the cells of a hydro carbon-assimilating yeast, tested in vitro with pepsin, was as high as 80% of that of the reference protein, milk casein, whereas that of the dried cells of the yeast was less than 50%. Viscosity was measured in regard to possible processed forms of the protein isolates. A few methods for disposing of the extraction residues were tested. The cultivation of microbial cells for food is especially suitable for Japan, where a population of over a hundred million has to live in a small area and where only about 16% of the total area is farm land and only 3% is meadow: Microbial cells can reproduce much faster than conventional food sources, such as animal and plant; they have high protein content; they can be produced without requiring any of the limited farm land available; and their production can be controlled easily, independent of climate. There are, however, some problems to be solved before microbial protein can be used as food. In order to enhance nutritive value, the products from microbial cells should be free from indigestibles such as cell walls; free from acute, subacute, and chronic toxicity; free from teratogenic and carcinogenic factors, which may be produced or accumulated in some microorganisms; and of high acceptability, that is, without strong color and unpleasant flavor. The author believes that separation of protein from the other cell materials is essential for meeting these conditions. In addition, it is hoped that the isolated proteins will have better properties for actual food processing. The generic name MIPRON (from "microbial isolated proteins") for the protein isolated from microbial cells has recently been proposed (3). This name emphasizes that any microorganism could theoretically be used as a protein food source if the above-mentioned conditions are satisfied and that the proteins must be isolated from other cell materials. The author has reported on isolation of proteins from microbial cells by freeze-thawing, toluene autolysis, autolysis-butanol (4), urea soaking (1), and alkali extraction (2). These methods can be used independently or in combination with each other or with presoaking the cells in an acid or alkali solution (1). The best method for isolation, however, varies with the type of microorganism, as shown in Table 1. Table 4, but a given volume of culture could produce more cells under bleaching conditions than under normal conditions, as shown in Table 5. The yield of protein per volume of bleached-cell culture was the same as or often larger than normal, as was efficiency in utilization of nitrogen from the medium. Table 3. Extraction of Chlorella pigments using conventional solvents. (mg/100g on dry basis) SHIHIRA-ISHIKAWA and HASE (6) reported that "glucose bleaching" of Chlorella protothecoides degenerated the lamella structure of chloroplasts, and certain changes in the cell membrane were also observed. The bleached cells of C. ellipsoidea in this experiment also exhibited a structural change in the cell wall region, as seen in the electron micrograph of Fig. 2 Fractionation of isolated proteins A procedure for isolating protein from microorganism cells is shown in Scheme 1. About half the extracted crude protein could be precipitated by adjusting pH of the extract to 4.0-4.2. The crude protein content of the lyophilized precipitate was 65-70%, and the preparation retained an odor specific to the dried cells of hydrocarbon-assimilating yeasts previously tested by the author. This protein content is not enough for the preparations to be called "protein isolate," and the odor is unacceptable for a protein source for human food. In order to overcome these shortcomings, the alkali extract was subjected to dialysis against water. After about 40 hr dialysis, white precipitates that could be removed by centrifugation were found inside the dialysis bags. The nitrogen content of the precipitate was always very low, and thus loss of protein into this fraction (Ppt-I in Scheme 1) was negligible. This fraction seems to be derived from the cell wall; the hydrolyzate of Ppt-I of yeast contained D-glucose and D-mannose as sugar components, determined by paper chromatography. Ppt-I PROTEIN ISOLATES FROM ALGAE, YEASTS, & MICROORGANISMS 7 must be removed as completely as possible in order to obtain a protein isolate of higher purity at a later stage of isolation. In addition to the characteristic odor described above, some nonprotein compounds could be removed by dialysis . Scheme 1. Protein isolation from microbial cells. Alkali extraction would be the most cumbersome step in the whole procedure of isolating proteins, especially for a large-scale preparation, because the high alkalinity of the extract, bulky residues, and strong foaminess of the solution make it impractical to use an ordinary continuous flow type centrifuge . The addition of ethanol to the extraction mixture made it possible to separate the residues by filtration; ethanol concentration up to 50% under these conditions resulted in very little loss of proteins. Ethanol was used successfully by the author in large-scale preparation of protein isolates for an animal feeding test. Fractional precipitation of protein with ethanol from the dialyzed extract of hydrocarbon-assimilating yeast was tested, and the results are shown in Table 6. a See Table 6 for the conditions . b CaCl2, BaCl2, or MgCl2 was added to 3.3%. Fehling solution was used for Cu++ (13.5ml per 100ml of the extract). c Based on the crude protein content of the dried cells. The effects of adding divalent cations to the dialyzed extract of hydrocarbon -assimilating yeast were investigated with the expectation that a cation might precipitate some polysaccharides as hemicellulose (Table 7). Although the amount of insolubles directly produced by the addition of divalent cations was very small, a fairly large amount of precipitation, Ppt-I (Me, D-H2O), occurred on the second dialysis, performed to eliminate excess cations, except with Mg++ which caused no precipitation in the second dialysis. The precipitates are of higher protein content, and more proteins could be obtained by the addition of ethanol or hydrochloric acid to the supernatant solution. Table 7 summarizes the results; among the divalent cations tested, Ca++ was the most suitable for treating food materials. Figure 3 shows a flow diagram of protein isolate preparation from hydrocarbon -assimilating yeast. Essential amino acid composition Essential amino acid patterns of the protein isolates were compared with those of the dried cells. Table 8 shows the ratio of content of each essential amino acid to total essential amino acids, i.e., A/E ratio and compares these ratios to those of whole egg, the reference protein (6). It is clear from these values that the first limiting amino acid in the cells and protein isolates of hydrocarbon-assimilat ing yeast is the sulfur-containing amino acid, and the amounts of the rest of the amino acids are close to those of whole egg. The shortage of the sulfur-containing amino acids in the cells and protein isolates of Chlorella (1) and Torula (1) were comparable with these of hydrocarbon-assimilating yeast. But the protein isolates prepared from those microbial cells are especially abundant in lysine (1). Thus, if the sulfur-containing amino acids are properly supplemented, those microbial proteins would be of high quality as human food. Digestibility One of the purposes of isolating protein from the microbial cells was increasing digestibility. Digestibility of the protein isolates was tested by in vitro procedures. Figure 4 shows that digestibility by pepsin of hydrocarbon-assimilating yeast is increased significantly by isolation of protein from the other cell materials, reaching the level of milk casein, the reference. Digestibility of the protein isolates from Chlorella and Torula by pepsin and trypsin was comparable to that of hydrocarbon-assimilating yeast by pepsin (1). Fig. 4. In vitro pepsin digestion of the protein isolate and the cells of a hydrocarbon assimilating yeast. Table 9. Protein efficiency ratio of proteins from hydrocarbon-assimilating yeast. Animal feeding tests Animal feeding tests were also carried out in order to assess the nutritional quality of the protein isolates. Table 9 shows that the protein efficiency ratio of the protein isolates from hydrocarbon-assimilating yeast supplemented with methionine compares favorably with the value for egg albumin and casein. A diet of protein isolates supplemented with methionine supports the growth of rats as the egg albumin diet does, whereas the protein isolates themselves are not suf ficient. No anomalous symptom was detected in the appearance of animals dur ing or after these feeding tests. Thus, no acute toxic factor was detected. Com parable results were obtained using the protein isolates from Torula yeasts (1). Physicochemical properties of the isolates: Viscosity and fiber formation It is thought necessary to elucidate rheological properties of the protein isolates with every regard to possible processed forms of the microbial isolated proteins. As is well known, a fibrous form of protein, often called spun protein, has been used (8) Disposition of the extraction residues The residues of hydrocarbon-assimilating yeast after alkali extraction are be lieved to be composed mainly of the cell wall materials, and their approximate composition was found to be as follows: polysaccharides, 70%; crude protein, 19%; ash, 1%; and moisture, 10%. They are insoluble in water, dilute acids, dilute alkalis, and organic solvents. Abandonment of the residues without pre treatment would become a source of severe water pollution. Salvage utilization or, at least, turning them into a chemically inactive form will be one of the most important problems to be solved in utilization of microbial cells for food. A few possible methods of residue disposition have been tested: Solidification. Kneading with a small amount of calcium hydroxide solidi fied the pasty extraction residues, and they became hard but brittle after drying. Though the detailed properties of the solid have not been examined yet, solidifica tion would make it easier to handle the residues. Resin cation. (i) Thermosetting resin. Treating the residues with formal dehyde should hydroxymethylate the amino group of the protein remaining in the residues, and an amine-type resin will be produced by the thermal hardening of the hydroxymethylated residues. Most of the polysaccharides probably act as fillers in the resin, but some may participate in cross-linkage formation in the resin. Carboxymethylation. The remaining saccharides in the extraction residues were carboxymethylated with monochloroacetic acid and sodium-hydroxide according to the conventional method of preparing sodium carboxymethyl cellulose. The product, a water soluble carboxymethylated preparation, was ob tained in relatively high yield (ca. 69%), and the viscosity of its solution was considerably lower than that of a commercial carboxymethyl cellulose prepara tion. Though the characteristics of those products have to be examined further and more studies have to be done, the results shown here give hope for disposition of the extraction residues.

v3-fos

2020-12-10T09:04:12.740Z

1973-08-01T00:00:00.000Z

237230782

s2

Production of Penicillic Acid and Ochratoxin A on Poultry Feed by Aspergillus ochraceus: Temperature and Moisture Requirements A strain of Aspergillus ochraceus Wilhelm, isolated from poultry feed, produced both penicillic acid and ochratoxin A. Studies demonstrating the ability of this fungus to colonize poultry feed and produce these two mycotoxins under various temperatures and moistures indicated that the interaction was complex. The optimal temperature for conidial development did not vary with moisture, but accumulation of both toxins did. A combination of low temperature, 15 or 22 C, and low moisture favored the production of penicillic acid, whereas high temperature, 30 C, and high moisture favored the production of ochratoxin A. The development of fungi proceeds only in an aqueous system whether the substrate is liquid or solid; thus, moisture is of major significance in determining the extent of fungal colonization. In addition, development is influenced by other factors such as temperature, light quality, and the chemical nature and availability of substrata. Studies of these factors suggest that temperature and moisture relations primarily determine the relative abundance of fungi on stored products (2,4,27,31). Knowledge of the manner in which these factors interact to influence the nature of secondary metabolites of fungi is needed, especially since many of these metabolites are toxic. Furthermore, quantitative data on the effects of these factors on the ecology of fungi on stored feed products are essential if we are to control their growth or predict their biochemical activities. While investigating the occurrence and distribution of possible toxigenic mycoflora in poultry houses and poultry feed, two strains of Aspergillus ochraceus were isolated, and both were found capable of producing penicillic acid and ochratoxin A. Penicillic acid, a carcinogenic lactone, first was isolated from Penicillium puberulum Bainier (1) but has since been reported from several species of Aspergillus and Penicillium (15). Ochratoxin A, a nephrotoxic dihydroisocoumarin, first was isolated from A. ochraceus Wilhelm (32) and is now known to be produced by other members of the ochraceus group (12,15) as well as by P. viridicatum Westling (33). The mean lethal dose (LD50) (subcutaneous) of penicllic acid for mice is 100 mg/kg, although a dose as low as 0.1 mg is carcinogenic (5); the LD50 (oral) of ochratoxin A is 100 to 200 ag/day-old cockerel and 20 mg/kg in rats (28). The toxicology of these two mycotoxins has been reviewed by Lillehoj et al. (15). Poultry feed is subjected to varying degrees of moisture during transportation and storage and while in the feed trough due to differences in relative humidity and the feeding habits of the birds. Consequently, a study was initiated to determine whether these two toxins could be produced on poultry feed at different moisture contents and under constant light as practiced in broiler houses. Such information would be significant because many investigations have indicated that poultry are susceptible to mycotoxins (9,11,16,26). Recently, the work of Choudhury et al. (3) demonstrated that graded levels of ochratoxin A caused effects ranging from delayed sexual maturity and reduced egg production at low levels (1 ppm) to mortality at high levels (4 ppm). In measuring the availability of water to microorganisms, the quantity of water activity (a,) or the equilibrium relative humidity often is used (25). In this paper a, will be used to describe the availability of water in feed under equilibrium conditions and is to be differentiated from some experiments in which moisture content was adjusted as a percentage weight of the feed. 155 MATERIALS AND METHODS Source of culture. The A. ochraceus strains (103 and 107) used in this investigation were isolated from feed and litter samples obtained in local broiler houses. On the semisynthetic medium (7) strain 107 produced higher yields of both penicillic acid and ochratoxin A and was, therefore, used in this study. The strain was maintained at 5 C on Czapek agar. For the preparation of spore suspensions used for inoculations, subcultures were made on Czapek agar supplemented with 20% sucrose (20). Moisture content. Flasks (125 ml) containing 50 g of poultry feed were stoppered with stainless-steel closures and autoclaved for 20 min at 121 C. After autoclaving, the sterilized feed samples were brought to the required moisture content by gradually adding sterile distilled water. Each gradual addition was accompanied by thoroughly shaking the sample to assure that the water content was as uniform as possible. Each sample was finally mixed for 15 min on a wrist action shaker. As a check for each of the required moisture contents, a 10-g sample was removed and the moisture was determined. All samples had an initial moisture content of 11.5% after autoclaving. Moisture contents were adjusted to the following final percentages: 14.0, 18.0, 23.0, 32.0, 42.0, 52.0, and 62.0. The feed was inoculated with spores from 10-day-old cultures and incubated for 12 days at ambient temperatures under constant light supplied by 40-W fluorescent lamps. Water activity and temperature. The required a,, in the feed was achieved and controlled by bringing the feed into water vapor equilibrium with solutions of NaCl (21). To determine the effects of a,, and temperature on toxin production, feed was ground in a Wiley mill (40-mesh screen) and air dried for 24 h. Samples (5 g) were placed in wide-mouth sampling jars (40 mm), capped, and sterilized for 15 min. The samples were then uncapped and transferred aseptically to desiccators. Each desiccator contained 250 ml of a NaCl solution of known water activity (Table 1) and each was evacuated and maintained at 15, 22, and 30 C for 2 weeks to permit rapid equilibration of the feed with the corresponding relative humidity. A subsequent experiment determined that the feed in the evacuated desiccator reached equilibrium after 8 days at each of the temperatures. The water sorption isotherm of the poultry feed is given in Table 1. After equilibration, spores of A. ochraceus were dusted onto the feed, and the sampling jars were suspended over 200 ml of the various salt solutions contained in 950-ml wide-mouth Mason jars. The jars were sealed and immersed in pans of water which were placed in constant-temperature rooms under constant light. The water in the pans was adjusted to one of the three temperatures prior to introduction of the Mason jars. The feed was analyzed after 2 weeks of incubation. At each aw the feed was examined under a binocular dissecting microscope and a compound microscope for growth and development of conidial heads. Toxin analysis. Penicillic acid and ochratoxin A were extracted from the moldy feed with chloroform and methanol (1:1, vol/vol) in a Waring blender for 3 to 5 min. The suspension was filtered and the resulting chloroform-methanol extract was worked up by the sodium bicarbonate procedure of Steyn et al. (29). The resulting extract was evaporated under reduced pressure and the volume was adjusted to 4 ml with chloroform. Penicillic acid was purified by preparative thinlayer chromatography on a Silica Gel G-254 by using chloroform as the moving phase. Crystallization from benzene gave a product (mp 86-87 C) which was identical to an authentic sample as determined by thin-layer chromatography, mass spectral data, and ultraviolet and infrared spectra. Penicillic acid was quantitatively determined in the final chloroform extracts as its trimethylsilyl derivative by using a Perkin-Elmer 900 gas chromatograph equipped with a flame ionization detector. The column was 6 ft by l/8 inch (182.88 by 0.318 cm) stainless steel and packed with 3% SE-30 Chromosorb W. Nitrogen flow rate was 45 ml/min. At a program rate of 8 degrees per min from 60 to 250 C, penicillic acid had a retention time of 11.5 min. Peak areas and retention times were measured with an Infotronics CRS-101 digital integrator. This procedure is similar to that described by Pero et al. (19), who demonstrated linearity of penicillic acid concentration with detector response. Ochratoxin A was initially identified by chromatographing a sample on Silica Gel G (24). The identity of ochratoxin A was confirmed by co-chromatography with an authentic sample on Silica Gel G thin-layer plates with several solvent systems (24,29,30) and by comparison of ultraviolet spectra in acid and base. The concentration of ochratoxin A was determined spectrophometrically at 333 nm after elution of the toxin from the plates with methanol (30). All experiments were done in triplicate and yields were reported as averages. RESULTS Toxin production at added moisture. Growth was not observed in feed samples at an initial moisture content of 11.5%. Microscope examination revealed that spores at this moisture level had not germinated. Growth and conidial development were observed at 14% moisture and higher. Although both mycotoxins were produced under constant light, their patterns of accumulation differed (Fig. 1). Maximum production of penicillic acid was reached at 23% moisture, whereas maximum production of ochratoxin A was reached at 42% moisture. Furthermore, near maximum production of penicillic acid occurred at 18% moisture, a level at which ochratoxin A production was still extremely low. The results also showed an interesting correlation between higher moisture content and toxin production. There was a marked decrease in the yields of penicillic acid at 42% moisture until, at 62%, the yield was about one-half the maximum. There was a similar pattern for ochratoxin A production, but the decrease began at a higher moisture level, i.e., 52%. Water activity and temperature. Table 2 illustrates the effect of temperature on the tolerance of low aw by A. ochraceus. At 15 C the fungus developed conidial heads only at the highest aw activity, but at 30 C it did so readily at all levels of aw. Generally, the optimal temperature for development of conidia as determined in this study was 30 C regardless of the aw. Although no accurate measure was used to quantitate the extent of growth and conidial development, samples with water activities of 0.90 and higher had more conidial heads than samples at lower aw. Furthermore, sclerotia were produced only at the lowest temperature (15 C) until moisture became a limiting factor. At an aw of 0.80 no growth occurred at 15 C, and sclerotia were produced at 22 C. These observations on the presence of mycelia, sclerotia, and conidial heads do not permit correlations with the quantity of toxins produced. The currently used methods for measuring fungal growth are not suitable for use on such a complex solid substrate as poultry feed. Generally, maximum production of both mycotoxins was temperature and moisture dependent ( Table 2). The accumulation pattern for each toxin, however, was different. Penicillic acid began to accumulate at an aw of 0.80 at 22 and 30 C, whereas ochratoxin A began to accumulate at an a w of 0.85 at 30 C. The optimal conditions for the production of each toxin were different. Maximum production of penicillic acid occurred at 22 C and an aw of 0.90 (Fig. 2 temperature on ochratoxin production. However, high yields of ochratoxin A have been reported on synthetic and semisynthetic media at 25 C (7,8). Apparently, low substrate moisture is optimum for the production of penicillic acid. Accumulation of the compound at low moisture and temperature may simply indicate that penicillic acid is synthesized more rapidly than ochratoxin A since, in shaken and stationary liquid culture, penicillic acid appears in the medium several hours before ochratoxin A (unpublished data). Apparently, the biosynthetic pathways for these two mycotoxins are controlled by different limiting factors. The accumulation of both mycotoxins decreased at 22 and 30 C as the moisture level increased to near saturation. Perhaps near saturating conditions on this substrate are least favorable for maximum growth, and thus these data reflect nothing more than a growth phenomenon. Reduced growth near saturation has been reported for all members of the A. glaucus group (2). The production of these mycotoxins then would be a consequence of suboptimal growing conditions; this possibility appears especially strong in the case of penicillic acid. Linear production of this toxin parallels the development of sclerotia, the presence of which is considered to represent suboptimal growing conditions (23). The favored production of other mycotoxins at suboptimal growth conditions supports the generality of this hypothesis (6,13,14,18,22). Since the volume of air available to the fungus in the jars was only about 600 ml, the differential production of the mycotoxins may simply reflect an aeration effect to which the two pathways have different affinities. However, results were parallel from the moisture experiment in which air was not limiting. Furthermore, similar experiments present evidence that oxygen is not a limiting factor under these conditions (10,17). Although the total amount of oxygen may not be limiting, the amount available in the feed under near saturating conditions may be. As the moisture increases, the air-filled porosity (texture) of the feed may change, creating a water-logged condition. Under such a condition reduced growth, 02, and high levels of CO, may affect the production of these two toxins. The fundamental effects of temperature and moisture on the production of these two toxins have not been explained entirely. Perhaps accumulation of these mycotoxins is not controlled by a simple temperature-humidity relationship. Generally, the controls and regulations of secondary metabolities are not well understood and therefore warrent investigations. This study demonstrates that, although growth of fungi may be barely visible on relatively dry poultry feed and during a relatively short incubation period, quantities of penicillic acid may be produced which are capable of inducing tumors in mice. The data further suggest that after long incubation and a cool temperature (15 C), lethal quantities of the toxin may be produced. It appears that ochratoxin A may become a problem in poultry feed under more obvious conditions of fungal decay where lethal levels of this compound may occur. Thus, A. ochraceus appears to produce ochratoxin A as near optimal conditions for conidial development are approached and to produce penicillic acid under suboptimal growth conditions. Studies of the possible interaction of other environmental factors which may be responsible for the accumulation patterns of these toxins are underway.

v3-fos

2016-05-12T22:15:10.714Z

1973-04-15T00:00:00.000Z

5870871

s2

14e session de la commission de génétique de la f.e.z. vérone 7. 8 et 9 octobre 1972 The investigation has been made to evaluate the economic consequencies of varying replacement rates and varying use of beef semen in a dual-purpose breed (Swedish Red and White Cattle). The investigation showed that crossbred calves (Charolais and Hereford sires, Swedish Red and White dams) are superior to purebreds (Swedish Red and White) in comercial beef production, which in economical terms amounts to about 200 sw. Cr. It was also found that there is a considerable capacity for beef crossing in dairy herds. When the replacement rate is 20 p. 100 , the percentage of cows available for beef crossing is 45 p. 100 . At the replacement rate 40 p. 100 , DETERMINATION OF THE STRUCTURE OF CATTLE PRODUCTION WITH UNEAR PROGRAMMING J. CZAKÓ. The aim of the investigations based on calculations and experimental findings was to determine which are the most effective productive types for the production of the two main products of cattle, milk and beef. In cattle breeding the industrial production processes have encouraged the forming of specialized productive types. The structure of the stock has to be planned in the light of the requirements. In the present study 10 populations were examined according to 9 objective functions. The comparisons of the populations were ca.rried out on the basis of products obtained with the same starch equivalent quantities. It can be stated that the optimal population structure generally consists of one milk producing and one beef producing stock. The most favourable result is from the population in which the heifers not needed for replacement are used for beef production by means of commercial crossing (Hungavofvies). Also those dual purpose populations were favourably classified in which the milk production required can be achieved with simple hybridization and beef production with triple crossing. The beef requirements in excess of the beef production from milk producing stock have to be produced by specialized beef cattle types. Modern breeding methods have the same role in the establishment of optimal population structure, as varied production types. The method of progress is not the construction of new breeds alone. Specialization in production on separation of technologies are at least as important as the forming of specialized types. Aufbauend auf ausführlichen Kalkulationen zur Ermittlung der ôkonomischen Gewichtung von Leistungseigenschaften beim Rind wurde versucht, die Auswirkungen verschiedener Kombinationen der Bestimmungsfaktoren des züchterischen Fortschritts auf die Effektivitât der Zuchtarbeit zu quantifizieren und eine aus ôkonomischer Sicht optimale Kombination dieser Faktoren zu finden. 2, Zürich (Switzevland). A study was made to determine whether it is economically worthwhile to include fat and/or protein tests in milk recording schemes under given milk pricing systems. Goal is maximizing net income in breeding programmes for milk and milk constituents in dairy cattle. Consumers and the dairy industry could determine the price relations for milk, fat and protein according to their future needs. These would apply for producers price of milk and ranking of breeding animals. Seven milk pricing systems were applied assuming that milk alone or fat and protein in different relations determine the price of milk. This gives the price differentials for the components that go into the index. Four sets of genetic parameters representing different populations were used for milk kg, fat kg, protein kg, fat p. ioo and protein p. 100 (Wm.cox et al., 1971 ). The calculations were made for a static population of izo 00 o cows under AI programmes and milk recording using discount cash flow procedures. The expected interest rate on invested money was taken as a measure for effectiveness assuming that the breeding programm is carried over twenty years. Costs per cow and year under present conditions in Switzerland are 41 . -sFr. If milk is paid for kilogrammes only it is not economic to carry out fat and protein tests. The higher expected fat yield as a result of fat testing and including in an index in addition to milk alone leads to the same interest rate as milk recording alone which means that costs for fat test are just compensated. This holds only if fat is the main worth determining component. Including protein in addition to milk and fat results in small additional yields because of the high genetic correlations between amounts of milk, fat and protein. The costs for protein testing lower the interest rate by 1/2 to i p. 100 . Even strong weighting of protein in the milk pricing system does not compensate costs.for protein testing under the above conditions. Including protein in breeding programmes is only justified if costs for fat and protein testing can be cut to 3 to 4 sFr. per cow and year (central laboratories, simultan analyzers) and if protein has adequate weighting in the pricing system. The analysis has shown that conclusions are not necessarily the same for sets of genetic parameters from different populations. While it seemed not worth to change the weighting from fat to protein for the danish data it can be economic for other populations. In model calculations concerning genetic and economic optimalization the effect of population structure, breeding systems and economic parameters on breeding plans of the dual purpose cattle was investigated. In the present examination the factors population size, proportion of cows which are milk recorded, capacity of testing stations for bulls on daily gain, intensity of selection for yearling performance, and the discount rate of interest were varied. Attempts were made to determine an advantageous breeding structure for three different breeding systems which maximized genetic improvement or economic return respectively. The income with regard to costs of breeding work was estimated from « breding population size » and « commercial used herd ». It was supposed that all cows are inseminated with deep-frozen semen. With respect to previous simulations the following aspects are to emphasize : -The population size is important for return of breeding plans. -If one assume operational breeding costs for milk recording the optimal proportion of the breeding population is depending on the number of cows in a population and on the choice of the discount rate per year. The value is 6 to 30 p. 100 . -It was shown that the return depends considerably on the genetic correlation between milk fat yield and monetary weighted net daily gain. -The relative contribution of the two-gene-paths a sires-sons » and « dams-sons &dquo; to the return is less than to the genetic improvement. COST STUDIES ON CATTLE BREEDING PROGRAMS P. H. P ETERSEN * , E. O VESEN ** and L. Gji5 L C HRISTENSEN ** . * * Institute of Animal Science, The Royal Veterinary and Agricultural University, Copenhagen. ** National Institute of Animal Science Rilighedsvej, 25, Copenhagen. The main breeding goals for the Danish dual purpose breeds are the improvement of butterfat yield and growth rate. In the breeding planning there is furthermore paid attention to other traits. This planning is based on the bull dam registration and selection and on the following characteristics of the system of testing, selection and differentiated use of bulls : performance testing and selection on growth rate, testinseminations from young bulls, storage of deepfrozen semen, progeny testing for milk productivity and milkability and selection of proven cow sires and bull sires. Special attention is paid to the economic evaluation of alternative breeding plans. The net returns are considered the best measure for the comparable economic efficiency of the breeding plans. The principles for the calculations of this measure are developed. The factors influencing the genetic gain and the net returns are divided in the following categories : biological factors, factors of breeding policy and market factors. The optimum combination of the factors of breeding policy under Danish conditions are given. It is found that the economic efficiency is almost the same from 20 to 4 o p. 100 elimination on growth rate at the performance testing. The influence of changes in the size of some of the market factors on the optimum breeding structure and on the economic efficiency of the original optimum plan under the supposed changed conditions is studied. It is concluded that the net returns of the original optimum breeding plan under the following changes : --Net income per one kg butterfat from 8 to 6 or I o dKr. The changes in the three first mentioned factors will affect the optimum breeding structure to some extent but the changes in the two last mentioned factors will not affect this structure to any noticeable extent. BETTER BREEDING PLANS R. D. P OLITIEK . -Department of Animal Husbandvy, Section Animal Breeding, Agvicultuval University, Wageningen (Netherlands). The breeding goal is to select the most profitable cow. A better breeding plan is a plan that can be realized. In the Netherlands, analyses of actual selection learned that the change to a better breeding plan took a period of 10 years. Modal calculations for different breeding plans revealed that it was profitable to use selected proven bulls on a very large scale (8 0 0 0o doses or more in a large population), B RASCAMP (Zeitsch. Tierziicht. Zuchtungs Biol., 1972 , in press). From an economic standpoint, the contribution to profit is e. g. SS z 7 p. 100 SD 37 p. 100 DS 2 6 p. 100 DD 10 p. 100 compared with the contribution to genetic progress : SS 45 p. 100 SD 25 p. 100 DS 25 p. 100 DD 5 p. 100 . Calves from selected parents express earlier their profit than calves from selected grandparents. In planning research at the new experimental farm with 200 cows (P. F. L.) it was decided to compare a random sample of calves born in I970 and 1971 in two main breeding districts of Dutch Friesians. A group of 102 heifers calved at an age of 2 years and gave an average milk production of 1 8 0 kg (a = 250 kg) in 100 days, fat-per cent : 4 , 03 (s =: o,a!), protein-per cent : 3 , 21 (a = 0 , 17 ), live-weight, 100 days after calving : 4 8o kg (a = 4 8 kg). Preliminary results gave a difference of nearly io p. ioo in milk yield between sub-populations, but no differences from heifers originating from high and low producing herds. These results can stimulate the introduction of better breeding plans. The next phase of this experiment, namely comparison of subpopulations from Dutch Friesian, British Friesian and Holstein Friesian, can give an extra stimulation for the realization of better breeding plans and may also affect the migration of breeding Nach einer Beschreibung der in Deutschland gebräuchlichen Methode der Zuchtwertschätzung auf Milch wird auf spezielle Probleme eingegangen, die sich aufgrund der Analyse der Population in Bayern ergeben haben. Diese Probleme sind mit dem genetischen Trend und durch Unterschiede in der genetischen Qualität der Vevgleichstieve umschrieben und werden heute in allen fortgeschrittenen Zuchtprogrammen diskutiert. Heritabilities were estimated for the following calf drop characteristics : The sex ratio, difficult calving (DC), stillbirth, perinatal mortality (PM) and a deleterious calving index (DCI), which combined DC and PM. The data contained above 50 ,ooo heifer and ioo 00 o cow calvings between the years 19 6 4 -1970 of the Israeli Friesian dairy breed. Each calving was identified by sire of calf (S) and its maternal grandsire (MG), all estimates were for S and MG, separately for heifers and cows. Three different models were deployed : a) A two-way simultaneous hierarchy for estimating the variance components of S and MG both on six years' data and separately for three consecutive two-year periods. b) A one-way model with sires nested in twelve year/management type blocks. c) Repeatability estimates by sires with more than 100 calvings. The calving by each sire were divided chronologically in subgroups of fifty and intraclass repeatabilities were estimated. This procedure simulates a situation of sire proofs. Empirical heritability estimates (he) were then derived from the repeatabilities (R) : In heifer calvings h.2 for DC, PM and DCI by S were : 4 . 5 p. ioo, 4 . 5 p. ioo and 6. 3 p. ioo respectively ; by MG : 2 .8 p. ioo, 2 . 3 p. ioo and 3 . 4 p. ioo. he in cow calvings for DC, PM and DCI by sire were : o. 9 , 1 . 4 and 1 . 7 and by MG : 0 . 5 , 0 . 2 and 0 . 5 . h 2 estimates by the alternative rrodels were similiar, but tended to be smaller. For 0 . 7 repeatabilities of sire tests for DCI 147 heifer calvings of mates and 275 calvings of daughters are required. In cow calvings 553 mates and i86 4 daughters. rg in DCI between heifer and cow mates was o. 5 z, vg between DCI of heifer mates and growth rate of sons : 0 . 32 and 305 FCM yields of heifer daughters : -0 . 42 . Inbreeding increased PM by appr. i p. ioo. After the first results of sire testing for calf drop characteristics, there was severe selection among sires for heifer inseminations and the incidence of DC in heifer calvings dropped from 8. 2 p. 100 in 19 6 4 -19 66 to 3 . 4 p. 100 in 19 68-1970 . h 2 of DCI dropped in the same periods from 6.o p. 100 to 2 . 5 p. ioo. It was postulated, that the phenomenon of low h 2 and immediate selection response may indicate a situation of only a few loci involved in the determination of calf drop characteristics. A similiar situation appears to exist in the sex ratio, h 2 was 0 . 4 , but a few sire families diverged consistently from the mean. h 2 of twinning by MG was 2 . 4 p. 100 . It was suggested to test AI sires, which were plusproven for yields by 200 heifer matings. The contemporary comparison (CC) is based on the assumption of genetically static populations. Sire selection on CC and the use of AI violate this assumption. A sire's breeding value, as estimated by CC, is based on the phenotypic deviation of his daughters (5 i ) from the average yield of their herdmates (HM k ) multiplied by the heritability, which is particular to the test group size (h!). The required adjustment is I/2 (S( k ) -S), which is the average deviation of the sires of the contemporaries in herd k from the original herd level. The calculation was described by BAR-A NAN (Der Tierziichter, 24, 35 , 1972 ). This procedure generates a matrix of estimates CD ij , which is the cumulative difference of sire i in test period j. The average over the index j gives the current best estimate of the sire's breeding value in comparison to the base population, and the average over the index i can be used to estimate the genetic progress due to the selection program. SIRE EVALUATION The within sire regression coefficients on time were obtained for CC and CD in 122 day daily kg FCM. The average yearly regression within sires of CC was -.i 3 8 kg daily FCM and CD + . 02 6 kg, the latter not significantly different from zero. In preliminary estimates of 305 day kg FCM, CD greatly reduced the negative trend in CC, but did not entirely remove it. The residual variances of CC and CD were similar. The CD estimates appear to be, for practical purposes, free of trends without reducing precision. The computing costs involved are negligible. The lecture discusses the fractionation of a metric character (milk proteins) in its discrete components. Differences in some Italian breeds of cattle are illustrated. Some Southern European breeds of cattle (Mavemma, Calabria and Modica in particular) are closer to zebu cattle, perhaps because some alleles confer better environmental adaptation. Second, it discuses galactopoiesis in a cow's population. Its time course can be represented by a harmonic oscillation having the year as period. The maximum ordinate varies with milk production level. Two deformations are described, one demographic in nature, the other due to micro-thermal variation. Galactopoiesis and food efficiency of food energy utilization is described. It tends to vary harmonically during the course of the year but in opposite fashion of galactopoiesis. The Italian national breeding plan was based on nucleus or inbreeding selection for abou-30 years. Fortunately, this has now been abandoned. However it is increasingly obvious that a lack of misunderstanding of the subject of selection has remained. Animal belonging to the nut cleus period were used to form the herdbooks, and the recording service, established for the purpose of nucleus selection, now works in function of herdbooks. Herdbook is so fully protected in this country that a few years ago it obtained the legal monopoly to produce and sell animals for reproduction in private and cooperative herds by natural service and artificial insemination. Herdbooks have contributed in the past in various countries to livestock production, but the author finds it is time to critically examine their services and costs, which are very high and in Italy are paid almost entirely by the state. From the genetic point of view it must be observed that for any population it is advisable to contain a high amount of genetic variability. Both deliberate inbreeding, used in the past, and the strong restriction in the number of parents tend to reduce such variability. To get over this problem, it seems that decision makers are accepting the suggestion that the recording service should be enlarged. But the author fears that this is planned as a means of increasing the number of herds and animals to be registred in the herdbook. The author is afraid because this is not the right avenue, and will have little or no utility. The avenue opened by his suggestion to enlarge the recording scheme is different, the principal aim is not that of culling low yielding cows, which is virtually wasteful, but the collection of data needed for progeny testing bulls for artificial insemination. There is of course the additional aim of helping all these herds to improve their environmental condition. Genetically speaking, the bull bears half the responsability for the production of low yielders, so better selection of bulls used in dairy herds is more effective and economic means of improving milk production and reducing costs, than the whole operation of registering after inspection and recording cows in herdbooks. Geneticists have no greater competence than any other educated men on question of political action, but it must be clear they do have a unique competence, as well as responsibility where genetics itself is concerned. OPTIMAL ESTIMATION OF BREEDING VALUES E. P. C UNNINGHAM . The success of modern AI dairy bull selection programmes has complicated the task of bull evaluation. The progeny of young bulls must now be tested in the presence of annual genetic change in the population, in competition with progeny of highly selected sires, and in a situation where farmers may be reluctant to use young bulls at all when top proven sires are available. These problems can be tackled by improving the statistical treatment of progeny test data or bv altering the physical structure of the testing method. Modern statistical developments in this area are briefly reviewed. They involve least squares estimation of bull breeding values in one or two stages from large-scale non-orthogonal data. By appropriate choice of constraints in solving the equations, the bulls can be evaluated in logical groups, and the estimates can be related to any chosen base. Most countries base their testing on the normal milk recording programme. In some, it has become necessary to concentrate young bull usage in special herds. This facilitates the use of modern statistical methods and eliminates some biases. A possible intensification of this system is suggested, whereby testing would be concentrated in a special test sub-population. Le Pig-Book sera fermé en 1957 . Les premiers reproducteurs inscrits au Pig-Book n'avaient pas de généalogie connue ; il était donc impossible d'évaluer la consanguinité pratiquée antérieurement. Se bornant à calculer la consanguinité d'individus dont au moins le père et le grandpère maternel sont connus, on constate que, pour les individus inscrits au Pig-Book de 1951 , 1952 , 1953 , 1954 et A partir des pedigrees, limités à cinq générations, de reproducteurs ayant participé aux concours généraux de 195 8, 19 62, 1966, 1970 While typing cattle sera for an allotypic specificity by double diffusion, a precipitation line was observed between two peripheral wells containing normal sera. Rocket-Ab (a bull which proved to be the source of the antibodies) and I o 35 (a calf). When Rocket-A b was tested against a serum sample of Rocket (Rocket-A g ) collected two years previously, a precipitation line developed between the wells containing the two samples of Rocket, thus suggesting the presence in Rocket-A b of antibodies (R Ab ) against an antigen (R Ag ) present in Rocket-A g . The reactive animals (z 3 out of 270 tested) had all the common characteristic of having an age varying from a few days to seven months, thereby suggesting the possibility that the antigen expression required for the animal not only the proper gene but also the proper age. Bloo3 factors A and C are controlled by allelic genes forming the AC system, anologous to the FV system in cattle. The B system is comprised of factors B-D-F-H-I and form several phenogroaps, like factors of the bovine B system. Factors J-O-Q-T-U belong to a third system (J system) and are linked by a subgroup relationship (as the E' factor series in cattle). Because of the lack of suitable family data, factors E-G-M have not been allocated within systems. Evidence was found that the antigenic specificity A in some animals is not located on the surface of erythrocytes withim them. To the authors knowledge, this situation has never been observed before. Three phenotypes determined by two codominant alleles (Alb A and Alb B ) have been detected in the albumin locus. Polymorphism of transferrins is also regulated by two codominant alleles (Tf D and TfE). The allotypic specificity A, identified by double immuno-diffusion (Ouchterlony technique) is common to both cattle and water buffalo. Specificities B and C have been identified by double immuno-diffusion, immuno-electrophoresis and passive hemagglutination. On double immunodiffusion slides, specificity D appears as a spur. The genetic mechanism controlling these foar specificities so far has not been studied. In many countries the system of improvement of livestock is based on the use of pedigree stock in which the breeding standards are much higher. If the differences in feeding and hasbandry conditions between improved and improving animals are very high it may be assumed that the discussed system of improvement of the animals will not produce the expected results. This trial is intended to reconstitute the system of improvement of livestock in some countries. Selection within litters of three mouse populations was carried out for live weight gains of animals between the third and sixth week of life. All the selected populations were of the same size, they comprised 10 parent pairs in four replications (together 40 dd + 40 °o) and the offspring Every experimental population had a corresponding unselected control population. One of the populations of mice was maintained on a feed with a 20 p. Too content of crude protein, the other on a feed with a 10 p. 100 content of crude protein, while in the third population females and their offspring were fed with the low-protein ( 10 p. 100 ) diet and mated with males from the first population maintained on the high-protein ( 20 p. 100 ) diet. This part of the trial has six selected generations. The second part of the trial was performed with mice obtained from a different experiment. Selection of the mice was carried out on the same principles as in the first part of this trial, only instead of weight gains, the weight of 5 week-old mice was taken into account. Animals were maintained on a diet identical to that given in the first part of the trial. After selection pursued over 13 and 14 generations (on the high-protein diet one extra generation was obtained) the females fed a low-protein 10 p. 100 diet were mated to males maintained on the high-protein ( 20 p. 100 ) feed, the offspring was then compared with the progeny of selected parents maintained permanently on the low-protein ( 10 p. 100 ) feed. Present findings clearly indicate the mating of females selected on low-protein ( 10 p. 100 of protein) food with males selected on high-protein ( 20 p. 100 of protein) food not to be successful. Reasons for and possibilities to many-sided progeny testing of AI bulls have been considered on the basis of Finnish experiences. The present routine testing in Finland includes milk and fat yield and estimated live weight of daughters. Large scale efforts have recently been made to develop progeny testing methods for the following traits : protein content of milk (one sample per daughter), carcass weight of young slaughter cattle (slaughterhouse data), milkkability, calmness, apetite and estrus symptoms (interview of herd managers), fertility (milkrecording and non-return statistics), frequency of stillbirth and frequency of diseases. In addition studies on the persistency of lactation and the frequency of paresislike leg faults have been considered. Preliminary estimates on correlations between progeny tests for different traits, as well as of correlations between results of performance tests and progeny tests were also given. Some interesting or surprising correlations were found in both cases. For example, the average calmness of daughters showed a significant positive correlation (ca 0 . 3 ) with the bulls own growth performance at the station, i. e. the correlation is as high as the average age-corrected carcass weight of offspring. The correlation between milkability and calmness tests was 0 . 4 . Fat content o daughters gave a negative (-o.i) and milk yield a positive (o.i) correlation with bull's own growth performance. Oxidation flavour is an off-flavour in milk which has been known as a problem in the dairy industry for many years. Ordinary market milk may show such oxidation faults after only 24 hours that itor products made from itcan hardly be accepted by most consumers. The problem is of increasing concern since structural rationalization has brought about bulk milk handring and a market chain longer both in time and distance. The milk is older when consumed and, therefore, the off-flavour has time to develop. (Furthermore, milk production is becoming more and more intense and this by itself seems to result in a greater sensibility to oxidation faults.) It is generally agreed that oxidized flavour of milk and dairy products is due to autooxidation of unsaturated fatty acids in the fat or phospholipid phases with the formation of mainly aldehydes and ketones, smelling and tasting badly. The disposition of milk for oxidized flavour is higher in winter than in summer milk, but several observations indicate that within season the disposition varies between different breeds, different herds, different cows in the same herd, or within the lactation period of the same cow. Until recently metal-induced oxidized flavour was the most important, as contamination of the milk with copper from milking machines, coolers, etc. was common. However, most of these contamination causes have now been eliminated and the importance of the natural content amount of copper as a possible cause of the accurrence of oxidized flavour has attracted the interest. As a measure of the oxidized flavour in milk the thiobarbituric acid (TBA) test has been introduced as an objective measure of the oxidative deterioration of the milk fat. It is based on the formation of a red pigment which is determined spectrophotometrically at 530 m> and which is the reaction product of malonic dialdehyde with the test reagents. The TBA test correlates well with the organoleptic quality of the milk, with 0 . 022 being the critical limit above which most people will recognize the milk as having oxidized flavour. In 19 68-6 9 an investigation on oxidized flavour was carried out on milk from cows at the Danish progeny test stations in order to study the genetic background of this character (TBA value), and its inter-relation to the copper content of the milk and the chemical make-up of the milk fat (refraction index). 10 8 2 cows were included, there of 25 progeny groups with 455 cows of Red Danish Cattle (RDM), 15 grogeny groups with 27 6 cows of Black and White Danish Cattle (SDM), 19 progeny with 351 cows of Danish-Jersey (J). In the course of the lactation 4 determinations of TBA, 2 determinations of refraction index and 2 determinations of copper content were carried out on individual milk samples. The cows were on average 50 days from calving at the first determination and about 135 days at the last. The refraction index was nearly constant through the period studied, whereas the TBA values and the copper content were decreasing with time from calving. SDM cows had milk with the lowest and RDM cows had milk with the highest TBA values. Jerseys were intermediate. The differences were significant, except between RDM and J. Significant differences were found between all breeds as to refraction index and copper content, Jevsey milk being especially characterized by low refraction index and high copper content. Based on calculations including all three breeds the coefficients of heritability for TBA values and copper content of the milk were found to be o.q ! o.i, while the coefficient of heritability for refraction index was estimated as low as 0 . 1 ! 0 . 1 . The phenotypic correlation between TBA values and copper content was found to be 0 . 5 . All other phenotypic correlations were not significant. Three significant genetic correlations were found. The genetic correlation between refraction index and daily butterfat yield was calculated to 0 . 57 , while it was o.66 between TBA-values and copper content, and -0 . 4 6 between refraction index and copper content. No significant correlations were found between TBAvalues and daily butterfat yield. The investigations indicate that it is possible through breeding measures to improve the quality of the milk as concerns oxidized flavour. They confirm a relation between natural copper content (ceruloplasmin ?) of the milk and the disposition to oxidization of the milk fat, but apparently it is not a simple cause-effect relationship. Blood samples studied on starch gel electrophoresis for Hb, Am, Alb and Tf variants, show two electrophoretic components for Hb (Hb 1 i and Hb,). but no polymorphism. Also for Am there is no genetic variation. Alb and Tf are on the contrary determined by two codominant autosomic alleles respectively. Each allele controls one band on starch gel, while Tf shows three large bands preceded by a weaker one. Frequencies of Alb A , Alb B , Tf B and Tf F were in our samples 0.329 7, 0.670 3, 0.2 3 o 8 and 0 . 7 6 9 2 respectively. Data are discussed in comparison with those obtained by other workers on the genetic foundation that the buffalo raised in Italy, origins from India. Pre-and post-albumins of serum in Buffaloes were studied with the technique of starch-gel electrophoresis of 66 0 buffaloes, reared in Campania and Latium, and belonging to seven flocks, was performed. The genetic equilibrium test for the pre-and post-albumin systems showed a deviation from the binomial distribution. It has been observed that sera from animals possessing the Tf E allele show a more intense yellow-brownish colour than those from animals not possessing this allele. Because differences of serum colour are mostly determined by different concentrations of the pigment bilirubin, although carotene and other pigments are contributing factors, the aim of this research was to study the relation between serum bilirubin content and transferrin types. For this purpose a random sample of 3 z8 sera from animals of the Piedmont, Brown alpine and Rendena breeds has been used. The results obtained from this study indicate that significant differences in serum bilirubin content exist between breeds. By grouping the figures according to phenotypes within breeds, significant differences have been found in the Piedmont and Rendena. In this respect it should furthermore be noted that, on average, transferrin types AE and DE from Piedmont and Brown alpine animals show consistently higher figures for bilirubin content. Although the sample examined in this research did not include phenotype Tf EE , which has a very low frequency in the breeds we have considered, the results could indicate the existence of a relation between transferrin types and serum bilirubin content. This phenomenon, which will be further investigated on a larger sample, is in agreement with the results obtained by N EETHLING et al. (Pyoc. Xlth Conf. E. S. A. B. R., Warsaw, 1968, i 7 i) who found that red cells from animals possessing the Tf E allele have a shorter half-life than those from animals of the 'I'f DD phenotype. The implication of these phenomena on the animal basal metabolic rate is discussed. Using electrophoresis in basic and acid buffers we analyzed 1 050 samples of Piedmont and 700 samples of Valdostana cattle. In Piedmont the gene frequencies were : locus ocs,, D = .oog, B = .8 17 and C = .i 7 8 ; locus fi, A, _ . 2 8 7 , A 1 = . 554 , B = . 123 , E = . 001 and C = . 035 : locus x, A = .6 15 and B = . 3 8 5 . This breed is characterized for the presence of fiE, that in acid buffer present the same mobility of !A1. In Valdostana breed the frequencies are : locus <xs,, B = .8 94 , C i o6 ; locus (3, A, = . 313 , A 1 = .753 and B = .035 ; locus x, A = .5 44 and B = . 4 66. The loci controlling polymorphism of as&dquo; and x casein are o structural a. The linkage disequilibrium, observed in the gametic association, was measured by 0 (the sign of this parameter indicate the direction of association). For the Piedmont we found there values : The first two appear in coupling while the others are in repulsion. In Progeny testing results in the field of AI-bulls for daily weight gain were analyzed both by contemporary comparison and least-squares procedures. i 5 -2o sons of each AI-sire are distributed at 2 -3 weeks of age to several private farms, where they are fattened to constant finish at about S oo kg. In 19 6 5 -19 66, io8 sons of 9 bulls were distributed to 12 farms according to a balanced incomplete block design (lattice-design). Contemporary comparison and least-squares analysis with sire and herd as main effects led to the same ranking of bulls. For practical reasons the balanced distribution of the progeny among herds was discontinued and, in 19 6 9 -1970 , 66 2 sons of 45 bulls were randomly distributed among 2 6 farms. In some of the farms, the calves were castrated. The following least-squares model was applied. In a second analysis, in addition to these effects, regressions on the circumference of chest of the mothers and on age weight at first weighing were fitted. The rank correlation between the bulls rated by the different methods amounted to : The regressions on maternal circumference of chest and on age at first weighing were not significant. The regression on weight at first weighing was significant and responsible for a substantial amount of variation in daily weight gain. Least squares procedures were prefered to contemporary comparison because the estimation of constants for bulls and for other effects was accomplished at the same time.

v3-fos

2020-12-10T09:04:12.757Z

1973-05-01T00:00:00.000Z

237230426

s2

Isolation of Salmonellae from Pork Carcasses Four hundred and twenty pork carcasses from four abattoirs were examined for the presence of salmonellae by use of swabbing-enrichment techniques and contact plate methods. Carcasses from only one abattoir were found to be contaminated by swabbing-enrichment (23.3%) and contact plate (17.9%) methods. The area of the skin side of the ham, near the anal opening, was determined to be the area to examine for isolating salmonellae from pork carcasses with the greatest frequency. The most frequently isolated species of salmonellae in this study were Salmonella derby, S. anatum, S. typhimurium, and S. indiana. The isolation of salmonellae from meat animals intended for food has been of interest since the first report on swine plague (2). Several authors have reported on the occurrence of salmonellae in meat products (4, 7-9, 11, 15, 26) and the use of various techniques to recover microorganisms, including salmonellae, from the carcasses of meat animals (11, 16, 18-20, 21, 26). Methods for assessing the bacterial content on the surface of raw foods are varied. Swabbir is one of the earliest and continues to be one u. the most widely used methods because of its versatility. Limitations of swabbing techniques were evaluated by Douglas (5) and Green and Herman (12). Variations of the swabbing techniques are discussed by Green et al. (13) and Walter (25). Dyett (6) proposed using a sharp, sterile knife to scrape the whole, exposed carcass surface and making an estimate of the number of microorganisms in the scrapings by a direct microscopy count or by . total viable count. Direct-surface agar plating was described by Angelotti and Foter (1), and application for sampling meat surfaces was described by L. ten Cate (3). Hall and Harnett (14) described a disposable plastic dish so constructed as to permit direct-contact plating of surfaces. The purpose of this study was to determine the incidence of salmonellae on pork carcasses in local abattoirs, to find the location on the carcasses most likely to be contaminated with salmonellae, and to report the species of salmonellae found. MATERIALS AND METHODS Four hundred and twenty pork carcasses from four different abattoirs were examined, after chilling, for the presence of salmonellae by swabbing an area approximately 25 cm2 on the skin surface of the ham near the anus and by placing a contact plate on the analogous area on the other half of the same carcass. Swabbing was accomplished by wetting a cotton swab in sterile, physiological saline, swabbing the designated area thoroughly, and then replacing the swab in the tube containing 10 ml of 0.85 M sterile saline for transportation to the laboratory. Samples (1 ml) from the tubes containing swab" were transferred into tetrathionate brilliant green (TET) broth (10) and incubated at 37 C for 24 h. After incubation, samples of the TET broth were streaked upon brilliant green agar (BGA, Difco) plates which were then incubated for 24 to 36 h at 37 C. Ctlonies showing typical growth of salmonellae on BGA plates (11) were inoculated into triple sugar-iron agar (Difco) and incubated at 37 C for 24 h, and those tubes showing positive reactions (10) were sent to the Southeaste.rn Salmonella Serotyping Laboratory, Atlanta, Georgia, for positive species identification. Contact plates, as described by Hall and Harnett (14), filled with BGA and bismuth sulfite agar (BSA; Difco) were applied to the surface of the carcasses in such a manner as to insure full and complete contact between the medium and the surface being examined. These plates were then transported to the laboratory and incubated at 37 C for 24 to 36 h, and suspected colonies from plates showing positive growth of salmonellae were treated as described above for the swabs in saline tubes. In a subsequent experiment, 91 pork carcasses from abattoir A were examined at three locations: (i) on the skin surface of the ham near the anus; (ii) on the skin of the jowl; and (iii) on the flesh si-le of the jowl, for a total of 273 samples. In this experiment, adjacent areas on the same carcass were selected for examination by swabbing and contact plates, thus eliminating variability between halves of carcasses. The isolates obtained were analyzed for salmonellae as described above. RESULTS AND DISCUSSION In a survey of pork carcasses from four different abattoirs, salmonellae were recovered from only one establishment (Table 1). None of the abattoirs surveyed obtained hogs from the same farms. Although they did receive hogs from the same general areas or locales on a regular basis, none of these areas overlapped. Galton et al. (9) concluded that there may be true regional variations and attributed these to (i) differences in the incidence of salmonellae infection in hogs, (ii) variation in the nature or control of processing procedures employed, or (iii) climatic factors affecting the viability or multiplication of salmonellae in the environment of abattoirs. Cross-infection of healthy pigs by infected pigs from individual farms may occur in the holding pens (11,17), leading to a continuing incidence at a particular abattoir. The most probable source of salmonellae is the animal from which the meats were obtained (4). Carcasses from abattoir A were examined on nine different occasions, or days, throughout a 12-month period, and salmonellae were found on all occasions. The greatest incidence of salmonellae was recovered from carcasses examined on sampling day 4. (Table 1). There was no apparent reason for the large number recovered at this particular sampling time. Total numbers of bacteria on all carcasses examined from abattoir A were in the order of 103 to 104 organisms per 25 cm2 of surface. No differences in total numbers were noted from those carcasses from which salmonellae were isolated. No salmonellae were recovered from abattoirs B, C, and D where 100, 65, and 34 carcasses, respectively, were examined. This indicates that the isolation of salmonellae from pork carcasses is not a simple, routine matter. Although no actual data were collected, it was noted that abattoirs B, C, and D were receiving hogs from geographic areas different from those of abattoir A. To determine their relative effectiveness in recovering salmonellae from pork carcass, the techniques of swabbing and the use of contact plates filled with BSA and BGA were compared. Results (Table 1) show that contact plates recovered salmonellae from 40 (17.9%) carcasses examined compared with 52 (23.3%) by swabbing and enrichment techniques. On only 3 sampling days were more salmonellae recovered by contact plates than by swabbing-enrichment techniques. On 5 other sampling days, more salmonellae were recovered by swabbingenrichment than by contact plates, and on three occasions salmonellae were not recovered at all by contact plates. Salmonellae were recovered by contact plates and swabbing from the same carcass in 12 instances. One important disadvantage of contact plates is that they are not representative of the entire carcass and only reflect the area that they touch. Either method of recovery may be satisfactory when the problem is of a gross nature. Recovery of salmonellae by contact plates seemed to depend upon the medium for recovery. Of 40 contact plates from which salmonellae were isolated, 26 of the isolations came from plates containing BSA and 14 came from plates containing BGA. Many of the plates containing BGA were overgrown. The increased efficiency of BSA or BGA for recovering salmonellae could be due to the greater inhibition of microflora by BSA. Taylor (23) found that, when the ratio of coliforms to salmonellae approached 50: 1, the appearance of a typical, well-isolated salmonella colony with its identifying characteristics for a given medium becomes the exception. In these overcrowded areas, the salmonellae are rarely able to disclose their distinguishing characteristics. However, Banwart and Ayres (2), by using pure cultures of salmonellae, found that BGA supported more luxuriant growth of the six species tested, whereas BSA was inhibitory to four. If a processor of pork carcasses desired to monitor the incidence of salmonellae on carcasses by the contact plate method, BSA would appear to be the medium of choice. BSA may require 24 to 48 h to develop characteristic growth of salmonellae. The incidence of salmonellae from pork carcasses processed by abattoir A, 17.9% by contact plates and 23.3% by swabbing-enrichment technique, is much lower than the figure of 56% reported by Weissman and Carpenter (26) for ISOLATION OF SALMONELLAE this same establishment. This decrease probably is due to extensive changes in slaughtering and processing techniques instituted in this particular plant in the interim between the periods of time when the two sets of data were collected. Recovery of salmonellae from three sampling locations on pork carcasses ( Table 2) was greater from the area examined on the skin side of the hams, near the anal opening, than from the skin or flesh side of the jowl for both methods of recovery, contact plate and swabbing-enrichment technique. No salmonellae were recovered by contact plates on either the skin or flesh side of the jowl, probably because of the difficulty of obtaining a satisfactory area for application of the plate in the case of the flesh or inside of the jowl. Salmonellae were recovered by swabbing-enrichment technique on both sides of the jowl, but with higher incidence of recovery from the skin side or outside. It might be expected that the jowl or neck area would be subject to greater contamination because of washings from the entire carcass fouling that area during processing. Koelensmid and van Rhee (16) found that water that drips from the skin of pork carcasses after singeing is not sterile. They isolated salmonellae from five samples of scrapings taken from 50 carcasses after inspection by veterinarians. In a study on beef, Mulcock (18) found the greatest number of bacteria on neck tissues (106) compared with sides (103) after incubation at 22 C for 5 days. Patterson (20) reported that, in sheep and cattle, contamination acquired during the butchering process is not spread evenly over the carcass. He found greater numbers on the brisket than on the foreleg or rump; yet he concluded that sites of heaviest contamination will vary from one abattoir to the next depending upon methods employed, washing, and other treatments used. Galton et al. (9) examined cultures from anal swabs from living and slaughtered hogs and swabs from sides of (26) concluded that no single area of a pork carcass was likely to be more contaminated than another, but suggested examining the area of the ham near the anal opening as the area of choice because of the possibility of fecal contamination. Results of this study ( Table 2) also indicate that this would be the most useful area to examine when monitoring pork carcasses for salmonellae. Table 3 lists the species of salmonellae isolated from pork carcasses in this study. The three most frequently isolated species, S. derby, S. anatum, and S. typhimurium, are common among isolates of salmonellae found in red meats. The presence of these organisms might be expected because the Center for Disease Control regularly lists these organisms among the 10 most commonly reported from nonhuman sources. However, isolation of S. indiana from red meats has been reported only once since 1967. Six isolations of S. indiana from swine have been reported during the same period. S. indiana is more commonly isolated from poultry and egg products (24). The serotypes isolated were not uniformly spread over the 9 sampling days. S. typhimurium and S. anatum were more frequently isolated on days 1 to 5. All of the S. indiana serotypes were isolated on day 4, whereas S. derby was isolated on days 5 to 9. Other serotypes listed were isolated on various days throughout the sampling period.

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2018-04-03T06:08:02.961Z

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Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species. which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species. Propionic acid is a major end product of the fermentation of plant polysaccharides by the rumen microbial population. Pure cultures of certain predominant rumen bacteria produce propionate directly from carbohydrates other than cellulose. For example, Selenomonas ruminantium and Megasphaera elsdenii both produce propionate from carbohydrates and lactate, the former by the succinate or randomization pathway (13) and the latter by the acrylate pathway (2). Several important species of rumen bacteria produce succinate as a major, pure-culture product of carbohydrate fermentation. Ruminococcus flavefaciens and Bacteroides succinogenes, two of the three major cellulolytic species in the rumen, produce succinate. Succinate, however, does not accumulate in the rumen ecosystem, but it is known to be produced and rapidly decarboxylated to propionate in the rumen. Quantitative studies have shown that succinate is a major precursor of propionate in the rumen (3). A likely explanation for the conversion of succinate to propionate is that a species like S. ruminantium decarboxylates succinate produced by other rumen organisms. Resting cell decarboxylation of succinate to propionate and CO, by bacteria that use the succinate pathway for production of propionate from carbohydrates or lactate is well established (10,11). S. ruminantium presumably would obtain energy for growth in the rumen by the conversion of carbohydrates to propionate, and the grown cells would then carry out what could be considered a resting cell decarboxylation of succinate, produced by other organisms, to propionate and CO2. The purpose of this study was to obtain experimental evidence for the decarboxylation of succinate produced by rumen cellulolytic bacteria when they are grown together with S. SCHEIFINGER AND WOLIN ruminantium. Initial studies were carried out by co-culturing B. succinogenes and S. ruminantium in media containing glucose or cellobiose, sugars that both species use as an energy source. It was subsequently found that S. ruminantium, a non-cellulolytic species, can grow together with the cellulolytic B. succinogenes on a medium containing cellulose as an energy source. B. succinogenes provides S. ruminantium with an energy source from cellulose, and the latter organism decarboxylates succinate produced by the former organism. The net result is a two-species cofermentation of cellulose to propionate, acetate, and CO. The results of these fermentation interaction studies provide evidence for the explanation of the mode of conversion of succinate to propionate in the rumen discussed above. MATERIALS AND MEFHODS Organisms and cell growth. B. succinogenes S-85 and S. ruminantium HD4 were used. Independent and combined cultures were usually grown at 37 C in 10 ml of medium in 18 by 150 mm rubber-stoppered test tubes. The atmosphere was CO, freed of trace amounts of 0, by passing over heated copper filings. A complex medium (4, 6) was slightly modified and used for routine transfers of both organisms. It was made by first adding the following ingredients and distilled H,0 to a final volume of 93 ml: Trypticase, 0.5 g; yeast extract, 0.1 g; dithiothreitol, 0.054 g; glucose, 0.2 g; cellobiose, 0.2 g; starch, 0.2 g; clarified rumen fluid, 20 ml; 4 ml each of minerals no. 1 (0.6% K,HPO,) and no. 2 (0.6% KH,PO0, 0.6% (NHJ),SO4, 1.2% NaCl, 0.24% MgSO4.7H,0, 0.16% CaCl, *2H20); and 0.1 ml of 0.1% resazurin. After adjusting the pH to 6.5 and autoclaving at 15 lb/in' for 15 min under CO, in a sealed flask, 5 ml of sterile 8% Na,CO, and 2 ml of sterile 2.5% cysteine-hydrochloride were added. The medium was then tubed under O,-free CO, for use. The procedures were essentially those previously described (4)(5)(6). In most experiments, the same medium was used except for the use of glucose, cellobiose, or cellulose as energy sources as indicated. A defined medium was used for some experiments, which was the same as the complex medium, except for the omission of rumen fluid, Trypticase, and yeast extract and the addition of vitamins, isobutyric, isovaleric, 2-methyl-butyric, and n-valeric acids as previously described (16). All cultures were incubated on a reciprocal shaker at 120 strokes per min. Direct counts. A Petroff-Hauser chamber was used. It was possible to enumerate both species in combined cultures because of their distinctly different morphologies. Manometric experiments. Cells for manometric analysis were grown for 24 h at 37 C in 100 ml of the complex medium, with 0.2% each of cellobiose and glucose, under an atmosphere of CO,. The cells were harvested by centrifugation (12,000 x g) for 5 min under CO, by using screw cap tubes and were suspended in approximately 5 ml of an anaerobic mineral salt dilution buffer. The dilution buffer previously described (16) was used, but was modified to delete the glucose and sodium sulfide and to contain 0.01 M dithiothreitol. Double-sidearm Warburg vessels (15 ml total volume) were used. The reaction mixtures contained 50 mM potassium phosphate buffer, at pH 6.5, approximately 1010 cells per flask, 2 jug of biotin per ml, and 10 mM sodium succinate in a final volume of 2.9 ml. The succinate, in 0.3 ml, was tipped in from one sidearm to start the reaction. After 40 min, the reaction was stopped by the addition of 0.1 ml of 6 N H,SO4 from the second sidearm, and the flasks were shaken for an additional 10 min to release and measure dissolved CO,. After centrifugation, the supernatant solutions were analyzed for acids (see below). Incubations were at 37 C in an atmosphere of argon. Fermentation analyses. Cellobiose was determined by the ferricyanide reduction method of Park and Johnson (12). Glucose was determined with glucose oxidase as described in Bulletin 510 of the Sigma Chemical Co. Culture supematant solutions were clarified by the Somogyi procedure (15) prior to analysis for glucose or cellobiose. The cellulose used was ball milled Whatman no. 1 filter paper in a 2% (wt/vol) aqueous slurry as described by Hungate (8). The concentration of the slurry was determined gravimetrically, and complete cellulose disappearance from cultures was estimated by microscopy observation of the disappearance of the cellulose particles. For fermentation acid analysis, 2 ml of culture supernant solution was acidified with 0.1 ml of 6 N H,SO, and centrifuged for 15 min at 15,000 x g to remove any precipitate. The silicic acid column and methods of Ramsey (14) were modified for batch collection (9). The solvents and elution order were (in milliliters) benzene, 56; CHCl,, 100; 1% tert-butanol (t-B) in CHCl, (C), 100; 2% t-BC, 200; 5% t-BC, 250; and 8% t-BC, 180. All solvents were equilibrated with HSO and used at room temperature. Samples were collected in graduated cylinders with 10 ml between batches to check for any trailing. The collection schedule was designed to obtain butyrate in the first 95 ml, propionate in the next 55 ml, acetate in the next 70 ml, formate in the following 240 ml, lactate in the next 165 ml, and succinate in the final 180 ml. The acids were titrated to a phenolphthalein end point by using 0.01 N ethanolic KOH. on March 23, 2020 by guest http://aem.asm.org/ Downloaded from culture of S. ruminantium was then centrifuged aseptically in a CO2 atmosphere, the cells were resuspended in the 48-h B. succinogenes culture, and the tubes were incubated for an additional 24 h. Succinate, but no propionate, was present in the B. succinogenes culture, and propionate, but no succinate, was present after incubation of the culture with the added S. ruminantium cells (Table 2). This experiment also showed that S. ruminantium decarboxylated succinate to propionate and that changes in the medium caused by growth of B. succinogenes did not prevent the decarboxylation. Identical results were obtained when glucose was the energy source for B. succinogenes. Concurrent fermentation of cellobiose or glucose by B. succinogenes and S. ruminantium. The question of whether both organisms could grow together and carry out a combined fermentation of carbohydrate to propionic acid was examined. When cellobiose or glucose are used, the two species are competing for energy source. If competition is significantly skewed in the direction of B. succinogenes, no significant growth of S. ruminantium will take place in the combined cultures, and the fermentation would essentially be the same as the independent B. succinogenes fermentation. If competition for substrate is strongly in favor of S. ruminantium, the fermentation would be the same as the independent S. ruminantium fermentation and the presumptive competitive cofermentation by the two species. Because of the inability to distinguish between an independent S. ruminantium fermentation and a truly competive cofermentation simply on the basis of product formation, the contribution of the individual species to the cofermentation process was estimated. This was done by determining cell numbers of each species in the combined culture and calculating the expected amounts of products produced by each species from their respective per cell activities in independent, single-species fermentation. Table 3 shows the results of independent and combined fermentations of cellobiose, and Table 4 shows the results obtained when glucose was the energy source. It can be seen that succinate, but no propionate, was produced by B. succinogenes alone and that propionate, but no succinate, was produced by S. ruminantium alone. In the combined cultures, propionate but no succinate was found, although significant amounts of succinate would have been expected on the basis of the independent activity of the concentration of B. succinogenes found in the combined cultures. The results strongly suggest that the species use cellobiose or glucose at similar rates when they are co-cultured under the conditions of these experiments. This results in a combined fermentation of cellobiose or glucose to propionate, acetate, and CO, without succinate accumulation. Decarboxylation The amount of propionate formed in the combined cultures was significantly greater than the amount expected on the basis of the amount of S. ruminantium present and was also greater than the amount expected on the basis of the estimated amount of succinate produced by B. succinogenes in the combined cultures. A possible reason for the larger than calculated amount of propionate obtained in the combined cultures has not been definitely established, but the discrepancy may be due to differences in product formation by B. succinogenes in single and combined cultures. Relatively good carbon recoveries were obtained in fermentation balance studies with the single S. ruminantium and the combined B. succinogenes-S. ruminantium fermentations, but not with B. succinogenes alone. In the single S. ruminantium fermentation, the only products were propionate, acetate, CO2 (calculated as equal to acetate), and small amounts of lactate. The combined fermentation yielded only propionate, acetate, small amounts of formate, and succinogenes alone produced succinate, acetate, and small amounts of formate, but significant amounts of carbon disappeared that could not be accounted for by the products or calculated CO2. Table 5 shows a comparison of fermentation balances for glucose. Similar results were obtained when cellobiose was used. These results suggest that either an unidentified product is produced by B. succinogenes alone which can be converted to propionate by S. ruminantium or that co-culturing of S. ruminantium and B. succinogenes prevents the formation of the unidentified compound by B. succinogenes. Fermentation of cellulose. B. succinogenes used cellulose as an energy source and fermented cellulose in the complex medium to succinate, acetate, formate, and CO, (Table 6). S. ruminantium grew only slightly in the same medium without degrading cellulose, but good growth of S. ruminantium was-obtained when it was co-cultured with B. succinogenes on the cellulose medium. No succinate was produced in the combined fermentation, and cellulose was fermented to propionate, acetate, and CO2 ( Table 6). As shown in Table 6, similar results were obtained when a defined medium was used, except that the base growth of S. ruminantium alone was eliminated. The carbon recovered in the synthetic medium (assuming CO2 equal to acetate minus formate) represented 94 and 110% of the original cellulose carbon for B. succinogenes alone and the mixture of B. succinogenes and S. ruminantium, respectively. There is probably some inaccuracy in the original cellulose concentration because SCHEIFINGER AND WOLIN APPL. MICROBIOL. 792 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from the cellulose was pipetted from a suspended slurry and the actual concentrations in the fermentation media were not measured. It appears, however, that most of the carbon of the cellulose was recovered in the indicated products. When grown alone, the amount of B. succinogenes cells per milliliter of synthetic medium was 6.5 x 108, and the respective concentrations of cells in the mixed culture were 4.4 x 108 for B. succinogenes and 1.0 x 108 for S. ruminantium. The combined cultures were serially transferred in the synthetic medium at 72-h intervals by using 0.5% inocula, and the combined culture fermentation of cellulose to propionate, acetate, and CO2 was maintained through at least seven serial transfers. DISCUSSION These experiments show that it is highly likely that the conversion of succinate to propio- nate in the rumen is carried out by bacteria that form propionate via the succinate pathway. S. ruminantium is probably a major factor in the conversion although, under certain circumstances, other species such as Veillonella alcalescens in the sheep rumen (10) may play a similar role. Dehority reported that high concentrations of rumen fluid caused the succinateproducing B. ruminicola to produce small amounts of propionate (7), but it was subsequently shown that the propionate is formed by the acrylate pathway (17). It is, therefore, highly unlikely that B. ruminicola is capable of decarboxylating succinate. The rate of succinate decarboxylation by resting cells of S. ruminantium was about 13.0 #mol per h per 1010 cells. The rate of conversion of succinate to propionate by bovine rumen contents was measured by Blackburn and Hungate (3) and was found to be approximately 1.6 pmol per h per g of rumen contents. By using the resting cell rate determined in these experiments it would have taken approximately 1.2 x 109 selenomonads per ml to account for the turnover number reported by Blackburn and Hungate. It is not possible to directly extrapolate from the cell suspension decarboxylating activity to the activity of the selenomonads in the ecosystem because of the differences in the conditions for succinate decarboxylation. The rate of succinate decarboxylation by cell suspensions leaves the question of whether bovine rumen selenomonads can account for all of the ecosystem conversion of succinate to propionate an open one. We estimate the cell suspension decarboxylating activity (on a dry-weight basis) of S. ruminantium HD4 to be about 87 times greater than that reported for propionibacteria (11), but only one-third of that reported for Veillonella (10). We suggest that the model presented in Fig. 1 is a fairly accurate representation of the microbial interactions that result in propionate tion pathway when starch or soluble carbohydrates are fermented in the ecosystem in addition to the interactions depicted in Fig. 1. S. ruminantium, depending on the strain, can ferment starch, lactate, and a variety of soluble carbohydrates to propionic acid directly. Nonstarch fermenting strains could feed off starch breakdown products, either sugars or lactate produced by starch-fermenting organisms. Microbial interactions that lead to propionate formation from starch and soluble sugars are probably more complex than those interactions involved in propionate formation from cellulose. The spin-off of carbohydrate from cellulose by major cellulolytic rumen bacteria to non-cellulolytic major rumen species has been logically assumed to be a significant means of providing energy to the latter species. To our knowledge, however, the present experiments represent the first direct demonstration of this type of interaction. The interaction between B. succinogenes and the HD4 strain on cellulose was duplicated with other selenomonas strains, both lactate and nonlactate-fermenting strains, and the results were essentially the same as with the lactate-fermenting HD4 strain. R. flavefaciens has also been substituted for B. succinogenes in the cellulose system with the HD4 strain with essentially similar results.

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Production of staphylococcal enterotoxins A, B, and C in colloidal dispersions. Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken. Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 ,g of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken. Most foods in which Staphylococcus aureus have multiplied and produced enterotoxins and which, when consumed, cause food poisoning are complex colloidal systems. The physical state, independent of the nutrient contribution, may affect the production of enterotoxin. The studies which have been reported previously have been focused on the production of high levels of enterotoxins. Casman and Bennett (2) observed greater enterotoxin A production in semisolid brain heart infusion plates than in liquid; however, gimkovicova and Gilbert (13) did not find large amounts of enterotoxin with this method, although, for one of two strains for which data are reported, there was a slight increase over that produced in broth alone. Neither of these two studies offers a direct comparison of effect of colloidal state itself. Membrane culture methods have been recommended (1,2,7,10) and may exert a surface effect as well as permit dialysis. In foods, the physical state of the substrate may exert an effect upon bacterial growth or enterotoxin production, or both, by changes in aeration, adsorption of metabolic products or nutrients, buffer action, alteration in rate of diffusion, alteration in available water, or changes in the bacterial dispersion and possibly ITechnical paper no. 3530, Oregon Agricultural Experiment Station. related differences in the growth pattern. However, the complex environment is difficult to study. Therefore, in the present study, numbers of colony-forming units and enterotoxin levels were compared in shaken and still cultures which had been varied in physical state by the addition of single colloidal ingredients to basic broth. MATERIALS AND METHODS Strains. The S. aureus S-6 culture, a strain that produces enterotoxins A and B, obtained from M. S. Bergdoll (Food Research Institute, Madison, Wis.), was preserved on porcelain beads (9) for use in the major portion of the study. Enterotoxin B-producing strain 243, enterotoxin A-producing strains 265-1 (from R. W. Bennett, Food and Drug Administration, Washington, D.C.) and 13N-2909 (a high producing mutant strain from S. J. Silverman, Fort Detrick, Frederick, Md.), and enterotoxin C-producing strain 361 (from M. Bergdoll) were used in a follow-up study which compared the NAK broth control with liquid and paste states produced with cornstarch and carrageenan. WOODBURN, MORITA, AND VENN placed the 4% NAK. The colloidal ingredients were cornstarch (Corn Products Refining Co., Argo, Ill.), agar (Noble special agar, Difco), carrageenan (Gelcarin, Marine Colloids, Inc., Springfield, N.J.), and low methoxyl pectin (LMP) (unstandardized product with no compounds added, Sunkist Growers, Corona, Calif.). Proportions used are given in Table 1. The final pH of each was 6.8. (Predetermined amounts of 0.1 N NaOH were added at the time of initial preparation or later, depending upon the colloid.) The pH was checked on random lots during the experiments. Consistencies of the dispersions (Table 1) were determined in standardizing procedures and at intervals during the experimental period. For liquid and paste, the Brookfield viscometer was used; for gels, the percentage sag was measured by taking the height of the gel before and 1 min after removal from the container. Water activity (a,) in each medium was calculated from the value for water potential at 27 C measured in duplicate samples by a thermocouple psychrometer (C-51 Sample Chamber, Wescor, Inc., Logan, Utah), standardized with KCl solutions of predetermined osmotic pressure. Preparation and storage conditions were carefully standardized for all replications. Fifty-milliliter amounts of each medium and of the NAK broth control were put into 300-ml triple-baffled shake flasks (Bellco Glass, Inc., Vineland, N.J.) for shaken samples and into plain 250-ml Erlenmeyer flasks for still samples and covered with gauze-cotton closures secured with clips. Dispersions with starch made by adding weighed quantities of cornstarch to basal medium which had been preheated to 80 C were heated to 90 C with constant stirring. Samples of 50 ml were autoclaved for 15 min at 121 C. For suspensions, 15 medium pearls (approximately 1.8 g) of tapioca (Manhattan Adhesives Corp., Brooklyn, N.Y.) were added to each flask before autoclaving. Agar and carrageenan were dispersed by heating in the basal medium, and were then distributed in 50-ml fractions. After being autoclaved for 15 min, the flasks were held at room temperature until pastes and gels were set (2 h for agar, 1 h for carrageenan). Pastes for both still and shaken samples were then shaken for 2 h. The use of LMP differed from that of the other colloids in that it was necessary to make a slurry of LMP in 95% ethanol before addition to the basal medium. Samples were calculated to be 50 g after adjustment of pH and calcium ion concentration. After 1 min of autoclaving at 121 C, the pH of each was adjusted to 6.8 with precalculated volumes of sterile 0.1 N NaOH. Sterile CaCl2 (5 ml) was added to the paste (0.05 M CaCl,) and gel (0.10 M CaCl2) dispersions. For suspensions thickened with agar, carrageenan, and LMP, 15 6-mm lengths of glass tubing (Exax Raschig Rings, Kimble Products, Toledo, Ohio) were added to each flask before autoclaving. Inoculation. Media, which were 24-h-old and had been held at 37 C a minimum of 15 h either shaken or still, were inoculated with 0.5 ml of a dilution of a 24-h NAK broth culture to give 10' organisms per ml. In the NAK-PHP series, two levels of inoculum, 8 x 103 and 6 x 105 colony-forming units (CFU)/ml, were compared. All flasks, except gels, were swirled to mix; the inoculum was spread over the surface of the gel. After inoculation, triplicate flasks of control NAK broth and duplicate flasks of liquid, suspension, and paste dispersions were incubated at 37 C for both still and shaken (gyratory water bath shaker, New Brunswick Scientific Co., New Brunswick, N.J.; 200 rpm) samples. All gel samples were incubated still. Sampling. Sampling was done at 5, 8, and 24 h. For all of the samples except the gels, 6-ml samples were removed at each of the times. A different gel sample was used at each sampling time. The gels were diluted 2:1 with NAK broth and then mixed by manual shaking except for the use of mechanical blending for cornstarch samples. CFU were determined by direct plating of appropriate dilutions on plate count agar (Difco). The sample was then heat treated at 50 C for 10 min to kill the staphylococci and was cooled, the pH was determined, and the sample was centrifuged for 30 min at 1,200 x g to obtain the supernatant fraction for enterotoxin studies. Starch samples were frozen and thawed twice before centrifugation to facilitate separation of a liquid phase. Enzymatic hydrolysis of agar, starch, and LMP dispersions before centrifugation did not increase enterotoxin recovery or detection in inoculated samples. Enterotoxin assay. Three methods of varying sensitivity were used for the determination of enterotoxin B, and two for enterotoxins A and C. Crowle's micro-slide gel double-diffusion technique as modified by Casman et al. (3) was used to estimate enterotoxin levels prior to Oudin assay and for samples containing too little enterotoxin to quantitate by the single gel-diffusion method. Samples giving negative results were concentrated ninefold with Aquacide (Calbiochem, Los Angeles, Calif.) and retested. The detection limit for the micro double-diffusion technique was 0.2 jg/ml. The reversed passive hemagglutination technique (RPH) adapted to a micro scale by Silverman et al. (12) was used to analyze those concentrated samples which were negative for enterotoxin B with the micro double-diffusion technique. Controls used in all cases included nonsensitized cells and uninoculated suspensions. Since the limit of detection of this method was approximately 0.0007 Ag/ml, a negative RPH sample was considered to be negative for enterotoxin. Development of this technique for enterotoxin A was unsuccessful, nor was the method used for enterotoxin C. The Oudin single gel-diffusion method as described by Hall et al. (6) was used for quantitative assay for the three enterotoxins. The sample which had been dialyzed against 4% NAK broth was placed on top of the prepared agar column, and the tube was incubated in a glass-walled water bath at 30 C. Measurements of the migration of the precipitate band were taken at 24, 48, and 72 h by using a cathetometer with a short-focus telescope. The K value (slope) was then compared to that obtained with known concentrations of enterotoxin (diluted in 4% NAK, pH 7.4). The minimal concentration of enterotoxin for which this method could be used reliably was 4 ug/ml. The purified enterotoxins A and B used for these assays were provided by E. J. Schantz (Fort Detrick, Frederick, Md.) and for enterotoxin C, by M. S. Bergdoll. Part of the antisera for enterotoxins A and B and all of that for enterotoxin C were provided through the courtesy of M. S. Bergdoll; much of the antisera for types A and B was produced in the investigators' laboratory. The titers of the antisera from both sources were the same. Statistical analyses. The experiment with strain S-6 was a replicated split-plot design with colloidal ingredients as main plots. One of the four colloids was chosen at random for each day's experimentation in each of two replications. Duplicate samples for each dispersion, and triplicate samples of the NAK broth, were analyzed with respect to CFU, pH, and enterotoxin. Shaken and still samples were analyzed separately at 5, 8, and 24 h. Least significant differences were used to test for differences between each ingredient-dispersion combination. Quantitative measures of enterotoxin were available for 8-and 24-h shaken samples and 24-h still samples. A logarithmic transformation was applied to CFU. Correlations were studied for the 8 and the 24 h data for shaken and the 24-h data for still samples. RESULTS For all of the physical states included, shaking during incubation greatly increased the total amount of enterotoxin at the end of 24 h as has been reported by others (4) and also resulted in earlier detectable enterotoxin. For the samples which were incubated in a thin layer without shaking, the physical state of the inoculated dispersions influenced the yield of enterotoxin. In general, pH and CFU followed trends in enterotoxin levels. Enterotoxin levels are expressed in the text on the basis of the minimal concentrations detectable by the assay methods if below that for which direct quantitation was possible. Data are presented on an unconcentrated basis in Tables 2, 3, and 4. The statistical analysis for the S-6 study is summarized in Table 5. The dispersions varied in viscosity (Table 1) depending upon the concentration of the colloidal ingredient, except for those with LMP in which the viscosity was controlled by the calcium level and carrageenan paste/gel which was controlled by physical treatment after sterilization. The a, was above 0.99 for all; the lower in the range of 0.996 to 0.992 being those made with NAK + PHP. Gels with NAK had an aw of 0.995. Shaken samples, strain S-6: enterotoxin levels. After 5 h of incubation, most shaken samples contained about 0.2 ,g per ml, and all but the carrageenan suspensions had at least (Table 2). Enterotoxin A was present at levels of 0.02 ,g/ml or slightly greater in about half of the samples. At 8 h, enterotoxin B levels did not differ (5% level) for the colloidal ingredients. However, samples containing colloids differed from the controls with differences (1% level) demonstrated among the states of dispersion. Enterotoxin B in the control NAK broth and in the colloidal media averaged 19 Mg/ml with the exception of pastes. Pastes prepared with carrageenan and LMP averaged 3 Mg/ml, whereas those prepared with cornstarch and agar averaged 12 ug/ml. Less efficient aeration may occur but both carrageenan, which gave the thickest paste (Table 1), and LMP, the thinnest, are lower in enterotoxin. A minimum of 0.02 ug/ml of enterotoxin A was present with more than half of the samples having about 0.2 Ag (Table 2). By 24 h the control NAK broth averaged 170 ug of enterotoxin B per ml. Colloidal ingredients and type of dispersion interacted ( Table 5). Concentration of enterotoxin was markedly lower in the paste than in the liquid or suspension except for an opposite trend with starch ( Table 2). Differences in replications were small except for LMP. A minimum of 0.2 ;ig of enterotoxin A per ml was present in all samples except for a few isolated carrageenan samples. Still samples, strain S-6: enterotoxin levels. In still samples, although enterotoxin B was present at 5 h, the amount was generally less than 0.02 ,g/ml (Table 3). After 24 h, the colloidal ingredient and type of dispersion each had a significant effect (1% level) ( Table 5). Enterotoxin B was significantly lower in the control NAK broth and in liquid states of colloidal dispersions, except for those with carrageenan. Gels had the highest concentration, from 47 gg/ml with cornstarch to 14 Ag/ml with LMP as compared to an average of 3 for the control broth (Table 3). Enterotoxin A production was also less rapid with still incubation (Tables 2 and 3). Only a few samples from both broth and cornstarch contained as much as 0.02 Mg/ml at 5 h, but the number was similar to that for samples positive for B; at 8 h, scattered samples contained 0.2 Mg/ml or above. After 24 h, nearly all samples had a minimum of 0.2 ug/ml. The effect of colloidal ingredient appeared to be greater than that of type of dispersion. Staphylococcal multiplication and pH changes. When samples were incubated without shaking as compared to shaken, the rate of increase in CFU was less (Tables 2 and 3). The difference was less after 24 h. Differences in pH were not appreciable at 5 h, about 1 pH unit lower at 8 h, and 2 pH units at 24 h, including the gels (Tables 2 and 3). In shaken samples, increase in CFU was rapid from the zero hour count of 106 per ml. The average count in NAK broth was 8.3 x 108 at 5 h and 1.3 x 1010at 8 h. Maximal counts had been reached prior to 24 h. The colloidal ingredient, as compared to the NAK broth, had no significant effect at 5 h but had a slight, although significant (5% level), depressing effect at 8 h. The addition of cornstarch or LMP as the colloidal ingredient had increased the 24-h CFU slightly (significant at the 1% level). The state of dispersion had little effect nor was there a significant interaction between dispersion and colloidal ingredient. Although pH differences were small, effects of the state of dispersion and the interaction with colloids were highly significant statistically. The colloids did not appear to differ until 24 h, when the LMP had the highest average number of CFU (2.8 x 1010 as compared to 7.6 x 109 for the control); pH was the lowest, 7.7. Other strains. Four additional strains were compared after 24 h still incubation in liquid and paste states with cornstarch as the colloid (Table 4). In addition, carrageenan was used for one strain. In samples incubated without shaking, increases in CFU, pH, and the production of enterotoxin were found for the pastes with the exception of CFU of strain 361. In the strain 361 study, the effect of colloid was greater than the effect of type of dispersion. Nutritive effects. Preliminary tests with phosphate buffer dispersions of the colloidal ingredients prior to these studies demonstrated IrA, AND VENN APPL. MICROBIOL. little nutritive effect as indicated by no or slight increases in CFU of inoculated staphylococci except possibly for the higher concentration of carrageenan. There was no enterotoxin production when the colloids had been added to buffer to provide the four types of dispersions. In addition, reducing sugars were determined for the starch dispersions, and then filter-sterilized glucose was added to adjust the control NAK broth and each starch dispersion to the level of the highest concentration found (17 mg/ml in the suspension and gel). Enterotoxin B was low in the still broth and liquid dispersion (average of 0.9 Mg/ml) and higher in the suspension and gel (20 and 13 gg/ml, respectively). A second broth, NAK-PHP, which was higher in protein content was used as the basis for liquid and paste cornstarch dispersions ( Table 6). The trends were little different from those with NAK broth, that with the higher proportion of cornstarch yielded larger quantities of enterotoxin. Limitations. It was more difficult to study the still systems because of the low levels of enterotoxin to be determined. If the concentration reported is below 0.4 Mg/mil, the value was estimated from dilution comparisons and the limits of detection of the methods used. In recovery studies, it was found that enterotoxin A decreased during the concentration procedure and, therefore, reported concentrations below 4 gg/mil are lower than the original. DISCUSSION It is evident that under conditions of forced aeration, there was no increase in enterotoxin production attributable to the addition of the colloidal ingredients. However, under more limited aeration (incubation still), paste and gel dispersions had increased production of enterotoxin. In preliminary investigations, gels surface-inoculated and those inoculated throughout the medium before gelation gave similar levels of enterotoxin. Colony form differs with the consistency of the growth medium; further study may delineate this or other physical factors as being important. Bacterial cells may adhere to colloid particles and thus encounter a different microenvironment (8). Haider et al. (5) in experiments with S. cerevisiae and A. niger observed more efficient utilization of glucose in the presence of montmorillonite. A protective effect has been attributed to several synthetic polymers substituted for part of the serum in media for selected human cell lines (11). Differences in a, appear not to be the cause since all were above 0.99 (14). Since foods represent colloidal systems and have no active aeration during storage, the production of enterotoxin may be favored by the colloidal matrix. However, not all colloids were equally effective.

v3-fos

2020-12-10T09:04:12.607Z

1973-11-01T00:00:00.000Z

237231027

s2

Preparation of Antimicrobial Compounds by Hydrolysis of Oleuropein from Green Olives Oleuropein, an intensely bitter glucoside, was isolated from green olives. Hydrolysis products obtained from oleuropein in sufficient quantity for further tests were: (i) β-3,4-dihydroxyphenylethyl alcohol prepared by acid hydrolysis of oleuropein; (ii) elenolic acid obtained by methanolysis of oleuropein, isolation of the intermediate acetal, and subsequent acid hydrolysis; and (iii) oleuropein aglycone formed by the action of β-glucosidase on the parent glucoside. Mass spectral verification of the isolated compounds and ultraviolet absorption data are given. Oleuropein and its aglycone had similar threshold levels for detection of bitterness, whereas elenolic acid and β-3,4-dihydroxyphenylethyl alcohol were not judged to be bitter. The bitter principle of olives, oleuropein, was named and studied by Bourquelot and Vintilesco (1). Later Panizzi et al. (8) showed that the oleuropein molecule contained glucose, fl-3, 4-dihydroxyphenylethyl alcohol, and an acid (Fig. 1). These workers suggested that this acid was identical to a hypotensive agent designated as elenolic acid (W. L. C. Veer, U.S. Patent 3,033,877, 1962; reference 10) which was prepared by hydrolysis of olive extracts with phosphoric acid. Fleming et al. (5) isolated a compound from green olives that appeared to have the antimicrobial properties noted earlier during the fermentation of brined olives (3,4). The compound, a bitter phenolic material, was considered to be an enzymatic degradation product of oleuropein (5). Others (8) proposed that oleuropein is hydrolyzed in vitro by fl-glucosidase into glucose and a bitter tasting aglycone. Because of the importance of a proper fermentation on the preservation of brined green olives, a complete understanding of oleuropein's role was needed. The present study was undertaken to develop a procedure to produce sufficient amounts of oleuropein and its hydrolysis products so that the chemical and antimicrobial properties could be determined. Effects of these compounds on selected species of bacteria and yeasts are reported in a separate paper (6). ' Paper no. 4108 of the Journal Series of the North Carolina State University Experiment Station, Raleigh, N.C. MATERIALS AND METHODS Extraction and purification of oleuropein. Oleuropein was extracted from steam-heated (100 C, 20 min), green, Manzanillo variety olives (500 g) as described earlier (5) except that the residue from the methanol extraction was shaken with hexane to remove lipids before extraction with ethyl acetate. The lipid-free ethyl acetate solution was evaporated to 20 ml, and the components were separated by counter-current distribution (CCD) with the solvent system of ethyl acetate: 0.1 M potassium phosphate, pH 4.5. By using absorption at 280 nm, the major band was located and collected, and the solvent was removed by evaporation. The isolate was a crusty, light-yellow material (7.2 g), consisting mainly of oleuropein. This dried extract was dissolved in methanol, and the oleuropein fraction was purified by preparative thin-layer chromatography (PLC) as described earlier (5) except that the solvent system was benzenemethanol-acetic acid (45:16:1). The oleuropein was purified further by CCD by using ethyl acetate as the mobile phase and distilled deionized water as the stationary phase. Oleuropein (2.01 g) purified in this manner was a light-yellow amorphous material. The final yield of purified oleuropein was about 0.4% of the weight of the pitted olives. Acid hydrolysis of oleuropein and isolation of the products. Purified oleuropein (500 mg) was hydrolyzed in 100 ml of 1 N H2S04 for 1 h at 100 C. The hydrolysate was cooled, adjusted to pH 2, and extracted with ethyl acetate. After drying, the solvent was removed in vacuo, yielding 304 mg of an oily product. The oil was dissolved in methanol and applied to thin-layer chromatography (TLC) plates coated with Silica Gel HF2,4. After development in benzene-methanol-acetic acid (45:8: 1), the plate was observed under shortwave ultraviolet (UV) light, and three compounds were noted. Compound 1 (R, 0.26) gave a positive reaction when sprayed with a phenolsensitive reagent (5). The R, was identical to that of authentic #l-3,4-dihydroxyphenylethyl alcohol. Compound 2 (R. 0.35) gave a faint blue color with the phenolic spray and was assumed to be the aglycone of oleuropein. Compound 3 (R. 0.43) gave a negative phenol test and had an R. value similar to that of elenolic acid. With the compounds tentatively identified, components of the oleuropein hydrolysate were separated by PLC by using the solvent system described above for analytical TLC. Each of the three zones was collected and dissolved in the appropriate solvent for purification by CCD. The solvent system used for CCD purification of the aglycone and elenolic acid was ethyl ether-water (compounds located by absorption at 224 nm), whereas ethyl acetate-water was used for fl-3,4-dihydroxyphenylethyl alcohol (located by absorption at 280 nm). Yields of the lyophilized isolated products were: fl-3,4-dihydrolyphenylethyl alcohol, 65 mg; aglycone, 14 mg; and elenolic acid, 15 mg. High-resolution mass spectra were obtained on each of these compounds. The glucose content in the hydrolysate, assayed by paper chromatography (5) followed by enzymatic glucose analysis (11), was 27.4% of the weight of the oleuropein sample. This value is similar to that previously reported (5) but less than the theoretical value of 33%. Macropreparation of compounds. Because direct acid hydrolysis of oleuropein gave very small amounts of elenolic acid and oleuropein aglycone, alternate methods were used to prepare quantities sufficient for microbiological studies. Elenolic acid was prepared by methylating a crude oleuropein extract with anhydrous methanolic hydrogen chloride. The resulting methyl-o-methyl elenolate (W. L. C. Veer, U.S. Patent 3,033,877, 1962) was isolated and converted into the free acid by hydrolysis in dilute mineral acid. Crude oleuropein (3.4 g dry weight) from an ethyl acetate extract of olives was dissolved in 10 ml of anhydrous methanolic HCl (5%), sealed in an ampoule under nitrogen, and heated at 65 C for 3.5 h. The methyl-o-methyl elenolate (1.06 g) was obtained from the reaction mixture by extraction with ethyl acetate, followed by a CCD separation using hexanemethanol, 1: 1, as the solvent system. The oily material (1.01 g) was dissolved in 5 ml of ether and added dropwise to 300 ml of 1 N H2S04, and the mixture was stirred at 70 to 80 C for 1.5 h. The solution was cooled and extracted with ethyl ether. The extract was washed several times to remove H2SO4, dried, and the solvent was evaporated, leaving 0.65 g of a colorless, oily material which TLC analysis indicated was primarily elenolic acid. This material was purified further by PLC and finally by CCD to give 0.150 g of pure elenolic acid. Oleuropein aglycone was prepared by enzymatic hydrolysis of oleuropein by using fl-glucosidase. A filter-sterilized 1% solution of pure oleuropein (500 mg) in 0.1 M sodium acetate buffer, pH 4.1, was mixed with one-ninth volume of filter-sterilized 2% fl-glucosidase solution (Sigma Chemical Co.) and incubated for 16 h at 32 C. The solution then was extracted with chloroform. After removal of the solvent, the resulting purple oil was purified by PLC followed by CCD. The purified aglycone was a lightbrown oil (78 mg). Analyses. Mass spectra were obtained at the facilities of the Research Triangle Institute on an AEI-MS-902 instrument. Elemental analyses and molecular weights were determined by Galbraith Laboratories, Knoxville, Tenn. UV spectra were recorded with a Cary model 15 instrument, and optical rotations were recorded with a Perkins Elmer model 141 polarimeter. CCD. CCD was performed with a 50-tube Post-Craig apparatus (H.O. Post Co., Middle Village, N.Y.). Solvents were reagent grade and redistilled prior to use. Water was distilled and deionized. TLC. Preparation of TLC and PLC plates using Silica Gel HF2.4 and Silica Gel PF2,4, respectively, was described previously, as was the procedure for developing the plates and detection of phenolic compounds (5). Compounds also were located on TLC plates by spraying with 50% H2SO and heating at 170 C for 30 min. PLC plates, after developing, were dried under nitrogen, compounds were visualized under UV light at 254 nm, and zones were collected (5). Reference compounds. A sample of elenolic acid as the calcium salt was provided by the Upjohn Company. A sample of ,B-3,4-dihydroxyphenylethyl alcohol was prepared by the Research Triangle Institute, N.C. Bitterness test. The bitterness of oleuropein and its aglycone was evaluated by a taste panel consisting of eight individuals. Whatman no. 1 filter paper was washed in ethyl alcohol, dried, and cut into 1-cm squares. The compounds were applied to the paper as ethyl alcohol solutions, and then the alcohol was removed by evaporation. Panelists were instructed to hold the paper squares on their tongues until they could detect bitterness or decide that no bitterness was present. They were given a blank square of paper first and then a series of papers containing increasing levels of the compounds. They were asked to discontinue the test at the first level where bitterness was detected and to describe any unusual characteristics of the bitterness. RESULTS AND DISCUSSION Purified oleuropein was obtained from green olives, portions were hydrolyzed, and the major fragments were isolated therefrom. Physical properties of the compounds isolated are summarized in Table 1. Oleuropein isolated by our procedure was essentially identical to that previously described (8), except that we observed a specific optical rotation value of -1780, whereas they reported -158°. The differences might be explained by the higher purity of our preparation. For example, aMolecular weight of oleuropein was determined by vapor pressure osmometry in tetrahydrofuran, and the elemental composition was determined by combustion. Molecular weights and elemental analyses of the other compounds were based on high-resolution mass spectral data. b Determined in ethanol. c Numbers in parentheses refer to percent relative abundance. dThe molecular ion peak, M+, was not obtained for oleuropein. when oleuropein was first described in 1908 (1), the specific rotation was -127°. In 1934 Cruess and Alsberg obtained a value of -145 to -1480. Each investigator since Bourquelot and Vintilesco (1) has used improved purification methods and each has reported higher specific optical rotation values. Our study confirms those of Panizzi et al. (8) and Cruess and Alsberg (2), who reported that oleuropein is hydrolyzed by fl-glucosidase. Shasha and Leibowitz (9) reported that the olive bitter principle is not attacked by the enzyme. The ability of fl-glucosidase to produce the aglycone from oleuropein is of considerable importance in olive fermentation due to the inhibitory nature of this moiety (6). A molecular weight of 540 for oleuropein was obtained by vapor pressure osmometry. This value agrees with that reported by others (8). The mass spectrum did not exhibit any fragments above 360 mass units, probably because of the nonvolatility of the oleuropein molecule. Some of the higher-molecular-weight fragments are given in Table 1. The mass spectrum for elenolic acid, whether from direct hydrolysis of oleuropein or from hydrolysis of methyl-o-methyl elenolate, was identical to the authentic reference compound. In addition, the mass spectrum of ,B-3,4-dihydroxyphenylethyl alcohol, from hydrolysis of oleuropein, was identical to that of the reference material. Some of the physical data are supplied in Table 1. Panizzi et al. (8) reported the formation of oleuropein aglycone on the basis of paper chromatographic examination of the products re-sulting from treatment of oleuropein with flglucosidase. No attempt was made by these workers to isolate or study the aglycone further. The structure given in Fig. 1, therefore, is tentative. Our preparation of the aglycone was subjected to several purification steps and is chromatographically pure (TLC). By the use of high-resolution mass spectrometry, we obtained an elemental composition of C 1H225O (molecular weight 378.132) for this compound, which corresponds to the product expected when glucose is hydrolytically cleaved from oleuropein. The highest-molecular-weight fragments and UV absorbance maxima are provided in Table 1. The structures given in Fig. 1 are those of Panizzi et al. (8) and other workers (W. L. C. Veer, U.S. Patent 3,033,877, 1962; reference 10). A recent paper, however, proposed a slightly modified structure for elenolic acid (7). The tentative structures are provided as an aid in observing the origin of oleuropein hydrolysis products. Oleuropein and its aglycone were bitter, the threshold levels for detection being about 50 sg for most of the panelists. Two individuals did not detect bitterness at a level of 200 Mg per paper square, which was the highest level tested. Although the threshold levels for detection of bitterness of both compounds were similar, some panelists described the taste of the aglycone as having a stinging, biting, or sharp sensation associated with the bitterness. Neither elenolic acid nor ,B-3, 4-dihydroxyphenylethyl alcohol was bitter at levels up to 200 Mg. Structures of oleuropein and its hydrolysis products as proposed by Panizzi et al. (8). The structure for the aglycone was not reported by these authors but was assumed from the structure of oleuropein. The names, elenolic acid and methyl-o-methyl elenolate, were applied by Veer (W. L. C. Veer, U.S. Patent 3,033,877, 1962; reference 10).

v3-fos

2020-12-10T09:04:17.074Z

1973-10-01T00:00:00.000Z

237233388

s2

Comparison of Methods for the Recovery of Virus Inoculated into Ground Beef Various methods for the recovery of virus inoculated into ground beef were investigated in an attempt to develop a sensitive system that could be used to detect viral contaminants in market foods. A 100-g sample, inoculated with poliovirus 1, was suspended in 150 to 900 ml of Eagle minimum essential medium, pH 8.5, and mixed in either plastic bags or plastic cups on a mechanical shaker. The particulate materials were removed by means of cheese cloth, glass wool, woven fiber glass, or low-speed centrifugation. Large volumes of fluid were concentrated by ultrafiltration. Microbiological contamination was controlled by high antibiotic concentrations or by filtration. Quantitative plaque-forming-unit recovery of the virus was determined by utilizing an agar overlay technique on Vero cell cultures. The data indicated that from 20 to 50% of the seeded virus could be recovered from a 100-g sample of ground beef. The glass wool and woven fiber glass methods were the most effective, with recovery of approximately 50% of the inoculated virus. In 1970, we reported the isolation of viruses from 3 of 12 market samples of ground beef. The viruses were identified as polioviruses 1, 2, and 3, and echovirus 6 (10). Approximately 50% of the beef in the United States is consumed in the form of ground beef or hamburger (6). The eating habits of our population are such that much of this beef is consumed in a rare to medium-rare state. The finding of viruses in a common food that is handled by both the processor and the consumer before cooking and is eaten in a semicooked condition indicated that this food could be of public health significance. Possibly such a food could be the carrier of agents in food-borne outbreaks of unknown etiology (2,3). The transport of viruses by foods may occur as frequently as meal time (1,2,4,8). A study was initiated to develop effective methods for the recovery of viruses from ground beef-methods that could be used in laboratories in much the same way as those used to test for bacterial content. In the study reported previously, 5-g samples of ground beef were analyzed. The small number of viruses found in the samples indicated that the probability of viral recovery would be better if a larger sample were examined. Therefore, in this study, methods were developed for the recovery of viruses from 100-g samples of ground beef. It was anticipated that such methods could also be applied to the recovery of viruses from other foods. MATERIALS AND METHODS Ground beef. Fresh ground beef was purchased from local retail markets on the same day that the samples were run. Samples (100 g) were weighed into sterile plastic bags, and 1 ml of virus suspension was added to the ground beef. The bags were sealed, and the contents were kneaded by hand for approximately 5 min. The previous study on the homogeneity and distribution of the virus in the ground beef indicated that this was an effective method for distributing the virus throughout the sample (10). Tissue culture. Vero monkey kidney cell cultures (ATCC, CCL#81) passage 125 were used as the source of tissue culture preparations. The culture was propagaged in 6-oz (170.1-g) prescription bottles; the confluent cell sheets were trypsinized, split 1 to 8, and recultured in sufficient quantities for viral growth studies. Growth medium. The growth medium used for tissue culture was a 1: 1 mixture of Leibovitz medium (L-15) and Eagle minimum essential medium (MEM) with Hanks salts containing 10% fetal bovine serum and 0.075% NaHCO3. The medium provided excellent growth and maintained the cells for 14 days without having to be changed. Plaque assay system. A previously reported viral plaque assay system was used (11). An agar medium overlay was used with monolayer (45 cm2) Vero cell cultures in 6-oz prescription bottles. 497 Virus. Poliovirus 1 (Mahoney) was passaged three times in primary cell cultures of Cercopithicus aethiops (African Green) monkey kidney cells. The cells were freeze-thawed three times, the debris was removed by centrifugation, and 1.2 ml of the virus suspension was placed into each of a number of 2-ml borosilicate glass ampoules and stored at -60 C. High-antibiotic MEM (HAMEM). MEM with non-essential amino acids in Hanks salts containing 2% fetal bovine serum, 2.5 mg MgCl2.6H2O per ml (12), and 100 ug of diethylaminoethyl-dextran sulfate (2 x 106 molecular weight) per ml was used for the elution of viruses from the ground beef. Antibiotics were added in the following concentrations per ml: 4,740 U of penicillin G, 5,000 gg of streptomycin sulfate, 250 gg of tetracycline hydrochloride, and 5.0 ;ig of amphotericin B. One normal NaOH was added to raise the pH to 8.5 for elution of the virus and also to prevent coagulation of the meat slurry (10). Glass wool and woven fiber glass methods. The methods using glass wool and woven fiber glass are similar and are described jointly. A 100-g sample of virus-containing ground beef and 100 ml of HAMEM were placed in a plastic bag. The bag and contents were shaken vigorously by hand, and the pH of the slurry was readjusted to 8.5. The plastic bag containing the slurry was placed on a mechanical shaker and shaken for 15 min. The contents were poured through 4 g of either glass wool or woven fiber glass, which had been placed in a funnel. The wool or fiber glass had been pretreated with 20 ml of HAMEM. The bag was rinsed with 30 ml of the HAMEM, and the rinse was added to the funnel. Approximately 100 ml of clarified meat slurry can be obtained after 1 h. A laminar vertical flow cabinet was used to prevent contamination of the sample during processing. The total recovered fluid was inoculated into 30 bottles of Vero cell monolayers; the bottles were incubated for 2 h at 36 C, and the Vero cell monolayers were then overlayed with agar medium. After the agar solidified, the bottles were inverted and incubated at 36 C. The plaques were counted and marked daily for 14 days. Potato ricer method. The preliminary processing of the 100-g sample was the same as that outlined for the glass wool and fiber glass methods. After being shaken for 15 min, the contents of the bag were poured into a commercial stainless-steel potato ricer. (The potato ricer is a hand-operated apparatus used to compress cooked potatoes through small holes in a metal container.) In this process the potato ricer was lined with four layers of cheese cloth pretreated with 20 ml of HAMEM. The bag was rinsed with 30 ml of the HAMEM; this fluid was added to the potato ricer. The liquid was squeezed from the meat-fluid mixture by pressure. The total recovered extract (100 to 110 ml) was inoculated onto 30 Vero cell monolayers and incubated for 2 h at 36 C. The cultures were processed as described above. Low-speed centrifugation method. The ground beef sample was placed in an 8-oz (226.8-g) plastic cup having a tight-fitting lid, and 100 ml of HAMEM was added. The cup was shaken vigorously by hand, the pH of the slurry was readjusted to 8.5, and the sample was then mixed on a mechanical shaker for 20 min. The pH of the slurry was readjusted to 8.5, and the sample was centrifuged for 20 min at 690 x g in a preparative centrifuge. After centrifugation, the supernatant fluid was decanted. The pellet was resuspended in 50 ml of HAMEM, shaken, and centrifuged a second time. The supernatant fluid was removed and combined with the first supernatant. The total fluid volume, approximately 110 to 120 ml, was inoculated onto 30 Vero cell monolayers, and the bottles were processed as described for the other methods. Ultrafiltration method. In previous studies with 1and 5-g samples, a meat and liquid ratio of 1:10 resulted in the extraction and recovery of a high percentage of virus from the ground beef. To simulate this study with a larger ground beef sample, 800 ml of HAMEM and a 100-g inoculated beef sample were placed in a plastic bag. The sample was mixed on a shaker for 15 min. The contents were poured into a potato ricer containing four layers of cheese cloth pretreated with 50 ml of HAMEM. The plastic bag was rinsed with 50 ml of HAMEM, and the fluid was poured into the potato ricer. The fluid was removed by pressure. Ten grams of diatomaceous earth (Celite 545) was added to the clarified slurry, and the fluid was filtered to remove bacterial contaminants and particulate material that would clog the 0.0075-Mim filter used to concentrate the virus. Three types of filters-(i) 0.45-mm cellulose acetate (Gelman Co.), (ii) 0.40-gm polycarbonate ("Nuclepore," General Electric Co.), and (iii) 0.45-gum silver (Selas Flotronics Co.)-were used. The filters were pretreated with fetal bovine serum immediately before use. It required from 1/2 to 8 h to process the sample, depending on the type of filter used. Each filtrate was concentrated by ultrafiltration with a protein-enrichment membrane (PEM) of 0.0075-um porosity (Gelman Co.) at 7 C. This process required from 8 to 16 h. The virus was eluted from the membrane with 60 ml of fetal bovine serum (9). Samples (10 ml) each were taken after passage through the filter and from the material eluted from the PEM. Dilutions (10-fold) were made, and 1 ml was inoculated into each of five bottles of Vero cell monolayers. Agar medium overlay, incubation, and the procedures for counting were the same as described before. RESULTS Two groups of experiments were done to compare various methods used to extract poliovirus from ground beef. In the first group, glass wool, woven fiber glass, the potato ricer, and low-speed centrifugation methods were evaluated for effectiveness in the clarification of a ground beef slurry and effectiveness of viral recovery. All the methods produced clarified suspensions suitable for inoculation onto cell sheets for viral plaque-forming units (PFU) enumeration. The results are shown in Table 1. Viral recovery data for the glass wool or woven fiber glass methods were similar, with mean recoveries of 48 and 49%, respectively. The potato ricer method, in addition to being quite cumbersome in application, produced the lowest viral recovery (19%) of the four methods analyzed. The glass wool, woven fiber glass, and potato ricer samples were processed in a laminar flow cabinet to prevent contamination of the product during handling. If adequate aseptic procedures are followed and the sample is covered with sterile aluminum foil, it is possible to process samples without a protective cabinet. However, when only one or two plaques are recovered, it may be difficult to prove that the virus came from the sample and was not a result of airborne contamination. The low-speed centrifugation method was developed in which the sample was processed in a relatively closed system. A 33% viral recovery resulted with this method. The four methods are compared in Table 2. In the second group of experiments, a comparison of the three bacterial retaining filters used as prefilters in the viral concentration study indicates that the use of the polycarbon- filter ate filter resulted in faster processing (30 to 60 min) as compared to the silver filter (3 h) and the cellulose acetate filter (8 h). About 37% of the virus was retained on the cellulose acetate filter, 24% was retained by the polycarbonate filter, and 11% was retained by the silver filter. The time required to concentrate the fluids on the ultrafilter was less than 8 h when the cellulose acetate prefilter was used, 16 h for the polycarbonate filter, and 20 to 24 h for the silver filter. Some bacteria and molds passed through the silver filter. Results of this study are presented in Tables 3 and 4. Clogging of the filters was reduced by preliminary clarification through cheese cloth and by the use of diatomaceous earth as a filter aid. In Tables 2 and 4, the mean recoveries and percent coefficient of variation were computed for each of the combinations described above. The percent coefficient of variation is defined as 100 X (standard deviation/mean recovery). A value of 20 to 25% might be considered good for the present virus work. A recovery of 50% of the initial virus load is considered adequate, considering the small viral inoculum for a 100-g sample. In the first series of experiments, the glass wool and woven fiber glass methods met the above criteria and perhaps merit further testing. The methods used in the second phase of the study were also satisfactory. Although the mean recoveries differ in the concentration step, the variation is large, as shown in Table 4. The distribution of the percent recoveries is unknown; however, it is assumed that the means are normally distributed. The following F test was performed to examine the null hypothesis that the three mean recoveries were equal. The test is performed at the a = 0.01 level. F = variation between groups/variation within groups. F2, 19 = 0.04318/0.01747 = 2.47. The value for F for these is 2.47. Since this value is less than the critical value (5.93), it is assumed that the mean recoveries could not be shown to differ. DISCUSSION In earlier studies, the addition of the ground beef slurry directly to the cell culture resulted in the mechanical stripping of the cell sheet. Various clarifying materials and methods were studied in an attempt to eliminate the particulate material. These investigations culminated in the development of the glass wool, woven fiber glass, potato ricer, and low-speed centrifugation methods. In all these methods, a highantibiotic medium, pH 8.5, was used to elute the virus from the ground beef and to control bacterial contaminants. The viral recovery data indicate that the glass wool and woven fiber glass methods both give recoveries of approximately 50%. The use of the low-speed centrifugation method resulted in recovery of 33% of the inoculated virus. All the methods appear to be effective for the recovery of virus from a 100-g sample of ground beef. A comparison of the three bacteria-retaining filters used in the viral concentration study indicates that the 0.45-am silver filter allowed for more effective passage of the virus (Table 4). These data confirm results reported by Hahn et al. (7). Hahn also demonstrated the inability of the silver filters to retain 100% of Serratia marcescens. It is possible that bacteria and particles of ground beef passed through the filter and interfered with recovery efficiency on the ultrafilter. Cliver (5) reported that treatment of the filter with serum before filtration greatly improved the filtration of the virus. Probably the 2% serum in the HAMEM plus the serum protein in the meat aided filtration. Hahn et al. (7) stated that the filterability was greatly enhanced by the protein extracts in the medium. If the concentration of virus in the sample is of such low magnitude that virus may be missed unless the total fluid volume is analyzed, concentration methods are of value. From the viewpoint of economics, the use of 4 or 5 culture bottles, as compared to 30, has a distinct advantage. However, the initial cost of the filter holders and the additional time required in the processing of the sample must be considered when selecting a method.

v3-fos

2018-12-07T13:31:54.021Z

1974-01-01T00:00:00.000Z

131081272

s2

Herbicides for Use on High pH Soils in the Wheat Fallow System in Southwest Kansas This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1974 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Herbicides for Use on High pH Soils in the Wheat Fallow System in Southwest Kansas Charles A. Norwood Garden City Branch Experiment Station Reduced tillage for dryland wheat involves the substitution of herbicides for tillage during the fallow period. Proper management of reduced tillage systems results in increased retention of straw, decreased erosion , increased retention of precipitation , and increased yield. Proper selection and use of herbicides will result in good weed control during fallow while minimizing the risk of injury to the subsequent crop. Injury to wheat will occur only from the misuse of herbicides. Most residual herbicides currently used in the wheat fallow system were not initially developed for wheat, so particular attention must be paid both to the· selection of the herbicides and the amount applied. Persistence of the herbicide depends on rainfall, soil moisture, texture, organic matter, and pH: Low soil moisture, coarse texture, low organic matter, and high pH will increase the persistence of herbicides. In western Kansas, injury to wheat occurs most frequently on high pH soils. Such soils contain significant amounts of calcium carbonate and are low in organic matter. : 1976: -1977: 1977: -1978: 1979: -1980: 1980: -1981 This experiment was conducted to study the effects of herbicides on weed control, crop injury, and yield of wheat grown in a wheat fallow system on a typical high pH soil. Procedure The soil type was a Colby silt loam with a pH of 8.0, free carbonates in the surface, and an organic matter content of 1.5% . Herbicide applications were made in July or August of each year to the stubble remaining after that year's wheat crop. Weeds emerging prior to application were controlled by contact herbicides that were mixed with the residual herbicide or by tillage before application if the weeds were large. In addition, some herbicides were applied the following spring, either to untreated stubble or to stubble treated the preceding summer. Precipitation occuring over the entire period is given in Table 1. Results The results from several studies initiated after harvest during the period 1976-1981 are reported. Results from 1976 and 1977. Weed control ratings were not recorded for the study initiated in 1976, but ratings made on June 12, 1978 for the study initiated in 1977 are given in Table 2. Weeds present were volunteer wheat, Russian thistle, redroot pigweed, and kochia. Since control was similar fo{ each species, individual ratings were not made. Weeds were just beginning to grow and there was little difference between the three treatments. Injury (% stand reduction) and yield of the 1978 and 1979 wheat crops are also included in Table 2. Considerable injury from the 1.0 lb rate of atrazine and 0.8 lb atrazine + 'Applied August 20, 1979 ;Augustll , 1980' AppliedApril22, 1980Apnl 29, 1981 mer of 1979 and the spring of 1980, along with the resulting weed control ratings, are given in Table 3 , while results from the 1980-81 fallow period are presented in Table 4. Grassy weeds present during both fallow periods were mainly volunteer wheat and some witchgrass. Broadleaved weeds included Russian thistle, kochia, and redroot pigweed, along with light infestations of prostrate pigweed and tansy mustard. There were relatively few broadleaves present in treatment on the first rating date of each year and all herbicides gave nearly perfect control. Grassy weed control exceeded 80% in 1980 and 90% in 1981 for all herbicides on the first rating date. Infestation of both grasses and broadleaves became more severe by the second rating date and the control given by several of the treatments diminished. The 0.5 lb rate of atrazine gave control only until early to mid-June in both years. The 1.0 lb rate of atrazine gave better control of broadleaves in 1980; In 1981 both the 0.8 and 1.0 lb rates gave better control than the 0.5 lb rate of atrazine. Control of witchgrass and volunteer wheat did not differ between rates of atrazine in either year. The tank mix containing 0.8lb atrazine and 1.6lb cyanazine gave better control of broadleaves in 1980 than 0.8 lb atrazine alone, but a significant difference did not occur In 1981. In other studies the main advantage of the atrazine + cyanazine tank mix has been better control of volunteer wheat following har- NS NS vest than with atrazine alone, particularly in years of above average rainfall. Good control was obtained with spring applications of cyanazine or metribuzin to previously untreated stubble . However, season-long control was not obtained since the plots required tillage between wheat harvest and winter freeze. The best and longest lasting weed control in both years was obtained with atrazine applied after wheat harvest followed by cyanazine or metribuzin the ne; spring. In 1981, control with 0.5 lb atrazine by cyanazine lasted into July , while at the higher rates of atrazine, control lasted into August. Weed control lasted longer into the fallow period of 1981 than 1980, probably because of different distribution of rainfall {only 0. 28 in occurred in June 1981, Table 1). As with the 1978-79 study, injury from atrazine occurred in only 1 of the 2 years {Table 5). A slight reduction in stand resulted from 0.8 lb atrazine in 1981 while a somewhat greater stand reduction resulted from the 1.0 lb rate of atrazine and the atrazine + cyanazine tank mix. The reduction in sta nd was considerably Jess than that occurring in 1978 , and no measurable reductions in yield occurred. Yields in 1981 were the lowest of the 4 years because of a freeze which occurred when the wheat was heading. New Herbicides. New herbicides for reduced tillage are being developed by the various chemical ( companies. One recently labeled herbicide is chlorosulfuron (Glean), manufactured by Du Pont. The results obtained so far indicate that chlorosulfuron gives weed control that lasts at least as long as atrazine. Chlorosulfuron was developed for cereal grains and will not injure wheat. Its main weakness seems to be a lack of control of volunteer wheat during fallow. A comparison of chlorosulfuron with other currently labeled herbicides will be published at a later date. Conclusions 'Atrazine or a cyanazine + atrazine tank mix applied in July or August following wheat harvest generally gave good weed control until at least mid-June of the following year. Following atrazine with cyanazine or metribuzin the next spring gave the longest period of weed control. Cyanazine and metribuzin applied in the spring to stubble not treated the preceding summer also gave good weed control, but necessitated tillage between harvest and winter freeze. Stand reductions due to atrazine carry-over occurred in 2 of the 4 years of this study, while a reduction in yield occurred in only 1 year. However, all plots having a stand reduction were weedy, often causing problems at harvest, and necessitating tillage for weed control immediately following harvest. Thus, on a high pH soil, such as the one in this study, the rate of atrazine used should not exceed 0.5 lb. active ingredient in order to minimize the possibility of injury. Agricultural Experiment Station, Manhattan 66506 Keeping up With Research 65 November 1982 Publications and public meetings by the Kansas Agricultural Experiment Station are available and open to the public regardless of race, color, na· tlonal origin, sex, or religion. 11·82-3M This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu.

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2019-04-04T13:09:56.131Z

1974-04-01T00:00:00.000Z

236888646

s2

Effect of Sodium Nitrite on Toxin Production by Clostridium botulinum in bacon Pork bellies were formulated to 0, 30, 60, 120, 170, or 340 μg of nitrite per g of meat and inoculated with Clostridium botulinum via pickle or after processing and slicing. Processed bacon was stored at 7 or 27 C and assayed for nitrite, nitrate, and botulinal toxin at different intervals. Nitrite levels declined during processing and storage. The rate of decrease was more rapid at 27 than at 7 C. Although not added to the system, nitrate was detected in samples during processing and storage at 7 and 27 C. The amount of nitrate found was related to formulated nitrite levels. No toxin was found in samples incubated at 7 C throughout the 84-day test period. At 27 C, via pickle, inoculated samples with low inoculum (210 C. botulinum per g before processing and 52 per g after processing) became toxic if formulated with 120 μg of nitrite per g of meat or less. Toxin was not detected in bacon formulated with 170 or 340 μg of nitrite per g of meat under these same conditions. Toxin was detected at all formulated nitrite levels in bacon inoculated via the pickle with 19,000 C. botulinum per g (4,300 per g after processing) and in samples inoculated after slicing. However, increased levels of formulated nitrite decreased the probability of botulinal toxin formation in bacon inoculated by both methods. Pork bellies were formulated to 0, 30, 60, 120, 170, or 340 ,ig of nitrite per g of meat and inoculated with Clostridium botulinum via pickle or after processing and slicing. Processed bacon was stored at 7 or 27 C and assayed for nitrite, nitrate, and botulinal toxin at different intervals. Nitrite levels declined during processing and storage. The rate of decrease was more rapid at 27 than at 7 C. Although not added to the system, nitrate was detected in samples during processing and storage at 7 and 27 C. The amount of nitrate found was related to formulated nitrite levels. No toxin was found in samples incubated at 7 C throughout the 84-day test period. At 27 C, via pickle, inoculated samples with low inoculum (210 C. botulinum per g before processing and 52 per g after processing) became toxic if formulated with 120 fg of nitrite per g of meat or less. Toxin was not detected in bacon formulated with 170 or 340 ,gg of nitrite per g of meat under these same conditions. Toxin was detected at all formulated nitrite levels in bacon inoculated via the pickle with 19,000 C. botulinum per g (4,300 per g after processing) and in samples inoculated after slicing. However, increased levels of formulated nitrite decreased the probability of botulinal toxin formation in bacon inoculated by both methods. Recent studies on canned, perishable, cured meat and wieners showed that increased nitrite levels decreased the probability of botulinal toxin formation (1, 4). However, the impact of nitrite upon toxigenesis differed somewhat between the two products. This difference is probably due, in part, to differences in formulation and processing employed in the manufacture of the two products. Bacon is formulated and processed differently from either canned, perishable, cured meat or wieners. Thus, results from the foregoing studies would not necessarily apply to bacon. This study was conducted as one of a series undertaken cooperatively by the American Meat Institute, the Food and Drug Administration, and the United States Department of Agriculture to determine the minimal level of sodium nitrite required in bacon for consumer acceptance and botulinal protection. The initial studies on-bacon were designed to investigate three distinct aspects and were conducted concurrently. Project I investigated the effect of nitrite level on product manufacture, chemical changes, product acceptance, off-flavor, growth of microbial spoilage organisms, and color. Project II examined the roles of nitrite level, cooking method, and ascorbates on the formation of N-nitrosopyrrolidine. Preliminary results from the first two projects have been reported (3). When completed, the first two projects will be reported as a separate paper. Project III, reported herein, investigated the degree to which nitrite retards or prevents growth of Clostridium botulinum in bacon. Additional research is now underway at the Food Research Institute, University of Wisconsin, to investigate the effect, if any, that high levels of sodium ascorbate have on the inhibition of botulinal toxin by sodium nitrite in bacon. MATERIALS AND METHODS Experimental design. The experimental variables are listed in Table 1. The design for the uninoculated portion of the experiment was a full replicate of a 6 x 6 x 2 (nitrite level x storage time x incubation temperature) factorial with one package for each treatment combination. A similar 6 x 6 x 2 (nitrite level x storage time x inoculum level) design was used for inoculated samples. There were five packages for each treatment combination for samples inoculated via the pickle solution and two replicates for samples inoculated with a sand-spore mixture after slicing. Inoculum. Spores of five type A (77A, 62A, 12885A, 33A, and 62A) and five type B (ATCC7949, 41B, 40B, 53B, and Lamanna B) strains of C. botulinum were used. Tubes of peptone colloid medium (Difco) modified by the addition of 0.1% glucose were inoculated with a heat-shocked spore suspension of the individual strains. After 16 h of incubation at 37 C, fresh tubes of the medium were inoculated, incubated for 4 h, and again transferred. After 4 h of incubation, these cultures were inoculated into the sporulation medium of Schmidt and Nank (5) consisting of 5% Trypticase (BBL), 0.5% peptone (Difco), and 0.05% sodium thioglycolate. The final cultures were incubated for 7 days at 37 C. Spores were harvested by centrifugation, washed several times, and suspended in sterile, distilled water. Samples of each suspension were heat-shocked (80 C for 15 min), and spore counts were determined by a three-tube most probable number procedure in modified peptone colloid medium (2). A single suspension was prepared containing equal numbers of spores of each strain. A portion of this spore mixture was heat-shocked, diluted, and added to the pickle solutions for inoculation of the bacon. A second portion of the heat-shocked spore suspension was mixed with sterile sand and dried under vacuum over phosphorus pentoxide at room temperature for inoculation of bacon after slicing. Preparation and inoculation of bacon. Raw, frozen pork bellies, curing ingredients, and water used for preparing pickle were from the same stocks used in the project I research and were supplied by Armour and Co. (Oak Brook, Ill.). Two bellies at each nitrite and spore inoculum level were pumped with curing pickle to an 11% gain and drained to an approximate 10% gain. The pickle contained sodium chloride (13.3%), sucrose (3.1%), tripolyphosphate (2.6%), sodium isoascorbate (0.23%), and the various concentrations of sodium nitrite and water. These concentrations of ingredients in the pickle were 10 times the levels desired in bacon. The drained bellies were smoked and processed to an internal temperature of 53 C over an 8.5-h period. Smoke from hardwood shavings was added during the initial 2.5 h. The processed bellies were held at -2.2 C for 36 h and sliced. The two bellies for each nitrite and spore inoculum level were divided into thirds. One slice from each third of the two bellies was placed in a Curpolene 200 (Curwood, Inc., New London, Wis.) pouch (six slices total, weighing 125 to 150 g), and the package was sealed under vacuum. The packages were stored at 7 or 27 C. Uninoculated bacon was prepared in the same manner except that spores were not added to the pickle. This bacon was handled and stored in the same manner as the inoculated bacon and was used for all chemical analyses. A portion of this bacon was inoculated with the dried sand-spore mixture after slicing. Toxin assay and determination of spore levels. The samples were randomized and labeled so that each package was designated for analysis at a specific time. However, samples which swelled prior to this time were removed from incubation and analyzed. The packages were weighed, and the entire contents of each package were blended for 1 min with an equal weight of gelatin phosphate buffer. The slurry was centrifuged, and 0.5 ml of the supernatant was injected into each of five mice (three unprotected and two protected with type AB botulinal antitoxin from Connaught Medical Research Laboratories, University of Toronto, Toronto, Canada). Death of the unprotected mice within 4 days and survival of the protected 2 mice were considered proof of the presence of botulinal toxin. Inoculum levels before smoking were determined by removing three plug samples (about 6.3 cm2/sample) from one of the two bellies at each nitrite level. The plug samples from each belly were composited for determination of inoculum level. Viable counts were determined on nonheat-shocked samples of bacon before and after processing and inoculated pickle solutions by the three-tube most probable number procedure. The pickle solutions were filtered through 0.45-,gm pore size filters. After rinsing the filters several times to minimize contamination by pickle ingredients, the filters were cut up, placed in phosphate buffer (pH 7.2), and agitated to dislodge the organisms, and counts per ml of pickle were determined. All bacon samples were blended as for toxin assay, and the slurry was diluted for determination of counts. Chemical analyses. A composite of nine plugs bored randomly from the bacon bellies was used for chemical analysis of bacon before and after heat processing. Nitrite and nitrate concentrations were determined as previously reported (1). RESULTS Samples of finished bacon at the various nitrite levels were analyzed for sodium chloride, moisture, fat, protein, and water activity. The range and average for these values, plus calculated brine concentrations, are shown in Table 2. Concentrations of nitrite found before smok- ing corresponded to formulated levels (Table 3). However, approximately 50 to 80% of the nitrite was lost during processing (i.e., heating to 53 C followed by holding for 36 h at -2.2 C). A further time-temperature-dependent reduction in nitrite occurred during storage. For example, Fig. 1 shows a best fitting (least squares) line fitted to predicted residual nitrite values for product formulated to 170 Ag of nitrite per g and held at 27 and 7 C. There was a geometric decline in nitrite levels with time at both temperatures. However, the rate of decrease was more rapid at 27 than at 7 C. Nitrate was not added to the system; however, analysis showed the presence of nitrate during processing and storage at 27 and 7 C ( Table 4). The amount of nitrate found was related to formulated nitrite levels. The range and logarithmic average counts of C. botulinum in the inoculating pickle, pumped bellies, and the finished bacon are shown in Table 5. These values represent counts across all nitrite levels. Although there was considerable variation, the counts were independent of the nitrite levels. Thus, exposure to nitrite, particularly the high levels in the pickle, had no effect on the inoculum. Three samples of bacon were analyzed per inoculum level after inoculating bacon with the sand-spore mixtures. The logarithmic averages were 40 and 3,400 per g with ranges of 18 to 86 and 1,800 to 4,600 per g for the low and high inoculum levels, respectively. Sixty samples of bacon inoculated via the pickle and 24 samples inoculated with the sand-spore mixtures were tested after packaging and without incubation. All were nontoxic, demonstrating that toxin was not carried into the product by the inocula. 14 28 54 84 7 14 28 54 84 0 0 19 36 15 16 35 18 0 10 10 NDa ND 30 0 29 63 26 22 35 10 0 17 18 ND ND 60 0 37 49 49 22 36 32 5 0 19 ND ND 120 18 48 58 130 25 64 62 58 31 21 26 16 170 35 70 102 135 34 103 67 73 23 35 26 33 340 35 80 63 149 60 137 82 112 217 51 32 16 a ND, Not determined. detected in bacon at all nitrite levels, but with decreasing frequency as the formulated level increased. All botulinogenic samples were proteolyzed. Samples stored for up to 84 days at 7 C were nontoxic and showed no evidence of proteolysis. Table 7 shows the numbers of botulinal toxic samples after holding bacon at 27 C when inoculated by the sand-spore mixture after slicing. As was true for samples inoculated before processing, increased nitrite levels in the bacon reduced the rate of toxin development and eventual number of toxic samples. Relatively few samples were confirmed to be toxic at 170 or 340 Ig of nitrite per g; however, there was at least one toxic sample at all nitrite levels studied. The total number of botulinogenic samples at the low inoculum, zero nitrite level was unexpectedly low.

v3-fos

2018-04-03T00:50:21.606Z

1974-05-01T00:00:00.000Z

26827697

s2

Proteinase Activity in Slow Lactic Acid-Producing Variants of Streptococcus lactis Variants of Streptococcus lactis that produce lactic acid slowly in milk were isolated by inducing plasmid loss in the wild type at 39 to 40 C. Such strains had lost most of their surface-bound proteinase activity and were designated prt-. The specific proteinase activities of S. lactis C10 prt+ whole cells and solubilized cell walls were 7 and 18 times, respectively, those of the prt- strain, but spheroplast lysates of prt+ and prt- strains contained similar proteinase activity. S. lactis H1 showed a similar relative distribution of activity between prt+ and prt- cellular fractions, although the overall level was lower. The limited growth in milk, characteristic of prt- strains, can be explained in terms of their low surface-bound proteinase activity. Streptococcus lactis and S. cremoris may spontaneously segregate slow variants that differ from the parent strain by their limited growth in milk (5). Such variants appear in susceptible cultures at high frequency (ca. 1%) and do not revert to the parental type (2,3,22). As slow variants can also be induced when the wild-type strain is treated with acridines or grown at high temperatures, this characteristic is believed to arise through loss of a plasmid (14). Evidence presented by a number of workers strongly suggests that slow variants are deficient in proteinase activity. Growth in milk is stimulated to wild-type levels by the addition of hydrolyzed casein (4), and in broth media both variant and parent strain show the same rates of growth and acid production (2). Milk cultures may contain up to 50% slow variants before the rate of acid production differs from that of the parent. This suggests a growth stimulatory interaction between the parent and the slow variant (14). Although "slowness" and relatively low proteinase activity have been correlated (2), there has been little direct evidence for proteinase deficiency in slow variants. Westhoff et al. (22) compared proteinase activity in whole and fractionated cells of S. lactis 3 and a slow acid-producing mutant. Quantitative differences in proteinase activity between the parent and mutant strains, assayed using either whole cells or cell fractions, were low (ca. 1.5-fold) and did not adequately explain the ' Present address: Department of Biology, McGill University, Montreal, Canada. growth characteristics of slow variants in milk. The mutant "intracellular" enzyme, however, did differ from that of the parent, and it was concluded that a different proteinase specificity was responsible for the limited growth of the mutant in milk (21). This study compares the proteinase activity of two strains of S. lactis (prt+) with that of slow variants derived from them (prt-). The behavior of the prt-strains in milk can be accounted for by the loss of most of their surface-bound proteinase activity. A portion of this paper was included in an M.S. thesis submitted by N lactis prt-strains were isolated at high frequency (up to 30%) after growth at 39 to 40 C. Prtclones developed as tiny colonies on citrate-milk agar (16) and were differentiated from the larger prt+ colonies on this medium. S. cremoris prt+ and prt-could not be differentiated on this medium. Optimal differentiation was obtained when the medium was autoclaved at 115 C for 15 min. prt-clones selected were lac+ on the medium of McKay et al. (9) and were sensitive to the same virulent phages as the parent. Skim milk was prepared from a single batch of spray-dried nonfat milk powder, reconstituted to 9.5% total solids and autoclaved at 10 lb/inch2 for 20 min. Plate counts were obtained using M,. agar (8) and T, broth (20) was used for cell preparation and growth experiments. Lactate determination. Lactate was measured as the lactate-ferric chloride complex at 400 nm (17). Cell fractionation and enzyme assay. The methods used were those of Thomas et al. (20). Cells growing logarithmically in T5 broth were harvested by centrifugation, lysed by enzymatic or mechanical methods, fractionated, and assayed for proteolytic activity using 125I-labeled casein as substrate. RESULTS Growth of prt+ and prt-in sterile milk and T5 broth. Doubling times of S. lactis C10 prt+ and prt-during exponential growth in sterile milk at 30 C were 60 and 72 min, respectively ( Fig. 1). 'ClO prt+, however, reached a maximum population of 2 x 10' colony-forming units (CFU)/ml in 7 h, whereas C10 prt-ceased exponential growth after 5 h and reached a maximum population density of 5.5 x 108 CFU/ml after 10 h. Both prt+ and prtstrains remained as diplococci throughout growth. Although the viable count of C10 prt-remained stationary after 10 h, acid production continued at a slow rate, and the milk reached pH 5.0 after 35 h of incubation. That is, C10 prt-cells continue to produce lactate while colony-forming units fail to increase (Fig. 2). The addition of trypsin-hydrolyzed casein (1 mg/ml) to cultures of C10 prt+ or C10 prt-growing in skim milk decreased the doubling time to 54 min for each strain, and both reached a maximum population density of 3 x 109 CFU/ml. In T5 broth, C10 prt+ and prtwere indistinguishable and showed the same growth rate (doubling time 38 min, maximum population 1.2 x 10' CFU/ml). This feature of growth was common to all prt-strains isolated in this laboratory and can be clearly seen in growth curves for S. Iactis H1 and S. cremoris R1 in T5 broth (Fig. 3). The slow acid-producing mutant of S. lactis 3 grew at a significantly slower rate than the wild type in T5 broth. Doubling times during logarithmic growth were 78 and 63 min, respectively. A prt-derivative of strain 3 was isolated and found to have identical growth characteristics to the parent strain in broth. Proteinase in whole cells and cell fractions. Intact cells of C10 prt+ exhibited about seven times the proteinase activity of C10 prt-, specific activities being 35.8 and 5.2 U per mg dry weight, respectively (Table 1). When the cell wall was removed under conditions that gave insignificant cell lysis (20), the majority of the prt+ proteinase activity was released. The Growth of S. lactis C10 prt+ and prt-in skim milk at 30 C. Two hundred-milliliter volumes of skim milk were inoculated with 2 ml of C10 prt+ and 8 ml of CIO prt-, respectively. Inocula were from 16-h, 22 C skim milk cultures. The inoculated milks were divided into portions and incubated. At intervals samples were removed for pH measurement (A, prt+; A, prt-); the culture was then chilled, diluted, and plated for colony-forming units (0, prt+; *, prt-). Fig. 1; lactate and colony-forming units were determined at intervals. same treatment removed less than half the prtactivity, the relative activity (prt+: prt-) being ca. 18: 1. No activity could be detected in membranes from either C10 prt+ or C10 prt-. The slightly higher intracellular activity de- Mid-log cells were washed twice in 0.2 M phosphate buffer, pH 6.4, suspended in spheroplasting medium (0.5 M sucrose, 20 mM MgCl2, 0.2 phosphate buffer, pH 6.4), and 3 ml phage-associated lysin was added. The suspension was incubated at 30 C for 120 min and centrifuged 35,000 x g, and the supernatant was assayed (20). c 35,000 x g pellet of solubilized cell walls, resuspended in buffer of equivalent volume to the original suspension. d Not detectable. tected on lysis of prt+, as compared with prtspheroplasts, is not considered significant. C10 prt+ and prtwere also fractionated after mechanical disintegration. The pellet containing cell walls and membranes (35,000 x A //^g , 10 min) contained 75% of the prt+ activity recovered in the component fractions but only 27% of the prt-activity. Specific activities were 18.6 and 1.3, respectively. Half of the prt-proteinase activity was associated with the cytoplasm (not sedimented at 157,000 x g, 120 min), whereas in the parent this fraction contained only one-tenth of the activity. Intact cells of S. lactis H1 prt+ (specific activity, 17.0) had less surface-bound proteinase activity than C10 prt+ (specific activity, 35.8), but the differences between prt+ and prtfollowed an identical pattern to that observed with C10. Specific activities of solubilized cell walls were 16.8 (prt+) and 3.6 (prt-); those of 3 4 the spheroplast lysates were 7.9 (prt+) and 10.7 (prt-). DISCUSSION The growth of prt-strains in sterilized milk and in broth follows the pattern established by other workers (2). prt-strains grow and produce lactic acid slowly in milk, but both characteristics can be restored to normal levels, or better, by supplementing the milk with casein hydrolysate (4). prt+ and prt-are indistinguishable when grown in rich broth media. The slow lactate increase in milk without increase in viable count was, however, of particular interest. Dissociation of acid production from net growth has been reported in other systems and is probably widespread. The phenomenon appears to be associated with conditions of cellular stress. Cultures of S. faecalis approaching the growth-limiting pH have been reported to cease dividing before acid production is inhibited (10). Lowrie et al. (7) have also observed that S. cremoris AM2 ceases to divide but continues to produce acid when growth is initiated at 30 C and the incubation temperature is raised to 37.8 C. Depletion of available nitrogen in milk cultures of prt-bacteria appears to be a further means by which this effect can be induced. Variants of lactic streptococci that produce acid slowly in milk have been isolated and studied in a number of laboratories (2,14,22). Although these can normally be isolated at high frequency, not all slow acid producers are of the prttype. It is not uncommon to find mutants that, for some other reason, grow more slowly in milk than the parent. These mutants also grow more slowly in broth, and the lacmutants are (9). It is essential therefore to screen putative prt-clones for growth rates in a broth medium where proteolytic activity is not essential for growth. The slow acid-producing strain of S. lactis 3 does not appear to be a prttype on the basis of its slow growth in T5 broth. This was confirmed when a prt-derivative of strain 3 was isolated that grew in broth at an identical rate to the parent strain. The enzyme assays clearly show the fundamental difference in proteinase activity between prt+ and prt-strains of S. lactis. The major portion of the proteinase activity in the parent strain has been shown to be localized near the cell surface using two methods of fractionation (20). Mechanical disruption and osmotic lysis both gave similar high levels of activity in fractions derived from the cell wall. In the present study, this activity has been found to be markedly reduced in prt-cells. S. lactis H1 had less total proteinase activity than strain C10 with a consequent reduction in relative activity between prt+ and prt-. The solubilized cell wall fraction from prt+, however, still had nearly five times the activity of the corresponding prt+ fraction. The low proteinase activity in prt-strains explains their limited growth in milk (see Fig. 1). It is likely that the maximum prt-cell count is determined largely by the initial amino acid and small peptide content of the sterilized milk. The specific activities of C10 prt+ and H1 prt+ spheroplast lysates showed that both parent strains carried a portion of their proteinase activity in fractions not associated with surface structures. The slight difference in levels of intracellular proteinase activity between prt+ and prt-in this respect is not considered significant. These intracellular enzymes may be responsible for degradation of peptides resulting from protein breakdown by the surface enzyme, as well as the turnover of endogenous nitrogen. Escherichia coli, for example, has at least eight distinct intracellular peptidases (18). Cells grown in broth were used for the present study due to the difficulty of harvesting bacteria from milk. The higher levels of available amino acids in broth may repress surface-bound proteinase activity, as has been reported with other extracellular proteinase systems (1,6,12,13). Hence, the differences found between prt+ and prt-strains in broth are probably an underestimation of the situation in milk. Westhoff et al. (22) reported that both "intracellular" and "membrane-associated" proteinase activities in S. lactis 3 were reduced by 30 to 35% in a slow acid-producing mutant. As the authors commented, such a difference might be expected to impair rather than prevent growth in milk. The only other difference between the two strains was an altered specificity of the intracellular enzyme (21). The present study shows that their conclusions cannot be applied to all slow lactic acid-producing strains of lactic streptococci. The loss of most of the surface-bound proteolytic activity accompanying the transition from prt+ to prt-is consistent with plasmid control of this character. Total proteolytic activity in lactic streptococci, however, is low compared with that of corresponding enzymes in some other bacteria (11). The proteinase character has not been recorded as a genetic marker in E. coli or Salmonella chromosome maps (15,19), however, and is possibly also plasmid linked in the enterobacteria. The fact that proteinase activity has been recognized and studied in the lactic streptococci is undoubtedly a consequence of the widespread use of milk as a culture medium for this group. The low level of cell-bound activity remaining in intact cells of C10 prt-is presumably determined by chromosomal genes as the possibility of significant cell lysis has been excluded (20). If the surface-bound proteinase is plasmid controlled, it is possible that it controls cellular activities other than proteinase synthesis. The transport of peptides has not been excluded, and further investigation will be required to clarify these points and physically identify the genetic element involved.

v3-fos

2020-12-10T09:04:12.305Z

1974-06-01T00:00:00.000Z

237235402

s2

Ultrastructure of a Thermotolerant Basidiomycete Possibly Suitable for Production of Food Protein The imperfect cellulolytic fungus Sporotrichum pulverulentum, which is commonly found growing in wood-chip piles, was grown in submerged culture on wheat shorts and other cereal flours. These substrates were broken down in 1 to 4 days at 30 to 40 C, and the mycelial mass was easily harvested by filtration. Scanning electron micrographs of hyphae in mycelial pellets are presented, and thin sections of conidia and hyphae were studied in a transmission electron microscope. Dolipores in septa of hyphae were observed, and cell walls are shown to be lamellar, which is characteristic of the Basidiomycetes. Actively growing hyphae are full of cytoplasm with numerous mitochondria, whereas old mycelial pellets contain highly vacuolated and almost empty cells. Large quantities of fruit bodies (sporophores) of many kinds of Basidiomycetes have long been eaten because they are nutritious and good tasting. Some higher fungi can be grown in submerged culture, but most of them grow slowly in conventional fermentors and only a few have been used for industrial production of food products (12). Other filamentous fungi, classified either as Ascomycetes or Fungi Imperfecti, are presently being investigated as unconventional sources of protein (13), and commercial production of "mycoprotein" by cultivation of a Fusarium sp. has recently been started (9). Before large-scale production of fungal biomass for human consumption is considered, it is necessary to prove that the product is nontoxic. Information on the systematic position of the organism studied is then useful. It is also important to determine how culture conditions influence the chemical composition and morphology of the organism. The protein content may vary considerably depending on culture conditions, but the nucleic acid content of fungi is usually lower than that of many bacteria, yeasts, and algae (13). Single-cell organisms may be easier to grow in submerged culture than fungi, which have a filamentous or pellet form of growth, but the latter can often be harvested by simple filtration. We have for some years studied the microbial degradation of cellulose and related polysaccha-rides. As part of this project, we have investigated the possibility of growing cellulolytic microorganisms on flours and meals of various cereals and other seeds. One such material is wheat bran, which contains 15 to 17% protein and about 70% carbohydrates, about 80% of which is cellulose and hemicellulose. Very large quantities of bran and other milling fractions containing bran are at present either used only as low-quality animal feed or wasted. The same applies to such products as rice bran, and it would be important if they could be converted to products of higher nutritional value. We have found that a thermotolerant, cellulolytic fungus can grow on various cereal flours, and preliminary experiments indicate that the mycelium has a high nutritional value. The organism we used is a wood-destroying, imperfect fungus that was isolated from a pile of wood chips by Nilsson (6). He found that it caused typical white-rot decay, and the organism was first classified as a Chrysosporium sp., but it has now been found (J. A. Stalpers, personal communication) to be identical with a fungus described as Sporotrichum pulverulentum by Novobranova (7). Its cellulolytic enzymes have been studied by Eriksson and Rzedowski (3), who purified several exo-and endoglucanases and other carbohydrases from cell-free fluids of submerged cultures of the fungus. This report describes some features of the ultrastructure of S. pulverulentum and effects 1142 of cultivation time on its cytoplasm. Studies on the effect of culture conditions on its growth morphology and chemical composition and the results of pilot-scale cultivation experiments are presented elsewhere. MATERIAL AND METHODS Fungal strain. The strain of S. pulverulentum that we studied was isolated in 1964 by Thomas Nilsson, College of Forestry, Stockholm, where it is maintained in the culture collection as strain P 127-1. It has also been deposited at Centralbureau voor Schimmelcultures, Baarn, The Netherlands, where it has the number CBS 671.71. Cultivation methods. Stock cultures were maintained on malt extract agar (Difco), on which large numbers of white conidia are formed. Scanning electron microscopy. Freeze-dried cell material was coated with evaporated gold before examination in a JSM-U3 scanning electron microscope operating at 25 kV. Transmission electron microscopy. Mycelial pellets from submerged cultures and pieces of agar containing hyphae were prefixed for 24 h in 2.5% glutaraldehyde buffered with Veronal-acetate buffer, pH 7. The material was further fixed in 2% potassium permanganate in water (4). After dehydration in a graded ethanol series, the material was impregnated with the plastic resin Epon 812, and this was polymerized at 60 C. Thin sections were cut with a diamond knife on an LKB ultramicrotome, and sections were post-stained with 2% aqueous uranyl acetate for 20 min at 60 C and with 6% aqueous lead citrate for 2 min at room temperature. Specimens were examined in a JEOL 100 B electron microscope operating with a double condensor at 60 kV. RESULTS Growth in liquid cultures. Conidia from agar cultures were used to inoculate Fernbach flasks containing a shallow layer of mineral salts medium with 1 or 2% wheat shorts. The cultures were incubated on a rotary shaker (150 rpm) at 30, 35, or 40 C. The spores germinated and formed small hyphal aggregates after about a day. In this medium and under these culture conditions, the small aggregates grew out into spherical pellets which increased in size during the following day with a concomitant decrease in pH to between 4 and 5. The pellets could easily be filtered off on a 30-mesh sieve giving an almost clear, slightly yellow filtrate which had a pleasant smell reminiscent of apples. The maximal growth temperature was between 40 and 45 C, but the fungus cannot be called thermophilic because it also grew below 25 C. Other milling fractions of wheat could be converted to fungal biomass at rates which depended on the fineness of the flour. It took 4 to 5 days at 35 C to degrade coarse wheat bran, whereas fine flours of rye and barley were degraded in a couple of days. Defatted rice bran, which consists of very fine particles, supported rapid growth and was completely degraded in about 2 days. Figure 1 illustrates a sample of freeze-dried mycelial pellets obtained on wheat shorts. Some small bran fragments were undegraded at the time of harvesting, but these could be washed away through a 30-mesh sieve. Growth on whole wheat flour gave smaller (1 to 2 mm), irregular hyphal aggregates, whereas very smooth, 4to 5-mm pellets were obtained on wheat bran. Electron microscopy. Figure 2 is a scanning electron micrograph of the interior of a mycelial pellet harvested from a 3-day-old culture on wheat bran. The mycelium is highly branched, and it can be seen at higher magnification ( Fig. 3) that many of the hyphae have collapsed to flat, fiber-like structures during the freeze-drying. Few conidia are formed in liquid cultures unless these are incubated for a long time or left standing at room temperature for some days. Figure 4 is a thin section of a young conidium attached to a hypha by means of a stalk cell which contains a supporting structure near the conidium. A cap-like structure can also be seen on top of the conidium, and its cell wall is still rather thin. When observed in the light microscope, most conidia are spherical, but some of them are dumbbell shaped (Fig. 5). This mature conidium has a thick, multilayered wall measuring almost 1 Mm, and it contains several nuclei and numerous mitochondria. Hyphae have thinner walls than the conidia, and old cells are often highly vacuolated. Figure 6 shows a thin section of a young hypha grown on malt agar. There is a distinct dolipore between the two cells and a peculiar membranous structure near the septum. The insert in Fig. 6 shows that the hyphal wall has a distinct lamellar structure, and pictures of old hyphae showed that these lamellae occasionally become partly separated from each other. Samples of small mycelial aggregates from 1 to 2 days old submerged on wheat shorts cultures contained a large proportion of hyphae which had an appearance similar to that of the hypha shown in Fig. 6, but very few of the septa had typical dolipores. Cultures which had been grown for more than 3 days on wheat bran contained 4-to 5-mm pellets in which the cells were more or less autolyzed. Figure 7 shows a thin section of some hyphae in a pellet grown on wheat bran for 5 days. DISCUSSION The increasing global demand for high-quality proteins for human food and animal feed has stimulated much work on large-scale production of microbial cells. Single-cell organisms are often easier to grow in submerged culture than filamentous fungi, but they are more difficult to harvest, and many of them contain so much nucleic acid that they cannot be used for human consumption. Many higher fungi are already accepted as a source of food in many parts of the world, but most of them grow slowly at rather low temperatures. Worgan (13) has recently discussed the relative merits of different substrates and general problems in the production of microbial protein. Cellulolytic fungi have earlier been tried for the conversion of paper and various agricultural wastes to animal feed (1,8,10), but many cellulolytic organisms grow very slowly or not at all on lignified plant material. Expensive pretreatments are therefore necessary in order to make such raw materials degradable. Some of the fungi tested, such as Trichoderma viride, Aspergillus fumigatus, and various Fusarium sp., may also produce mycotoxins under certain conditions. The fungus that we studied has some properties which suggest that it may become an interesting new source of protein. It grows rapidly at relatively high temperatures and can, degrade a variety of cheap carbohydrates such as cellulose, hemicellulose, and starch, and it can even grow on lignified tissues. Its systematic position remains somewhat uncertain as long as it must be classified among the Fungi Imperfecti, but our electron micrographs show that it must be an imperfect conidial stage of a basidiomycete. The presence of the characteristic dolipores in the septa of hyphal cells is thus a characteristic feature of Basidiomycetes (2). The lamellar structure of the hyphal walls is also considered to be typical of this group of fungi (5). Furthermore, only Basidiomycetes are known to cause white-rot attack on wood. It is possible that S. pulverulentum is closely related to the higher fungi of the family Polyporaceae, of which many are eaten. We have as yet only carried out preliminary feeding experiments with mycelium grown on wheat shorts, but no harmful effects were noticed when the fungus was given as sole source of protein to young rats, and their rate of growth was normal for over 3 weeks. More extensive feeding tests must, of course, be carried out to prove that the fungus is wholesome. Brans and other milling fractions of cereals contain cellulose, hemicellulose, and starch in varying proportions, and they are interesting raw materials for mycoprotein production. They are available in very large quantities and can easily be stored. Several technical problems must be solved before a fungus can be grown on a large scale on such partly insoluble substrates. Many fungi grow either in filamentous or pellet form, depending on the nature of the inoculum, composition of the medium, or physical conditions (11). It is important to be able to control these factors so that a maximal protein content is obtained in the mycelium. Our electron microscope studies show that large pellets of S. pulverulentum contain a high proportion of partly autolyzed cells, and it has been found that the protein content of these cells is only about 20%. It is therefore obviously desirable to grow the organism under conditions which favor the formation of small hyphal aggregates.

v3-fos

2018-04-03T00:26:23.239Z

1974-04-01T00:00:00.000Z

12073733

s2

Preparation of free heat-resistant ascospores from Byssochlamys asci. When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores. When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores. Byssochlamys fulva and B. nivea are heatresistant fungi that cause spoilage in canned and bottled fruit juices and juice mixes and in some canned fruit. First noticed in England in the early 1930s, these fungi have since caused spoilage outbreaks in Europe, Africa, North America, and Australia. In the processing of fruits and juices, the heat treatment is kept mild to avoid flavor damage, and Byssochlamys can survive the heat treatment and grow in the finished product. It is believed to cause no health hazard; it is a matter of the esthetics (2). These fungi are ascomycetes with spherical, eight-spored asci. The ascospores are believed to be the heat-resistant stage in the life cycle (1). Electron micrographs show a thin, structureless membrane surrounding the ascospores in the ascus (7), but the light microscope usually shows only the ascospores in their typical arrangement without any surrounding structure. MATERIALS AND METHODS Production of asci. The asci were produced by growing B. fulva strain NRRL 3493 on a thin layer of unacidified Difco potato dextrose agar for about 30 days at 30 C. The mycelium was then scraped, and the resulting mixture of hyphae and asci was suspended in water, from which the hyphae settled out so that the asci could be decanted as previously described (3). In our experience, conidiospores are rarely if ever seen with the asci. In this treatment, some of the asci were broken and their ascospores were released. To obtain a uniform spore preparation, it was therefore necessary to break the intact asci into ascospores or to separate them from the free ascospores. Spore counts. To determine the relative numbers of asci and ascospores in a population, differential counts were made with a phase microscope at x600. Relative frequencies of damaged and undamaged spores were determined in the same way. Dormant ascospores (either within the ascus or solitary) are refractile when seen under the phase microscope (3). For heat resistance determinations, counts of viable asci and ascospores were made by plating on unacidified potato dextrose agar. These counts are not very precise, because Byssochlamys forms very thin, rapidly spreading colonies, which are difficult to see when small. As a result, we planned for about 30 colonies per plate and made several replications to partly compensate for the low numbers of colonies. Nevertheless, the precision is lower than in most plate counts of bacteria. Heat resistance. Heat resistance was determined by using a thermal death-time flask (4, 5). Grape juice was preheated to 86 C and continuously stirred in a heated, three-neck flask. It was inoculated at zero time, after which samples were withdrawn at intervals for plate counts. This method is suitable for heat resistance determinations at temperatures below 100 C. RESULTS AND DISCUSSION Preparation of free ascospores. Asci were subjected to several treatments (Table 1, treatments 1-4) in order to break them into ascospores. None were completely successful, although treatments 2 to 4 could be used if the asci and ascospores could be subsequently separated. It is also reported that blending in a microhomogenizer (Sorvall) broke only 10% of the asci (9). Sublethal heating with stirring in a thermal death-time flask at 86 C (the method described above for heat resistance) caused almost complete breaking of the asci into ascospores in 5 min. However, without stirring, the asci remained intact, and others have reported (9) that asci remained intact when heated. Evidently the heat breakage of asci occurs under special conditions but is not a general phenomenon. Furthermore, ascospores heated at this temperature were heat shocked or heat damaged, and some probably were killed. Separation of free ascospores from asci by filtration has been reported (6). To test this method, we sonicated a suspension of asci and passed it through a Teflon filter membrane (30-60 ,um pore size; Chemware 75-X) to remove debris and then through a 75-M membrane (10-20 jim pore size). This should have been just small enough to retain the asci while passing the ascospores. In fact, although a few ascospores came through the filter, it plugged up so quickly that the yield was extremely low. Saccharomyces asci have been broken into their constituent ascospores (8) by incubation in a solution of Pronase (Calbiochem, Los Angeles, Calif.) followed by passage through a pressure cell (Aminco French pressure cell). When Byssochlamys asci were suspended in water and passed through the pressure cell, 99% or more of the ascospores were liberated from the asci (Table 1, treatment 6). This result was obtained regardless of the presence of Pronase, which was therefore omitted from further trials. The liberated ascospores formed large aggregates that could not be dispersed by shaking. Addition of 1 or 0.1% Tween 80 to the suspension before the pressure cell treatment prevented this, except for a few small groups of two to several ascospores after some treatments. When the treated ascospores were examined by microscopy, it was noted that some showed evidence of damage. Usually these had surface defects. A few had lost their refractility, thus showing evidence of internal change. A trial of several pressures showed that apparent damage to the ascospores was much more frequent at high pressures (Table 2). However, the plate counts were about the same throughout the pressure range, suggesting that the visually observed damage was superficial. Nevertheless, to minimize the possibility of damage, the pressure range of 4,000 to 9,000 lb/in2 is recommended. Theoretically, plate counts of the free ascospores should be almost eight times as high as those of the untreated asci. Actually they are about four times as high. This is probably because some of the free ascospores are dormant and fail to grow in time to be counted on the plates. If germinating asci are observed by microscopy, it can be seen that the eight ascospores never germinate simultaneously. Thus, when they are separated and plated, some do not form colonies soon enough to be counted. For the data shown in Table 2, samples were subjected to a flow rate out of the pressure cell of about 3 ml/min. At 9,000 lb/in2, a flow rate of 20 ml/min had the same effect. At 3,800 lb/in2, the 20 ml/min rate may have been somewhat less effective. The flow rate was 3 ml/min elsewhere in these experiments, although a higher rate would probably be acceptable for treatment of large amounts of material. Heat resistance of free ascospores. To show that ascospores retain their heat resistance after this treatment, we compared treated and untreated spores. The free ascospores had about the same heat resistance as the asci from which they came, as shown by a thermal death-time plot for untreated asci and treated ascospores at 86 C (Fig. 1). Each line is the most probable (best least squares) fit to the corresponding points. That the lines have nearly the same slope shows that the treatment did not greatly change the heat resistance of the spores. As previously stated, plate counts on Byssochlamys lack precision, and this probably accounts for the scatter in the points.

v3-fos

2020-12-10T09:04:20.904Z

1974-11-01T00:00:00.000Z

237232200

s2

Rapid Detection of Bovine Mycoplasma Antigens by Counterimmunoelectrophoresis Twelve reference strains of mycoplasma and acholeplasma previously reported to have been recovered from cattle were tested against hyperimmune rabbit serum by counterimmunoelectrophoresis. This technique detected antigen by the formation of precipitin lines with antibody within 1 h and promises to be a useful technique for detecting and identifying mycoplasma isolates in either pure or mixed cultures. Twelve reference strains of mycoplasma and acholeplasma previously reported to have been recovered from cattle were tested against hyperimmune rabbit serum by counterimmunoelectrophoresis. This technique detected antigen by. the formation of precipitin lines with antibody within 1 h and promises to be a useful technique for detecting and identifying mycoplasma isolates in either pure or mixed cultures. Mycoplasma isolates are presently classified on the basis of cultural, biochemical, and antigenic characters (8). The antigenic relationships of bovine mycoplasmatales have been examined by means of metabolic inhibition (3), immunodiffusion, growth precipitation, and growth inhibition (2). However, these techniques require at least 12 h to 8 days for the detection of mycoplasma antigens or antibodies. Therefore, a faster, reliable, serological method was sought. Counterimmunoelectrophoresis is widely used in many laboratories to detect hepatitisassociated antigen (4) and other viral and bacterial antigens and antibodies. We applied this technique for the rapid detection of bovine mycoplasmatales antigens with hyperimmune rabbit serum as the source of antiserum, and favorable results were obtained. The strains used in the production of antisera are listed in Table 1. Strains B12PA and B142P were supplied by J. L. Al-Aubaidi (1), and the other strains were supplied by the National Collection of Type Cultures (London, England). The antigens and hyperimmune antisera were prepared according to the methods of Langford and Leach (7). The antigens used in the counterimmunoelectrophoresis were suspended to a final protein concentration of 10 mg/ml. The counterimmunoelectrophoresis tests were carried out on glass plates (75 by 50 mm) covered with 10 ml of 1% Ionagar with 0.02% sodium azide in tris(hydroxymethyl)aminomethane-barbital-sodium barbital buffer (pH 8.8, ionic strength 0.025). Three sets of parallel rows of wells, 3 mm in diameter, spaced 7 mm apart were cut in the agar. The antigen was placed in the wells on the cathode side, and antiserum was placed in the anode wells. Electrophoresis was carried out at room temperature at 250 V. Figure 1 illustrates a pattern of counterimmunoelectrophoresis. A single, weak precipitin line generally became visible within 15 min of starting electrophoresis; however, stronger or multiple lines, or both, appeared at the termination of electrophoresis, usually 1 h. Table 1 shows the reactions of antigens of 12 reference strains with homologous and heterologous antisera. All of the antigens tested reacted with their homologous antisera: Antigens prepared from Mycoplasma bovigenitalium, Acholeplasma laidlawii, M. bovirhinis, and M. bovoculi cultures reacted only with -their homologous antiserum. However, A. modicum and Mycoplasma sp. Group 7 (Leach) antigens cross-reacted with anti-M. bovigenitalium serum, but the reverse reactions did not occur. Similarly, such oneway cross-reactions were observed with M. agalactiae var. bovis antigen against anti-M. bovirhinis serum; with A. modicum antigen with anti-Leach Group 7 antiserum; and with Leach Group 7 with anti-M. alkalescens antiserum. These one-way cross-reactions may be explained as follows. (i) M. bovigenitalium may contain common antigens with A. modicum and Leach Group 7 in sufficient amounts to elicit an antibody production in rabbits. However, the antigens are not sufficient in quantity to react with the heterologous antisera, or (ii) A. modicum or Leach Group 7 have antigen(s) which react with anti-M. bovigenitalium antisera, but the antigen(s) do not elicit enough antibody response to react with M. bovigenitalium antigen. Thus, the concentration of cross-reacting antigen and the amount of cross-reacting antibody might determine the a Antisera were prepared in rabbits using organisms grown in Difco PPLO broth supplemented with boiled yeast extract (10% vol/vol of a 25% aqueous extract) and 20% rabbit serum, except for M. dispar, which was grown in the media described by Gourlay and Leach (5) in which the fetal calf serum was replaced by rabbit serum. Serological test antigens were grown in the same media supplemented with horse serum and fetal calf serum, respectively. (-), Negative reaction. one-way or reciprocal cross-reaction. It is interesting to note that M. dispar antigen reacted with anti-M. agalactiae var. bovis serum when the antiserum was prepared by immunizing the rabbit with whole cell antigen, whereas when the antiserum was prepared by immunizing the rabbit with a cell membrane antigen preparation from M. agalactiae var. bovis, M. dispar antigen did not react with it. This result probably indicates the specificity of the membrane preparation. Several workers have studied cross-reactions between mycoplasma species using double immunodiffusion (2, 6). Kenny (6) observed recip-rocal cross-reaction among M. gallinarum, M. arginini, and M. gateae. These organisms are the arginine-positive, glucose-negative species. On the other hand, Erno and Jurmanova (2) observed reciprocal cross-reaction among M. arginini, M. gateae, and the other arginine-positive, glucose-negative species M. alkalescens. However, these three organisms did not crossreact with anti-M. gallinarum, and M. gallinarum did not react with anti-M. gateae, which observations are contrary to the findings of this study and of Kenny (6). In the present study, reciprocal cross-reactions were observed among M. alkalescens, M. arginini, M. gateae, and M. gallinarum (Table 1). It was observed that when three different rabbit antisera against M. arginini were reacted with these four arginine-positive, glucose-negative species, our first serum reacted only with the homologous strain. After further immunization of the rabbit, the serum reacted more intensely with the homologous strain and also cross-reacted with M. gateae. However, anti-M. arginini serum (supplied by H. Erno) reacted with all of these four strains. This shows that the amount of antibody is a factor in determining one-way cross-reaction or reciprocal cross-reaction. In the present experiment, it was generally observed that the homologous reactions were stronger and usually consisted of more lines than were observed in the heterologous reactions. In the counterimmunoelectrophoresis, antigen which shows a negative charge and migrates anodally will react with antiserum. Antigen which does not migrate or migrates cathodally will not show precipitin lines, contrary to double immunodiffusion. It was demonstrated that counterimmunoelectrophoresis is a more sensitive test than growth inhibition and growth precipitation tests. Anti-M. agalactiae var. bovis rabbit antiserum was titrated using these three tests. By the counterimmunoelectrophoresis test, antibody was detected up to serum dilution of 1:40, whereas by growth inhibition and by growth precipitation tests, the antibody was detected only up to serum dilution of 1:5. This counterimmunoelectrophoresis is simple and rapid; therefore, it could be utilized as a preliminary method for detecting and identifying bovine mycoplasmas recovered from biological specimens either in pure or in mixed cultures. It may be used in conjunction with or as a replacement for other cultural, biochemical, or serological tests used. In a preliminary experiment, mycoplasma isolates were cultured in 25 ml of broth media, and the cultures were concentrated by centrifugation 2 to 3 days later. The pellets were suspended in saline and reacted against a battery of hyperimmune, rabbit anti-mycoplasma typing sera in counterimmunoelectrophoresis tests. The results of this experiment indicate that such a test is feasible. The counterimmunoelectrophoresis test also promises to be a useful confirmatory test for classifying mycoplasma isolates.

v3-fos

2020-12-10T09:04:17.148Z

1974-04-01T00:00:00.000Z

237233280

s2

Requirement for and Sensitivity to Lysozyme by Clostridium perfringens Spores Heated at Ultrahigh Temperatures The requirement of ultrahigh temperature (UHT)-treated Clostridium perfringens spores for lysozyme and the sensitivity of heated and unheated spores to lysozyme were studied. The UHT-treated spores requiring lysozyme for germination and colony formation originated from only a small portion of the non-UHT-treated spore population. This raised a question of whether the requirement for lysozyme was natural to the spores or was induced by the UHT treatments. However, these spores did not require lysozyme for germination before UHT treatment, which confirmed that the requirement for lysozyme had been induced by the UHT treatment. Only 1 to 2% of the spores were naturally sensitive to lysozyme; therefore, the mere addition of lysozyme to the plating medium did not permit the enumeration of all survivors. Treatment of UHT-treated spores with ethylenediaminetetraacetate (EDTA) sensitized the spores to lysozyme and increased by 10- to 100-fold the number of survivors that were detected on a medium containing lysozyme. Under the heating conditions used, spores that were naturally sensitive to lysozyme and spores that required EDTA treatment were equally heat resistant. The recovery of severely heated Clostridium perfringens spores was greatly improved if the enumeration medium was supplemented with lysozyme (1,3,4). Lysozyme did not increase the colony counts of heat-activated spores, which suggested that the requirement for lysozyme was not natural to the spores but had been induced by the heat treatments (injury). For C. perfringens spores heated at ultrahigh temperatures (UHT), however, the spores responding to lysozyme in the recovery medium were derived from only a small portion (1 to 2%) of the non-UHT-treated spore population (1). Had these spores naturally required lysozyme for germination, a 1 to 2% difference between the colony counts of heat-activated spores enumerated on a medium with or without lysozyme would not have been detected by the agar plate count method (plating duplicate samples). It was not certain, therefore, that the spores had actually been injured by UHT treatment. When UHT-treated C. perfringens spores were enumerated on a medium containing lysozyme, the time-survivor curves were biphasicconcave (1). Such curves are typically observed for the inactivation of spore populations composed of two types of spores. The biphasic l Paper no. 4224 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. time-survivor curves were observed only when lysozyme was present in the medium and when lysozyme germinates C. perfringens spores (3,4), which suggested that the two types of spores differed in their sensitivity to lysozyme or in the heat resistance of their outgrowth systems. The distinction is vital to the development of thermal processes capable of lowering to an acceptable level the number of viable C. perfringens spores in a food. Unless all injured survivors are sensitive to lysozyme, the mere addition of lysozyme to the recovery medium is inadequate for their detection. The findings presented here show that: (i) the requirement for lysozyme was induced by heating; (ii) within a spore population, the spores differed in their sensitivity to lysozyme; and (iii) enumeration of all survivors required that the heated spores be sensitized to lysozyme prior to enumeration on a medium containing lysozyme. MATERIALS AND METHODS Maintenance of test organisms, composition and preparation of media, preparation of spore suspensions, heat treatments, and methods for enumeration of surviving spores have been described (1). Germination of lysozyme-sensitive spores in a complex medium. An aqueous suspension (10' spores/ml) of C. perfringens NCTC 8798 spores was heated at 75 C for 20 min to activate the spores and was then divided into two portions. Portion A was held in ice. Portion B was centrifuged at 12,100 x g for 20 min, the supernatant was removed, and the spores were suspended in Trypticase (15 g/liter)-yeast extract (10 g/liter) broth -(TYB). This medium was similar to the Trypticase-yeast extract-citrate-sulfite agar (TYCS) plating medium used for the enumeration of survivors (1). The spores were held at 35 C for 1 h, heated at 75 C for 20 min to kill germinated spores, washed twice with water, and resuspended to the original volume. Both portions were heated at 105 C in capillary tubes, and survivors were enumerated on TYCS plus lysozyme (18 U/ml) (Sigma Chemical Co., St. Louis, Mo.). Germination of lysozyme-sensitive spores by lysozyme. Non-heat-activated strain 8798 spores were suspended in 50 mM sodium phosphate buffer (pH 7) with or without 18 U of lysozyme per ml. The spores suspended in each medium were incubated at 45 C for 1 h and then heated at 75 C for 20 min to kill any germinated spores and heat activate the ungerminated spores. The spores were then washed twice, suspended in distilled water, heated at 105 C, and enumerated on TYCS plus lysozyme (18 U/ml). EDTA sensitization of UHT-treated spores. Duplicate capillary tubes, each containing 0.05 ml of UHT-treated spore suspension, were crushed in 100 ml of 10 mM sodium ethylenediaminetetraacetate (EDTA; pH 9.5) and held at 45 C for 1 h (2). The spores then were diluted in 0.1% peptone water (plus 0.02% Antifoam B, Sigma Chemical Co. St. Louis, Mo.) and enumerated on TYCS with or without lysozyme (18 U/ml). RESULTS AND DISCUSSION Injury during UHT treatment. We reported (1) that UHT-treated C. perfringens spores requiring lysozyme for colony formation appeared to derive from only 1 to 2% of the non-UHT-treated spore population. This portion of the total population was so small that it was not certain if the requirement for lysozyme was induced by UHT treatment (injury) or was natural to the spores. If these spores naturally required lysozyme for germination and colony formation, a medium containing lysozyme should be effective for the enumeration of all survivors. However, if these spores were injured during UHT treatment, the remaining spores also may have been injured but were not detected on TYCS plus lysozyme. The mere incorporation of lysozyme in the enumeration medium may not be sufficient for the detection of all survivors. To determine whether spores requiring lysozyme after UHT treatment were able to germinate normally before UHT treatment, heatactivated strain 8798 spores were incubated in TYB, heated to kill germinated spores, and then UHT treated. Spores incubated in water instead of TYB were treated similarly. Colony counts of non-UHT-treated spores incubated in water or TYB were 1.3 x 10'/ml and 7 x 107/ml, respectively, indicating that 94% of the spores had germinated in TYB (Fig. 1). If 1 to 2% of the spores had been unable to germinate normally in TYB (i.e., required lysozyme for germination), they should constitute 17 to 33% of the spores remaining after this extensive germination. This change in the composition of the spore population would be reflected in the time-survivor curves, because only the second phase of the curves represented a response to lysozyme (1). However, the time-survivor curves for spores held in TYB or water were identical, indicating that the relative composition of the spore suspension was unchanged by germination in TYB. The spores that required lysozyme after UHT treatment had been able to germinate normally before UHT treatment. This confirmed that the spores had been injured by the UHT treatments and suggested that other spores also may have been injured but were not detected on TYCS plus lysozyme. Basis for the biphasic time-survivor curves. The time-survivor curves were biphasicconcave only when lysozyme was used in the enumeration medium (1). Lysozyme reportedly germinated injured C. perfringens spores (3,4). If lysozyme germinated only some of the spores, the biphasic survivor curves must represent the rapid injury of spores not sensitive to lysozyme and the slower inactivation of the outgrowth system of spores sensitive to lysozyme. Alternatively, if all of the spores are sensitive to lysozyme, the two portions of the survivor curves reflect inactivation of the outgrowth systems in spores that differ in heat resistance. In the latter case, many of the heated spores germinated by lysozyme would not complete outgrowth, and the number of spores germinated by lysozyme should be up to 100-fold greater than the number of survivors enumerated on a medium containing lysozyme. To test this, strain 8798 spores were heat activated and then UHT treated at 105 C for 2.5 or 5 min. Survival and germination activity of non-UHT-treated and UHT-treated spores were determined. The data in Table 1 show that UHT treatment greatly reduced the number of spores able to germinate in TYB. Germination of UHT-treated spores in TYB plus lysozyme, however, was no greater than in TYB without lysozyme, and the extent of germination in either medium was similar to the level of survival measured on TYCS plus lysozyme. This indicated that the majority of the UHT-treated spores were not sensitive to lysozyme and suggested that the biphasic nature of the time-survivor curves was based on differences among the spores in their sensitivity to lysozyme rather than on differences in the heat resistance of their outgrowth systems. Results of an earlier study (1) suggested that a portion of strain 8798 spores was naturally sensitive to lysozyme. The spores required heat activation for normal germination, and germination of a small percentage of the spores by lysozyme was indicated by the higher colony counts of non-heat-activated spores on TYCS with lysozyme than on TYCS without lysozyme. The experiment was repeated with 10 replicates for each medium so that the results could be statistically analyzed. In the absence of lysozyme, 3.7% of the spores formed colonies; with lysozyme in the medium, 5.9% of the spores germinated and formed colonies. This increase of 2.2 percentage units was statistically significant of P = 0.025 (t test, degrees of freedom = 13) and was similar in magnitude to the percentage of heat-activated spores that had appeared to be unique in their sensitivity to lysozyme or the heat resistance of their outgrowth systems (1). In another experiment, unheated strain 8798 spores were incubated in a lysozyme-phosphate buffer mixture to allow the germination of any Clostridium perfringens NCTC 8798 spores to lysozyme on subsequent inactivation kinetics and recovery on TYCS plus lysozyme (18 U/ml). VOL. 27, 1974 lysozyme-sensitive spores prior to heat activation and UHT treatment. Spores held in phosphate buffer without lysozyme served as a control. The lysozyme pretreatment did not affect colony counts of heat activated-non-UHT-treated spores (data not presented) or the inactivation kinetics of spores not responding to lysozyme in the plating medium (represented by the first phase of the survivor curves, Fig. 2). However, for lysozyme-pretreated spores, the first phase of the survivor curve extended to a lower level of survivors before the response to lysozyme in TYCS (indicated by the second phase of the time-survivor curve) was observed. Extrapolation of the second phase of each survivor curve indicated that 97% of the spores that normally would have been germinated by lysozyme in TYCS already had been germinated by lysozyme during pretreatment. This confirmed that a small portion of the unheated spores was naturally sensitive to lysozyme, and suggested that it was recovery of the surviving fraction of these spores that was enhanced by the use of lysozyme in TYCS. Generally, bacterial spores are resistant to lysozyme and require some sensitization treatment (5). Bacillus megaterium ATCC 9885 spores appear to be the only exception (6). However, natural sensitivity to lysozyme by a very small portion of a spore population, as shown here for C. perfringens NCTC 8798 spores, would be easily overlooked. The natural sensitivity of B. megaterium ATCC 9885 spores may not be as unique as the large percentage of the spores having this trait. To confirm that the biphasic time-survivor perfringens spores were sensitized lysozyme by EDTA (2). The EDTA treatment did not influence the recovery of spores on TYCS lacking lysozyme, but increased by up to 100-fold the number of survivors detected on TYCS plus lysozyme (Fig. 3). This was observed for strain 8798 spores heated at 105 or 120 C (Fig. 3) and for two other strains of C. perfringens spores ( Table 2). The time-survivor curves for EDTAtreated spores enumerated on TYCS plus lysozyme were linear and unbroken, which indicated that all of the spores had been sensitized to lysozyme and that the biphasic nature of the time-survivor curves resulted from the resistance of many of the survivors to lysozyme. When survivors were enumerated on TYCS plus lysozyme, the D values (decimal reduction times: times required for a 90% decrease in the number of viable spores) for spores requiring EDTA treatment were similar to those for spores naturally sensitive to lysozyme. This indicated that, for the particular heating conditions used, the two types of spores were equally heat resistant. The results demonstrate that many of the C. perfringens spores surviving UHT treatment were injured and required lysozyme for germination and colony formation, but the majority were not sensitive to lysozyme; maximal recovery of survivors required that the heated spores be sensitized to lysozyme prior to enumeration. In the absence of such a treatment, more than 90% of the survivors were not detected, and the actual heat resistance of these spores was unknown. The usefulness of lysozyme for the recovery of injured spores may not be limited to C. perfringens spores. Alderton, Chen, and Ito (Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 17, 1973) reported that lysozyme increased the recovery of heated C. botulinum spores. As with C. per-fringens spores, however, the time-survivor curves were linear and unbroken when survivors were enumerated on a medium lacking lysozyme, but were biphasic-concave when lysozyme was used in the medium. This suggests that the effective use of lysozyme for the enumeration of injured C. botulinum spores, or spores of other species, may require that the spores be sensitized to lysozyme before the survivors are enumerated.

v3-fos

2020-12-10T09:04:23.016Z

1974-08-01T00:00:00.000Z

237233013

s2

Evolution of Dimethylselenide from Soils Alcohols, carbonyl compounds, and fatty acids were formed in two glucose-amended soils incubated under argon, but dimethylselenide was evolved under argon only from one, a selenium-rich clay, after the addition of selenite and glucose. Substantial quantities of dimethylselenide were released from the four soils tested when they were incubated with glucose and selenite in air. No dimethylselenide was produced in the selenium-rich clay soil in air if it received glucose but no selenite. Fungi of several genera are able to form dimethylselenide in axenic culture, and species of Scopulariopsis, Penicillium, and Aspergillus have been demonstrated to be capable of synthesizing this product from inorganic selenium compounds (2,3). At least one coryneform bacterium also can convert inorganic selenium to dimethylselenide in vitro (J. W. Doran and M. Alexander, unpublished data). Nevertheless, apart from the demonstration of the evolution of trace quantities from samples of amended sewage (3), nothing is known about dimethylselenide formation in natural ecosystems. The potential for such an activity is suggested by a report that volatile selenium is released from soil apparently as a result of microbial action (1), but the identity of the compound (or compounds) was not determined. Selenium is present in many soils as selenate and selenite, and is probably present in organic compounds derived from plant tissues and microbial cells (6). In some regions, it is present in such high concentrations that plants accumulate the element to levels that are toxic to animals consuming the plants. On the other hand, the soils of many regions are deficient in this element, and, inasmuch as selenium is essential for livestock, its addition to the land is under consideration. Because volatilization would change the quantity in soil that might be available for assimilation by forage plants and also might lead to the presence of selenium in the atmosphere, possibly to account for its transport to remote areas such as Antarctica (8) and the ice sheets of Greenland (7), a study was initiated to assess whether this compound might be discharged from soil. MATERIALS AND METHODS Samples of four soils were used: a selenium-rich clay soil from South Dakota containing approximately 30 ppm of selenium, 2.5% organic matter, and with a pH of 7.2; Honeoye silt loam from New York, with a pH of 5.5 and 5.6% organic matter; Croghan loamy sand from New York, with a pH of 5.9 and 4.6% organic matter; and a sandy loam with a pH of 5.9 and 0.5% organic matter from the Sonoran Desert near Tucson, Ariz. The soils were air-dried and passed through a 2-mm sieve prior to use. The Croghan loamy sand had been stored air dry for approximately 15 years, and the other soils had been stored for periods ranging from 6 months to 2 years. A 10-g sample of soil was placed in a 50-ml glass bottle equipped with an inlet and an outlet tube. The soil was amended with water only or with a solution containing 100 mg of glucose or 100 mg of glucose and 10 mg of NaSeO, in sufficient water to bring the soil to field capacity. The inlet tube of the sample bottle was connected to a gas manifold, and the outlet was connected to a stainless steel tube 15 cm long by 3 mm outer diameter. The steel tube contained Porapak QS (100 to 120 mesh). A stream of high-purity grade argon or pure-grade air (Union Carbide, Linde Division) was passed through the sample bottles to sweep the volatile compounds present in the head space into the Porapak trap, where they were retained. The flow rate was regulated at 1 to 2 ml/min by means of a needle valve placed between the sample bottle and the Porapak trap. The needle valve and a clamp placed between the sample bottle and gas manifold allowed the sample to be isolated when the trap was removed for analysis. The volatile metabolites retained in the trap were analyzed with a gas chromatograph-mass spectrometer, Perkin-Elmer model 270, at regular intervals. The trap was inserted into the injector port of the gas chromatograph and connected to the chromatographic column. The injector port was heated at 250 C to inject the contents of the trap into the chromatographic column (1.83 m by 3 mm outer diameter), which contained Chromosorb 101 (100 to 120 mesh). The column temperature was maintained at 25 C for 4 248 min, and then it was programmed to 100 C at a rate of 32 C/min and from 100 to 250 C at a rate of 6.5 C/min. The mass spectrum obtained for each compound was compared with the mass spectra of authentic compounds for the purposes of identification. RESULTS AND DISCUSSION Soils receiving water but no other amendment did not produce detectable amounts of volatile compounds under either air or argon. When the seleniferous clay soil was amended with glucose or glucose and sodium selenite and incubated under argon, however, ethanol, n-propanol, nbutanol, acetone, methyl ethyl ketone, methyl propyl ketone, acetic acid, and butyric acid were found at various times during a 48-day incubation period. In addition to these compounds, traces ( <2 ug) of dimethylselenide were found when sodium selenite was included in the amendment. Similar experiments with the silt loam yielded ethanol, n-and isopropanol, n-butanol, acetone, methyl ethyl ketone, ethyl acetate, ethyl butyrate, and butyl butyrate. Dimethylselenide was not detected, and the only significant difference in products between the silt loam receiving glucose alone and the same soil receiving glucose and selenite was the generation of n-hexanol when the selenium salt was added. All four soils amended with glucose and NaSeO and incubated under a stream of flowing air evolved dimethylselenide. The quantities of this metabolite evolved from the four soils during a 48-day incubation period are shown in Table 1. The initial rate of evolution was more rapid in the silt loam and the seleniferous clay than in the other soils, but a considerable amount of dimethylselenide appeared in the sandy loam late in the incubation period. Little evolution was evident from the loamy sand. The amount of selenium recovered as dimethylselenide was approximately 2% of that added with the seleniferous clay, sandy loam, and silt loam, and the recovery was about 0.3% with the loamy sand. Ethanol and acetone were also found in the headspace over the silt loam, seleniferous clay, and loamy sand during the first 15 days, but only dimethylselenide was detected in the headspace thereafter. No volatile organic compounds other than dimethylselenide were detected in the sandy loam. When the seleniumcontaining South Dakota clay was amended with only glucose and incubated in air, ethanol, acetone, iso-propanol, and methyl ethyl ketone but no dimethylselenide were detected in the first 7 days, and no volatile compounds were evident in the next 53 days. The results suggest that the microbial methylation of selenium is potentially widespread. This activity was found in the seleniferous soil and also in other soils when a readily available carbon source and sodium selenite were added. Because the seleniferous clay did not evolve the alkyl selenium product when amended with glucose only, the evolution must require that soils contain the element in a suitable concentration or in a form which is readily utilized by the microorganisms responsible for methylation. Volatile organic selenium compounds other than dimethylselenide were not detected by the procedures employed, yet they may be generated under certain circumstances. The ecological significance of the finding that a volatile methylated selenium compound can be formed in soil is not clear. In this regard, it is noteworthy that selenium is found in the atmosphere above the geographic South Pole (8) and in the ice sheets of Greenland (7), and it is plausible to suggest that the element reaches these remote sites as a result of the discharge of dimethylselenide from soils in the tropics or temperate zone. Indeed, a parallel may be drawn with dimethylsulfide, which has recently been proposed to be the major volatile carrier of sulfur in the natural sulfur cycle (4). If dimethylselenide is, in fact, a common metabolite evolved from natural ecosystems, it would also be of great importance to learn more of its toxicity, especially in view of the known hazards of methylmercury and trimethylarsine. McConnell and Portman (5) reported that dimethylselenide had a low mammalian toxicity, the median lethal dose to mice and rats being greater than 1.0 g/kg when given intraperitoneally, but of significance in the context of the present investigation would be the toxicity of inhaled dimethylselenide. Furthermore, the identities of the organisms responsible for the formation of dimethylselenide in soil have yet to be determined, although it is known that fungi of diverse genera are able to bring about selenium methylation in culture media. It is clear, therefore, that additional studies of the ecological significance, the microbial formation, and possibly the toxicology of dimethylselenide are warranted. ACKNOWLEDGMENT This investigation was supported by grant NGR 33-010-127 from the National Aeronautics and Space Administration. We thank A. N. MacGregor and 0. E. Olson for providing us with soil samples and Scott Smith for his able technical assistance. @

v3-fos

2018-04-03T01:50:50.244Z

1974-11-01T00:00:00.000Z

31297207

s2

Chemical disinfection of holding-tank sewage. A number of chemical disinfectants were evaluated for their bactericidal and virucidal effectiveness in holding-tank sewage. It was found that the disinfection efficiencies of formaldehyde, benzalkonium chloride, cetylpyridinium chloride, and methylene blue were markedly improved if the pH of the sewage was raised from 8.0 to 10.5. When formaldehyde, benzalkonium chloride, and methylene blue were tested with either 2-week holding times with no sewage additions or 10-day holding times with daily sewage additions, disinfection effectiveness was maintained as long as the sewage pH was kept at 10.5 and the disinfectant concentration was kept at 100 mg/liter or more. Calcium hypochlorite, zinc sulfate, and phenol were found to be relatively ineffective disinfectants for holding-tank sewage. Holding tanks and chemical toilets are widely used for small-scale waste containment and disposal in a variety of situations where on-site waste treatment systems are impractical, such as on aircraft, boats, and trains and in camps, parks, mobile homes, and construction sites. The trend toward increased use of such facilities and vehicles and a possible ban on waste discharges from vessels in navigable waters (3) indicates that the use of chemical toilets and holding tanks will continue to increase in the future. Although a variety of disinfectants and "sanitizers" are available for use in such systems, few studies have been reported in the literature on their disinfection efficiency in holding-tank or chemical-toilet wastes, particularly for enteric viruses. The purpose of this study was to evaluate a number of chemicals for their bactericidal and virucidal activity in holding-tank and chemical-toilet sewage and to develop conditions for their efficient use. MATERIALS AND METHODS Sewage. To simulate chemical-toilet and holdingtank sewage, which is generally stronger than typical domestic wastewater, a simulated sewage with a suspended-solids concentration of about 1,000 mg/liter was used in this study. The sewage contained per liter: 3.5 g (wet weight) of fecal material, 10 ml of urine, 0.25 g of toilet tissue, 0.4 ml of liquid hand soap, 100 ml of domestic raw sewage, and 885 ml of dechlorinated tapwater. The components were com-I Present address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, N.C. 27514. bined and blended for 1 min, and the mixture was used immediately. Virus and virus assays. Type 1 poliovirus strain LSc was selected as representative of the enteric viruses potentially present in sewage and was used exclusively in this study. The virus was assayed in baboon kidney cell cultures by the plaque-formingunit (PFU) method as previously described (4). Bacteria and bacteria assays. The sewage used in this study had a heterogeneous population of bacteria which was largely contributed by the fecal material and domestic raw sewage it contained. Treated-and untreated-sewage samples were assayed for total viable bacteria on tryptone glucose yeast agar plates (1). Bacteria concentrations were expressed as colony-forming units (CFU) per milliliter. Disinfectants. The following germicidal agents were used in this study: methylene blue, histological grade; phenol, certified grade; formaldehyde, reagent grade; and zinc sulfate, reagent grade (all the above from Fisher Scientific, Fair Lawn, N.J.); benzalkonium chloride (BAC) (Sterling Drug, Montvale, N.J.); calcium hypochlorite (HTH; Olin Chemical, Stamford, Conn.); and cetylpyridinium chloride (CPC), practical grade (MC/B Chemical, Norwood, Ohio). Those chemicals not already in a liquid form were prepared for use in experiments as concentrated stock solutions (wt/vol) in sterile distilled water. Treatment of samples for bacteria and virus assay. Sewage samples taken for bacteria and virus assay were treated when they were obtained, either to SOBSEY ET AL. RESULTS Effect of disinfectant concentration and pH on bactericidal and virucidal activity. It has been known for some time that increasing pH above physiological levels improves the bactericidal activities of many quaternary ammonium compounds (2,6). In addition, previous studies in our laboratory (Wallis et al., unpublished data) have shown that the bactericidal and virucidal activities of methylene blue and certain other dyes are improved at alkaline pH levels. Therefore, in the initial screening of chemical agents for germicidal activity in holding-tank sewage as a function of disinfectant concentration, the experiments were conducted at both the natural pH of the sewage (pH 8.0) and at pH 10.5. In these experiments, poliovirus was added to pH 8.0 raw sewage to give an initial virus concentration of about 106 PFU/ml. The sewage was divided into 2 volumes, one of which was adjusted to pH 10.5 with Ca(OH)2. Each volume was further divided into 100-ml samples, and disinfectants were added to sam-ples to give the concentrations shown in Tables 1 and 2. One volume each of pH 8.0 and pH 10.5 sewage received no disinfectant and served as a control. The samples were held at 25 C in loosely capped bottles, and after 24 h, pH values were measured and bacteria and virus assays were made (Tables 1 and 2). HTH was highly bactericidal and virucidal throughout the entire concentration range tested at both pH 8.0 and 10.5. At pH 8.0, BAC, CPC, methylene blue, and formaldehyde demonstrated increasing bactericidal effectiveness with increasing concentration, whereas at pH 10.5 they were highly bactericidal at even the lowest concentrations tested. Formaldehyde demonstrated appreciable virucidal activity throughout the entire concentration range at both pH levels tested. BAC, CPC, and methylene blue were not highly virucidal at pH 8.0, but were effective at higher concentrations at pH 10.5. At both pH levels, phenol demonstrated no appreciable germicidal activity for the entire concentration range tested, and zinc sulfate was somewhat bactericidal only at the highest concentration tested a Based on bacteria concentrations in control samples receiving no disinfectant. Control bacteria concentrations averaged 3 x 107 colonies/ml at pH 8.0, and 2.5 x 10" colonies/ml at pH 10.5. "After 24 h, the pH levels in the samples initially adjusted to pH 10.5 had appreciably decreased. and exhibited no virucidal activity. The increased bactericidal and virucidal effectiveness observed for some of these agents at pH 10.5 was not simply due to the effects of high pH alone, because control sewage samples at the same pH showed appreciable levels of bacteria and persistence of virus. A series of experiments was conducted to further investigate the effect of increasing pH on the bactericidal activities of formaldehyde, BAC, CPC, and methylene blue using a minimal disinfectant concentration. The virucidal effectiveness of formaldehyde was also evaluated because the results of the previous experiment indicated that it was effective for both bacteria and viruses at a minimal concentration. In the experiments with formaldehyde, poliovirus was added to pH 8.0 raw sewage to give an initial concentration of about 3 x 104 PFU/ml. In all of the experiments, the pH 8.0 sewage was divided into four samples of 200 ml each, and the samples were adjusted to pH 9.0, 10.0, and 10.5 with Ca(OH)2. The samples were further divided into 2 volumes of 100 ml each, and 1 volume was dosed with disinfectant to give a concentration of 100 mg/liter. The samples were held at 25 C in loosely capped bottles, and after 24 h pH levels were measured and bacteria and virus assays were made. The results of these experiments for formaldehyde, BAC, CPC, and methylene blue are shown in Tables 3, 4, 5, and 6, respectively. The bactericidal activities of 100-mg/liter concentrations of formaldehyde, BAC, and methylene blue at pH 10.0 and 10.5 were markedly improved over their activities at pH 8.0 or 9.0. CPC was highly bactericidal at pH 10.5 but not at pH 8.0, 9.0, or 10.0. At all pH levels tested, a 100-mg/liter concentration of formaldehyde was highly virucidal. At a 100-mg/liter concentration, CPC, BAC, and methylene blue were shown to be ineffective against viruses in the simulated TALE 3 (Tables 1 and 2). However, since this simulated sewage prepared for these studies contained an anionic detergent, the quaternary compounds would have been inactivated under these conditions. Because a 100-mg/liter concentration of formaldehyde in raw sewage gave extensive virus inactivation in 24 h at pH levels ranging from 8.0 to 10.5, it could not be determined from the results of this experiment whether increasing pH enhanced virus inactivation. Therefore, an experiment similar to the one described in the previous paragraph was performed with formaldehyde at a concentration of 25 mg/liter. The results of this experiment ( activity of formaldehyde were markedly improved as the pH was increased in the alkaline range. Effect of holding time on bactericidal and virucidal activity. To determine the ability of chemical disinfectants under alkaline conditions to sustain bactericidal activity for a prolonged period and to determine the degree and rate of virus inactivation with minimal disinfectant concentration, a series of experiments was conducted in which samples of pH 10.5 raw sewage containing 30-and 100-mg/liter concentrations of formaldehyde, BAC, methylene blue, or HTH were held at room temperature for 14 days and were periodically assayed for bacteria and virus. Raw sewage at pH 10.5, which contained no disinfectants, was used as a control. In these experiments, raw sewage which had been adjusted to pH 10.5 with Ca(OH)2 was dosed with poliovirus to give an initial concentration of 106 PFU/ml. The sewage was then divided into 100-ml volumes, and the disinfectants were added to these samples to final concentrations of 30 and 100 mg/liter. A volume of pH 10.5 sewage containing no disinfectant served as a control. The samples were held at 25 C in loosely capped bottles, and samples were taken periodically for virus and bacteria assay. The pH levels of the samples were maintained at 10.5 by periodic adjustment with Ca(OH)2 if necessary (Tables 8 and 9). Both the 30-and 100-mg/liter concentrations of BAC and methylene blue and the 100-mg/ liter concentration of formaldehyde were capable of keeping bacteria concentrations well below those in control sewage for the entire 14-day period. However, some bacterial regrowth was apparent in the samples with 30 and 100 mg of BAC per liter by days 4 and 11, respectively. The bactericidal effects of BAC were more rapid at a concentration of 100 mg/liter than at 30 mg/liter, as indicated by the longer persistence of bacteria in the latter. Although both concentrations of formaldehyde and HTH were at first highly bactericidal and virucidal, extensive bacterial regrowth occurred in both HTH samples and in the 30-mg/l formaldehyde sample after several days. Virus inactivation rates in samples containing BAC and methylene blue were somewhat greater than that for control sewage but were relatively slow as compared with those for samples containing HTH or formaldehyde. Virus inactivation was somewhat more rapid with methylene blue than with BAC. Effect of daily sewage addition on the bactericidal activity of formaldehyde, methylene blue, and BAC. An experiment was conducted to determine the effects of incremental additions of sewage or sewage plus disinfectant on the ability of formaldehyde, methylene blue, and BAC to sustain bactericidal activity for a prolonged time period. Initially, 100-ml samples of raw sewage, adjusted to pH 10.5 with calcium hydroxide, were dosed with formaldehyde, methylene blue, or BAC to give concentrations of 1,000 and 100 mg/liter. The samples containing 1,000 mg of disinfectant per liter were given daily 100-ml volumes of fresh pH 10.5 sewage, and the samples containing 100 mg of disinfectant per liter were given daily 100-ml volumes of fresh pH 10.5 sewage with 100 mg of disinfectant per liter. The samples were held at 25 C and assayed for bacteria just prior to the daily sewage additions. Two types of pH 10.5 control sewage samples were used: one received no additional sewage and the other received a daily 100-ml volume of fresh pH 10.5 sewage. The experiment was conducted for 10 days, and a total of nine daily sewage additions were made. All samples were maintained at pH 10.5 by periodic adjustment with calcium hydroxide. Bacterial growth was extensive in both control sewage samples, with concentrations near the 107 CFU/ml level throughout most of the experiment (Table 10). In all samples containing disinfectants, bacteria concentrations were substantially lower than those in controls. This was true of those samples initially containing 1,000 mg of disinfectant per liter and receiving daily doses of sewage and of those samples initially containing 100 mg of disinfectant per liter and receiving daily doses of sewage with TABL 9. Effect of holding time on the virucidal activity offormaldehyde, BAC, methylene blue, and HTH 100 mg of disinfectant per liter. Although some bacterial growth occurred in a number of the disinfectant-containing samples as the experiment progressed, it was never extensive, and bacteria concentrations were kept well below the concentrations in control samples. DISCUSSION In raw sewage adjusted to pH 10.5, formaldehyde, methylene blue, and BAC were found to be effective disinfectants for periods of at least 14 days. In addition, the bactericidal effectiveness of these agents in raw sewage could be maintained for a period of at least 10 days when raw sewage was added daily, as long as the pH was maintained at about 10.5 and the disinfectant concentration was maintained at 100 mg/liter or more. HTH, although initially highly germicidal, had insufficient stability to be effective for more than a few days. The lack of stability of HTH, as well as its properties of corrosiveness in solution and strong oxidizing power which made it difficult to store and handle, detract from its suitability as a holdingtank or chemical-toilet disinfectant. Zinc sulfate and phenol demonstrated poor bactericidal and virucidal effectiveness in both pH 8.0 and 10.5 raw sewage at relatively high concentrations, and therefore appear to be unsuitable disinfectants for holding tanks and chemical toilets. In general, the results of this study suggest that at pH 10.5, formaldehyde, methylene blue, BAC, and CPC would be suitable holding-tank and chemical-toilet disinfectants. Although the germicidal property of alkaline formaldehyde has been previously suggested (A. R. White, U.S. Patent 2, 077, 060, 1937), quantitative studies on its bactericidal and virucidal effectiveness were not reported. One possible advantage of formaldehyde over some other disinfectant chemicals for holding tanks and chemical toilets is that, upon dilution and at less alkaline pH levels, it can be biodegraded in natural aquatic environments and in biological wastewater treatment systems. Formaldehyde is capable of being used as a substrate by bacteria either directly at low concentrations (5) or after its chemical conversion by a variety of reactions with various naturally occurring chemicals (7) to either formate or hydroxymethyl derivatives. Thus, the use of formaldehyde as a holding-tank disinfectant would be particularly attractive in situations where biodegradability is essential. Although the quaternary ammonium compounds BAC and CPC and the dye methylene blue were somewhat less efficient germicides than was formaldehyde, they were effective in pH 10.5 sewage at relatively low concentrations, and they may be useful in holding-tank and chemical-toilet situations where biodegradability is not a critical factor or where formaldehyde would not be sufficiently stable. In addition, these agents are not highly volatile or irritating to the skin or mucous membranes as is formaldehyde, thus making these agents safer and easier to handle. It should be noted that the germicidal properties of quaternary ammonium compounds such as BAC and CPC are adversely affected by soaps and anionic detergents. Therefore, it is likely that the soap in the simulated holding-tank sewage used in our experiments decreased the bactericidal and virucidal effectiveness of these agents. In fact, it has been shown that in the absence of high concentrations of soaps and anionic detergents the bactericidal and virucidal effectiveness of CPC is markedly improved at pH 10.5 (Wallis et al., unpublished data). The observation that formaldehyde, the quaternary ammonium compounds BAC and CPC, and the dye methylene blue were more efficient germicides at pH 10.5 than at 8.0 has important practical application in the disinfection of holding-tank and chemical-toilet sewage, because the use of any of these disinfectants in combination with enough base to give a pH of 10.5 greatly improves their germicidal efficiency, thereby reducing the quantity of chemical required for disinfection. The enhanced germicidal effectiveness of these agents at alkaline pH levels may have practical applications other than sewage disinfection.

v3-fos

2020-12-10T09:04:23.045Z

1974-11-01T00:00:00.000Z

237230909

s2

Characterization of Lac+ Transductants of Streptococcus lactis A phage-mediated transducing system was used in studying certain physiological characteristics of S. lactis C2 wild type, lactose-negative mutants, and lactose-positive transductants. Lac- mutants, obtained by acriflavine treatment of the wild type, were similar to the wild type in all characteristics tested except they lacked β-D-phosphogalactoside galactohydrolase (β-Pgal) and could not transport [14C]lactose; they also had approximately 10% of the proteolytic ability than wild-type cells. The lactose-fermenting characteristic was transduced from the wild type to Lac- mutants. The Lac+ transductants obtained were similar to the wild-type parent with respect to lactose fermentation and level of β-Pgal activity (0.186 U of protein per mg). These transductants, however, had not regained full proteolytic ability and were similar to the Lac- mutant in this respect. Lactic acid production of the transductants in milk was approximately two-thirds that of the wild type. Data suggest that both the lactose-fermenting and proteolytic characters are carried on extrachromasomal particles (plasmids). McKay et al. (11,12,13) have shown that the lactose-fermenting character of Streptococcus lactis C2 can be transduced from a wild-type (Lac+) strain to a Lac-mutant. The phage responsible for this transduction is a lysogenic phage carried in S. lactis C2 and is ultraviolet inducible. In studying the properties of the wild-type and lactose-fermenting transductants, three characteristics were examined: enzyme activity of the lactose-hydrolyzing enzyme, fl-D-phosphogalactoside galactohydrolase (fl-Pgal); proteolytic ability; and the rate of lactic acid production. MATERIALS AND METHODS Microorganisms and media. Lactic streptococci used were obtained from the stock collection maintained by the Department of Microbiology, Oregon State University. Cultures were regularly maintained in sterile nonfat milk (NFM) containing 11% solids. Lactic broth (5) with lactose as the sole carbohydrate (LLB) served as the growth medium for the routine assay of fl-Pgal. On occasion, glucose was substituted for lactose in this medium (GLB). The survey of organisms and determination of optimal assay conditions were carried out in this broth. For induction 'Technical Paper no. 3848, Oregon Agricultural Experiment Station. 'Present Address: Bureau of Water Works, 1800 S.W. 6th Ave., Portland, Ore. 97021. experiments, lactic broth was prepared with a reduced level of yeast extract (1 g/liter) and without carbohydrate. After sterilization of the medium, a filter-sterilized solution of the carbohydrate to be tested was added to a final concentration ranging from 0.001 to 0.005 g/ml. In all experiments involving broth, the organism was transferred three times over 36 h at 32 C in the appropriate broth before beginning the experiment. Preparation of toluene-acetone-tested cells. Toluene-acetone (1:9)-treated cells were normally used when small numbers of cells were being assayed for enzyme activity. Cells were harvested, washed, and resuspended in 0.05 M sodium phosphate buffer at pH 7.0 to an optical density of 0.30 at 420 nm, and quantitated in terms of milligrams of cell (dry weight). This insured that a standard amount of cells would be solvent treated for each experiment. The procedure of Citti et al. (2) was followed when treating the cells with toluene-acetone, except cells were shaken vigorously for 10 min after addition of the solvent. The resultant suspension was assayed for enzyme activity. The assay mixture contained 0.5 ml of toluene-acetone-treated cells and 2.0 ml of the chromogenic substrate. Incubation was at 37 C for 10 min. The reaction was stopped by the addition of 2.5 ml of 0.4 75_3 M sodium carbonate. The release of ortho-nitrophenol (ONP) was measured colorimetrically at 420 nm, and micromoles of ONP released was determined from a standard curve. Cells were removed by centrifugation before absorbancy was measured. One unit of enzyme was equivalent to 1 qmol of ONP liberated from ONPG, or ONPG-6-PO4, per 10 min in the case of the solvent-treated cell assay. Specific activity was expressed as units per milligram of cell (dry weight). Cells grown in milk. The method of Hull (8) was used to determine the degree of proteolysis that had occurred in 13-h-old cultures. Results were expressed as micrograms of tyrosine per milliliter released in the milk. Lactic acid production by Lacmutants and Lac+ transformants growing in milk was measured as follows: cultures (18-h) of organisms were inoculated into 50 ml of NFM and incubated at 31 C for 6 h. Samples (10 ml) were taken at 1-h intervals and the pH was determined with a Corning model 12 pH meter. Nearly equal numbers of cells were used as inoculum. ,B-Pgal activity in milk was assayed as previously described (16). Isolation of lactose-negative mutants. Cultures of S. kactis C2 and 7962 were exposed to acriflavine using a modified procedure of McKay et al. (12). One drop of a 12-h-old culture of cells grown in LLB was added to fresh LLB containing 0, 1.0, 3.0, or 6.0 sg of filter-sterilized acriflavine per ml. Cultures were incubated at either 21 or 31 C for 24 h. After this period, 1-ml samples were removed, and the appropriate dilution was spread on indicator plates which were held for 24 to 48 h at 31 C and examined for the appearance of Lacmutants. Two types of indicator plates were used: lactic agar with lactose as the sole carbohydrate containing 0.004% of bromocresol purple, on which Lac+ colonies appeared yellow and Lac white, and a modification of an agar developed by Morse and Alire (17). It consisted of tryptone, 20.0 g; yeast extract, 5.0 g; gelatin, 2.5 g; lactose, 10.0 g; sodium chloride, 4.0 g; sodium acetate, 1.5 g; ascorbic acid, 0.5 g; tris(hydroxymethyl)aminomethane buffer (Sigma 7-9), 1.3 g; dihydrocholic acid, 1.5 g; neutral red, 0.075 g; agar, 15.0 g; and water to 1 liter. The final pH was 7.2 and Lac + colonies appeared red on this medium, whereas Lacmutants were white or clear. Suspected Lac mutant colonies were picked, and inoculated into glucose broth and incubated at 31 C for 24 h. These cultures were then checked for their ability to grow in LLB and assayed for ,-Pgal activity. Induction of prophage in S. lactis C2. Cells of S. lactis C2, 7962, and S. cremois HP were subjected to ultraviolet irradiation to induce phage lysis using the procedure of McKay and Baldwin (11). Transduction. The lysates obtained from ultraviolet induction were filter-sterilized through a 0.45-jim membrane filter (Millipore Corp.). The recipient cells were Lacmutants obtained from the acriflavine treatment already described. The mutant was grown for approximately 4 h in GLB. The cells were then harvested and resuspended in 2 ml of lactic broth containing no sugar but with 5.0 x 10-' M calcium carbonate added. This suspension contained approximately 6.8 x 108 cells. The lysate was mixed (1:1) with the recipient and incubated at 31 C. Samples were removed at intervals and spread on LLB agar with 0.004% of bromocresol purple added. Plates were incubated at 31 C for 24 to 48 h and examined for the appearance of Lac+ transductants. Lac+ colonies were selected from these plates and examined further. Characterization of Lac+ transductants and Lac-mutants. Selected transductants and mutants were examined for drug resistance markers, carbohydrate fermentation patterns, a-gal and fl-Pgal activity, lactic acid production in lactic broth and milk, and proteolytic activity in milk. Drug resistance was determined using Dispens-o-Disks (Difco) containing the antibiotic to be tested. Streptomycin, ampicillin, carbenicillin, gentamicin, kanamycin, chloromycitin, tetracycline, and furadantin were the antibiotics used. The culture to be tested consisted of log-phase cells suspended in overlay agar and spread on GLB agar plates. The disks were applied to the overlay agar after spreading. Fermentation patterns were determined by using the API system for lactobacilli (Analytab Products, Inc., New York). ,-gal and fl-Pgal activity was determined as described above as were lactic acid production and proteolytic activity. RESULTS Effect of acriflavine treatment on S. lactis. Cells of S. lactis C2 and 7962 were treated with acriflavine (1.0 to 6.0 ,ug/ml) and examined for the appearance of Lacvariants (Table 1). S. lactis C2 yielded Lacvariants at a rate of approximately 3%; S. lactis 7962, however, did not yield any Lac-variants, even though it was more sensitive to acriflavine than C2 with respect to the number of surviving cells. Lacvariants were not observed to occur spontaneously in either C2 or 7962. Comparison of S. lactis C2 wild type and Lacmutants. Lacmutants isolated from S. tactis C2 by acriflavine were compared with the parent strain for a number of characteristics. Table 2 lists the results of this comparison. Lac mutants were unable to transport and ferment lactose, but were only slightly impaired in galactose fermentation. The Lacmutants also lacked detectable fl-Pgal activity even when growing on galactose. The wild-type and mutant cells were the same in all other reactions; they were sensitive to ampicillin, carbenicillin, gentamicin, kanamycin, chloromycitin, tetracycline, and furadantin. The wild-type and the Lacmutants were both resistant to streptomycin. Both cell types gave positive arginine hydrolysis reactions and did not produce diacetyl. Acriflavine treatment failed to cure the wild type with respect to lysogeny; ultraviolet treatment of both the Lac and wildtype cells resulted in lysis of the cultures. Lysates of these Lac-cells, however, did not yield Lacmutant, indicating that the lac marker had been irrevocably lost and that Lac-was not due to a point mutation. Comparison of wild type, Lacmutants, and Lac+ transductants. In examining the Lac+ cells obtained by transduction, it was found that they had regained the ability to ferment lactose and also contained the same levels of P-Pgal as the wild type. These results are summarized in Table 3. The transductants were indistinguishable from the wild type in all characteristics examined except one; they were unable to produce lactic acid at the same rate as the wild type when growing in NFM. Figure 1 shows lactic acid production of the wild type, a Lacmutant, and transductants (designated T8 and T9) when grown in milk. All initial inoculations were 1% and contained approximately 5.0 x 10" organisms per ml. After 6 h at 30 C, the mutant had lowered the pH only about 0.15 U and had a cell concentration of 7.5 x 107. The wild type lowered the pH about 1.1 10g. T8 lowered the pH 0.7 U. This difference in rate of acid production between the wild type and the transductants could not be demonstrated when the cells were growing in LLB as shown in Fig. 2. Here the Lacmutant first lowered the pH slightly and then raised it again; final cell concentration was 9.0 x 107. When the Lacmutants were grown in GLB, the pH was lowered over 2.0 U and a final cell concentration of 6.9 x 108 was achieved. The rates of acid production of the wild type and transductants T9 and T8 were essentially the same, the pH being lowered 2.0 U at final cell concentrations of approximately 4.0 x 108. Because it appeared that the Lacmutants could ferment glucose and produce lactic acid at a high rate when grown in broth, it was thought that the addition of glucose to NFM might restore the lactic acid production of the Lac mutants in milk. shown: T8 (A), Lac-mutant (0), and wild type (0). given in Fig. 3. Addition of 1% glucose to NFM restored the ability of the Lacmutant to produce acid at a rate similar only to the slow transductant, T8. The presence of 1% glucose in milk did not appreciably affect the rate of acid production of the wild type or Lac+ transductants. Earlier studies into the nature of lactic acid production by the Lacmutants and transductants indicated that the proteolytic ability of these organisms had been impaired (18,20,21). Proteolytic activity of the Lacmutant, Lac+ transductant T8, and the wild type was, therefore, examined during growth in milk. Samples were assayed at 5 h when acid-producing differences between cell types was obvious. Table 4 summarizes the results. Wild-type cells growing in NFM exhibited considerable proteolytic activity as expressed by the liberated tyrosine. Both the Lacmutant and Lac+ transductant, however, exhibited very little proteolytic activity. Even when glucose (1%) was added to the milk, the proteolytic ability of the Lacmutant and the Lac+ transductant was not significantly increased; in fact, glucose appeared to inhibit proteolysis by the wild type. Table 1 indicate that while S. lactis 7962 is much more sensitive to acriflavine treatment with respect to survivors than C2, no Lac mutants could be isolated from 7962; Lacmutants were readily obtained from C2, however. The lactose utilization system of S. lactis 7962 has been compared to that found in Escherichia coli (14). Acriflavine causes mutations by eliminating extrachromosomal deoxyribonucleic acid that may be present in the appear that the enzyme (fl-Pgal) of this organism is carried extrachromosomally and thus subject to elimination by acriflavine treatment. In this regard, it is noteworthy that plasmids have been identified in S. lactis (3,7,19). The Lacmutants obtained by acriflavine treatment of S. lactis C2 were examined to see if other mutant characteristics could be identified in these organisms. This comparison was complicated by the fact that S. lactis C2 wild type is an extremely fastidious organism requiring a complex medium for growth. Thus, most mutations could be lethal. By identifying other mutations linked with the lactose-fermenting region, mapping of this region would be possible. Results given in Table 2 indicate that with the exception of the ability to ferment lactose and proteolytic activity (including lactose transport and fl-Pgal activity) the mutants examined were indistinguishable from the wild type. Results given in Of special interest where the Lac-mutants were concerned was their ability to grow on galactose and yet not produce any ,B-Pgal. Since Molskness et al. (16) have shown that free galactose is a potent inducer of ,B-Pgal in lactic streptococci, the question arises as to what the role of this carbohydrate is during induction. Lactic streptococci have been shown by Lee et al. (9) to use free galactose by the Leloir Pathway, where the phosphorylated product is galactose-1-phosphate and not galactose-6-phosphate. In S. aureus, galactose-6-phosphate (a product of hydrolysis of lactose-6-phosphate) has been shown to play a role in the induction process (18). Studies to determine the actual inducer in Streptococcus organisms have been hampered by the fact that, unlike staphylococci, streptococci are impermeable to phosphorylated compounds. Our attempts to increase the permeability of the cell membrane and still leave the cell viable have been unsuccessful. Upon initial examination, all the Lac+ transductants isolated from bromocresol indicator plates were indistinguishable from the parent S. lactis C2 wild type ( Table 3). The transductants had regained full f-Pgal activity and once again could ferment lactose in LLB at a rate that was essentially the same as the wild type. When the transductants were examined for lactic acid production in milk, however, it was found that the transductants were somewhat slower in the rate at which they lowered the pH (lactic acid production). Slow lactic acid production in milk is usually an indication of poor proteolytic ability, though the cells are still able to ferment lactose (4,22). Thus, cells of poor proteolytic ability for the purposes of discussion might be designated Lac+ Prtto designate lactose fermentation but reduce proteolytic activity, whereas the wild type exhibits a Lac+ Prt+ phenotype. Upon examination of the Lacmutants, it was found that these cells were indeed Prt-. It is important to note that in the lactic streptococci, Prt-cells cannot be detected when grown in lactic broth. This is due to the large amount of small peptides already present in this medium. It is only when the cells are grown in milk and forced to hydrolyze casein to get their required amino nitrogen that the Prt-phenotype can be demonstrated. In these experiments, Lacwas the only mutant phenotype selected. Prtwas not selected due to the lack of a screening method for this character. Yet as a result of this selection, all the Lacmutants obtained were also Prt-; Lac-Prt+ types probably also exist, but were not found in this study. These data, along with a recent report by Pearce et al. (20), indicate that certain proteolytic enzymes of these organisisms are carried on plasmids. These plasmids, like the lactose-fermenting character, are lost during acriflavine treatment. Westhoff et al. (21,22,23) have characterized both intraand extracellular proteases in lactic streptococci. The nature of the lost protease responsible for the Prtphenotype found in this study is not known but presumably it is the surface-bound activity described by Pearce et al. (20). It is well known that lactic streptococci are unstable with respect to proteolytic activity. Strains become slow lactic acid producers, as a result of losing their proteolytic ability, at a rate of about 1% based on colony isolation of plated cultures. The Prtstrains obtained by this type of spontaneous mutation, however, are still Lac+. On the other hand, spontaneous mutation to Lachas never been reported or observed in this laboratory, indicating that it occurs at a very low frequency. The difference in stability of the lactose and the proteolysis characters suggests that they are carried on different plasmids. A hypothetical model of the lactose and proteolytic genes in S. lactis C2 might be constructed as follows: genes responsible for lactose fermentation are carried on one plasmid. The genes responsible for proteolytic activity are carried on a second plasmid and transduced independently of the lactose marker. Thus, when transductants are selected by the Lac+ phenotype alone, they have not necessarily regained their proteolytic ability. This would mean that when acriflavine treatment is used in obtaining Lac-mutants, Prt-mutants are formed concurrently. Indeed all the Lacmutants examined up to this time have also been Prt-. The effect that acriflavine had on the Prt+ to Prtmutation rate independent of the lac characteristic is yet to be determined. Results of these studies indicate at least two areas where genetic manipulation might be used to improve lactic fermentation by lactic streptococci. One is the use of transduction to stabilize the Prt characteristic. Macrina and Balbinder (10) showed that when a mutant F' plasmid obtained from Salmonella typhimurium was transduced into E. coli it exhibited unique stability. If stability of this nature could be developed for the Prt characteristic in streptococci, the high rate of appearance of slow acid-producing mutants would be greatly reduced. The second area where genetic manipulation could be used involves lactose fermentation itself. In results presented elsewhere (T. A. Molskness, Ph.D. thesis, Oregon State University, 1974), it was shown that in S. cremoris HP lactic acid production in broth lags until f-Pgal reaches a certain level. This lag is not as long in cells inoculated into glucose broth, presumably because induction of f-Pgal is not critical in this case. From this it seems that it would be possible to increase acid production in an organism by creating stable merodiploids for f3-Pgal. A higher level of this enzyme would decrease the lag time for growth and lactic acid production, and thus decrease the production time for fermented dairy products. Merodiploids have been constructed in E. coli K-12 (6) and the level of p3-gal has been determined. It was found that the merodiploids contained approximately twice as much ,-gal as found in the haploid cell; also Brenchley and Magasanik (1) have shown that a lac plasmid of Klebsiella aerogenes could be transferred to E. coli and Salmonella typhimurium, and that segregants losing the plasmid grew on lactose at only 50% the rate of the plasmid-containing strains and they contained only one-tenth to one-fifth as much A-gal. To create merodiploids in lactic streptococci, however, a method of selecting for the merodiploid cell must first be developed. Current methods of plating would not work since these methods only select for ability to ferment lactose and not the amount of fl-Pgal present in the cell.

v3-fos

2018-04-03T06:18:03.441Z

1974-01-01T00:00:00.000Z

46117989

s2

Effect of Sodium Nitrite on Toxin Production by Clostridium botulinum in bacon Pork bellies were formulated to 0, 30, 60, 120, 170, or 340 ,ig of nitrite per g of meat and inoculated with Clostridium botulinum via pickle or after processing and slicing. Processed bacon was stored at 7 or 27 C and assayed for nitrite, nitrate, and botulinal toxin at different intervals. Nitrite levels declined during processing and storage. The rate of decrease was more rapid at 27 than at 7 C. Although not added to the system, nitrate was detected in samples during processing and storage at 7 and 27 C. The amount of nitrate found was related to formulated nitrite levels. No toxin was found in samples incubated at 7 C throughout the 84-day test period. At 27 C, via pickle, inoculated samples with low inoculum (210 C. botulinum per g before processing and 52 per g after processing) became toxic if formulated with 120 fg of nitrite per g of meat or less. Toxin was not detected in bacon formulated with 170 or 340 ,gg of nitrite per g of meat under these same conditions. Toxin was detected at all formulated nitrite levels in bacon inoculated via the pickle with 19,000 C. botulinum per g (4,300 per g after processing) and in samples inoculated after slicing. However, increased levels of formulated nitrite decreased the probability of botulinal toxin formation in bacon inoculated by both methods. Pork bellies were formulated to 0, 30, 60, 120, 170, or 340 ,ig of nitrite per g of meat and inoculated with Clostridium botulinum via pickle or after processing and slicing. Processed bacon was stored at 7 or 27 C and assayed for nitrite, nitrate, and botulinal toxin at different intervals. Nitrite levels declined during processing and storage. The rate of decrease was more rapid at 27 than at 7 C. Although not added to the system, nitrate was detected in samples during processing and storage at 7 and 27 C. The amount of nitrate found was related to formulated nitrite levels. No toxin was found in samples incubated at 7 C throughout the 84-day test period. At 27 C, via pickle, inoculated samples with low inoculum (210 C. botulinum per g before processing and 52 per g after processing) became toxic if formulated with 120 fg of nitrite per g of meat or less. Toxin was not detected in bacon formulated with 170 or 340 ,gg of nitrite per g of meat under these same conditions. Toxin was detected at all formulated nitrite levels in bacon inoculated via the pickle with 19,000 C. botulinum per g (4,300 per g after processing) and in samples inoculated after slicing. However, increased levels of formulated nitrite decreased the probability of botulinal toxin formation in bacon inoculated by both methods. Recent studies on canned, perishable, cured meat and wieners showed that increased nitrite levels decreased the probability of botulinal toxin formation (1, 4). However, the impact of nitrite upon toxigenesis differed somewhat between the two products. This difference is probably due, in part, to differences in formulation and processing employed in the manufacture of the two products. Bacon is formulated and processed differently from either canned, perishable, cured meat or wieners. Thus, results from the foregoing studies would not necessarily apply to bacon. This study was conducted as one of a series undertaken cooperatively by the American Meat Institute, the Food and Drug Administration, and the United States Department of Agriculture to determine the minimal level of sodium nitrite required in bacon for consumer acceptance and botulinal protection. The initial studies on-bacon were designed to investigate three distinct aspects and were conducted concurrently. Project I investigated the effect of nitrite level on product manufacture, chemical changes, product acceptance, off-flavor, growth of microbial spoilage organisms, and color. Project II examined the roles of nitrite level, cooking method, and ascorbates on the formation of N-nitrosopyrrolidine. Preliminary results from the first two projects have been reported (3). When completed, the first two projects will be reported as a separate paper. Project III, reported herein, investigated the degree to which nitrite retards or prevents growth of Clostridium botulinum in bacon. Additional research is now underway at the Food Research Institute, University of Wisconsin, to investigate the effect, if any, that high levels of sodium ascorbate have on the inhibition of botulinal toxin by sodium nitrite in bacon. MATERIALS AND METHODS Experimental design. The experimental variables are listed in Table 1. The design for the uninoculated portion of the experiment was a full replicate of a 6 x 6 x 2 (nitrite level x storage time x incubation temperature) factorial with one package for each treatment combination. A similar 6 x 6 x 2 (nitrite level x storage time x inoculum level) design was used for inoculated samples. There were five packages on January 9, 2021 by guest http://aem.asm.org/ Downloaded from for each treatment combination for samples inoculated via the pickle solution and two replicates for samples inoculated with a sand-spore mixture after slicing. Inoculum. Spores of five type A (77A, 62A, 12885A, 33A, and 62A) and five type B (ATCC7949, 41B, 40B, 53B, and Lamanna B) strains of C. botulinum were used. Tubes of peptone colloid medium (Difco) modified by the addition of 0.1% glucose were inoculated with a heat-shocked spore suspension of the individual strains. After 16 h of incubation at 37 C, fresh tubes of the medium were inoculated, incubated for 4 h, and again transferred. After 4 h of incubation, these cultures were inoculated into the sporulation medium of Schmidt and Nank (5) consisting of 5% Trypticase (BBL), 0.5% peptone (Difco), and 0.05% sodium thioglycolate. The final cultures were incubated for 7 days at 37 C. Spores were harvested by centrifugation, washed several times, and suspended in sterile, distilled water. Samples of each suspension were heat-shocked (80 C for 15 min), and spore counts were determined by a three-tube most probable number procedure in modified peptone colloid medium (2). A single suspension was prepared containing equal numbers of spores of each strain. A portion of this spore mixture was heat-shocked, diluted, and added to the pickle solutions for inoculation of the bacon. A second portion of the heat-shocked spore suspension was mixed with sterile sand and dried under vacuum over phosphorus pentoxide at room temperature for inoculation of bacon after slicing. Preparation and inoculation of bacon. Raw, frozen pork bellies, curing ingredients, and water used for preparing pickle were from the same stocks used in the project I research and were supplied by Armour and Co. (Oak Brook, Ill.). Two bellies at each nitrite and spore inoculum level were pumped with curing pickle to an 11% gain and drained to an approximate 10% gain. The pickle contained sodium chloride (13.3%), sucrose (3.1%), tripolyphosphate (2.6%), sodium isoascorbate (0.23%), and the various concentrations of sodium nitrite and water. These concentrations of ingredients in the pickle were 10 times the levels desired in bacon. The drained bellies were smoked and processed to an internal temperature of 53 C over an 8.5-h period. Smoke from hardwood shavings was added during the initial 2.5 h. The processed bellies were held at -2.2 C for 36 h and sliced. The two bellies for each nitrite and spore inoculum level were divided into thirds. One slice from each third of the two bellies was placed in a Curpolene 200 (Curwood, Inc., New London, Wis.) pouch (six slices total, weighing 125 to 150 g), and the package was sealed under vacuum. The packages were stored at 7 or 27 C. Uninoculated bacon was prepared in the same manner except that spores were not added to the pickle. This bacon was handled and stored in the same manner as the inoculated bacon and was used for all chemical analyses. A portion of this bacon was inoculated with the dried sand-spore mixture after slicing. Toxin assay and determination of spore levels. The samples were randomized and labeled so that each package was designated for analysis at a specific time. However, samples which swelled prior to this time were removed from incubation and analyzed. The packages were weighed, and the entire contents of each package were blended for 1 min with an equal weight of gelatin phosphate buffer. The slurry was centrifuged, and 0.5 ml of the supernatant was injected into each of five mice (three unprotected and two protected with type AB botulinal antitoxin from Connaught Medical Research Laboratories, University of Toronto, Toronto, Canada). Death of the unprotected mice within 4 days and survival of the protected 2 mice were considered proof of the presence of botulinal toxin. Inoculum levels before smoking were determined by removing three plug samples (about 6.3 cm2/sample) from one of the two bellies at each nitrite level. The plug samples from each belly were composited for determination of inoculum level. Viable counts were determined on nonheat-shocked samples of bacon before and after processing and inoculated pickle solutions by the three-tube most probable number procedure. The pickle solutions were filtered through 0.45-,gm pore size filters. After rinsing the filters several times to minimize contamination by pickle ingredients, the filters were cut up, placed in phosphate buffer (pH 7.2), and agitated to dislodge the organisms, and counts per ml of pickle were determined. All bacon samples were blended as for toxin assay, and the slurry was diluted for determination of counts. Chemical analyses. A composite of nine plugs bored randomly from the bacon bellies was used for chemical analysis of bacon before and after heat processing. Nitrite and nitrate concentrations were determined as previously reported (1). RESULTS Samples of finished bacon at the various nitrite levels were analyzed for sodium chloride, moisture, fat, protein, and water activity. The range and average for these values, plus calculated brine concentrations, are shown in Table 2. Concentrations of nitrite found before smok- ing corresponded to formulated levels (Table 3). However, approximately 50 to 80% of the nitrite was lost during processing (i.e., heating to 53 C followed by holding for 36 h at -2.2 C). A further time-temperature-dependent reduction in nitrite occurred during storage. For example, Fig. 1 shows a best fitting (least squares) line fitted to predicted residual nitrite values for product formulated to 170 Ag of nitrite per g and held at 27 and 7 C. There was a geometric decline in nitrite levels with time at both temperatures. However, the rate of decrease was more rapid at 27 than at 7 C. Nitrate was not added to the system; however, analysis showed the presence of nitrate during processing and storage at 27 and 7 C ( Table 4). The amount of nitrate found was related to formulated nitrite levels. The range and logarithmic average counts of C. botulinum in the inoculating pickle, pumped bellies, and the finished bacon are shown in Table 5. These values represent counts across all nitrite levels. Although there was considerable variation, the counts were independent of the nitrite levels. Thus, exposure to nitrite, particularly the high levels in the pickle, had no effect on the inoculum. Three samples of bacon were analyzed per inoculum level after inoculating bacon with the sand-spore mixtures. The logarithmic averages were 40 and 3,400 per g with ranges of 18 to 86 and 1,800 to 4,600 per g for the low and high inoculum levels, respectively. Sixty samples of bacon inoculated via the pickle and 24 samples inoculated with the sand-spore mixtures were tested after packaging and without incubation. All were nontoxic, demonstrating that toxin was not carried into the product by the inocula. 14 28 54 84 7 14 28 54 84 0 0 19 36 15 16 35 18 0 10 10 NDa ND 30 0 29 63 26 22 35 10 0 17 18 ND ND 60 0 37 49 49 22 36 32 5 0 19 ND ND 120 18 48 58 130 25 64 62 58 31 21 26 16 170 35 70 102 135 34 103 67 73 23 35 26 33 340 35 80 63 149 60 137 82 112 217 51 32 16 a ND, Not determined. detected in bacon at all nitrite levels, but with decreasing frequency as the formulated level increased. All botulinogenic samples were proteolyzed. Samples stored for up to 84 days at 7 C were nontoxic and showed no evidence of proteolysis. Table 7 shows the numbers of botulinal toxic samples after holding bacon at 27 C when inoculated by the sand-spore mixture after slicing. As was true for samples inoculated before processing, increased nitrite levels in the bacon reduced the rate of toxin development and eventual number of toxic samples. Relatively few samples were confirmed to be toxic at 170 or 340 Ig of nitrite per g; however, there was at least one toxic sample at all nitrite levels studied. The total number of botulinogenic samples at the low inoculum, zero nitrite level was unexpectedly low.

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2018-04-03T04:24:56.315Z

1974-05-01T00:00:00.000Z

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Acid protease production by fungi used in soybean food fermentation. Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested-wheat bran, wheat, and soybeans-wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal. Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested-wheat bran, wheat, and soybeans-wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal. Aspergillus, Rhizopus, Mucor, and Actinomucor have long been used in Oriental food fermentations (3). Extensive studies have been made on soy sauce and miso fermentations carried out by Aspergilli, but not on those food processes involving Rhizopus, Mucor, and Actinomucor. During the last decade, pure culture fermentation methods for making tempeh from soybeans by Rhizopus oligosporus (2,8) and sufu from soybeans by Mucor dispersus and Actinomucor elegans (12) were developed. We have also expanded our effort to explore the enzymes produced by these fungi, partly to find out more about the role of such enzymes in these fermentation processes and partly to discover commercially useful enzymes. Our previous studies revealed that R. oligosporus (14), M. dispersus (13), and A. elegans (unpublished data) all produce acid type proteases, whereas most fungal proteases have a pH optimum around neutral or alkaline. Although these fungi grew abundantly in submerged culture containing soybeans, wheat, or wheat bran, their enzyme yields were unsatisfactory. The production of proteases by R. oligosporus decreased as the concentration of substrate increased (16). The protease produced by M. dispersus NRRL 3103 (formerly M. hiemalis) was bound to the mycelial surface (13). Addition of sodium chloride or other 901 ionizable salts in the growth medium for this fungus increased the enzyme in the culture filtrate. The total enzyme yield, however, was limited because fungal growth was suppressed by the added salt. The low enzyme production by these fungi in submerged culture was not unexpected, because fermentation processes carried out by these fungi are usually in solid state. Before World War II, solid state fermentation, generally known as the "bran process," was almost universally employed for the production of fungal enzymes. Deep-tank fermentation, however, has since replaced this technique in the West. Success in obtaining high yields of secondary metabolites by some fungi on solid substrates (4) and difficulties encountered in increasing enzyme yields of the fungi under investigation in submerged culture prompted us to return to our work with solid substrates. The present study was undertaken to find a set of conditions for protease production by R. oligosporus, M. dispersus, and A. elegans on solid substrate. MATERIALS AND METHODS Cultures. R. oligosporus NRRL 2710, M. dispersus NRRL 3103, and A. elegans NRRL 3104 were maintained on slants of potato-dextrose agar at 4 C. Before each experiment, the organisms were transferred to slants that then were incubated at 25 C for 7 days. ACID PROTEASE PRODUCTION Spore suspensions for inoculation were prepared by adding 3 ml of sterilized distilled water to each slant and vigorously shaking the culture for 1 min. Fermentation. Each 300-ml Erlenmeyer flask containing 10 g of wheat bran and 4, 8, or 15 ml of water was mixed and allowed to stand for 1 h at room temperature with frequent shaking. The flasks were autoclaved at 120 C for 20 min, cooled, and inoculated with 0.2 ml of inoculum. The cotton plugs were covered with aluminum foil to prevent evaporation, and flasks were incubated stationary at 15, 20, 25, or 32 C for varying lengths of time. The fermentation mass was extracted with 100 ml of 2% sodium chloride solution at room temperature for 1 h with frequent stirring, followed by centrifugation. The supernatant was used as the source of protease. Two experiments were carried out for each set of conditions. The average values of two runs will be presented. Assay of proteolytic activity. Proteolytic activity was measured according to the hemoglobin digestion method described by Anson (1). Reaction mixture containing 1 ml of 1% denatured hemoglobin in 0.05 M citrate buffer of pH 2.5 and 1 ml of properly diluted culture extract was incubated at 38 C for 20 min. The reaction was stopped by the addition of 3 ml of 5% trichloroacetic acid. The undigested hemoglobin was removed by filtration, and the acid-soluble products were determined spectrophotometrically at 280 nm. One unit of protease is defined as the amount of enzyme that yields a change in optical density at 280 nm equivalent to 1 Mmol of tyrosine per h at 38 C. RESULTS Moisture content of wheat bran medium. The initial moisture content of autoclaved wheat bran media containing 10 g of bran and 4, 8, or 15 ml of water was determined by drying at 110 C for 24 h. The average values obtained from three experiments were 35, 50, and 63%, respectively. Visual growth as affected by incubation temperature and moisture of wheat bran. Quantitative determination of growth on solid substrates, such as wheat bran, is difficult. Therefore, only subjective observations on growth are presented in Table 1. Of the three wheat bran media studied, the medium having the lowest moisture, 35%, did not support good growth of R. oligosporus or A. elegans at any of the temperatures investigated, although growth usually was noticeable after 2 weeks of incubation. M. dispersus grew fairly well on wheat bran of 35% moisture at 25 and 32 C, but it grew slower at 20 C. On the other hand, wheat bran media containing 50 and 63% moisture provided all three fungi with an environment for luxuriant growth. Rates of growth, R. oligosporus grew rapidly on moist media of 50 and 63% moisture. Abundant growth was observed after 1 day at 32 C, 2 days at 25 C, and 4 days at 20 C. M. dispersus did not grow as rapidly as R. oligosporus at the highest temperature, 32 C, but it surpassed the growth of R. oligosporus at the lower temperature of 20 C. A. elegans also grew well at low temperature, and it preferred to grow on the medium with 63% moisture. Production of acid protease by R. oligosporous NRRL 2710. The amounts of acid protease produced by R. oligosporus grown on wheat bran containing 50 and 63% moisture and at three different temperatures are summarized in Fig. 1. The data on enzyme production by the fungus on 35% moisture bran are not presented, because the activities were low throughout the 2 weeks of incubation. As indicated in Fig. 1, enzyme activities reached a maximum after 2 to 3 days at 32 C, 3 to 4 days at 25 C, and 5 to 7 days at 20 C. After that, the activities decreased. The rate of inactivation, however, seemed to be slower at 20 C than at the other two temperatures. A large shift in culture pH values was noted. An initial pH value of 5.7 usually rose to above 7 as enzyme production reached its maximum and continued to rise gradually. Although the fungus appeared to grow as well on wheat bran containing 63% moisture as on that of 50%, the amount of enzyme recovered from media of 50% moisture was much greater than that from media of 63% moisture. Under the conditions investigated, R. oligosporus grown on wheat bran of 50% moisture for 3 to 4 days at 25 C yielded the highest amount of protease. Our results also indicated that the moisture content of the medium was a more important factor than incubation temperature. Production of acid protease by M. dispersus NRRL 3103. As stated before, M. dispersus grew fairly well on bran containing 35% moisture; the amount of enzyme produced, however, was insignificant during the 2 weeks of incubation. Thus, only the results obtained from the growth media containing 50 and 63% moisture are presented in Fig. 2. When M. dispersus was grown on either 50 or 63% moisture bran, it produced equally impressive amounts of enzyme at growth temperatures of 20 or 25 C, but significantly less at 32 C. Production of enzyme by the fungus grown on 50% moisture bran reached a steady maximum after 4 to 6 days at 25 C and 7 to 9 days at 20 C, and then gradually decreased. At 63% moisture, the protease yield was maximum at about 3 days at 25 C and 7 to 8 days at 20 C, and then rapidly decreased to about zero. It is apparent that M. dispersus can tolerate a broad moisture range for enzyme synthesis. The enzyme, however was increasingly susceptible to denaturation as the percentage of moisture increased. Therefore, excellent yields of enzyme by M. dispersus can be recovered from bran containing 50 to 63% moisture after 3 to 4 days of growth at 25 C, or for longer incubation time at lower temperatures. The pH of extract from fermented wheat bran at 63% moisture was above 7 after 2 weeks of incubation; whereas at 50% moisture, it was around 6 except when the culture was incubated at 32 C. Here the pH also reached 7 after 2 weeks. Production of acid protease by A. elegans NRRL 3104. Of the three fungi studied, A. elegans required the lowest temperature for growth and enzyme production. Like R. oligosporus and M. dispersus, this fungus did not grow well on 35% moisture bran nor did it produce meaningful amounts of enzyme. However, when the moisture of bran was high enough for good growth, the organism showed a marked degree of moisture tolerance for enzyme production as indicated in Fig. 3. The enzyme yield, on the other hand, was greatly affected by temperature. The fungus grew luxuriantly at 25 C yet produced low enzyme yield. In this respect, A. elegans behaved similarly to M. dispersus. Unlike M. dispersus, a marked pH shift from 5.6 to 7.9 of A. elegans culture extracts was observed under all the conditions investigated, and the enzyme decreased rapidly regardless of the moisture content of the media. Based on this study, the optimal conditions for protease production by A. elegans grown on wheat bran were 50 to 63% moisture with 3 days of incubation at 20 C or 4 to 5 days of incubation at 15 C. Effect of substrate and culture method on protease production. Like wheat bran, soybeans or wheat did not provide a good growth condition for R. oligosporus, M. dispersus, and A. elegans unless the moisture level of these materials was around 50%. The protease yield per unit substrate produced by these fungi grown on solid state and in submerged culture is presented in Table 2. Wheat bran generally was a superior substrate for enzyme production by these fungi regardless of the culture method employed. Solid culture fermentation was a better method than the submerged culture fermentation. DISCUSSION Many members of the order Mucorales are known to be hydrophytes. They require a relative humidity of 90% or greater for growth and grow best at humidities near 100%. However, little is known regarding the relationship between their growth conditions and enzyme yields. The three fungi investigated in this study, representing three genera in Mucorales, were found to be hydrophytes and fast-growing. R. oligosporus is used for tempeh fermentation. M. dispersus and A. elegans are used for sufu fermentation. The substrates for these fermentation processes have moisture contents of about 55 and 80%, respectively. Therefore, the high moisture requirement by those fungi for growth is expected. The relationship between enzyme yield and the three environmental factors investigated varied with the organism, but they all yielded greater amounts of enzyme at temperatures lower than their optimum growth temperatures. Similar findings with respect to protease production by other fungi were reported by Max-VOL. 27, 1974 Submerged culture: 10 g of substrate, soybean meal, wheat flour, and wheat bran in 100 ml of water for R. oligosporus, and in 100 ml of 0.5 M NaCl for M. dispersus and A. elegans. Incubated on a reciprocating shaker at temperature and time same as for solid culture. well (6) using Aspergillus oryzae, and Yamamoto (17) using Aspergillus sojae. The effect of temperature on enzyme production observed in this study also emphasized the importance of some common practices in solid culture fermentation, i.e., frequent turning of the growth mass or use of thin layers of solid substrates. Otherwise, the heat resulting from active growth will increase the incubation temperature and affect the enzyme production. As with most extracellular enzymes produced by many microorganisms, maximum yield by the three fungi studied was usually reached about the time of maximum growth. However, the enzymes were rapidly inactivated except when M. dispersus was grown on wheat bran containing 50% moisture at 20 and 25 C. Although many enzymes are often destroyed by protease produced in the same culture, the disappearance of protease observed in this study probably was not due to self-digestion. Previously, we have reported (15) that no degraded products of self-digestion acid protease isolated from culture filtrate of R. oligosporus could be detected when the enzyme preparation was incubated at 28 C for 25 h. The inactivation of the protease was explained as being caused by an alkaline shift in pH. Acid proteases produced by R. oligosporus and M. dispersus are very unstable as the pH approaches 7 (13,14). The rapid disappearance of the proteases limited the harvest time. On the other hand, this trait can benefit the yield and purification processes for other useful enzymes produced by the same organisms. There are conflicting reports on the importance of aeration for the production of enzymes by microorganisms. Richou and Kourilsky (7) found that, in general, aeration was unfavorable for protease formation by various microorganisms. Our study also suggested that aeration may not be an important factor for enzyme production by the three fungi investigated. We noted that the very moist substrate and dense mycelial growth resulted in a tight mass that restricted aeration. The finding that a solid substrate method for protease production by these fungi was superior to the commonly used liquid culture method was not unexpected, mainly because these fungi have been traditionally used in.solid substrate fermentation. There have been few comparisons by these two methods of enzyme yield per unit substrate. Recently, Tsujisaka et al. (11) found that Aspergillus niger produced more lipase on solid medium containing wheat bran and calcium carbonate than in liquid media. It is, however, surprising to find that soybeans were not as good a substrate for protease production as was wheat bran. The fact that these fungi are fast growing and require high moisture levels to grow and synthesize enzymes might eliminate the danger of contamination by such toxin-producing fungi as Aspergillus flavus and Aspergillus ochraceus, which have been reported (5,9,10) to require low moisture levels (calculated at 33%) for toxin synthesis. This should improve the feasibility of using Mucor crude enzyme preparation in food and feed industries.

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1974-01-01T00:00:00.000Z

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Salmonella in natural animal casings. Destruction of salmonellae in inoculated and naturally contaminated natural animal casings was studied. Salmonellae were effectively destroyed (99.999%) in inoculated hog casings after exposure for 24 h to saturated brine at pH 4.0 and 10.0 adjusted with acetic acid and sodium hydroxide, respectively. Treatment of inoculated hog and sheep casings in saturated brine or saturated brine with citric acid was not nearly as effective as brine containing acetic acid or sodium hydroxide. Salmonellae in naturally contaminated hog casings were virtually eliminated after 21 days of storage in crystalline sodium chloride. Salmonella in sheep and hog casings were eliminated after 7 days of storage in crystalline salt. Treatment of naturally contaminated hog casings with glycerin-salt or sorbitol-salt solutions was not as effective in destroying salmonellae as treatment with crystalline salt. Destruction of salmonellae in inoculated and naturally contaminated natural animal casings was studied. Salmonellae were effectively destroyed (99.999%) in inoculated hog casings after exposure for 24 h to saturated brine at pH 4.0 and 10.0 adjusted with acetic acid and sodium hydroxide, respectively. Treatment of inoculated hog and sheep casings in saturated brine or saturated brine with citric acid was not nearly as effective as brine containing acetic acid or sodium hydroxide. Salmonellae in naturally contaminated hog casings were virtually eliminated after 21 days of storage in crystalline sodium chloride. Salmonellae in sheep and hog casings were eliminated after 7 days of storage in crystalline salt. Treatment of naturally contaminated hog casings with glycerin-salt or sorbitolsalt solutions was not as effective in destroying salmonellae as treatment with crystalline salt. Natural casings are considered as processed meat products, and as such must be free of Salmonella. Therefore, the processing applied to natural casings must effectively destroy this organism. The raw natural casings are the intestinal tracts of animals and, therefore, would be that part of the carcass most likely to be contaminated with Salmonella. The basic processing of natural casings involves the removal of fecal material and washing. The casings may then be wet packed in saturated brine or packed dry with crystalline salt. Natural casings are decontaminated in two of the process steps. First is the physical removal of bacteria through washing, and second is the destruction of bacteria by high concentrations of salt. The natural level of contamination and efficiency of the washing procedure as well as the temperature of storage in salt will determine the length of storage time required to rid the casings of Salmonella. Sanitation in handling of unsalted casings also has an effect on the numbers of salmonellae that contaminate the unsalted material. Investigators at the University of Zurich (3) found that salmonellae survived in inoculated salted casings after 27 days of storage. The casings were not studied beyond 27 days. Zabel (D. V. M. thesis, Free Univ. of Berlin, 1959) reported that salmonellae in inoculated casings treated with dry salt and soda were destroyed after 11 days of storage. Several attempts have been made to develop processes which destroy salmonellae more rapidly than occurs with salting alone. Paddock (U.S. Patent 2,673,804) patented a process involving a combination of acetic acid and hydrogen peroxide to improve the texture and appearance of tripe. This process may also serve to destroy salmonellae. Bickel (U.S. Patent 2,735,776, 1956) preserved sausage casings in a solution of 2 to 10% tartaric acid and sodium hexametaphosphate. Swift and Co. (C. A. Rinehart and L. B. Jensen, U.S. Patent 2,966,415, 1960) obtained a patent for the use of peracetic acid for bleaching casings. Unfortunately, none of these processes was evaluated for the ability to destroy salmonellae even though the chemical agents used are known to have bactericidal effects. The Danish Meat Research Institute (2) described a procedure involving the salting of casings in 4% lactic or 4% tartaric acid for 8 h followed by a cold-water rinse and then immersion in 1% sodium tripolyphosphate for 12 h. The procedure was effective in killing bacteria and has been approved for use in Denmark. The casings apparently were not weakened by the process. In our studies, the effect of pH-adjusted brine solutions on the survival of salmonellae inoculated into natural casings was evaluated. In addition, the destruction by salt and brine solutions of naturally occurring salmonellae in casings was examined. MATERIALS AND METHODS Sources of casings. All casings used in these studies were cleaned, finished, and unsalted. Hog and sheep casings for inoculation with salmonellae were obtained from two processors in the Midwest. Naturally contaminated hog and sheep casings were obtained from three processors, one each in the East, Midwest, and West. Naturally contaminated beef casings were obtained from a single Midwestern processor. Preparation of inoculum. Five Salmonella strains of the serotypes C, (0) b (H), G (0) z29 (H), E, (0) 1 complex (H), C, (0) G complex (H), and B (0) G complex (H) were used. The salmonellae were freshly isolated from low-moisture food samples. The cultures were grown separately for three transfers at 24-h intervals with incubation at 35 C. Equal volumes of each culture were mixed and then added at the rate of 1 to 1,000 ml of sterile phosphate-buffered distilled water at pH 7.2. Each casing for inoculation was attached to a sterile 50-ml funnel outlet and filled with the inoculum, which then was allowed to drain through the casing. After inoculation of the lumen, the casings were dipped in the inoculum to contaminate the outer surface. Test I: treatment of inoculated casings with pH-adjusted brine solutions. Saturated sodium chloride solutions were prepared, and acetic acid or sodium hydroxide was added so that the initial pH of of the brine solutions was 3.0 and 12.4, respectively. Hog casings were treated with regular saturated brine as a control and with pH-adjusted brine solutions. The inoculated hog casings were cut into about 20 segments (5 cm) and introduced into 500 ml of each of the pH-adjusted solutions. The ratio of casings to brine was 1:1 (wt/vol). The pH values of the casingbrine mixtures were 4.0 and 10.0 for the acetic acidand sodium hydroxide-containing brine solutions after 15 h at 25 C. Test II: treatment of inoculated casings with acidified salt. Citric acid was dry blended with salt so that addition of water to obtain a saturated solution provided a pH of 4.0. A control of regular salt was used for comparison. The dry citric acid-salt blend and regular salt were supplied by the Morton Salt Co., Chicago, Ill. Inoculated hog and sheep casings were passed through acidified or regular salt crystals. Excess salt was allowed to drop off. The inoculated and salted casings were then introduced at the rate of 30 segments (5 cm) into 500 ml of the corresponding saturated salt solution. Three inoculated casings of each type (hog and sheep) were not exposed to salt to determine the initial level of contamination. Sampling of inoculated casings. The hog casings from test I were sampled immediately after inoculation and after 15 and 48 h exposure to the brine solutions. In addition, the brine solutions were sampled 15 and 48 h after the casings had been added. The hog and sheep casings from test II were held at 25 C and sampled after 1, 2, 4, 8, 12, 24, 36, 48, and 74 h of exposure to the brine. One control and two-acid treated samples (sheep and hog) were removed at each time period. Test III: treatment of naturally contaminated casings. Thirty samples each of hog and sheep casings and 10 beef casings were used. The samples were drawn from the processing lines in early morn-ing, noon, and late afternoon for 5 days. Each casing sample was packaged individually and frozen immediately by the processors. Each sample weighed approximately 100 g. Prior to testing, the casings were thawed overnight at 6 C. After thawing, 11 g of each casing was weighed into a sterile Waring blender jar by cutting through the mass of casing with sterile scissors. These samples were used to determine the natural level of salmonellae prior to treatment. The remaining portion of each hog and sheep casing sample was then divided into three equal smaller portions for treatment. One portion was packed in a sterile plastic bag with sodium chloride at a ratio of 30 g of casing to 200 g of salt. The second portion was packed with 250 ml of glycerin-salt solution (25% glycerin, 22.7% salt) and the third portion was packed with 250 ml of sorbitol-salt solution (20.9% sorbitol, 19.3% salt). Beef casings were stored in salt only. Immediately after packing, the casings were placed at 6 C for the entire test period. Samples were removed from storage for analysis after 7, 14, and 21 days. Each treated casing was sampled by weighing 11 g into a sterile Waring blender jar. Microbiological testing. Bacteriological media used in these studies were obtained from Difco, Detroit, Mich. Casings from tests I and II were analyzed by blending an 11-g sample of casing with 99 ml of sterile buffer in a sterile Waring blender for 1 min. From this 1:10 dilution, three 10-ml portions of the sample were introduced into three tubes of singlestrength nutrient broth. Further dilutions were prepared to analyze the samples for Salmonella by the most-probable-number technique. Salmonellae were also determined by using direct plating on brilliant green agar. Total coliforms were determined by direct plating of appropriate dilutions with deoxycholate agar. Total aerobic plate counts were determined with standard plate count agar. The colonies on these plates were counted after 24 or 48 h of incubation at 35 C. The naturally contaminated casings from test III were analyzed for salmonellae only. The 11-g sample weighed into the sterile blender jar was blended with 99 ml of lactose broth containing 1% tergitol anionic-7 (Union Carbide, Chicago, Ill.) for 1 min at high speed. The blender contents were then removed to a sterile bottle, and serial dilutions were made in lactose broth with tergitol to yield single subsamples of 1, 0.1, and 0.01 g. A single 10-g sample of each casing was weighed directly into 90 ml of lactose broth. For all samples, inoculated and naturally contaminated, Salmonella analysis was completed by inoculation of the pre-enrichment broths to tetrathionate broth containing brilliant green dye. The tetrathionate broth cultures were incubated for 24 h at 35 C and then streaked onto differential agars. Salmonella-suspect colonies were identified by appropriate biochemical and serological techniques (4). For the inoculated casings, however, most salmonellae numbers were determined by direct plating on brilliant green agar with biochemical and serological confirmation of suspect colonies. RESULTS Results from test I using inoculated hog casings treated in saturated brine at pH 4.0 and 10.0 indicated that these treatments effectively reduce the level of Salmonella in the casings by greater than 99.999% after 15 h at 25 C (Table 1). After 48 h, no salmonellae were detectable in either the casings or brine solutions at either pH. This represents a reduction of greater than 99.99999%. Results from test II show that the treatment of hog and sheep casings in regular saturated brine or saturated brine with citric acid was not nearly as effective as that observed with the acetic acidor sodium hydroxide-containing brine solutions (Tables 2, 3). In the citric acid-adjusted brine, salmonellae were isolated after treatment for 72 h. The results indicate that citric acid-treated salt was no more effective in destroying salmonellae than saturated brine. The data from test III for casings naturally contaminated with salmonellae and treated with crystalline salt are presented in Table 4. Hog casings were the most heavily contaminated of the types tested. Salmonellae were virtually eliminated from the hog casings after 21 days of storage at 6 C. Salmonellae in the sheep and beef casings were eliminated after 7 days in salt packs. The bactericidal effect of glycerin-salt or sorbitol-salt solutions was not as great as that of salt alone for the hog casings (Tables 5, 6). After 21 days of storage, salmonellae were detected in 1 of the 30 10-g samples in both solutions. No salmonellae were detected after 7 days in the sheep casings stored in the solutions. DISCUSSION The bactericidal properties of acetic acid, beyond its influence as an acidulant, are obvi-ous when the destruction of salmonellae by acetic acid-and citric acid-adjusted brine solutions are compared. The buffering capacity of the casings was considered in the initial adjustment of the brine pH with acetic acid, but was not taken into account with citric acid since the commercial source of citric acid-adjusted salt was used as supplied. It has been shown previously that acetic acid more effectively inhibits salmonellae than other commonly used acidulants such as hydrochloric, citric, tartaric, gluconic, malic, lactic, and succinic acids (1). Obviously, this information implies that the cidal effects are due to more than pH alone. The effect of the sodium hydroxide at the initial sampling period appeared greater than acetic acid, but this greater kill was due to the lag time in analyzing the alkaline-treated casings. The discrepancy between the total count results and Salmonella numbers for untreated casings at the initial sampling period in Table 1 was the result of holding the samples overnight before analyzing for total bacteria. This illustrates the destructive capacity of brine alone. The treatments involving pH adjustment of the brine were performed on artificially contaminated casings. Information pertaining to the natural levels of Salmonella contamination must be used in evaluating bactercidal processing steps. The segment of the study involving naturally contaminated samples revealed that the levels of' salmonellae are relatively low. If' casings are treated with acidic brine adjusted with acetic acid or packed in salt, the food poisoning significance of natural casings would be minimal as compared to other raw meat and poultry products. For rapid treatment of' casings, acetic acid brine would render naturally contaminated casing virtually free of salmonellae within 24 h. Storage of casings thus treated in salt would contribute to further reduction in the numbers of salmonellae. If a processor chooses to use the more traditional approach of salting alone, casings could be expected to be Salmonella negative after 2 to 3 weeks. The use of glycerinsalt or sorbitol-salt solutions would have nearly the same effect as dry salting, but the manipulations involved in handling the casings would be considerably greater. There would be essentially no Salmonella risk with natural casings moving in international trade because of the lengthy exposure of the casings to saturated brine solutions. There may be some risk entailed in using casings fresh from the cleaning and finishing process, since no salting or other kill treatment would be employed. In conclusion, the appropriate treatment of natural casings contaminated by salmonellae with acetic acid or sodium hydroxide pHadjusted brine solution or crystalline salt would effectively destroy salmonellae. Such treated

v3-fos

2020-12-10T09:04:13.105Z

1974-01-01T00:00:00.000Z

237235378

s2

Salmonella in Natural Animal Casings Destruction of salmonellae in inoculated and naturally contaminated natural animal casings was studied. Salmonellae were effectively destroyed (99.999%) in inoculated hog casings after exposure for 24 h to saturated brine at pH 4.0 and 10.0 adjusted with acetic acid and sodium hydroxide, respectively. Treatment of inoculated hog and sheep casings in saturated brine or saturated brine with citric acid was not nearly as effective as brine containing acetic acid or sodium hydroxide. Salmonellae in naturally contaminated hog casings were virtually eliminated after 21 days of storage in crystalline sodium chloride. Salmonella in sheep and hog casings were eliminated after 7 days of storage in crystalline salt. Treatment of naturally contaminated hog casings with glycerin-salt or sorbitol-salt solutions was not as effective in destroying salmonellae as treatment with crystalline salt. Destruction of salmonellae in inoculated and naturally contaminated natural animal casings was studied. Salmonellae were effectively destroyed (99.999%) in inoculated hog casings after exposure for 24 h to saturated brine at pH 4.0 and 10.0 adjusted with acetic acid and sodium hydroxide, respectively. Treatment of inoculated hog and sheep casings in saturated brine or saturated brine with citric acid was not nearly as effective as brine containing acetic acid or sodium hydroxide. Salmonellae in naturally contaminated hog casings were virtually eliminated after 21 days of storage in crystalline sodium chloride. Salmonellae in sheep and hog casings were eliminated after 7 days of storage in crystalline salt. Treatment of naturally contaminated hog casings with glycerin-salt or sorbitolsalt solutions was not as effective in destroying salmonellae as treatment with crystalline salt. Natural casings are considered as processed meat products, and as such must be free of Salmonella. Therefore, the processing applied to natural casings must effectively destroy this organism. The raw natural casings are the intestinal tracts of animals and, therefore, would be that part of the carcass most likely to be contaminated with Salmonella. The basic processing of natural casings involves the removal of fecal material and washing. The casings may then be wet packed in saturated brine or packed dry with crystalline salt. Natural casings are decontaminated in two of the process steps. First is the physical removal of bacteria through washing, and second is the destruction of bacteria by high concentrations of salt. The natural level of contamination and efficiency of the washing procedure as well as the temperature of storage in salt will determine the length of storage time required to rid the casings of Salmonella. Sanitation in handling of unsalted casings also has an effect on the numbers of salmonellae that contaminate the unsalted material. Investigators at the University of Zurich (3) found that salmonellae survived in inoculated salted casings after 27 days of storage. The casings were not studied beyond 27 days. Zabel (D. V. M. thesis, Free Univ. of Berlin, 1959) reported that salmonellae in inoculated casings treated with dry salt and soda were destroyed after 11 days of storage. Several attempts have been made to develop processes which destroy salmonellae more rapidly than occurs with salting alone. Paddock (U.S. Patent 2,673,804) patented a process involving a combination of acetic acid and hydrogen peroxide to improve the texture and appearance of tripe. This process may also serve to destroy salmonellae. Bickel (U.S. Patent 2,735,776, 1956) preserved sausage casings in a solution of 2 to 10% tartaric acid and sodium hexametaphosphate. Swift and Co. (C. A. Rinehart and L. B. Jensen, U.S. Patent 2,966,415, 1960) obtained a patent for the use of peracetic acid for bleaching casings. Unfortunately, none of these processes was evaluated for the ability to destroy salmonellae even though the chemical agents used are known to have bactericidal effects. The Danish Meat Research Institute (2) described a procedure involving the salting of casings in 4% lactic or 4% tartaric acid for 8 h followed by a cold-water rinse and then immersion in 1% sodium tripolyphosphate for 12 h. The procedure was effective in killing bacteria and has been approved for use in Denmark. The casings apparently were not weakened by the process. In our studies, the effect of pH-adjusted brine solutions on the survival of salmonellae inoculated into natural casings was evaluated. In addition, the destruction by salt and brine solutions of naturally occurring salmonellae in casings was examined. MATERIALS AND METHODS Sources of casings. All casings used in these studies were cleaned, finished, and unsalted. Hog and sheep casings for inoculation with salmonellae were obtained from two processors in the Midwest. Naturally contaminated hog and sheep casings were obtained from three processors, one each in the East, Midwest, and West. Naturally contaminated beef casings were obtained from a single Midwestern processor. Preparation of inoculum. Five Salmonella strains of the serotypes C, (0) b (H), G (0) z29 (H), E, (0) 1 complex (H), C, (0) G complex (H), and B (0) G complex (H) were used. The salmonellae were freshly isolated from low-moisture food samples. The cultures were grown separately for three transfers at 24-h intervals with incubation at 35 C. Equal volumes of each culture were mixed and then added at the rate of 1 to 1,000 ml of sterile phosphate-buffered distilled water at pH 7.2. Each casing for inoculation was attached to a sterile 50-ml funnel outlet and filled with the inoculum, which then was allowed to drain through the casing. After inoculation of the lumen, the casings were dipped in the inoculum to contaminate the outer surface. Test I: treatment of inoculated casings with pH-adjusted brine solutions. Saturated sodium chloride solutions were prepared, and acetic acid or sodium hydroxide was added so that the initial pH of of the brine solutions was 3.0 and 12.4, respectively. Hog casings were treated with regular saturated brine as a control and with pH-adjusted brine solutions. The inoculated hog casings were cut into about 20 segments (5 cm) and introduced into 500 ml of each of the pH-adjusted solutions. The ratio of casings to brine was 1:1 (wt/vol). The pH values of the casingbrine mixtures were 4.0 and 10.0 for the acetic acidand sodium hydroxide-containing brine solutions after 15 h at 25 C. Test II: treatment of inoculated casings with acidified salt. Citric acid was dry blended with salt so that addition of water to obtain a saturated solution provided a pH of 4.0. A control of regular salt was used for comparison. The dry citric acid-salt blend and regular salt were supplied by the Morton Salt Co., Chicago, Ill. Inoculated hog and sheep casings were passed through acidified or regular salt crystals. Excess salt was allowed to drop off. The inoculated and salted casings were then introduced at the rate of 30 segments (5 cm) into 500 ml of the corresponding saturated salt solution. Three inoculated casings of each type (hog and sheep) were not exposed to salt to determine the initial level of contamination. Sampling of inoculated casings. The hog casings from test I were sampled immediately after inoculation and after 15 and 48 h exposure to the brine solutions. In addition, the brine solutions were sampled 15 and 48 h after the casings had been added. The hog and sheep casings from test II were held at 25 C and sampled after 1, 2, 4, 8, 12, 24, 36, 48, and 74 h of exposure to the brine. One control and two-acid treated samples (sheep and hog) were removed at each time period. Test III: treatment of naturally contaminated casings. Thirty samples each of hog and sheep casings and 10 beef casings were used. The samples were drawn from the processing lines in early morn-ing, noon, and late afternoon for 5 days. Each casing sample was packaged individually and frozen immediately by the processors. Each sample weighed approximately 100 g. Prior to testing, the casings were thawed overnight at 6 C. After thawing, 11 g of each casing was weighed into a sterile Waring blender jar by cutting through the mass of casing with sterile scissors. These samples were used to determine the natural level of salmonellae prior to treatment. The remaining portion of each hog and sheep casing sample was then divided into three equal smaller portions for treatment. One portion was packed in a sterile plastic bag with sodium chloride at a ratio of 30 g of casing to 200 g of salt. The second portion was packed with 250 ml of glycerin-salt solution (25% glycerin, 22.7% salt) and the third portion was packed with 250 ml of sorbitol-salt solution (20.9% sorbitol, 19.3% salt). Beef casings were stored in salt only. Immediately after packing, the casings were placed at 6 C for the entire test period. Samples were removed from storage for analysis after 7, 14, and 21 days. Each treated casing was sampled by weighing 11 g into a sterile Waring blender jar. Microbiological testing. Bacteriological media used in these studies were obtained from Difco, Detroit, Mich. Casings from tests I and II were analyzed by blending an 11-g sample of casing with 99 ml of sterile buffer in a sterile Waring blender for 1 min. From this 1:10 dilution, three 10-ml portions of the sample were introduced into three tubes of singlestrength nutrient broth. Further dilutions were prepared to analyze the samples for Salmonella by the most-probable-number technique. Salmonellae were also determined by using direct plating on brilliant green agar. Total coliforms were determined by direct plating of appropriate dilutions with deoxycholate agar. Total aerobic plate counts were determined with standard plate count agar. The colonies on these plates were counted after 24 or 48 h of incubation at 35 C. The naturally contaminated casings from test III were analyzed for salmonellae only. The 11-g sample weighed into the sterile blender jar was blended with 99 ml of lactose broth containing 1% tergitol anionic-7 (Union Carbide, Chicago, Ill.) for 1 min at high speed. The blender contents were then removed to a sterile bottle, and serial dilutions were made in lactose broth with tergitol to yield single subsamples of 1, 0.1, and 0.01 g. A single 10-g sample of each casing was weighed directly into 90 ml of lactose broth. For all samples, inoculated and naturally contaminated, Salmonella analysis was completed by inoculation of the pre-enrichment broths to tetrathionate broth containing brilliant green dye. The tetrathionate broth cultures were incubated for 24 h at 35 C and then streaked onto differential agars. Salmonella-suspect colonies were identified by appropriate biochemical and serological techniques (4). For the inoculated casings, however, most salmonellae numbers were determined by direct plating on brilliant green agar with biochemical and serological confirmation of suspect colonies. RESULTS Results from test I using inoculated hog casings treated in saturated brine at pH 4.0 and 10.0 indicated that these treatments effectively reduce the level of Salmonella in the casings by greater than 99.999% after 15 h at 25 C (Table 1). After 48 h, no salmonellae were detectable in either the casings or brine solutions at either pH. This represents a reduction of greater than 99.99999%. Results from test II show that the treatment of hog and sheep casings in regular saturated brine or saturated brine with citric acid was not nearly as effective as that observed with the acetic acidor sodium hydroxide-containing brine solutions (Tables 2, 3). In the citric acid-adjusted brine, salmonellae were isolated after treatment for 72 h. The results indicate that citric acid-treated salt was no more effective in destroying salmonellae than saturated brine. The data from test III for casings naturally contaminated with salmonellae and treated with crystalline salt are presented in Table 4. Hog casings were the most heavily contaminated of the types tested. Salmonellae were virtually eliminated from the hog casings after 21 days of storage at 6 C. Salmonellae in the sheep and beef casings were eliminated after 7 days in salt packs. The bactericidal effect of glycerin-salt or sorbitol-salt solutions was not as great as that of salt alone for the hog casings (Tables 5, 6). After 21 days of storage, salmonellae were detected in 1 of the 30 10-g samples in both solutions. No salmonellae were detected after 7 days in the sheep casings stored in the solutions. DISCUSSION The bactericidal properties of acetic acid, beyond its influence as an acidulant, are obvi-ous when the destruction of salmonellae by acetic acid-and citric acid-adjusted brine solutions are compared. The buffering capacity of the casings was considered in the initial adjustment of the brine pH with acetic acid, but was not taken into account with citric acid since the commercial source of citric acid-adjusted salt was used as supplied. It has been shown previously that acetic acid more effectively inhibits salmonellae than other commonly used acidulants such as hydrochloric, citric, tartaric, gluconic, malic, lactic, and succinic acids (1). Obviously, this information implies that the cidal effects are due to more than pH alone. The effect of the sodium hydroxide at the initial sampling period appeared greater than acetic acid, but this greater kill was due to the lag time in analyzing the alkaline-treated casings. The discrepancy between the total count results and Salmonella numbers for untreated casings at the initial sampling period in Table 1 was the result of holding the samples overnight before analyzing for total bacteria. This illustrates the destructive capacity of brine alone. The treatments involving pH adjustment of the brine were performed on artificially contaminated casings. Information pertaining to the natural levels of Salmonella contamination must be used in evaluating bactercidal processing steps. The segment of the study involving naturally contaminated samples revealed that the levels of' salmonellae are relatively low. If' casings are treated with acidic brine adjusted with acetic acid or packed in salt, the food poisoning significance of natural casings would be minimal as compared to other raw meat and poultry products. For rapid treatment of' casings, acetic acid brine would render naturally contaminated casing virtually free of salmonellae within 24 h. Storage of casings thus treated in salt would contribute to further reduction in the numbers of salmonellae. If a processor chooses to use the more traditional approach of salting alone, casings could be expected to be Salmonella negative after 2 to 3 weeks. The use of glycerinsalt or sorbitol-salt solutions would have nearly the same effect as dry salting, but the manipulations involved in handling the casings would be considerably greater. There would be essentially no Salmonella risk with natural casings moving in international trade because of the lengthy exposure of the casings to saturated brine solutions. There may be some risk entailed in using casings fresh from the cleaning and finishing process, since no salting or other kill treatment would be employed. In conclusion, the appropriate treatment of natural casings contaminated by salmonellae with acetic acid or sodium hydroxide pHadjusted brine solution or crystalline salt would effectively destroy salmonellae. Such treated

v3-fos

2020-12-10T09:04:20.703Z

1974-10-01T00:00:00.000Z

237233932

s2

Concentration of Enteric Viruses from Water with Lettuce Extract A method for recovering enteroviruses, adenovirus, and reovirus from water with lettuce extract is described. Lettuce extract at pH 8.5 was added to the sample and the pH was reduced stepwise with hydrochloric acid to 4.0 to 4.5. The flocculent lettuce-extract particles, and adsorbed virus, were readily removed from solution by low-speed centrifugation. Electron microscopy suggests that, under conditions suitable for adsorption, virus particles are coated with the lettuce-extract colloid. sample and the pH was reduced stepwise with hydrochloric acid to 4.0 to 4.5. The flocculent lettuce-extract particles, and adsorbed virus, were readily removed from solution by low-speed centrifugation. Electron microscopy suggests that, under conditions suitable for adsorption, virus particles are coated with the lettuce-extract colloid. A number of methods for the recovery of virus from water have been described recently (1,(4)(5)(6)(7). Virus adsorption followed by elution has been the principal approach for virus concentration. In an earlier report (2), we described a method for enterovirus recovery using lettuce floc. The work has been extended to include two other enteric viruses, reovirus 1 and adenovirus 7a. Lettuce extract was prepared as described previously (2). The extract is a clear amber-colored colloidal suspension at pH 5.5 or above. A floc forms below pH 5.5 and is readily sedimented by low-speed centrifugation at pH 4.0 to 4.5. The dry weight of the floc varied from 1.5 to 3.0 mg/ml depending on the batch. Approximately 50% of the dry weight was protein, as determined by the method of Lowry et al. (3). Adsorption to lettuce extract of coxsackievirus types B4 and B5, echovirus type 7, poliovirus type 1 (Sabin), reovirus type 1, and adenovirus type 7a was examined as follows. Virus concentrations of 100 or 1,000 plaqueforming units (PFU) in 0.1 ml of phosphate-buffered saline were added individually to samples containing 10 to 1,000 ml of distilled water. Final virus concentrations varied from 0.1 to 100 PFU per ml of sample. A similar inoculum was added to duplicate samples of 2.5 ml of growth medium to serve as virus controls. A 10% volume of lettuce extract was added to the water samples at pH 5.0, 6.0, 7.0, 8.0, or 8.5. Samples were adjusted to pH 4.0 to 4.5 in 0.5 to 1 log steps by dropwise addition of HC1. After centrifugation at 1,000 x g for 10 min, the pellets were dissolved by adding NaOH; 0.05 ml of 1 N NaOH dissolved the 0.4to 0.8-ml pellet obtained from a 200-ml sample. Water samples larger than 200 ml were divided, centrifuged, and finally recombined after dissolution of the pellets. The sample was diluted to 2.5 ml with concentrated medium 199 to give single strength medium and then was assayed on a single monolayer of cultured cells as described previously (2). HEp-2 cells were used for the coxsackievirus and poliovirus, Vero cells were used for echovirus, and primary African green monkey kidney cells were used for reovirus and adenovirus. Enterovirus plaques were read after 3 days, adenovirus and reovirus after 8 days. Results of preliminary experiments indicated that virus in the dissolved pellet alone or with added 10% serum would not adsorb to cell monolayers. Incorporation of medium 199, Earle, or saline with or without 10% serum, however, permitted infection of monolayers. Coxsackievirus B4, B5, echovirus 7, poliovirus 1, and adenovirus 7a were efficiently concentrated from water with colloidal lettuce extract at pH 6.0 or higher. Flocculent extract at pH 5.0 was less efficient in concentrating adenovirus than enteroviruses; reovirus required a pH of 8.0 for effective concentration (Fig. 1). Quantitative recovery of all viruses tested was accomplished by adding lettuce extract at pH 8.5 to the water sample followed by the dropwise addition of HCl in 0.5 to 1 log steps to pH 4.0 to 4.5. Virus inputs varying from 0.1 to 100 PFU per ml of sample were concentrated from water volumes of 10 to 1,000 ml. Concentrates from volumes as great as 500 ml could be assayed in one plastic dish without apparent toxicity to the cell monolayer. Less than 1% of the virus input remained in the supernatant fluid as unadsorbed virus. Mixed as well as unmixed populations of reovirus and adenovirus were adsorbed. For electron microscopy, viral suspensions were diluted with 2% potassium phosphotungstate at pH 6.8 and spread on pure carbon or carbon-coated Formvar electron microscope grids. The carbon surface was rendered hydrophilic by a brief treatment (ca. 40 s) of exposure to ionized air in a Plasmod unit (Tegal Corp., Richmond, Calif.). The negatively stained viruses were then examined at a magnification of 60,000 to 100,000 in a Siemens Elmiscop 101. Electron photomicrographs showed that virus was coated with colloidal particles or aggregates of them at optimal pH levels. Capsomers of reovirus were sharply defined at pH 6.0, but were indistinct at pH 8.5 due to adsorbed lettuce-extract colloid (Fig. 2). Adenovirus was well defined at pH 4.5 but hazy at pH 6.0 (Fig. 3). Controls of reovirus and adenovirus without lettuce extract at similar pH measurements were all sharply defined. Colloidal particles were a few nanometers in diameter; some aggregates of the particles were as large as 100 nm. By following the procedure outlined above, enteroviruses, adenoviruses, and reoviruses are efficiently removed and concentrated from water.

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2020-12-10T09:04:12.767Z

1974-05-01T00:00:00.000Z

237235356

s2

Proteinase Activity in Slow Lactic Acid-Producing Variants of Streptococcus lactis Variants of Streptococcus lactis that produce lactic acid slowly in milk were isolated by inducing plasmid loss in the wild type at 39 to 40 C. Such strains had lost most of their surface-bound proteinase activity and were designated prt-. The specific proteinase activities of S. lactis C10 prt+ whole cells and solubilized cell walls were 7 and 18 times, respectively, those of the prt- strain, but spheroplast lysates of prt+ and prt- strains contained similar proteinase activity. S. lactis H1 showed a similar relative distribution of activity between prt+ and prt- cellular fractions, although the overall level was lower. The limited growth in milk, characteristic of prt- strains, can be explained in terms of their low surface-bound proteinase activity. Streptococcus lactis and S. cremoris may spontaneously segregate slow variants that differ from the parent strain by their limited growth in milk (5). Such variants appear in susceptible cultures at high frequency (ca. 1%) and do not revert to the parental type (2,3,22). As slow variants can also be induced when the wild-type strain is treated with acridines or grown at high temperatures, this characteristic is believed to arise through loss of a plasmid (14). Evidence presented by a number of workers strongly suggests that slow variants are deficient in proteinase activity. Growth in milk is stimulated to wild-type levels by the addition of hydrolyzed casein (4), and in broth media both variant and parent strain show the same rates of growth and acid production (2). Milk cultures may contain up to 50% slow variants before the rate of acid production differs from that of the parent. This suggests a growth stimulatory interaction between the parent and the slow variant (14). Although "slowness" and relatively low proteinase activity have been correlated (2), there has been little direct evidence for proteinase deficiency in slow variants. Westhoff et al. (22) compared proteinase activity in whole and fractionated cells of S. lactis 3 and a slow acid-producing mutant. Quantitative differences in proteinase activity between the parent and mutant strains, assayed using either whole cells or cell fractions, were low (ca. 1.5-fold) and did not adequately explain the ' Present address: Department of Biology, McGill University, Montreal, Canada. growth characteristics of slow variants in milk. The mutant "intracellular" enzyme, however, did differ from that of the parent, and it was concluded that a different proteinase specificity was responsible for the limited growth of the mutant in milk (21). This study compares the proteinase activity of two strains of S. lactis (prt+) with that of slow variants derived from them (prt-). The behavior of the prt-strains in milk can be accounted for by the loss of most of their surface-bound proteinase activity. A portion of this paper was included in an M.S. thesis submitted by N lactis prt-strains were isolated at high frequency (up to 30%) after growth at 39 to 40 C. Prtclones developed as tiny colonies on citrate-milk agar (16) and were differentiated from the larger prt+ colonies on this medium. S. cremoris prt+ and prt-could not be differentiated on this medium. Optimal differentiation was obtained when the medium was autoclaved at 115 C for 15 min. prt-clones selected were lac+ on the medium of McKay et al. (9) and were sensitive to the same virulent phages as the parent. Skim milk was prepared from a single batch of spray-dried nonfat milk powder, reconstituted to 9.5% 933 total solids and autoclaved at 10 lb/inch2 for 20 min. Plate counts were obtained using M,. agar (8) and T, broth (20) was used for cell preparation and growth experiments. Lactate determination. Lactate was measured as the lactate-ferric chloride complex at 400 nm (17). Cell fractionation and enzyme assay. The methods used were those of Thomas et al. (20). Cells growing logarithmically in T5 broth were harvested by centrifugation, lysed by enzymatic or mechanical methods, fractionated, and assayed for proteolytic activity using 125I-labeled casein as substrate. RESULTS Growth of prt+ and prt-in sterile milk and T5 broth. Doubling times of S. lactis C10 prt+ and prt-during exponential growth in sterile milk at 30 C were 60 and 72 min, respectively ( Fig. 1). 'ClO prt+, however, reached a maximum population of 2 x 10' colony-forming units (CFU)/ml in 7 h, whereas C10 prt-ceased exponential growth after 5 h and reached a maximum population density of 5.5 x 108 CFU/ml after 10 h. Both prt+ and prtstrains remained as diplococci throughout growth. Although the viable count of C10 prt-remained stationary after 10 h, acid production continued at a slow rate, and the milk reached pH 5.0 after 35 h of incubation. That is, C10 prt-cells continue to produce lactate while colony-forming units fail to increase (Fig. 2). The addition of trypsin-hydrolyzed casein (1 mg/ml) to cultures of C10 prt+ or C10 prt-growing in skim milk decreased the doubling time to 54 min for each strain, and both reached a maximum population density of 3 x 109 CFU/ml. In T5 broth, C10 prt+ and prtwere indistinguishable and showed the same growth rate (doubling time 38 min, maximum population 1.2 x 10' CFU/ml). This feature of growth was common to all prt-strains isolated in this laboratory and can be clearly seen in growth curves for S. Iactis H1 and S. cremoris R1 in T5 broth (Fig. 3). The slow acid-producing mutant of S. lactis 3 grew at a significantly slower rate than the wild type in T5 broth. Doubling times during logarithmic growth were 78 and 63 min, respectively. A prt-derivative of strain 3 was isolated and found to have identical growth characteristics to the parent strain in broth. Proteinase in whole cells and cell fractions. Intact cells of C10 prt+ exhibited about seven times the proteinase activity of C10 prt-, specific activities being 35.8 and 5.2 U per mg dry weight, respectively (Table 1). When the cell wall was removed under conditions that gave insignificant cell lysis (20), the majority of the prt+ proteinase activity was released. The Growth of S. lactis C10 prt+ and prt-in skim milk at 30 C. Two hundred-milliliter volumes of skim milk were inoculated with 2 ml of C10 prt+ and 8 ml of CIO prt-, respectively. Inocula were from 16-h, 22 C skim milk cultures. The inoculated milks were divided into portions and incubated. At intervals samples were removed for pH measurement (A, prt+; A, prt-); the culture was then chilled, diluted, and plated for colony-forming units (0, prt+; *, prt-). Fig. 1; lactate and colony-forming units were determined at intervals. same treatment removed less than half the prtactivity, the relative activity (prt+: prt-) being ca. 18: 1. No activity could be detected in membranes from either C10 prt+ or C10 prt-. The slightly higher intracellular activity de- Mid-log cells were washed twice in 0.2 M phosphate buffer, pH 6.4, suspended in spheroplasting medium (0.5 M sucrose, 20 mM MgCl2, 0.2 phosphate buffer, pH 6.4), and 3 ml phage-associated lysin was added. The suspension was incubated at 30 C for 120 min and centrifuged 35,000 x g, and the supernatant was assayed (20). c 35,000 x g pellet of solubilized cell walls, resuspended in buffer of equivalent volume to the original suspension. d Not detectable. tected on lysis of prt+, as compared with prtspheroplasts, is not considered significant. C10 prt+ and prtwere also fractionated after mechanical disintegration. The pellet containing cell walls and membranes (35,000 x A //^g , 10 min) contained 75% of the prt+ activity recovered in the component fractions but only 27% of the prt-activity. Specific activities were 18.6 and 1.3, respectively. Half of the prt-proteinase activity was associated with the cytoplasm (not sedimented at 157,000 x g, 120 min), whereas in the parent this fraction contained only one-tenth of the activity. Intact cells of S. lactis H1 prt+ (specific activity, 17.0) had less surface-bound proteinase activity than C10 prt+ (specific activity, 35.8), but the differences between prt+ and prtfollowed an identical pattern to that observed with C10. Specific activities of solubilized cell walls were 16.8 (prt+) and 3.6 (prt-); those of 3 4 the spheroplast lysates were 7.9 (prt+) and 10.7 (prt-). DISCUSSION The growth of prt-strains in sterilized milk and in broth follows the pattern established by other workers (2). prt-strains grow and produce lactic acid slowly in milk, but both characteristics can be restored to normal levels, or better, by supplementing the milk with casein hydrolysate (4). prt+ and prt-are indistinguishable when grown in rich broth media. The slow lactate increase in milk without increase in viable count was, however, of particular interest. Dissociation of acid production from net growth has been reported in other systems and is probably widespread. The phenomenon appears to be associated with conditions of cellular stress. Cultures of S. faecalis approaching the growth-limiting pH have been reported to cease dividing before acid production is inhibited (10). Lowrie et al. (7) have also observed that S. cremoris AM2 ceases to divide but continues to produce acid when growth is initiated at 30 C and the incubation temperature is raised to 37.8 C. Depletion of available nitrogen in milk cultures of prt-bacteria appears to be a further means by which this effect can be induced. Variants of lactic streptococci that produce acid slowly in milk have been isolated and studied in a number of laboratories (2,14,22). Although these can normally be isolated at high frequency, not all slow acid producers are of the prttype. It is not uncommon to find mutants that, for some other reason, grow more slowly in milk than the parent. These mutants also grow more slowly in broth, and the lacmutants are one such group (9). It is essential therefore to screen putative prt-clones for growth rates in a broth medium where proteolytic activity is not essential for growth. The slow acid-producing strain of S. lactis 3 does not appear to be a prttype on the basis of its slow growth in T5 broth. This was confirmed when a prt-derivative of strain 3 was isolated that grew in broth at an identical rate to the parent strain. The enzyme assays clearly show the fundamental difference in proteinase activity between prt+ and prt-strains of S. lactis. The major portion of the proteinase activity in the parent strain has been shown to be localized near the cell surface using two methods of fractionation (20). Mechanical disruption and osmotic lysis both gave similar high levels of activity in fractions derived from the cell wall. In the present study, this activity has been found to be markedly reduced in prt-cells. S. lactis H1 had less total proteinase activity than strain C10 with a consequent reduction in relative activity between prt+ and prt-. The solubilized cell wall fraction from prt+, however, still had nearly five times the activity of the corresponding prt+ fraction. The low proteinase activity in prt-strains explains their limited growth in milk (see Fig. 1). It is likely that the maximum prt-cell count is determined largely by the initial amino acid and small peptide content of the sterilized milk. The specific activities of C10 prt+ and H1 prt+ spheroplast lysates showed that both parent strains carried a portion of their proteinase activity in fractions not associated with surface structures. The slight difference in levels of intracellular proteinase activity between prt+ and prt-in this respect is not considered significant. These intracellular enzymes may be responsible for degradation of peptides resulting from protein breakdown by the surface enzyme, as well as the turnover of endogenous nitrogen. Escherichia coli, for example, has at least eight distinct intracellular peptidases (18). Cells grown in broth were used for the present study due to the difficulty of harvesting bacteria from milk. The higher levels of available amino acids in broth may repress surface-bound proteinase activity, as has been reported with other extracellular proteinase systems (1,6,12,13). Hence, the differences found between prt+ and prt-strains in broth are probably an underestimation of the situation in milk. Westhoff et al. (22) reported that both "intracellular" and "membrane-associated" proteinase activities in S. lactis 3 were reduced by 30 to 35% in a slow acid-producing mutant. As the authors commented, such a difference might be expected to impair rather than prevent growth in milk. The only other difference between the two strains was an altered specificity of the intracellular enzyme (21). The present study shows that their conclusions cannot be applied to all slow lactic acid-producing strains of lactic streptococci. The loss of most of the surface-bound proteolytic activity accompanying the transition from prt+ to prt-is consistent with plasmid control of this character. Total proteolytic activity in lactic streptococci, however, is low compared with that of corresponding enzymes in some other bacteria (11). The proteinase character has not been recorded as a genetic marker in E. coli or Salmonella chromosome maps (15,19), however, and is possibly also plasmid linked in the enterobacteria. The fact that proteinase activity has been recognized and studied in the lactic streptococci is undoubtedly a consequence of the widespread use of milk as a culture medium for this group. The low level of cell-bound activity remaining in intact cells of C10 prt-is presumably determined by chromosomal genes as the possibility of significant cell lysis has been excluded (20). If the surface-bound proteinase is plasmid controlled, it is possible that it controls cellular activities other than proteinase synthesis. The transport of peptides has not been excluded, and further investigation will be required to clarify these points and physically identify the genetic element involved.

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Soil Ecology of Coccidioides immitis at Amerindian Middens in California Outbreaks of coccidioidomycosis and isolation of Coccidioides immitis have been reported from Amerindian middens. This study was undertaken to determine the most important ecological component(s) for the occurrence of C. immitis at archeological sites. Soils from 10 former Indian villages with no prior history of coccidioidal infection were collected and cultured. The physicochemical properties of the midden soils were compared with nonmidden soils and positive soils. The following theories for the sporadic distribution of the pathogen in the soil of the Lower Sonoran Life Zone were considered: (i) the Larrea tridentata (creosote bush) association, (ii) the preference for saline soils, (iii) isolation near rodent burrows, and (iv) animals as possible agents of dispersal. Results showed that a high percentage of the midden soils contained C. immitis, whereas none ofthe adjacent, nonmidden soils yielded the fungus. Physicochemical analyses revealed that the dark color and alkaline pH of the midden soils were due to past organic contamination. Repeated isolations were made from soils with low to moderate alkalinity. Alkalinity and sandy texture were consistent features of all soils in this study. However, the lack of any reports of nonsandy infested soils possibly indicates that the sandy texture and alkalinity may be factors in the distribution of this fungus. The organic content, soil parent material, and color were not important in the soil ecology. L. tridentata was not significant in the macroflora at the infested sites surveyed. Samples collected without reference to rodent burrows yielded a high percentage of recoveries. Animals, although not the major natural reservoir, cannot be ignored as possible factors in the ecology of C. immitis. Our knowledge of the ecology of Coccidioides immitis is more advanced than for any other respiratory mycotic disease agent (1). However, as Huppert has pointed out, "A major gap in our knowledge of C. immitis is why the fungus should be so limited in its natural distribution" (10). Others have reported on the uneven, yet consistent, occurrence of the fungus in the soils of the Lower Sonoran Life Zone (15,29). At one site, an area of approximately 2.5 m2 has been positive from 1954 through this study, whereas the surrounding soil has yielded only rare positives. The sporadic occurrence of the fungus in nature seems to contradict its laboratory physiology. It grows rapidly on all common media at temperatures of 20 to 30 C and is not exacting in its nutritional requirements. Growth occurs between pH 3.5 and 9.0 and on clay through sandy soils (13). The fungus is known to infect mam-'Present address: Department of Plant Pathology, University of California, Riverside, Calif. 92502. mals, reptiles, fish, and amphibians (30) and thrives on parts of many desert plants (23). Various theories have been advanced to explain the spotty distribution of C. immitis in the soils of endemic regions. Emmons (9) felt that the carcasses, sputa, urine, feces, and purulent materials of infected rodents were the sources of the pathogen in soil. Maddy (14) emphasized the presence of Larrea tridentata (creosote bush) at his isolation sites in Arizona. Egeberg and Ely (6) recovered the fungus from 13.6% of soil samples taken within 5 ft (15.24 m) of animal burrows, whereas only 3.4% of the samples collected further away were positive. Egeberg and his associates (7,8) reported that high soil salinity was related to increased recovery of C. immitis and the suppression of antagonists. Swatek (27) noted that repeated isolations had been made from old Indian campsites and suggested a possible relationship between the fungus and the increased organic content of the soil on these sites. The purpose of these studies was to define the most important ecological components for C. immitis infestation at former Amerindian habitation areas. Studies were undertaken to determine the relationship between the fungus and the midden soil (areas rich in charcoal, obsidian chips, and other evidence of domestic contamination) and the differences between midden and adjacent, nonmidden soil. MATERIALS AND METHODS Soil collection. Soils were collected aseptically from former Amerindian village sites having no known history of human infection in areas endemic for coccidioidomycosis. Villages in the widely separated Kern and Madera counties of California were chosen for this study, along with control midden soils from sites culturally positive for C. immitis from San Diego, Kern, Butte, and Merced counties (see Fig. 1 .). Amerindian habitation sites may be delineated by various criteria. In this study they included: (i) soil darkened by domestic contamination near watersources (some only seasonal) and, (ii) macroscopic artifacts of human occupation such as obsidian flakes or tools; bedrock mortars or metate stones; "housefloor" depressions of semisubterranean homes; soapstone, shell or glass beads; human remains and petroglyphs. Counties in California from which Amerindian, archeological soils originated. C. immitisinfested control soils (four) or sites (two) were from Butte, Kern, Madera, and San Diego counties. Two sites implicated in human infection were sampled in Fresno and Madera counties. Ten random sites with no known coccidioidal histories were chosen in Kern (three) and Madera (seven) counties. Additionally, cooperating archeologists collected soils from four random sites in Madera County. Determination of points for soil sampling within the middens was made by the use of grids or traverses. Collections of adjacent, nonmidden soils were made at the nearest area which resembled the midden in exposure, drainage and foliage. Soil isolation. The technique of soil isolation employed was basically the double-pour, antibioticfortified, yeast extract agar method (28). Modifications included using 10 g of soil in 90 ml of sterile, distilled water in milk dilution bottles and agitating the 1-in-10 soil suspensions for 1 min and again at 20 and 40 min, and then leaving them undisturbed for 20 min. The plates were incubated at ambient room temperature (23 to 27 C) in a humidified chamber for 3 to 5 weeks. The colonies were examined macro-and microscopically for similarities to C. immitis with consideration for the wide morphologic range possible (11). All fungi demonstrating arthroaleuriospores (20) or racquet cells, or both, were subcultured on Sabouraud dextrose agar. If, on examination of the subculture, it was felt there was still a resemblance to the pathogen, intraperitoneal inoculation of mice was carried out with an aqueous spore suspension. Only fungi which produced spherules and endospores in mice were reported as C. immitis. Soil characterization. Selected midden and adjacent soil samples were compared physicochemically. Carefully mixed individual or composite (combined by equal portions) samples were passed through a no. 10 mesh screen. They were subjected to the following analytical scheme (manufacturers' procedures were followed unless noted): (i) color was determined on dry and paste portions by using Munsell soil color charts (Munsell Color Company, Inc.); (ii) textural classification was made by use of stainless-steel, heat-sterilizable soil sieves; (iii) measurements of pH and Eh were made with a Beckman G pH-meter on 1:5 double distilled water, soil extracts, or soil pastes; (iv) electrical conductivities were determined with a model RC-16B2 Industrial Instruments, Inc., conductivity bridge on pastes and 1: 5 extracts; (v) levels of principle ions were delimited with a Simplex soil testing kit (Edwards Laboratory), and (vi) organic and inorganic carbon and organic nitrogen determinations were made by wet combustion and micro-Kjeldahl techniques, respectively, on powdered (to pass 400 mesh), autoclaved soils (2,12). Soil comparisons. Comparisons of positive midden soils were made with analyses of other infested soils reported in the literature (8,19), by personal communications and on soil samples received. The following persons provided either physicochemical data or samples of C. immitis-infested soils: J. L. Converse RESULTS Soil isolation. Ten sites of former Amerindian villages, with no known histories of coccidioidal infection, were chosen to represent the test middens. Two sites (4-Mad-117 and Fre-SFSC-1) associated with past infection, whose soils had never been studied mycologically, were included as additional controls. Table 1 summarizes the histories of infection and verification of prolonged human occupation at the midden sites surveyed. Results of the soil isolation of C. immitis are tabulated in Table 2 Soil characterization. The results of most of the physicochemical analyses of midden and adjacent, nonmidden soils are compiled in Table 3. The levels of principle ions in the 23 midden and adjacent soils may be summarized as follows: Mn2+, less than 1 ppm; K+, 15 to 20 ppm; Ca2+, less than 40 to 150 ppm; Mg2+, less than 2 to 6 ppm; Al3+, less than 3 ppm; Fe3+, less than 2 ppm; NH,+, up to 2 ppm; NO3-, less than 2 to greater than 25 ppm; NO2-, less than 1 to less than 3 ppm; P03-, 0.5 to 5.0 ppm; Cl-, less than 20 ppm; and SO,-, less than 150 to 600 ppm. Soil comparisons. Comparisons of positive midden soils with other C. immitis-infested soils in the literature, by personal communication and analyses, revealed that: (i) sandy-textured soils occurred in 98.0% of 51 samples (conflicting results were discovered for three of four soils studied by both us and Orr [19], and only the results of the mechanical analyses were used), (ii) alkaline soils occurred in 96.7% of 62 soils, (iii) organic carbon values for 12 soils ranged from 0.21 to 1.80% weight, (iv) organic nitrogen values for 21 soils ranged from 0.029 to 0.190% weight, (v) total organics for 47 soils ranged from 0.39 to 3.13% weight, and (vi) electrical conductivity values for 56 soils ranged from 37 to 27,000 x 10-6 mhos/cm at 25 C. C. immitis has been isolated from soils developed from diverse parent materials: granitic, volcanic, and sedimentary rocks (either stream or ocean derived) and alluvium or lacustrine deposits. DISCUSSION It is evident that a single factor does not determine the distribution of C. immitis in the soil of the Lower Sonoran Life Zone. Emmons has refuted his original hypothesis that rodents are the major reservoir of the pathogen in nature (1) and this is supported by the reports of Swatek et al. (29). Yet, Sorensen (24) has indicated that spherules and endospores protected by body fluids might survive long enough in the soil to allow mycelial growth. Maddy and Crecelius (16) reported the establishment of the fungus in soil with infected animal tissues. During the present study it was noticed that animal activity was increased on the midden sites (221 burrows were counted on eight 188-M2 plots at six different middens compared to 146 on an equal number of adjacent plots). Animals, although not the major reservoir, cannot be ignored as possible factors in C. immitis dispersal. Correlation of the distribution of C. immitis and the macroflora of the positive sites was unproductive. The sites ranged from sparsely vegetated deserts through oak woodlands with scattered pine (Fig. 2). Creosote bush was observed near only 1 positive site (IK-2) out of the 11 included in this survey. The repeated isolation of C. immitis from areas having vegetation markedly different than that of L. tridentata regions should broaden the search for C. immitis-macrofloral associations in nature. Riker (23), in her report on the growth of the pathogen on parts of desert plants, described the inhibition caused by creosote bush. She also noted that the parts of several plants, among them six Opuntia spp. (prickly pear cactus), supported abundant growth and sporulation of the fungus. Campins (5) and Mayorga (18) observed the same genus in endemic areas of Guatemala, Honduras, and Venezuela. The genus is also common in endemic regions of the United States and Mexico. It is possible that the fungus does form alliances with the macroflora, but they may be casual rather than distribution affecting. Egeberg and Ely (6) published a report that incriminated the soil near rodent burrows as a source of C. immitis in nature. Midden samples taken without reference to animal burrows in this research yielded 9.5% positives (of 325 samples), which would indicate that other factors may also be important. Elconin et al. (8) found a positive correlation between the recovery of C. immitis and high soil salinity. Repeated isolations (32 positive samples) from sites in this study revealed markedly VOL. 27,1974 less salinity (114 to 1,856 x 10-6 mhos/cm at 25 C) and may indicate that the high salinity might have been a local phenomenon, which, rather than limiting its distribution, represents a more halotolerant extension of the fungus physiology. Swatek's contention (27) that C. immitis infestation is enhanced in the soils of former Amerindian villages has been borne out by this study. However, it appears that sandy texture and alkalinity were more important than the organic content of the soil in this relationship. The pathogen was recovered from 8.1% of 395 soils cultured. Considering just the midden soils, 9.8% were positive, or 8.7% after subtracting the control soils. Soils of 5 of the 10 random midden sites contained the pathogen. Additionally, 1 of 4 random sites, from which soils were collected by cooperating archeologists, was positive. Mention should also be made that the Santee site (29) was another positive midden with no prior history of C. immitis isolation. One of the two sites suspected of causing human infection, 4-Mad-117, yielded the fungus. Historically, random soil samples from en-demic areas have yielded between 2.0 and 3.4% positives for C. immitis (6,9,27). Considering these figures, the percentage of positives reported in this study would appear to be significant. The lack of any positives among the nonmidden soils supports this contention. The epidemiological impact of the association of C. immitis and midden soils will be presented elsewhere. Darkening of the soil was a very stable character of the midden sites and was undoubtedly influenced by past human habitation. During cyclic periods of occupation, the soil was the final receptacle of charcoal, wood, thatch, domestic scraps, human wastes, and burials. Over as much as 1,500 years (at CaMad-173 and the Inyokern Cave), this amounted to a considerable localized increase in organic contamination. Soil color may be related to drainage, aeration, chemical content, and climate. In the midden soils, which were sandy, well drained, low in soluble iron and manganese, and situated in temperate, semiarid to arid regions, the color must be attributed directly to organic materials and charcoal. The soil was alkaline at the middens surveyed. In general, except for IK-3 (which was in an alkaline area), the pH of the surrounding soil was lower. This was due to the accumulation of ash minerals and organic debris. Soil alkalinity is directly related to the intensity and duration of human occupation as revealed in a study of a Chowchilla River Valley site where a proportional increase among pH, artifact yield, and depth of the midden was found (17). Preliminary studies (by D. Rosenberg and J. Kelly, Department of Anthropology, California State University, Long Beach) indicated a similar pattern at CaMad-173. Assessment of organic contamination at contemporary sites of human activity (since 1850) was not part of this study, but it was a contributing factor at two of the sites investigated. CaMad-173 had hydraulic gold mining and ranching activities until the present, and IK-2 had the foundations of a small building on it. Campers, sheepherders, and transients have been observed on some of the sites during this research. All the positive soils studied exhibited an Eh range of 88 to 266 +mV (uncorrected). The Eh-pH milieu occupied by C. immitis in nature appears to be naturally segregated when compared by the method of Baas-Becking (3) with adjacent, nonmidden and random Southern California soils (Fig. 3). Midden soils tended to be higher in carbonates (0.05 to 1.55%) and phosphates (1.0 to 5.0 (29); however, the samples included here did not yield the pathogen. ppm) than adjacent soils (0.02% and 0.5 to 1.8 ppm, respectively). Except for sites liable to seasonal flooding (Fre-SFSC-1, IK-2, and IK-3), all middens demonstrated strong to violent effervescence when tested with 10% hydrochloric acid. Bone and shell, rich in these ions, were part of the aboriginal contamination of the middens. These components may have ecological significance as buffering agents. Comparisons of infested soils revealed physicochemical characters similar to those reported by Cameron in studies of California desert soils (4). Possibly, ecologically limiting factors for C. immitis may include soil pH and texture, whereas nonlimiting factors would include color, organic content, salinity, and soil parent materials. Stotzky (25,26) reported that a positive correlation existed between the presence of clay minerals and the isolation of Histoplasma capsulatum from soil, but no conclusions could be formed concerning C. immitis. Analyses of soils collected for this study revealed that some contained montmorillonite, but the correlation with the presence of the pathogen was not as good as obtained with H. capsulatum (Stotzky, personal communication). It is evident that C. immitis is a physiologically versatile organism, yet the contradiction remains that is has a spotty distribution in nature to the limit of present soil isolation FIG. 2. The macroflora of sites positive for C. immitis ranged from sparsely vegetated deserts (Inyokern Cave, above) to oak woodlands with scattered pine (4-Mad-118, below). techniques. The limiting factor may be the competitive saprophytic ability of the fungus. Observations of its behavior in the mixed cultures of soil isolation plates and comments of other workers support the hypothesis that C. immitis may be a poor competitor for nutrients and biological space. Another consideration is that the consistent soil physicochemical characters determined in this study may well be those most favorable for control of its microbial competitors. Manipulation of these conditions should be considered for the control of the saprophytic phase of C. immitis. The results of this research indicated that C. immitis is strongly associated with Amerindian middens in California. Comparisons of infested soils in the literature and in the laboratory revealed that the chief factors for this association were the presence of alkaline and sandy soils. At the midden sites surveyed, the soil alkalinity and color were due to past accumulation of domestic contaminants.

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1974-10-01T00:00:00.000Z

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Isolation of Salmonella enteritidis Serotype Agona from Eutrophic Regions of a Freshwater Lake Salmonella enteritidis serotype Agona, which is associated with animal feeds containing fish meal, was isolated consistently from waters influenced by sewage containing poultry processing wastes. In recent years Salmonella sp. have been isolated from various fresh and marine waters contaminated by urban sewage. Spino (12) obtained Salmonella as far as 70 miles (ca. 112.6 km) downstream from a source of treated urban sewage effluents. Claudon et al. (4) found 12 serotypes of Salmonella, mainly serotypes Anatum, Typhimurium, Thompson, and Derby, in Lake Mendota, a major recreational lake near Madison, Wisc. The presence of the salmonellae was related to contamination by agricultural and urban runoff waters. Several investigators have examined surface waters in North Georgia, a major poultry center. Hendricks (8) Mississippi were among the more common of 29 different serotypes obtained. Serotypes Cubana and Heidelberg were the predominant salmonellae isolated from the environment around a Georgia chicken processing plant (9). Lake Sidney Lanier, located about 45 miles (ca. 72.5 km) north of Atlanta, Ga., is a 38,000acre recreational lake which also serves as a water source for metropolitan Atlanta. Portions of this young, man-made reservoir which receive sewage effluents, including wastes from poultry processing plants, are eutrophic and support annual blooms of blue-green algae and algophorous amoebae (5). The established association of salmonellae with poultry products suggested that the lake waters be examined for salmonellae to aid in further detecting the influences of sewage on the eutrophication processes. In the summer of 1972, Moore swabs (10) were positioned at four stations in Flat Creek and its embayment area, a region receiving urban sewage effluents: at one station in the center of the lake, at one station on Two Mile Creek, a stream draining rural residences on the opposite side of the lake from Flat Creek; and at two stations in Balus Creek, which received textile mill effluents. Physical and chemical characteristics of the lake and a map of the collection area are presented elsewhere (5). Two Mile Creek and the center of the lake contained relatively clean water characterized by a biochemical oxygen demand of less than 1 mg/liter and a fecal coliform number of less than 1/100 ml. The Moore swabs (two per station) were left submerged for 1 week during July, August, and September. After collection, the swabs were placed in sterile plastic bags and iced for transportation. With the exception of the use of the API system for presumptive identification of salmonellae isolates, the methods of isolation and identification were identical to those of Claudon et al. (4). Briefly, one swab from each station was introduced into tetrathionate enrichment broth while the other was incubated in selenite brilliant green sulfa broth. After 24 and 48 h of incubation at 41.5 C, the broths were streaked onto brilliant green, Salmonella-Shigella, and bismuth sulfite agars. Selected isolates were inoculated to triple sugar iron agar (Difco) and lysine iron agar. Isolates typical of Salmonella were identified by the API system and serotyped according to the methods of Edwards and Ewing (6). Twenty-one different serotypes were isolated. Fifteen were obtained from the Flat Creek area, with 12 of these found exclusively in this region (Table 1). Serotype Agona was the only Salmonella isolated from all stations in Flat Creek on all collections. Its only other occurrence was in a single sample from the embayment station. Six different serotypes were obtained at the embayment station. Aside from serotype Agona, only serotypes Albany and Minnesota were common to both the embayment and Flat Creek. No salmonellae were obtained from the center of the lake. Three serotypes, Weslaco and Derby obtained from Two Mile Creek and Eimsbuettel from Balus Creek, were isolated exclusively from their respective areas. The limited distribution of certain serotypes may reflect differential host-free survival indexes of the various serotypes. Densities of fecal coliforms were determined for waters collected from all stations on each collection date according to the membrane filter method of Geldreich (7). In general the higher densities of fecal coliforms (up to 5,000/ml) were found at those sites yielding the greatest variety of serotypes. One exception was at a station located about 100 yards (ca. 91.4 m) below a (8). eFound in Georgia waters by Hoadley (9). ' Found in Georgia waters by Schneider et al. (11). sewage treatment plant on Flat Creek. Salmonellae were isolated, but no fecal coliforms were obtained. No fecal coliforms were obtained outside Flat Creek, its embayment area, and the Balus Creek site. The common presence of serotype Agona in the Flat Creek area is significant. In previous studies of Georgia waters, a single isolation of this serotype is noted (2). Agona, rare before 1969, is of increasing epidemiological significance. In 1973 serotype Agona was the seventh most common human isolate of Salmonella submitted to the Center for Disease Control for identification (1). Clark et al. (3) associated serotype Agona with fish meal (an ingredient of poultry feeds). Hence, the worldwide emergence of Agona as a significant agent of salmonellosis is currently linked to the need and broad use of fish meal as a protein supplement in animal feeds. Its occurrence in Flat Creek is due presumptively to its presence in poultry wastes. It can be expected that this occurrence of serotype Agona in the feeder streams of a recreational lake will ultimately result in a broader base for its epidemiology.

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1974-12-01T00:00:00.000Z

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Antagonism of Lactic Streptococci Toward Staphylococcus aureus in Associative Milk Cultures The inhibition of growth of Staphylococcus aureus by lactic streptococci in associative cultures in milk was not due to hydrogen peroxide produced by the streptococci. Dialyzed whey from the milk culture of lactic streptococci was more inhibitory than dialyzed whey from milk acidified with lactic acid, indicating that material other than lactate was also involved. Analyses of cation and anion exchange fractions from the dialyzed whey showed that only the neutral fraction was inhibitory. The inhibition of growth of Staphylococcus aureus by lactic streptococci in associative cultures in milk was not due to hydrogen peroxide produced by the streptococci. Dialyzed whey from the milk culture of lactic streptococci was more inhibitory than dialyzed whey from milk acidified with lactic acid, indicating that material other than lactate was also involved. Analyses of cation and anion exchange fractions from the dialyzed whey showed that only the neutral fraction was inhibitory. We have shown (6) that commercial lactic streptococcus starter cultures are antagonistic toward staphylococci and salmonellae during associative growth in milk. The intensity of the antagonism varied among starter cultures and could not be correlated with the rapidity of acid production. Furthermore, the antagonism was still evident when the milk was automatically maintained at pH 6.5. Others (2,8,11) have also shown that the inhibition of staphylococci by lactic streptococci in milk is not entirely due to low pH. Certain strains of lactic streptococci have been reported to produce antibiotics (4,7). Others also produce peroxide (5), which has been implicated in the inhibition of Staphylococcus aureus (3). Volatile fatty acids produced by some lactic acid bacteria can also cause inhibition of undesirable bacteria in foods (9,12). The mode of action involved in the inhibition of food-borne pathogens by the lactic streptococci may include one or more of the aforementioned possibilities. A more thorough understanding of factors involved in the antagonism should make it possible to utilize the lactic streptococci more efficiently in controling food-borne pathogens. The purpose of this investigation was to characterize the substance(s) produced in milk by lactic streptococci which inhibits food-borne pathogens. The multiple-strain cultures of lactic streptococci used in these experiments were obtained from a commercial culture supplier. The S. aureus B925 was from the North Carolina State University Food Microbiology culture collection. The cultures of streptococci were maintained by subculturing in litmus milk using 1% inocula and incubation at 22 C for 18 h. S. aureus was subcultured in sterile 10% nonfat milk solids using 1% inoculum and incubation at 32 C for 18 h. All cultures were stored in a refrigerator between subcultures. The required volume of sterile 10% nonfat milk solids was inoculated with approximately 10' colony-forming units (CFU) of S. aureus per ml and asceptically divided into two flasks. One flask was additionally inoculated (1%) with an 18-h milk culture of lactic streptococci. The samples were then incubated 6 h at 32 C, after which requisite dilutions of both were plated (spread technique) onto selective media. Mannitol salt agar was used for enumerating S. aureus. The plates were incubated 24 h at 37 C. Percentages of inhibition were determined using the following formula: % inhibition = [(CFU/ ml in control) -(CFU/ml in sample inoculated with streptococcus) ]/(CFU/ml in control) x 100. To determine if hydrogen peroxide was responsible for the antagonistic interaction, catalase (Nutritional Biochemical Corp., Cleveland, Ohio) was added (30 U/ml) to a duplicate set of milk cultures prior to incubation. The acidity of 1 liter of an 18-h (22 C) milk culture of lactic streptococci was determined as percent lactic acid by titration with 0.1 N NaOH. The titratable acidity of 1 liter of sterile 10% nonfat milk solids was adjusted to that of the cultured milk using 30% lactic acid. Each was centrifuged 30 min at 4,080 x g to remove the curd. The supernatant fluid (whey) was collected, and 700 ml from each sample was dialyzed against 1,400 ml of distilled water at 5 C for 24 h. The dialysis tubing (Fisher Scientific Co.) was approximately 3.2 cm wide (flat) and was prepared for use by soaking in hot (100 C) distilled water. The tubes were long enough so that each could contain approximately 50 ml of whey. The dialysates were concentrated at 45 C under vacuum to the VOL. 28, 1974 original volumes of whey, adjusted to pH 6.5 with NaOH, filtered through sterile membrane filters (0.45-Mm pore size; Millipore Corp.) into sterile flasks, and stored at 5 C until assayed. Samples of whey-dialysate were prepared for ion exchange chromatography in a similar manner, except the pH was not adjusted nor were the samples filter sterilized. Samples (100 ml) of dialysate were passed over an Amberlite IR 120 cation (H+ form) exchange column (bed volume, 200 ml). The samples were washed through the column with distilled water until the effluent was negative to the molisch test. The flow rate was 2 ml/min. The column was then eluted with 6.5 bed volumes of 2 N NHOH. The NHOH was removed from the eluate by repeated evaporation (at 45 C under vacuum) and dilution. Both effluent and eluate were concentrated by evaporation to a final volume of approximately 5 ml, the pH was adjusted to 6.0 with NaOH or HCl, and the volume was adjusted to 10 ml with distilled water. Both samples were passed through a sterile membrane filter (0.45-gm pore size; Millipore Corp.) and stored at 5 C in a sterile container. Five milliliters of the concentrated effluent fraction from the cation exchange column was applied to an Amberlite IR 400 anion (Clform) exchange column having a 100-ml bed volume. The sample was washed through the resin with distilled water (2 ml/min) until the effluent was negative to the molisch test. The effluent fraction was concentrated to 5 ml and adjusted to pH 6. The column was eluted with 10 bed volumes of 2 N acetic acid. The acetic acid was removed by repeated evaporation (at 45 C under vacuum) and dilution. The final volume was adjusted to 5 ml at pH 6.0. Both anion effluent and eluate fractions were passed through sterile membrane filters (0.45-Am pore size; Millipore Corp.) into sterile containers and stored in a refrigerator. Sterile 20% nonfat milk solids was inoculated with approximately 2 x 103 CFU of S. aureus B925 per ml. The inoculated milk was dispensed in 1.5-ml portions into sterile screwcapped test tubes (13 by 100 mm). The desired dialysate or ion exchange fraction from lactic streptococcus milk cultures were asceptically added to the tubes (1.5 ml each). Tubes were also prepared for the appropriate control dialysates and ion exchange fractions. The comparative effect of an aqueous solution of sodium lactate (pH 6.0) was evaluated in a similar manner. The lactate was at the same concentration as in the dialysate samples. All tubes were incubated 6 h at 32 C. The samples were then placed in an ice-water bath and plated on Trypticase soy agar (BBL). The plates were incubated 24 h at 37 C. The data presented in Table 1 show the inhibitory effect of three lactic streptococcus cultures on S. aureus B925 during a 6-h incubation period. Catalase had no effect on the inhibition of S. aureus. Dialysate from the whey of a milk culture of lactic streptococcus D was inhibitory to S. aureus B925 (Table 2). Since the preparation of the control whey dialysate involved adjusting milk to the same acidity as that of milk culture D with lactic acid, these data show that inhibition of S. aureus B925 by lactic streptococcus D was not entirely due to the presence of lactic acid. An aqueous solution of sodium lactate (pH 6) equal in lactate concentration (1.1%) to the dialysate samples was also evaluated. More growth occurred in this sample than in either of those containing the dialysate samples. However, the amount of growth was less in the sample containing lactate than in a sample to which only water was added, indicating that lactate was responsible for part of the inhibition. Throughout the remainder of the study, dialysates or fractions thereof from both milk cultures of lactic streptococci and milk acidified with lactic acid were compared in order to study inhibition due to factors other than lactate. Assay of fractions obtained from cation exchange chromatography of the dialysate samples revealed that the inhibitory substance(s) produced by streptococcus culture D was associated with the effluent ( Table 3). The number of CFU per milliliter in the sample containing the cation effluent fraction of whey dialysate from streptococcus D was only 32% of that in the sample containing the effluent from the control dialysate. More growth was observed in the sample containing the eluate fraction from culture D than in the sample containing the control eluate. Analyses of fractions obtained by anion exchange chromatography of the cation effluent fractions revealed that the inhibitory material was in the anion effluent fraction ( Table 3). The amount of inhibition was greater than that observed from the cation effluent fraction. The anionic material in the eluate fraction from whey dialysate of culture D allowed more growth of the staphylococci than did the eluate from the control. Ion exchange fractions from dialysates of several additional batches of whey from control milk and milk cultures of the lactic streptococci had similar effects on the staphylococci. Results from a previous study (6) showed that the inhibition of staphylococci and salmonellae by lactic streptococci was not caused entirely by an acidic environment created during growth of the streptococci. Certain lactobacilli have been shown to produce sufficient hydrogen peroxide to inhibit the growth of staphylococci (1) and Pseudomonas species (10). The lactic streptococci when grown in milk produce auto-inhibitory levels of hydrogen peroxide (5). Haines and Harmon (3) reported that hydrogen peroxide was involved in the inhibition of S. aureus by lactic streptococci in associative culture in a broth medium. However, in the present study peroxide was apparently not produced in suffi-cient quantities by the lactic streptococci to inhibit S. aureus B925 in associative milk cultures. Sodium lactate, at a concentration equal to that in the culture and control whey dialysates, exerted some inhibitory action on S. aureus B925. This indicated that lactate produced by the streptococci during growth was partially responsible for inhibiting the pathogen. The lower growth response of S. aureus in milk containing whey dialysate from the lactic streptococcus milk culture than in milk containing the control dialysate suggested that an inhibitor(s) other than lactate was involved in the antagonistic action. The dialyzability of the inhibitor(s) indicated that it had a relatively small molecular weight. Results from analyses of fractions obtained from ion exchange chromatography revealed that the inhibitor(s) was neutral (i.e., not absorbed by cation or anion exchange resins). The inhibitor(s) did not resemble nisin (4) or diplococcin (7), which are produced by some lactic streptococci. Nisin should be absorbed by cation exchange resins (4) and diplococcin is isolated only by extraction directly from cells of the streptococci (7). The antagonistic action of the lactic streptococci toward S. aureus in milk cultures apparently results from several factors. After sufficient growth of the streptococci has occurred, certainly the low pH and the lactic acid produced during growth are somewhat inhibitory. Other inhibitors produced by the lactic streptococci in milk are also important, one of which is a neutral material of low molecular weight.

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2018-04-03T01:01:44.202Z

1974-10-01T00:00:00.000Z

27712095

s2

Microbial Response to Drought in a Texas Highplains Shortgrass Prairie The population of the microbial flora of a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) prairie near Amarillo, Texas, was studied during 1971 after a severe drought. Bacteria, fungi, and algae were estimated by plate count and terminal dilution procedures. Rates of grass and paper decomposition were determined. The microbial flora of soil associated with bovine-grazed grass did not differ significantly from the flora associated with ungrazed grass, either qualitatively or quantitatively. During drought, a greater number of fungi were found in soil associated with prickly pear than in that associated with blue gramma grass. The microbial biomass decreased one full log between the surface and a depth of 50 cm, and the percentage of anaerobes increased with depth. The maximum numbers of fungi and algae detected were 8 x 105 and 6 x 104/g respectively. A linear relationship existed between the microbial biomass and soil moisture. The maximum number of aerobic, heterotrophic bacteria detected was 1.5 x 108 viable cells per g of soil. Clark the ecology grasslands. The biomass in the top 30 cm of a by direct microscope with 30 76 g/m2 on a basis. soil extract dilution plates showed 4 x 107 bacteria/g in the 0- to 10-cm layer (during April) with a linear decrease of total number with depth. The population of the microbial flora of a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) prairie near Amarillo, Texas, was studied during 1971 after a severe drought. Bacteria, fungi, and algae were estimated by plate count and terminal dilution procedures. Rates of grass and paper decomposition were determined. The microbial flora of soil associated with bovine-grazed grass did not differ significantly from the flora associated with ungrazed grass, either qualitatively or quantitatively. During drought, a greater number of fungi were found in soil associated with prickly pear than in that associated with blue gramma grass. The microbial biomass decreased one full log between the surface and a depth of 50 cm, and the percentage of anaerobes increased with depth. The maximum numbers of fungi and algae detected were 8 x 105 and 6 x 104/g respectively. A linear relationship existed between the microbial biomass and soil moisture. The maximum number of aerobic, heterotrophic bacteria detected was 1.5 x 108 viable cells per g of soil. Clark and Paul (4) have reviewed the sparse literature on the microbial ecology of native grasslands. The biomass in the top 30 cm of a Canadian grassland is estimated by Babiuk and Paul (2), by direct microscope counts with fluorescein isothiocyanate, to be 30 to 76 g/m2 on a dry weight basis. Aerobic soil extract dilution plates showed 4 x 107 bacteria/g in the 0-to 10-cm layer (during April) with a linear decrease of total number with depth. This study provided a taxonomic and physiological activity profile of the microbial population of the soil during the 1971 growing season after a year of severe drought in a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) grassland. The contribution of prickly pear to the total ground cover varied from 3 to 11%. The significance of the cactus is due to its tendency to grow in thick clones. During the dry season, it may at times appear to be the predominant ground cover, because of greatly reduced amounts of grass. Minor amounts of buffalograss (Buchloe dactyloides Nutt.), sand dropseed (Sporobolus cryptandrus Torr.), and purple three-awn (Aristida purpurea Nutt.) prevail throughout the undisturbed grassland. There were specific questions of interest in this study. (i) Are there qualitative or quantitative differences between the microbial flora of soil associated with either bovine-grazed grass or ungrazed grass? (ii) Do the microbial flora associ-ated with grass ground cover differ from that of prickly pear? (iii) Is there any change in total population or type with increasing depth? (iv) Is the microbial biomass linked with available soil moisture? MATERIALS AND METHODS Site description. At Texas Tech University Research Farm (Amarillo, Texas), a moderately grazed site of 32 hectares and a 14-hectare site which had been ungrazed for 5 years were selected, approximately 6.8 km apart at an elevation of 1,180 m. The weather is extremely variable, characterized by strong prevailing winds, dry winters, and sharp temperature changes (9,12). Precipitation of only 14 cm was received at the site during 1970; the average is 56 cm. During 1971, the following amounts of precipitation were received: in April, 1.5 cm; May, 1.3 cm; July, 8.8 cm; August, 9.8 cm; September, 6.3 cm; and October, 3.5 cm. The soil at the sites is classified as Pullman silty loam (6). Each of the sites was subdivided into two replicate sites. Each replicate was further subdivided into 200 quadrats (2.74 by 2.74 m), which were arranged in two parallel rectangles (5.49 by 137.2 m) with an access alley inbetween. Bacterial population studies. Two cores (2.5 by 50 cm) were taken from each of six plots, chosen at random from each replicate of both the grazed and ungrazed sites at each sampling period, for a total of 48 cores. The cores were obtained with a hollow soil sample tube hydraulically pressed into place. Two complete additional sets of cores were obtained when the sites were subdivided for comparison of prickly pear and grass ground cover. Each core was divided MICROBIAL RESPONSE TO PRAIRIE DROUGHT into five zones, or depths, from 0 to 5 cm, 5 to 10 cm, 10 to 20 cm, 20 to 30 cm, and 30 to 50 cm. The sections from each core were placed separately in sterile plastic bags, sealed, and immediately stored in an ice chest. All 12 samples of each zone from each replicate were blended together in 90 ml of sterile, physiological saline for 3 min, and appropriate serial dilutions were prepared. Pour plates were prepared with five replicates at each of at least three dilutions. Plate counts were made from the maximal colony development after incubation at 30 C. Aerobic heterotrophs were assayed (15) with standard methods agar (SMA; tryptone glucose yeast agar from BBL). Anaerobes were assayed with anaerobic agar (BBL) and with incubation under prepurified nitrogen; the methylene blue confirmed anaerobiosis. Aerobic and-anaerobic sporeformers were assayed by heat-shocking the appropriate dilutions at 85 C for 20 min before plating. Fungi were cultivated in rose bengal streptomycin agar (7). The population of algae was estimated by the dilution-frequency method (1). The total populations of different nutritional types were also determined by the dilution-frequency method, with the basal salts media of Lockhead and Chase (8). Three additions were made to this set of media: (i) Phenol red broth base (BBL) was chosen to approximate the SMA; (ii) a 1.0 g amount of microcrystalline cellulose (Calbiochem, Los Angeles, Calif.) per 100 ml was added to one set of the basal medium; (iii) the chitin basal salts medium of Campbell and Williams (3) was used to determine chitinase activity. The most common colony types from each horizon were selected from the SMA and classified according to nutritional type as described previously (8). Decomposition. Rates of litter decomposition were evaluated with squares (8 by 8 cm) of Whatman no. 1 filter paper or 8-g samples of blue-stem hay (Andropogon gerardi Vitman) sewn lengthwise in 1-mm mesh nylon net bags. These bags were placed horizontally on the soil surface. Blue-stem hay was used throughout the U.S. grassland study as a standard litter material. Filter paper samples were placed both on the surface and at a depth of 5 cm in the soil. Five samples were used per replicate per treatment of each sample. The samples were incubated at 50 C, and dry weights were obtained. The total sample was ashed, and the excess ash weight was subtracted from the sample weight to correct for soil contamination of the sample. Respiration. The open end of round metal cans (12.6 cm in diameter by 17.0 cm high) were inserted 2 cm into the soil, and carbon dioxide was absorbed in 10.0 ml of 0.1 M potassium hydroxide, which was placed in open vials in the cans for an average period of 16 h. The cans were shaded with plastic covers, and five samples per replicate were taken at each period and compared to unexposed 10.0-ml samples of base. Trapped carbon dioxide was estimated by automatic titration with 0.05 M potassium biphthalate. Physical measurements. Pooled cores from each horizon were weighed to 0.1 g. Moisture content was determined gravimetrically on 10.0-g samples by drying at 105 C for 24 h. Ash content was determined from samples ashed at 600 C for 6 h. Statistical analysis. The total plate count data were analyzed for covariance with a prewritten computer program. Significant differences were identified by contrasts. The grazed-grass plate count data means were analyzed separately for covariance, and mean differences were then compared by the method of Tukey (14). RESULTS The results of viable cell counts of bacteria, fungi, and algae are presented in Fig. 1 through 4. Both the viable cell and/or spore counts in the 0-to 5-, 10-to 20, and 20to 30-cm zones roughly followed the 5to 10-or 30-to 50-cm zones, with appropriate corrections for the effect of depth. The computer-based analysis of all data sets for bacteria identified highly significant (1%) effects due to date, treatment, and zone depth. A marked difference (99% confidence) was found between the number of fungi associated with grass versus that associated with prickly pear ground cover. The effect of depth on the number of observed fungal colonyforming units was significant at the 99% confidence limit. To clarify the above results, the data obtained from the grazed grass site were analyzed separately. Highly significant effects were identified for the numbers of aerobic heterotrophs due to depth but not to the date. The means from both the 0-to 5-and 5to 10-cm zones differed significantly (95% confidence) from the means of the 10-to 20-, 20-to 30-, or 30-to 50-cm zones. The difference between the 0to 5and 5-to 10-cm zones was not significant. Analysis for covariance of the total population of aerobic heterotrophs to a soil depth of 50 cm identified a highly significant effect due to the date. Comparison of means showed that the total population of aerobic heterotrophs on 30 September differed significantly from the populations on 4 August, 29 June, and 8 June. The total population on 2 September differed significantly from those on 29 June and 8 June 1971. In the top two horizons, the 0-to 5-and 5-to 10-cm zones, the number of fungal colony-forming units associated with prickly pear cacti decreased nearly one-half log during the period from May until August. The number of fungi associated with grass ground cover was consistently smaller than the number associated with the prickly pear cacti (during the spring and early summer). During this same period, many of the cacti were heavily infested with Coccidae scale insects. The computer analysis of data from both sites placed a 99% confidence limit on this effect. There was a significant effect due to Microbial populations and moisture in the 5to 10-cm soil horizon during 1971. Symbols: A, ungrazed site (U), grass ground cover; 0, grazed site (G), grass ground cover; and 0, prickly pear ground cover (pp), results averaged from grazed and ungrazed sites. the date of sampling on the number of fungal colony-forming units at the grazed site. There was a highly significant fungal-colony population change associated with increasing depth. The number of fungal colony-forming units in the 0to 5-and 5to 10-cm soil depths differed significantly from the numbers associated with any other depth. It was not determined whether the fungal colony-forming units were due primarily to spores or to mycelial fragments. Limited data did not permit evaluation of algal population changes as the season progressed. The number of algae in this arid soil was greater than expected. The average populations per gram of wet soil in the 0to 5-cm horizon on the following dates were: on 29 June 1971, 9 x 103; 2 September 1971, 0.3 x 103; and 30 September 1971, 50 x 103. Highly significant decreases in the average populations of aerobic bacteria, actinomycetes, anaerobic bacteria, and the aerobic fungi ( Fig. 3) were observed with increasing depth. Because the latter data might be misleading because of the presence of many faculatively anaerobic organisms, dilutions were heated to kill the vegetative cells and plated in the appropriate medium. The number of aerobic sporeformers tended to decrease with depth, but the number of anaerobic or faculative sporeformers did not. Additional data will be required to firmly establish this relationship. Actinomycete-type colonies were counted on the same plates as the bacteria, but represented only 2% of the colonies in the top horizon and 4.5% in the bottom horizon. The medium was not ideal for growth of the actinomycetes, and these data provide only preliminary estimates. In some ways SMA was an unfortunate choice for the plating medium, since it is too rich to allow the growth of many of the soil bacteria. However, its choice by U. S. International Biological Program permits the comparison of results at several sites; the usual soil extract agar would vary with the soil used for its preparation. There are, in addition, several different formulas for soil extract agar. To evaluate partially the influence of SMA on the results, terminal dilution studies were conducted with several media (8). The dilutions were prepared from the same soil suspensions used for the plate count studies. Only slightly higher counts were obtained by using a basal salts medium supplemented with amino acids. Cellulose in the basal salts medium resulted in slightly lower counts. Apparently, the SMA allowed adequate growth of the bacteria from this soil. The most common colony types on SMA were selected and cloned from samples of each depth collected on 30 June 1971. These were compared by using basal salts medium plus various supplements, including soil extract (8). All but two of the bacteria were gram-positive rods and 12 of these were Bacillus species. Large variations existed in the nutritional requirements of these organisms; some required soil extract, amino acids or yeast extract for growth. Several cultures were inhibited by the addition of growth factors, yet all 18 isolates grew well in the presence of both soil extract and/or yeast extract. No clearly identifiable changes in nutritional requirements were associated with soil depth. The moisture content and bulk density of the soil increased with depth (Fig. 5). The ash content varied only slightly in the hundreds of samples examined and averaged (95.2 ± 4.0 g on a dry weight basis). The ash content increased slightly with depth. During May and June, soil samples collected at 0 to 5 cm below prickly pear had 0.5% more moisture than samples collected beneath grass. The biomass of platable, aerobic heterotrophs on the grazed site to a depth of 50 cm on 8 June 1971 is estimated at 3 x 1012 bacteria per square meter, or approximately 3.3 g/m2 (1.1 x 10-12 g/cell; 10). Analysis of covariance indicated that significant (a = 0.01) effects were associated with date, treatment, and depth, but not moisture. The difference between the number of bacteria associated with the grazed and ungrazed site was not significant at 1%, but it was significant at the 5% level for the 0-to 20-cm zone. There was a marked difference (a = 0.10) between the number of fungi associated with grass versus fungi associated with prickly pear ground cover. The negative relationship between soil moisture of individual horizons and their bacterial populations seemed questionable as populations increased after rains during July and August. A linear correlation was found to exist between the aerobic bacterial populations and total moisture per 50 cm of core (Fig. 4). This effect was identical to that for date of sampling. Decomposition rates were significantly different for surface versus subsurface placement and may have depended on moisture. During June, grass litter samples lost 17.0% in weight. Filter paper samples which were placed on the surface lost an average of 1.2% by weight, and subsurface samples lost an average of 15.2%. During August, the grass lost 23.1% of its weight, the surface filter paper lost 2.4%, and the subsurface filter paper 23.9%. Soil carbon dioxide evolution estimates (Table 1) indicate no significant differences between the two sites, but soil respiration increased as the season progressed. DISCUSSION There were minor but consistent differences in microbial populations on the two sites, resulting in the significant covariant. These differences never exceeded one-half log and were of only minor importance. Sampling difficulties made it inadvisable to continue at the ungrazed site after September. Statistical analysis indicated a highly significant difference between the microbial flora associated with grass ground cover and that associated with prickly pear. Data in Fig. 1 showed that more fungi were associated with the prickly pear ground cover during the early summer (in the first two horizons), when the moisture content of the soil was lowest and cacti were diseased. Shade provided by cacti may have allowed the soil to retain a greater moisture content during early summer than soil under the sparse grass. Comparison of soil moisture values for grass and prickly pear indicates that the soil in the first horizon had 0.5% higher moisture content under the prickly pear. This differential was true during June, but decreased during the remainder of the season. The greater fungal population during the dry weather may have resulted both from stored moisture in the cacti and/or higher soil moisture under the cacti. The populations of both bacteria and fungi decreased significantly in number with depth (Fig. 3). This was verified by statistical analysis. Though the total number of aerobes and anaerobes decreased with depth, analysis of the data in Fig. 3 showed that the percentage of anaerobes doubled from the top to the bottom horizon. This is in agreement with the theory that oxygen must diffuse from the soil surface downward, and that the rate of diffusion might be slow in this very hard, unworked soil. The decrease in numbers of aerobic bacterial and fungal colony-forming units with depth would be expected. There is no direct evidence that oxygen is limiting at greater depths in this soil. Such effects are usually associated with watersaturated soils. The primary producers (grass, algae, and prickly pear cacti) were at the surface. Thus, nutrients produced by photosynthesis diffused downward and decreased with depth. The ash content of the soil increased slightly with depth, which may indicate a lower percentage of organic matter at greater depths, as was found at the Amarillo Experiment Station (5). The experimental plate count data provide no information about the autotrophic bacteria. Terminal dilution studies indicated no shift with depth in nutritional types. The same results were obtained from the nutritional groupings of organisms selected from SMA. The latter data is biased, because SMA favors heterotrophic metabolism. All of the pure cultures grew in basal salts medium, supplemented both with soil and/or yeast extracts. A large percentage of the aerobic heterotrophic bacteria were Bacillus species. Values of 20% have been (4). Twelve out of 18 clones were Bacillus species, and in plate counts a preponderance of the colonies were Bacillus (Fig. 3). This may be due to lack of moisture or simply to spore survival despite insufficient water activities for vegetative cell growth. The estimate of' biomass (aerobic, heterotrophic bacteria) is a very low 3.3 g/m2 in June and 6.7 g/m2 in November. Babiuk and Paul (2) calculated that the active biomass based on plate counts was only '41 that of direct counts. However, they concluded that the plate count procedure may be a better estimate of metabolizing cells in the soil. Our results were in accord with a very dry soil and a maximum carbon dioxide evolution of 0.7 g per m2 per 24 h ( Table 1). Several workers obtained similar plate counts with a peak of 20 to 75 x 106/g of soil at the 0-to 5-cm depth at this and associated sites in 1970 (12). The maximum plate count in this study was 150 x 106/g of soil at the 0-to 5-cm depth. Reuss (11) reported that another worker, Doxtader, estimated the total dry weight biomass of' bacteria plus fungi to a depth of 30 cm at a grassland site in Colorado to vary from 51 to 82 g/m2. Babiuk and Paul obtained counts of 4 x 107 to 27 x 107 bacteria per gram in the 0-to 10-cm layer on a Canadian grassland soil, by using soil extract agar. These counts compare very favorably with the results of this study. Since our figures did not include the true anaerobes, actinomycetes, autotrophs, and the fungi, and also were based on the wet soil weight, the estimate of biomass was very low. If Babiuk and Paul's estimate (2) of the relationship between viable cell and direct count is applicable, the maximum biomass in our experiments may have been as much as 100 g/m2. On the basis of individual samples from each horizon, no correlation between moisture and the number of viable bacteria was demonstrated by statistical analysis. Yet, one would expect such a correlation if for no other reason than the dependency of grass on available moisture. Since the herbage is the primary producer in the system, the number of active bacteria should be linked to the growth of the grass or other plants. The data were reexamined. The sum of the total viable cell counts of aerobic heterotrophs for 0 to 50 cm was directly proportional to the sum of the available moisture (Fig. 4). Lack of moisture altered the grassland drastically. The soil at the Texas Tech University Research Farm had not been studied extensively. Results obtained at the Amarillo Experiment Station, which also has Pullman soil, indicated that the moisture tensions during this study were always in excess of 15 bar. The wilting point for grass on the Pullman soil is approximately 14 bar (5). The soil moisture was always lowest in the first 20 cm (Fig. 5), exactly where one would expect the greatest microbiological and plant activity. The top 5 cm was often dust, with much of the remaining soil brittle and dry to touch. The water activities may have been below the critical points for growth of some bacterial species. The parched, sparse spring and summer vegetation changed to a luxuriant grass cover in the fall, accompanied by increased microbial biomass. The decomposition studies provide an indication of the soil activity. Though cattle-grazing influence upon the microbial flora of this grassland was negligible, it must exist directly below their excrement. The influence of the ruminants on the decomposition of litter is not known, beyond their eating the decomposition samples. Jack rabbits ate tongue depressors marking the locations of buried samples during the spring' During June, surface grass samples lost an average of 17% in weight. The average litter biomass of the blue-gramma grass during June of 1970 (13) was estimated at 106 to 181 g/m2; thus, if the 17% decomposition is representative, then 18 to 31 g of litter decomposed per m2 during the month, or 0.6 to 1.0 g per m2 per day. The carbon dioxide evolution measured during this period was 0.26 g per m2 per 24 h. The two figures are not greatly disproportionate. Decomposition, respiration, and microbial biomass changes were all apparent responses to rainfall and available moisture. The effects of extreme drought were the controlling factors in this grassland ecosystem.

v3-fos

2018-04-03T00:40:34.317Z

1974-08-01T00:00:00.000Z

21130120

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Storage of stock cultures of filamentous fungi, yeasts, and some aerobic actinomycetes in sterile distilled water. Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable. Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable. Several methods have been proposed for maintaining culture collections of fungi. Among these, dispersal of spores in sterile soil (1), sterile mineral oil overlays (3), deep freezing (4), ultra-low temperature freezing (9), and lyophilization (7) are the most favored. With the exception of the sterile soil and sterile mineral oil overlay techniques, the other methods involve time and expensive equipment. Castellani (5,6) reported maintenance of several cultures of human pathogenic fungi and yeasts in sterile distilled water for 12 months without any apparent changes in their morphology or physiology. A slightly modified version of Castellani's method, wherein physiological salt solution was substituted for distilled water and screw-capped bottles were used in place of cotton-plugged test tubes, was described by Benedek (2). Hejtmankova-Uhrova (8) reported successful maintenance of 73 strains of fungi belonging to 13 genera in sterile distilled water for 12 months. The present study comprises four independent experiments. They were initiated at different times to evaluate Castellani's technique of maintaining fungal cultures in sterile distilled water. The results of these experiments are presented. MATERIALS AND METHODS Four hundred and seventeen isolates of 147 species belonging to 66 genera were included. Of these genera, 48 were of filamentous fungi, 15 were yeast genera, and 3 were aerobic actinomycetes. The viability of fungal cultures stored in sterile distilled water was tested by four independent workers. Even though many of the species and genera investigated were common to all four experiments, the individual isolates selected by each worker differed in each experiment. Some of the genera were represented by several species, and each species in turn included several isolates. In some cases, on the other hand, the genus and species were represented by a single isolate. The first experiment was initiated in 1969, and covered only 16 isolates of 16 species belonging to 15 genera. The viability of these isolates maintained in sterile distilled water was tested only once, that is, after 60 months of storage. No attempt was made to revive these cultures between 1969 and 1974. Similarly, the second and third experiments were started in 1970 and 1972, and their results were read only once. The second experiment included 18 isolates of 17 species belonging to 12 genera. The third experiment covered 48 species belonging to 27 genera. Fifty-three isolates were included in the third experiment. The cultures stored in sterile distilled water from these two experiments were revived at the same time that those from the first experiment were cultured for viability. The fourth experiment, which was started in 1973, included a greater number of cultures than were used in the three previous experiments. One hundred and twenty-six species of 58 genera were represented by 330 isolates. These 330 water cultures were revived after 12 months along with the other cultures from the three previous experiments. Cultures were inoculated onto slants of potato dextrose agar in screw-capped tubes (20 by 150 mm) and were incubated at 25 C for 2 weeks. Six to seven milliliters of sterile distilled water was pipetted aseptically onto each 2-week-old culture. The spores WATER-STORED CULTURES OF FUNGI AND YEASTS ferred to a sterile glass 1-g vial. The cap of the vial was tightened to prevent evaporation of the water. The labeled vials then were stored at 25 C on laboratory shelves. incubated at 25 C for 3 weeks and were observed periodically for growth. Those cultures that did not grow by the end of 3 weeks were retested with the same procedure. When no growth was observed after the second subculture, the isolates were recorded as not viable. RESULTS AND DISCUSSION It became clear from the results of the four experiments (Tables 1 to 4) that the viability of the isolates ranged from 92 to 94%. None of the cultures were found to be contaminated by bacteria or other fungi. All the yeast cultures survived. In addition, several fungi that are notoriously difficult to maintain over an extended period, such as Aureobasidium pullulans, showed a better survival rate when stored in water than on conventional media. About 6 to 8% of the 417 cultures did not survive storage in water. These belonged to the following genera and species: Madurella mycetomi, Paracoccidioides brasiliensis, Trichophyton concentricum, T. gallinae, T. megninii, T. verrucosum, and T. yaoundii. All of these species are poor sporulators. On routine media like Sabouraud dextrose agar or potato dextrose agar, the above-mentioned species do not sporulate regularly. A reexamination of the storage vials of these species revealed that the inoculum had been too scanty in most cases. When inocula were adequate in regard to size, and consisted of several wefts of hyphae, even some of the poorly sporulating or nonsporulating species like T. violaceum, T. schoenleinii, and M. ferrugineum survived storage very well. To insure success, care must be taken to select actively sporulating isolates and to suspend adequate amounts of spores and pieces of hyphae in sterile distilled water for storage in vials. Care must also be taken not to allow the water to evaporate from the vials. Some of the revived cultures of species with perfect forms were tested for their mating ability after storage in water. The species like Arthroderma ciferrii and Petriellidium boydii readily formed ascocarps in abundance on oatmeal salts agar. In case of heterothallic species like Arthroderma uncinatum and Nannizzia incurvata, numerous ascocarps were formed on oatmeal salts agar when the revived cultures were crossed with the opposite mating type strains. In the case of other imperfect species, the revived cultures were comparable to the originals with respect to morphology and sporulation. The storage in water showed supression of pleomorphic changes in many cases, and, in cases of perfect species, showed no loss of mating competence. Results of the present study clearly show that the method of maintaining fungal cultures in sterile distilled water for extended periods of time is simple, inexpensive, and reliable. The method offers many advantages for laboratories that maintain small culture collections for reference or teaching purposes. The water culture technique is simple, as is the technique for reviving these cultures. The storage space required for the vials is minimal, and, since the vials are stored at room temperature, expensive refrigeration is not needed. The revival technique is less messy than that for cultures stored under mineral oil.

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2018-04-03T06:03:43.930Z

1974-10-01T00:00:00.000Z

45507299

s2

Identification of major high-boiling volatile compounds produced during refrigerated storage of haddock fillets. The two major high-boiling volatile compounds produced during refrigerated storage of haddock fillets were found by gas chromatography and mass spectroscopy to be phenethyl alcohol and phenol. The major genera of bacteria responsible for the spoilage of refrigerated fish have been studied extensively. Little work, however, has been reported on the major high-boiling metabolites produced in fish tissue during refrigerated storage. Wong et al. (3) reported the formation of benzene, toluene, and several ketones in cod held at 0 C. This study documents the formation of phenethyl alcohol and phenol as the major high-boiling volatile components produced in haddock fillets stored at 2 C. MATERIALS AND METHODS Isolation of volatile components from haddock. A 30-g amount of fish tissue was placed into a stainless-steel centrifuge tube (50 ml capacity), 15 ml of Mazola corn oil was added, and the mixture was homogenized with a glass rod. After thoroughly blending in this manner, the oil phase was separated by centrifugation at 17,000 x g for 20 min. A 10-ml volume of the oil was then used for molecular distillation (2). The sample container was immersed in an oil bath at 80 C, and the oil sample was stirred continuously by a Teflon-coated magnetic stirring bar. The volatiles were collected from the oil by distillation under a vacuum of approximately 10-3 torr, and condensed onto a liquid nitrogen-cooled cold finger (2). After 2 h of distillation, the cold finger was removed, and about 10 ml of anhydrous diethyl ether was used to rinse the collecting surface. Prior to gas chromatography analysis, the ether was concentrated to 20 Mliters in a vial with a gentle flow of nitrogen. A volume of 2 Mliters was used for injection into gas chromatography columns. Separation and identification of the major volatile components. A Perkin-Elmer model 881 dualcolumn gas chromatograph equipped with a flame ionization detector was used with a 1 mV full-scale Honeywell model W recorder for analysis of collected volatiles. After various columns were tested, a 12-ft (ca. 3.7 m) column packed with Carbowax 20M was used for initial separation of the volatile components. I Paper no. 1007 of the Massachusetts Agricultural Experiment Station, University of Massachusetts at Amherst. Column temperature was maintained at 145 C, and prepurified nitrogen (Airco) was used as the carrier gas. Using a combined gas chromatograph and mass spectrometer unit, the mass spectra of the major high-boiling volatile components were obtained. The effluent from an Aerograph 1200 gas chromatograph was admitted via a heated line to a Bieman Helium separator and then to the ion source of an Hitachi-Perkin Elmer RMU-6A mass spectrometer. The identification of volatile compounds was verified by comparing their mass spectra and gas chromatography retention times on two different 12-foot columns (Carbowax 20M and diethylene glycol succinate). Bacterial count on haddock fillet. The viable bacterial count on the haddock fillet used above was determined on pour plates of Trypticase soy agar without dextrose (BBL) incubated at 2 C for 6 days. RESULTS AND DISCUSSION Increase of volatile components during refrigerated storage of haddock. A market fresh haddock fillet of high organoleptic quality was found to contain few high-boiling volatile compounds at zero time storage (Fig. 1). After 5 days of storage, a number of high-boiling volatile peaks were present in relatively low concentration. On day 9 of storage, a major peak, designated peak 40, was present. On day 15, coincident with the development of a maximal bacterial population on the tissue (Fig. 2), peak 40 had decreased notably and a third peak, designated peak 44, predominated. On day 20 of storage, peak 40 was only barely detectable, whereas peak 44 had greatly increased. Identification of the major high-boiling volatile compounds. Peaks 40 and 44 were identified by mass spectroscopy as phenethyl alcohol and phenol, respectively ( Fig. 3 and 4). Authentic phenethyl alcohol (C6H5CH2CH2OH, Eastman Organic Chemicals) and phenol (Fisher Scientific Co.) were used for verification. Both the mass spectral data and gas glycol succinate column. Phenol had a retention time of 49 min on the Carbowax column and 13 min on the diethylene glycol succinate column. The genus Achromobacter was found to be responsible for phenethyl alcohol production and is the subject of an accompanying report (1).

v3-fos

2018-12-12T09:04:36.552Z

1974-01-01T00:00:00.000Z

55487690

s2

EFFECTS OF EARLY-CUTTING MANAGEMENT ON FORAGE YIELD AND QUALITY OF ALFALFA IN NORTHEAST KANSAS Alfalfa cutting management has been a topic of interest and concern in Kansas and other regions of the United States. Current recommendations for cutting schedules in Kansas are based on a combination of plant and crown-bud development. This is critical because Kansas has a highly variable climate, and alfalfa forage is harvested during both shortand long-day periods throughout the production season. Depending on the point of the cutting cycle and the existing environmental conditions, the "first regrowth at the crown" harvest indicator can occur at or prior to the 10%-bloom stage throughout the season. The maturity stage at which alfalfa is initially harvested in the spring is important for several reasons. The new growth initiates from the crown buds, which depletes total nonstructural carbohydrate (TNC) root/crown reserves. Depending on the previous year's fall-cutting management and severity of winter and/or spring weather, an alfalfa field can be stressed early in the spring. Also, insect and 105 disease damage to the alfalfa stand compounds the level of stress. These factors can greatly weaken and consequently reduce plant populations depending on management of the first cutting. If the stand is injured, forage yield and quality will decline. Finally, timeliness of the first cut will ultimately determine the total number of seasonal harvests and subsequent tonnage and quality possible for a growing season. We investigated the impact of harvesting first-cutting alfalfa at various maturity stages on forage yield and quality. Procedure This study was established in a producer's field near Keats, in northeast Kansas, with a 5-year-old stand of `Kansas Common' alfalfa grown under rain-fed conditions on a Reading silt loam. The study began in early spring 1990 and concluded after the first killing freeze in fall 1991. Identical first-cutting treatments were repeated in each year. The experimental design was a randomized complete block with four replications. The initial cycle of trts took approximately 8 weeks to complete. Subsequent cuttings were harvested when regrowth was observed at the crown or at the 10%-bloom stage. Plots were 5 x 22.5 ft. Average plant height in each plot was recorded prior to harvesting. Plots were cut with a 3 ft. sicklebar mower at a 2 1 / 2 -inch stubble height. Forage yields were estimated by weighing the fresh forage from the entire plot area and converting to lbs. per acre dry weight. A randomly hand-picked subsample of approximately 1 lb. was obtained from each plot, oven dried to a constant weight, and used for both determination of dry matter (DM) content and quality analysis. Dried samples were prepared for Near Infrared Reflectance Spectroscopic (NIRS) analysis by grinding through 1-mm screens in a Wiley mill followed by a Udy mill. NIRS analysis provided data for percent crude protein (CP), acid detergent fiber (ADF), and neutral detergent fiber (NDF) on a 100% DM basis. ADF content was used to calculate percent digestible dry matter (DDM), and NDF content was used to calculate percent dry matter intake (DMI). In addition, the latter two calculations were used to calculate percent relative feed value (RFV). Total crude protein (TCP) was calculated by multiplying the forage yield times the CP. For the year-end totals, the TCP figure was yield weighted for the entire season of production. Results The first-cutting yields in 1990 (Table 1) increased as maturity advanced from trts 1 through 8. In contrast, most of the quality parameters (i.e., DDM, DMI, RFV, CP) declined. Total crude protein (for the first cutting only) was greater for trts 2 through 4 and 8 than for trts 1 and 7. Treatment 8 had higher TCP levels because the regrowth of what would have been the second cutting was cut with the initial harvest. However, this extremely late cutting (8 weeks after the first trt) tended to be low in quality and reduced the number of seasonal harvest opportunities. Total seasonal yield data were collected to study the impact of first-cutting management on subsequent cuttings. The 1990 total forage yields for all trts varied less than 1 / 2 ton, with the exception of trt 7, which had the lowest yield. The "secondcutting" crown regrowth under trt 7 was approximately 6-to 8-inches tall, indicating that root reserves were at a critically low level when the trt was initiated. This observation along with the detrimental effects of self-shading help explain the low seasonal yields for this trt. Total seasonal forage quality data for 1990 indicated advantages for initially harvesting alfalfa at trt 4. Treatment 7 was lower in DDM than trts 1 through 5. There was a significant advantage in DMI and RFV for trt 4 compared to trts 1 and 2 and trts 6 through 8. The TCP was higher for trt 1 than all other trts, except trts 3 and 4. In 1991, the study was continued on the same plots to investigate the effects of early-cutting management in the subsequent year. The first-cutting yield (Table 2) of trt 4 was higher than those of earlier trts; however, it was lower than those of the remaining trts. Treatment 1 was lower in yield, DDM, DMI, and RFV than trts 2 and 3. In general, the quality parameters, excluding TCP, declined with advancing maturity after trt 2. The TCP (for the first cutting only) in 1991 was lowest for trt 1. Treatments 2 and 3 were lower in TCP than the balance of the trts. These results for lbs. of CP per acre for the early-cut trts are clearly paralleled by the differences in yield. Total alfalfa yields for the 1991 season were higher for trts 3 through 6 and 8 than for trt 1. In addition, trts 5 and 6 were higher-yielding than trt 7. Total seasonal forage quality data for 1991 indicated that, with the exception of trt 6, the first four trts were higher in DDM than later-cut trts. Treatment 1 was lower in DMI than trts 2 through 4. A decline in RFV occurred with advancing maturity after trt 2. The TCP for the 1991 season was higher for trts 3 through 6 than for trt 1. 1990, first regrowth at the crown occurred at one-tenth bloom. 9 Critical 6-to 8-inch regrowth present 10 Vegetative stage was cut five times, early-bud through 50%-bloom stages were cut four times, and fullbloom and green-seedpod stages were cut three times during 1990. In 1991, first regrowth at the crown occurred at one-tenth bloom. 9 Critical 6-to 8-inch regrowth present 10 Vegetative through late-bud stages were cut five times, and the remaining maturity stages were cut four times in 1991. This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu. Summary Short-term yield advantages occurred for first cutting at the green-seedpod stage (trt 8), and short-term quality advantages occurred for first cutting at the vegetative stage (trt 1). These advantages became minimal or nonexistent when the total seasonal production was considered. The first-regrowth stage (trt 4) appears to be the compromise to obtain high alfalfa yields and quality. Second-year data showed the impact of physiological stress caused by early-harvest management. First cutting at the vegetative stage gave no short-term superiority in quality and showed a severe yield depression. Likewise, second-year yields for the early-bud stage (trt 2) were relatively low. First cutting at the green-seedpod stage in year 2 showed similar, yet less extreme, trends than in year 1. For 2 consecutive years, first cutting at the full-bloom stage (i.e., 6-8" second regrowth; trt 7) showed season-long declines in yield and quality. Conclusions Two years of results gathered from an established stand of `Kansas Common' indicate that the window for harvesting firstcutting alfalfa should be between late-bud (i.e., just prior to regrowth; trt 3) and 50%-bloom stage (i.e., less than 6" regrowth present; trt 6) in order to maintain high production levels over multiple years. Many of the responses to early-harvest management are directly related to changes in stand persistence. These changes should be considered in the overall management scheme, because they will ultimately determine longevity of the alfalfa stand.

v3-fos

2018-04-03T02:39:25.834Z

1974-04-01T00:00:00.000Z

34452351

s2

Comparative denitrification of selected microorganisms in a culture medium and in autoclaved soil. The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated. The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated. Extrapolation of results from pure microbial studies to the natural environment has often been questioned. However, due to the complexity of a natural ecosystem such as soil, laboratory studies serve as the only feasible method of monitoring or controlling all the inherent variables. For example, in the nitrogen cycle, it is difficult to determine if the denitrification process is partially obscured by nitrifying organisms or by non-biological factors. Therefore, it is desirable to design experiments that indicate what effect the introduction of new variables has on the microbial process under investigation. The basic knowledge of the denitrification process was reviewed extensively some years ago (6,7), and, although much research has been performed in the meantime, essentially no new findings have been reported. However, since denitrification can be undesirable from an agricultural viewpoint (2) or desirable in the removal of nitrate-nitrogen from the environment (8), the factors regulating this process need to be further clarified in order to utilize the potential of denitrifying microorganisms for practical purposes. In this paper, the denitrifying characteristics of selected bacteria in an artificial growth medium and in an autoclaved soil system were compared to clarify whether the change from a chemically defined liquid to the solid and complex structure of soil causes an essential change in denitrification patterns. I Paper no. 4408 in the Journal Series of the Pennsylvania Agricultural Experiment Station. MATERIALS AND METHODS Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis, from the culture collection of the Department of Microbiology at the Pennsylvania State University, and three soil isolates designated as isolates A, D, and H (5) were used. The bacteria were transferred from nitrate agar (Difco) to liquid Giltay medium and grown for 2 days at 30 C before they were used for inoculation of autoclaved soil or liquid media. Giltay medium (1), adjusted to a pH of 7.0 with NaOH, was used for both the stock culture solution and liquid media experiments. The soil used in these experiments was a Hagerstown silt loam soil, pH 7.0; the indigenous NO--N concentration was 75 uig/g of soil; organic carbon was 1.8%; and the sand, silt, and clay content was 8.5%, 63.4%, and 28.1%, respectively. Soil (20 g) was placed into incubation flasks, and 10 ml of distilled water containing 6 mg of NO--N was added. The soil was sterilized by autoclaving three times for 30 min over a 2-to 3-day period with at least a 12-h interval. The soil samples were inoculated with 1.0 ml of the selected stock culture. Liquid cultures containing 50 ml of Giltay medium were inoculated with one loop of bacterial culture. Anaerobic samples were incubated in 125-ml bottles sealed with a one-hole rubber stopper containing a septum for gas sampling (Applied Science Laboratories, Inc., State College, Pa.). Anaerobic conditions were attained by flushing the bottles with helium until all air was removed, as indicated by gas chromatographic analysis. Uninoculated control samples were assayed after different time periods to determine possible atmospheric contamination. Soil was incubated aerobically in 125-ml Erlenmeyer flasks closed with foam tube plugs. The flasks were placed in a closed incubation chamber, which was continuously flushed with filtered air. To minimize drying of the soil, a large pan of water was placed 674 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from COMPARATIVE DENITRIFICATION OF MICROORGANISMS in the chamber to increase the humidity. Aerobic liquid samples were incubated on a rotary incubation shaker revolving at 200 oscillations per min. All samples, aerobic and anaerobic, were incubated at 30 C for 3 days. The experiments were repeated two or three times, and several replicates were evaluated for each specific treatment. Gas samples were taken from the anaerobic bottles through the septum with a gas-tight syringe and injected into a gas chromatograph (Varian Aerograph, model 1820). Each sample was split equally into two parallel columns (3 mm outside diameter) of Porapak Q (600 cm; 50-80 mesh), which indicated peaks of CO2 and N20, and molecular sieve 5A (450 cm; 45-60 mesh), which separated 0, and N2. The columns were maintained at 50 C, and dual thermal conductivity cells at 200 C served as detectors. The carrier gas, helium, flowed at a rate of 40 ml/min, and the filament current was 200 mA for the Porapak Q and 150 mA for the molecular sieve 5A column. The quantity of gases was determined by the use of an integrator and calculated by comparisons with pure standard gas samples. Nitrate was measured with a nitrate electrode (Orion Research Inc., Cambridge, Mass.), and nitrite was determined calorimetrically by the a-naphthylamine-sulfanilic acid procedure (3). RESULTS After incubation for 3 days, it was apparent that the disappearance of nitrate was slower in the soil than in the liquid Giltay medium (Fig. 1), but the essential features of nitrate transformation by the various bacteria were not affected under the two conditions, with the exception of P. aeruginosa. Isolates A and D produced a considerable amount of nitrous oxide in the culture medium as well as in autoclaved soil, but the intermediary formation of nitrite could not be observed. On the other hand, S. marcescens and isolate H showed similar characteristics, although the marked formation of NO2in Giltay medium was not so apparent under soil conditions. The growth of P. aeruginosa was very rapid in the culture solution, as indicated by increasing turbidity. At the same time, practically all NO8which disappeared was recovered as N2. However, when the Pseudomonas species was cultivated in soil, the NO,was used more slowly, and, although N, was the predominant gas, it was also possible to detect NO2and some N20. B. subtilis was also included in these experiments, although this bacterium is normally not considered to be a denitrifier. It was found that, both in autoclaved soil and in the culture solution, some NO,was transformed, and it could be recovered as NO,and N2. Simultaneously with the release of nitroge- nous gases, the formation of CO, was observed, and the results of one representative experiment are shown in Table 1. Isolate H and S. marcescens, which accumulated a considerable amount of NO2in the culture solution, showed a strong production of CO2 when compared with the other bacteria, but the weaker NO.,--transforming ability in soil was complemented with a relatively smaller formation of CO2. This fact is especially noteworthy if it is compared with the bacteria that did not accumulate NO2in Giltay medium: their production of CO2 was more extensive in autoclaved soil than in the culture medium, although less NO,disap- The small amount of CO, formation found in the sterile control samples appears to be of non-biological origins, e.g., CO2 may be produced by decarboxylation of organic compounds or decomposition of free carbonates (9). When incubation occurred under aerobic conditions, there was practically no disappearance of NO,in soil inoculated with the various bacteria ( Table 2). Only isolate H produced a considerable amount of NO,-, but its origin was not further clarified. A strong decrease in NO,was observed when P. aeruginosa or isolate H was cultivated aerobically in the culture solution. Since gas sampling and analysis under aerobic conditions, in which a continuous exchange of air took place, were not sensitive enough for measurement of change in the content of nitrogenous gases, no clear conclusions in relation to this phenomenon could be drawn. However, it was very obvious that all other bacteria did not reduce NO3in soil or in culture solution if air or 02 was continuously exchanged by flushing or shaking of the incubation flasks. The complete inhibition of denitrification was demonstrated only under highly aerobic conditions. In an experiment in which isolate A was cultivated in a closed vessel that was flushed with pure oxygen, air, or helium prior to incubation, the denitrification was obviously related to the decrease of available oxygen (Table 3). The reduction of NO3and its use during the denitrification process were clearly indicated by the corresponding formation of N20. A similar result was obtained when isolate A was inoculated in sterilized soil. Since growth in soil was slower during 3 days of incubation, there was no or very little disappearance of nitrate in an oxygen or air environment, respectively, whereas, in a helium environment, the missing NO3could be recovered as N20. DISCUSSION Stimulated by the recent concern with environmental pollution, much interest has been shown in enhancing the denitrifying abilities of microorganisms and, thereby, reducing the danger of nitrate contamination of groundwater. To study the denitrification process and the major influencing factors, it is necessary to have well-controlled conditions as well as pure microbial cultures for obtaining conclusive results. However, since the conditions designed in the laboratory have to be quite different from those existing in the microenvironments within the soil, the application of these results is questionable. The comparison of the activity of denitrifying microorganisms in a liquid growth medium and after inoculation into sterilized soil is an attempt to determine whether incubation under various environmental conditions will significantly affect the denitrifying potential of certain microbes. Numerous bacterial species can denitrify in culture media, but there are no reliable data demonstrating how the same organisms behave in soil. It was even stated that the major part of denitrification in nature is performed by microbes that are different from those investigated essentially under laboratory conditions (10). Beijerinck and Minkman (4) found that the most numerous denitrifying organisms under average soil conditions were some Bacillus species, and, therefore, they assumed that these would also be the most important denitrifiers. Woldendorp (11) found that a change of the denitrifying population occurs after the addition of nitrate to soil: Bacillus species were originally the dominant denitrifiers, but gramnegative rods were mainly responsible for denitrification after incubation with nitrate. Woldendorp also compared pure cultures of gramnegative rods with Bacillus species and concluded that soil conditions are not favorable for denitrification by the latter type of organisms. In our investigation, it was shown that the characteristics of bacteria isolated from soil and laboratory cultures of denitrifiers did not show an essential change in their denitrifying behavior, with the exception of P. aeruginosa, whether observed in a culture medium or in autoclaved soil. Growth and also denitrification were slower during a similar time period in soil than in a liquid solution, and some intermediates of the nitrate reduction process appeared in different quantities in the two media. The increased accumulation of nitrite in the liquid medium of S. marcescens and of isolate H could be correlated with the faster reduction of nitrate and a subsequent inhibition in further transformation to nitrogen gas. There is no doubt that the different media and their influence on growth and other metabolic activities can change the speed of reactions and may have a secondary influence on the denitrifying patterns of the microbes, but the results of the reported experiments demonstrated that the features of selected denitrifiers are, for most bacteria, comparable in autoclaved soil and liquid media. Stotzky (10) emphasized that, since "soil is the most complex microbial habitat," the study of processes under consideration may require various levels of experimental complexities: it may be necessary to move back and forth between in vitro model systems and soil in situ to obtain knowledge of microbial activity in a soil ecosystem.

v3-fos

2014-10-01T00:00:00.000Z

1974-04-15T00:00:00.000Z

1057370

s2

Selection in dual-purpose cattle populations: effect of beef crossing and cow replacement rates SUMMARY The discounted gene-flow method is used to calculate the effects of varying cow replacement and beef crossing rates on the breeding policy appropriate for a dual-purpose cattle population. It is shown that increasing beef crossing and increasing cow replacement rates both increase the number of expressions of a bull's dairy genotype following one insemination while the expressions of his beef genotype are little affected. The result of this is that (i) a high level of beef crossing is efficient, (2) more emphasis should be given to dairy traits in selecting bulls, (3) the return for investment in dairy bull testing and selection is enhanced. INTRODUCTION Dual purpose bulls for use in AI should be selected for a weighted function of their additive genetic merit for the dairy and beef traits of interest in the population concerned. In a previous paper (M C C LIN TOC K and CuNNiN&HAM, 197 1) it was shown that the selection objective can be defined to reflect the real economic value of the bull's total genotype as expressed through one insemination. This requires that each beef or dairy trait be weighted by a factor which is the product of two elements : a) the financial value of a unit of production for the trait; b) the number of standardised expressions of the bull's genotype for the trait, which will follow from one insemination. The financial element depends on production costs and market returns. In any particular population, it will be relatively fixed. The relative degrees of expression ( 1 ) Present Address : Milk Marketing Board, Thames Ditton, Surrey, England. of the bull's beef and dairy genotype depend primarily on the probability that the calf born from an average insemination will become a dairy cow. This probabiliy in turn depends on two aspects of population structure : the amount of crossing with beef bulls and the replacement rate of dairy cows. In this paper, we examine the way in which these factors affect the definition of the breeding objective, and through it the whole breeding strategy in dual purpose cattle populations. METHODS We define as « dual-purpose » any population in which the cows are milked and the male and surplus female progeny are reared for beef. Population size is presumed constant. All calves are reared either as cow replacement or as beef animals for sale at about two years of age. Heifers calve at two years of age, and 8 5 progeny are reared per hundred cows per year. All cows are bred artificially either to selected dual purpose bulls of their own breed, or to bulls of a specialised beef breed. Dual-purpose bull selection is based on an index which is calculated assuming heritabilities of 0 . 2 for milk yield and 0 . 3 for growth rate. Progeny group sizes are 10 for beef progeny and 40 for dairy progeny. The genetic correlation of milk yield and growth was given values of -0 . 2 , o.o and 0 . 2 , though the main results are presented only for the zero correlation. Selection procedures for beef breed bulls are not considered, but it is assumed that (a) the cost per bull is not greater than that for a dual purpose bull, and (b) that the progeny of beef breed bulls are at least as good for beef production as those of dual-purpose bulls. Since the main components of dairy and beef merit are milk yield and growth rate, we assumed that bulls are selected solely on these traits. Selection is on a conventional index which incorporates up to 15 items of information, ranging from a beef performance test on the bull's grandsire to a dairy progeny test on his daughters (table i). The objective is to select those bulls which excel for a total genotype defined as where B and D are the bull's genotypes for growth rate and milk yield, and v and w are relative net economic weights. As shown by M C CLINTOcx and Currrnrrcxnm ( 1973 ) the appropriate economic weights are where E b and E d are the numbers of standard discounted expressions of the bull's beef and dairy genotypes following one insemination, and C b and C d are the financial values of a unit increase in beef and milk production. In our calculations, we have taken the phenotypic standard deviation for each trait as the unit of measurement. Our financial values C b and C d , are therefore per standard deviation for beef ( 6b ) and milk (a a ) respectively. If the net financial returns per kg of beef (i. e. per kg liveweight at a fixed age) and milk are F 6 and F d , then Since only two traits are involved, it is convenient to take the economic weight for beef as unity, and that for milk as Thus, the relative economic weights for the two traits can be treated as the product of three separate ratios. The ratio crdjcr b depends on the scale of milk and beef production per animal. We have assumed that all beef animals are reared to about two years of age, or about 500 kg liveweight. Growth rate has a coefficient of variation of about io p. IOO , so that this implies a value of a b = 50 kg approximately. Milk yield has a coefficient of variation of about 20 p. IOO . Average production per lactation varies in European countries from 2 500 to 5 00 o kg, giving a range of s a from 500 to i o 0 o kg. The ratio c rd jcr b can therefore vary from about 10 to 20 , depending on the scale of milk production per cow. In any particular population the ratio will be fairly constant, and in these calculations we have taken a value relevant to Irish conditions of 12 . 5 We discuss later the possible effect of variation in this ratio. The value of the financial returns on beef (F b ) and milk (F d ) are best expressed as the cash return over feed cost for each additional kg of liveweight or milk yield. Costs other than feed are per animal or per lactation, and not per unit of production. In Irish conditions, the ratio of net returns for beef and milk is about 6, that is to say that the net return on I kg of liveweight (F 6 ) equals the net return (F d ) on six kg of milk. Irish feed costs for milk production are exceptionally low by European standards. The value of 1/ 6 is therefore likely to be at the lower end of the range of values which the ratio F d/ F b will have in European countries. In our present calculations, we have used a value of F a/ F b = 1/7 . The effect of variation in this ratio is discussed below. In any particular population, therefore, the ratio of net economic weights for dairy and beef traits will depend mainly on the ratio E d/ E b . The genetic consequences of a single insemination are summarised in the factors E 6 and E d . The standard unit in which they are calculated is defined to be one progeny expression of the trait in the year in which the insemination is carried out. All future expressions are adjusted for the generation in which they occur, discounted for the time interval separating them from the insemination, and weighted by the probability of their occurrence. E b and E d represent the sum of these discounted consequences of the insemination for beef and dairy traits respectively. We have called this series of adjustments the o Discounted Gene Flow Method *, and its derivation is described in more detail elsewhere (M C C LIN -TOCK and CUNNINGHAM, r9!3). The probabilities involved are functions of the following three parameters of population structure : L : the average number of lactations per cow ; K : the proportion of cows crossed to beef bulls ; S : the number of animals surviving to maturity per successful insemination. The probability that the insemination leads to a lactation by a descendant in generation g is then L g (i -K)-l . The probability of a beef progeny is (SL(i -K) -1 )/L(i -K), and the probability of a beef descendant in a later generation is (SLi)/L g ( l -K). If r is the discount rate in percentage units and N a gy is the number of possible dairy descendants in generation g and year y, then the total discounted dairy progeny equivalents from one insemination is where G and Y are the number of generations and years considered. Similarly, the number of discounted beef progeny equivalents is Note that in this case we separate the actual beef progeny, which can occur in year three, from the other beef descendants which arise via a dairy daughter. The summation over generations therefore begins at g = 2 . The calculation and evaluation of the selection indexes involved was carried out using the general index program Selind (CurrrrcrrcxaM, i9!o). I . -General The object of this study is to clarify the effect of varying beef crossing and cow turnover rates on the breeding strategy for dual-purpose cattle. These factors enter into En and Ea, and therefore into the net economic weights which should be used to define the balance of beef and dairy traits in the breeding objective. In order to trace the effects of beef crossing and cow turnover rates, it is therefore necessary to fix as far as possible the other factors involved. We have taken a value of 12 . 5 for the ration aa/6 a and z/ 7 for the ratio F/a!b. The ratio of the dairy and beef genetic consequences of an insemination, Ea/E b , is quite complex. However, many of the factors involved can be taken as fixed. The survival rate per successful insemination will be fairly constant in any particular population, and we have taken S = o.8 5 . The discount rate has been taken as r = 8 p. 100 . We have fixed Y at 10 years. This is reasonable in the operational sense that the economic gain from a particular insemination in the 10 hears following that insemination is likely to satisfy both the users of A. 1. and the A. I. authorities who must evaluate the investment in bull testing and selection. In addition, genetic predictions over many generations tend to be uncertain, and 10 years involve four generations of descendants of the bull. However, we should also check whether the effect of the insemination in question is largely compelte within the 10 years, or whether it is still in a stage of rapid development. In figure i we have plotted the cumulative numbers of beef and dairy expressions for 10 years following one insemination for a population with 4 lactations per cow, and for crossbreeding values of K = 0 . 2 o and q.o p. 100 . It can be seen that the number of beef expressions (E b ) increases little after the first few years. The number of dairy expressions is still increasing at year io, though at a declining rate. We are therefore justified in limiting consideration to the 10 years following the insemination. If we chose to include additional years, the effect would always be to increase the ratio of dairy to beef expressions. The pattern of cumulative discounted consequences of an insemination is similar for populations with 3 , 5 and 6 lactations per cow. . -Effect of population structure The way in which the actual numbers of standard discounted beef and dairy expressions vary with varying beef crossing (K) and cow turnover (L) rates is shown in figure 2 . The number of beef expressions is essentially the same whatever the level of crossbreeding. The reason for this is that a large proportion of the net discounted beef expressions comes from an actual beef progeny. As K increase, a larger proportion of heifer calves per dairy insemination are required as herd replacements, and the probability that such an insemination leads directly to a beef progeny is therefore reduced. However, since the probability that the insemination leads to a replacement female is simultaneously increased, the probability of beef expressions in later generations via the female, is also increased, which offsets the corresponding reduced probability of a progeny beef expression. The result is that the total number of discounted beef expressions is relatively stable, whatever the breeding structure of the population. In contrast, the number of dairy expressions incrases steadily as K increases. The result is that the ratio Ea/En also increases nearly linearly with K. The effect of increasing K is therefore to increase the weighting on milk relative to beef in the selection objective. This has widespread consequences for the breeding pro-gramme : it increases the emphasis on milk in bull selection, increases the total return on investment in testing, and increases the proportion of this return which comes via milk. The effect of increasing the rate of cow turnover is similar to, though less marked than the effect of increasing beef crossing. The fewer lactations per cow, the greater the proportion of heifer calves required for herd replacement, and the greater the probability that a dairy insemination will result in a replacement female. For a population of constant size, the limits to beef crossing are dictated by the rate of cow turnover, and by the net reproductive performance of the population. These maximum levels of K are given in table 2 . The actual numbers of discounted beef and dairy progeny equivalents per insemination for the full range of beef crossing and cow replacement rates which are feasible in practice is given in table 3 . The ratios of E dJ E b for varying beef crossing and cow replacement rates are also given in table 3 . Since we have for the moment considered the ratio of phenotypic standard deviations fixed at 12 . 5 and the ratio of economic values fixed at 1/7 , the ratio of net economic weights to use in selecting bulls, w/v, is a direct function of >!a/!b. This is shown in table 3 . Once the breeding objective has been defined in this way, it is possible to calculate the economic effectiveness of a programme of bull selection using a given array of information. The measure of effectiveness is the economic value of the genetic merit conferred on the population by a single insemination. This is where i is the selection differential on a standard normal distribution and 0 '1 is the standard deviation of the index used for bull selection. Provided the same selection differential is applied in all cases, the relative gains from selection are then simply the relative standard deviations of the index. For convenience, we have given the gain for the most effective situation a value of 100 and scaled the others down from that. In table 3 , the relative economic value of an insemination is given for each combination of K and I,. Note that since the same information is used and the same level of selection applied in each case, these figures represent the relative economic returns for a constant level of investment in testing and selection. Whatever economic gain is conferred with each insemination is composed of gain due to genetic improvement for milk yield and gain due to improvement of growth rate. It is of some interest to know what proportion of the total gain is attributable to improvement in each trait. In table 3 we have shown the percentage of total economic gain which is due to improved growth rate for each combination of number of lactations and degree of croossbreeding. The method for calculating these percentages is given by CurrrmrrGHAM (i 97 o). There is some uncertainty about the genetic relationship between milk and beef traits in dual-purpose cattle populations (MASON et al., 1971 ). We therefore gave the genetic correlation of milk yield and growth rate values of -0 . 2 and !-0 . 2 , and recalculated the relative genetic return per insemination for each of these situations. The result was, as might be expected, that the net gain was slightly less for the negative and slightly more for the positive correlation. The change in each case was about 2 . 5 p. 100 . A negative correlation also slightly increases the share of total economic gain from selection which is due to improvement in milk yield. However, the effects of variation in crossbreeding percent and cow turnover rates were barely affected, and the general conclusions regarding population structure therefore hold for this range of genetic relationships between milk and beef. DISCUSSION In order to develop the results given here, we have had to relate the calculations to a particular population, to the extent of fixing the ratio of standard deviations and the ratio of financial margins for milk and beef production. (Their product is in fact the ratio of the net value of one phenotypic standard deviation in milk yield to the net value of one phenotypic standard deviation in liveweight). In our case, it was 1 .8. However, this ratio is not likely to differ greatly in other populations. As GRnv!ExT ( 19 66) has shown, the ratio of gross returns for I kg milk and I kg liveweight in European countries covers a relatively narrow range of I : 5 . 3 to I : 8.i. The standard deviation for liveweight that we have used should be fairly generally applicable. That for milk is low for many countries. However, as the yield per cow increases, and with it the standard deviation in yield, the feed cost per unit of production is also likely to increase, thus reducing the margin over feed cost. So any increase in 6 a will tend to be offset by a reduction in Fa, thus stabilising the product of the two ratios. The only result which can be affected by this factor is the ratio of economic weights in the selection objective, and through it the percentage of total gain which becomes via improvement in milk and beef (table 3 ). The relative economic return per insemination for different population structures (table 3 ) is essentially inde-pendent of change (particularly upward change) in the product Fd &mdash; ' All the other hb 6b results are quite independant of it. The general conclusions which we draw from these results should therefore be applicable over a wide range of situations. The higher the cow replacement rate (i. e. the fewer lactations per cow), and _ the higher the percentage of cows bred to beef bulls, the greater will be the probability that a dual-purpose insemination will result in a dairy animal. This probability is the key to the other results. As it increases, so does the probability of dairy descendants in later generations. The result is that the number of discounted dairy progeny equivalents per insemination increases rapidly. However, the probability of an initial dairy progeny has little effect on the discounted beef progeny equivalents per insemination. The reason for this is that the beef consequences of the insemination are expressed either in an immediate progeny or through the beef descendants in later generations which arise via a dairy daughter. The probabilities of these two outcomes are complementary : as one goes up the other must come down. The result is that irrespective of the probability of the initial insemination leading to a dairy daughter, the total effect in terms of discounted beef progeny equivalents is relatively static. As beef crossing (K) increases, a higher proportion of the heifer calves resulting from dairy inseminations will be required for herd replacement. Thus the probability of a dairy insemination leading to a lactating daughter is increased. The result is that the ratio of expressions moves very much in favour of dairy traits. This has a parallel effect on the ratio of economic weights for dairy and beef traits in the selection objective. We therefore find that both the ratio of expressions and ratio of economic weights increase rapidly as beef crossing increases and more slowly as the number of lactations per cow goes down ( fig. 2 and table 3 ). Note that while the absolute number of beef and dairy expressions depends on the rate of cow turnover, their ratio is, within rounding errors, the same for all levels of cow turnover provided beef crossing is at its maximum. As the balance of economic weights moves in favour of milk, the total financial return per insemination increases fairly rapidly. The reason for this is basically that the economic value of the increased number of dairy expressions is being added to the nearly constant economic return from the beef expressions. In table 3 , the relative returns per insemination are given by number of lactations per cow and by proportion of crossbreeding. Since in these calculations we have assumed that the information used and cost incurred for each bull selected is the same for all situations, those conditions which increase the return per insemination also increase the total return on the investment in bull testing and selection. One effect of changes in the relative economic weights in the breeding objective will be to alter the value of the different items of information (table i) that can be used in selecting dual purpose bulls. As the ratio of economic weights shifts in favour of milk, the value of data on the growth rate of the bull and his relatives declines, while dairy records increase in value. The lowest relative economic weights that we have encountered are w/v = 1 . 72 (no beef crossing and six lactations per cow). The most valuable item of information in bull selection here is his dairy progeny test__ net genetic gain is reduced by R = 33 p. ioo if this is ignored. The next most valuable item is his beef progeny test (R = io p. l oo), followed by his beef !erform:an.£!_t!!t_J...R = 2 p. ioo). As the ratio of economic weights moves to its maximum value of w/v = 3 . 7 8 (maximum beef crossing, 5 lactations per cow), the utility of these items changes considerably : the dairy progeny test is now of overwhelming importance (R = 41 p. 100 ), while the beef progeny (R = 2 p. 100 ) and performance (R = 0 . 5 p. ioo) tests contribute little to total gain. It is therefore evident that the population structure, and in particular the amount of beef crossing, has a great deal to do with the kind of testing programme that is appropriate. The cow turnover rate depends mainly on the intensity of milk production. and would be very difficult to alter. The proportion of cows bred to beef bulls, however, can easily be modified from one year to another. The proportion of beef crossing in current practice ranges from 5 0 P . 100 in Ireland (GuNrmrrGHnM et at., 1971 ) to !8_p. 100 in Britain (MILK MARKETING BOARD, 1970 ), 27 p. 100 in France (F R E-BLING et G AILLARD , ig6g) and practically zero in the Scandinavian countries (L INDSTR 6 M , 1970 ). Our results suggest that the most efficient dual-purpose breeding programme requires the maximum amount of beef crossing that the population can tolerate. This conclusion is reinforced by some quite separate advantages that follow from a certain amount of beef crossing. If a farmer must request a beef or a dual-purpose insemination for each cow, he is likely to select his best cows for dualpurpose inseminations. This compels him to a greater degree of selection in the dams of his replacement heifers than would otherwise be the case. In countries with a distinct commercial beef cow population ( 30 p. 100 of all cows in Ireland and in Britain), the use of beef breed bulls on the dual-purpose cows in dairy herds provides a steady supply of beef X dual-purpose heifers as replacement cows for the beef herds. These should have three advantages over heifers bred within the beef herd : lower cost, higher milk yield and more heterosis for reproductive and maternal ability. Finally, if even partial control of the sex ratio becomes practicable in A. I. the advantages of beef crossing on the dairy cow population will be increased even further. In the final column of table 3 , the percent of total return per insemination due to genetic improvement in growth rate is shown. If cow turnover rate is fast i. e. 3 lactations per cow, beef never accounts for more than about io p. 100 of the total gain. With a longer cow life, it can contribute up to 24 p. ioo of the gain. However, its contribution rapidly drops off as the beef crossing percentage increases. The general conclusion is that either a fast rate of cow turnover, or a high level of beef crossing increases the probability that an insemination will lead to a dairy animal, that this gives dairy traits a high value in the selection objective, and that as a result, the total return from that insemination, and the proportion of that return due to milk improvement is greatly increased. This chain of cause and effect is shown in figure 3 . ---_-m __ --!! CONCLUSIONS I . The most efficient breeding programme for milk and beef in a dual-purpose population will require the maximum amount of crossing with beef breed bulls that is compatible with the provision of cow replacements. 2 . If all other factors are constant, the effect of increasing the level of crossbreeding by I p. 100 is to increase the economic value of the genetic merit conferred with each dual-purpose insemination by approximately i p. 100 . This effect is greatest where an appreciable amount of crossbreeding is already being practised. 3 . A second effect of increasing the percentage of cows bred to beef bulls is to greatly increase the emphasis which should be given to milk production in the selection of dual-purpose bulls. 4 . The percentage of total economic gain from dual-purpose bull selection which is accounted for by improvement of milk yield varies from 75 p. 100 to nearly 100 p. 100 . If 20 p. 100 or more of the cows are bred to beef bulls, the percentage is always over 8 0 p. 100 . 5 . A high cow replacement rate has an effect similar to a high level of beef crossing, i. e., it increases the emphasis on and return via milk. 6. The effects of level of crossbreeding and cow replacement rate on both the total gain from selection, and on the balance of gain via milk and beef, comes for the most part through their effects on the number of expressions 'of a bull's genotype for milk and beef. 7 . A high level of beef crossing and/or a high cow replacement rate greatly enhances the return on investment in the testing and selection of dual-purpose bulls.

v3-fos

2018-04-03T02:42:28.265Z

1974-03-01T00:00:00.000Z

34644158

s2

Production of Flavine-Adenine Dinucleotide from Riboflavine by a Mutant of Sarcina lutea A study was made to develop a new method for the production of flavine-ade- nine dinucleotide (FAD) from riboflavine and adenine by a mutant of Sarcina lutea deficient in the enzyme adenosine deaminase. It was found that this strain could convert exogenously supplemented riboflavine to extracellular FAD. The yields of FAD were increased by addition of D-cycloserine in the culture medium. The culture conditions for FAD production were investigated under the addition of D-cycloserine, and increased production of FAD was observed with the addition of an appropriate amount of thiamine, acetate, and sodium ion. The yield of 0.7 g/liter was obtained in the optimal culture in 5 days. Accumulated FAD was readily isolated by adsorption chromatography and ion-exchange chromatography in a 70% yield. The use of flavine-adenine dinucleotide (FAD) as a biochemical and nutritional agent has been recently increasing, and the chemical, biochemical, and fermentative methods have been reported for its production. In the past, FAD was produced by extraction from the mycelium of Eremothecium ashbyii (10) or by the chemical synthesis from flavine mononucleotide (FMN) and adenosine monophosphate (AMP) (2). We previously showed that a large amount of FAD accumulated in culture fluid when a strain of Sarcina lutea was cultured in the medium supplemented with FMN, a wellknown precursor of FAD, and adenine (7). During the course of these studies, it was found that riboflavine was more favorable than FMN as the precursor for FAD production because of its low cost and easy separation from FAD. This paper deals with the fermentative method for FAD production from riboflavine and adenine. MATERIALS AND METHODS Organism. A purine-requiring and adenosine deaminase-less mutant ATCC 21881, which was derived from Sarcina lutea IFO 1099, was used for the fermentative experiments. Fermentation experiments. Unless otherwise noted, fermentations for FAD production were carried out as follows. The compositions of seed medium and fermentation medium are shown in Table 1. Seed media were distributed in 30-ml amounts to 500-ml flasks, sterilized, and inoculated with one loopful of cells of the S. lutea mutants. Cultures were incubated at 30 C. After 24 h of incubation, 0.5 ml of the seed medium was combined with 20 ml of the respective fermentation medium in a 500-ml shaking flask. All cultures were incubated at 30 C with reciprocal shaking (120 rpm, 8-cm stroke). After 24 h of incubation, a 2.5-ml solution of 0.5% adenine and 0.5% riboflavine was added, and incubation was continued for an additional 2 to 4 days. Culture broth was heated at 80 C for 3 min and centrifuged. The supernatant was employed in the determination of products. Methods of analysis. The assay of FAD and FMN was carried out by the manometric method (7) and the fluorometric method (11). Determination of growth was carried out as follows. The culture broth was diluted 80-fold the original volume with water, and the optical density at 660 nm was measured with a photometer (Hitachi EPO-B type). An absorbance of 1.00 represented 1.6 mg of dry cells per ml. Sucrose Kulka (3). easily isolated without separating FMN and ured by the method of Hirata and FAD since only traces of FMN were formed nine, hypoxanthine, AMP, and from riboflavine. Accordingly, it was predicted determined by measuring their that riboflavine might be a more favorable 260 nm after extracting their spots precursor for FAD production. tograms with 0.01 N HCl. Paper Screening of detergents. It was shown that is carried out on Toyo filter paper some detergents vent system containing isobutyric some amino stimulated fermentative pro-,al water (5:3, vol/vol). duction of amino acids (8) and nucleic acidwas purchased from Boehringer related compounds (6). The benefit of adding heim, GFR), adenine was pur-detergents to the medium is the increase in cell Co., Ltd. (Tokyo), and ribofla-permeability. The results of Fig. 1 show that the 'okyo Tanabe Seiyaku Co., Ltd. rate-limiting step of FAD synthesis may be chemicals used were of the best riboflavine kinase (adenosine 5'-triphosphate previously demonstrated that formation would be stimulated and yields of active precursor for FAD pro-FAD would be substantially increased. Thus, ever, a large amount of FMN the effect of detergents on FAD overproduction ged in the final culture broth ersion a t h e ofinal FMNuinturot was investigated. Cationic surfactants, such as cetyltrimethylammonium bromide (CTAB) or are the chemical structures of cetylpyridinium chloride inhibited both growth milar to one another, but their and FAD production at 0.01% concentration ysical characteristics are quite ( Table 2). Growth inhibition could be reversed the separation of a large by lowering the concentration of the addition, from FAD in the culture broth but FAD production could not be increased. rder to avoid the disadvantage Almost all antibiotics, such as penicillin and e decided to use riboflavine, a streptomycin, showed the same result as ionic in the biosynthetic pathway of surfactants. Overproduction of FAD occurred serine was specific for FAD oversynthesis. The fact that the addition of D-cycloserine inhibited growth showed that FAD overproduction was not due to an effect of D-cycloserine on growth, but was due to some other unknown mechanism. In media with D-cycloserine, the amount of FAD accumulated in the culture broth was twice that accumulated in medium without D-cycloserine. Effect of addition time of D-cycloserine on FAD production. The correlation between FAD production and addition time of D-cycloserine was studied in detail (Fig. 2). Growth inhibition was observed when D-cycloserine was added within 24 h after inoculation, but FAD production was effectively stimulated. On the other hand, when it was added later, FAD production was appreciably depressed in spite of no inhibition of growth. This result suggests that FAD production is increased under conditions causing growth inhibition. Accordingly, the addition time of D-cycloserine should be at the early stage of FAD fermentation. When D-cycloserine was supplied within 24 h after inoculation, growth obtained was less than half of the maximal growth. Effect of D-cycloserine concentration on FAD production. The effect of D-cycloserine concentration on FAD production is shown in Fig. 3. Experiments were made to determine an optimal concentration of D-cycloserine at zerotime addition. As seen in Fig. 3, addition of D-cycloserine at the concentration of 80 ug/ml gave the optimal yield of FAD, but growth was nearly 60% of the maximal growth without added D-cycloserine. S. lutea was markedly 0.6r Ag/ml strongly inhibited growth but that of 40 gg/ml permitted normal growth. Formation of FAD from FMN or riboflavine by non-growing cells. To examine how D-cycloserine stimulates the accumulation of FAD from riboflavine, activities of FAD formation in the cells grown on the basal medium with D-cycloserine and in the cell supernatant were compared with those grown without D-Cycloserine ( Table 3). The cells grown on the medium without D-cycloserine formed a small amount of FMN, but not FAD, from riboflavine, while they synthesized a relatively large in the cells grown on the medium supplemented with D-cycloserine, FAD was equally synthesized either from FMN or riboflavine in reasonable yields. The cell supernatant had no activities of FAD formation, regardless of the addition of D-cycloserine to medium. These results suggest that the stimulation of FAD formation from riboflavine may not be caused by the leakage of enzymes from the cells, but by the improvement of permeabilities of substrate or products. Effect of culture conditions on FAD production. The culture conditions for FAD production were investigated under the addition of D-cycloserine. (i) Effect of precursors. Various kinds of precursors were added to the culture medium at 24 h after inoculation. As shown in Table 4, it is apparent that this strain has a de novo pathway for FAD formation because a small amount of FAD was made without addition of precursor. Guanine, a well-known precursor of riboflavine, inhibited growth and was not effective for FAD production, whereas adenine and riboflavine stimulated FAD production without inhibiting growth. But separate addition of adenine and riboflavine did not stimulate FAD production compared with their simultaneous addition. Accordingly, it is essential for FAD overproduction that adenine and riboflavine are simultaneously added to culture medium. Growth inhibition by guanine was reversed by adding adenine, but the amount of FAD accumulated was about 120 ug/ml, which was the same amount as that accumulated by the single addition of adenine. (ii) Effect of aeration. The effect of aeration was studied by changing the volume of medium in the flasks. Maximal production of FAD was obtained with 25 ml of medium or less in a 500-ml flask, which is equivalent to an oxygen absorption rate greater than 2.95 mmol/min (Table 5). (iii) Effect of thiamine. Table 6 demonstrates the effect of thiamine concentration on FAD production. A low concentration of thiamine supported good growth but not FAD overproduction. There was also a similar tendency with regard to growth and FAD production at a high level of thiamine. The optimal concentration of thiamine was limited to a narrow range which was about 0.5 ,ug to 2.0 ,ug/ml. (iv) Effect of sodium ion. Sodium ion was also found to exert a considerable effect on FAD production. Experiments were carried out to examine the changes during fermentation with or without sodium ion (Fig. 4). When sodium ion was removed from basal medium, growth was enhanced, but FAD production was strongly inhibited. It was estimated that the inhibition of sugar assimilation caused by the removal of sodium ion repressed ATP formation. As the result, FAD production was inhibited. (v) Effect of acetate. We reported that acetate stimulated FAD formation from FMN (7). A similar result was obtained when using riboflavine (Fig. 5). Omission of acetate from the medium reduced the FAD yield by about 80%. When an optimal concentration of acetate was added, rapid assimilation of sugar and no for- x, pyruvate. mation of pyruvate was observed. The definite mechanism responsible for this phenomenon has not been established. Changes occurring during fermentation. An example of the chemical changes which occurred during the fermentation of FAD under optimal conditions is given in Fig. 6. After an initial lag period of approximately 12 h, the logarithmic phase proceeded for a long period, and rapid consumption of sucrose was accompanied by growth. As growth slowed down, FAD production started and reached a maximum of about 700 gg/ml at 5 days. The characteristic pH change during the growth phase was a feature of this fermentation process. The rise in pH suggested the start of logarithmic phase, and the pH rose as high as 8.5. Assimilation of acetate and formation of ammonium ion from peptone contributed to the pH rise. After growth ceased, the pH decreased to 6.5 -7.0 and remained constant during the latter part of fermentation. During fermentation, only traces of FMN formed in the medium. Accordingly, FAD in the fermentation broth was easily isolated by an ordinary procedure using Florisil and ion-exchange resins in a 70% yield. Infrared spectrum of the product was identical with that of authentic FAD, and no other fluorescence compound was detected by paper chromatography. DISCUSSION Many purine-pyrimidine-related substances are produced by fermentation methods. These include a number of nucleotides, nucleosides, and their analogues. Practical methods for the synthesis of nucleotide derivatives from the corresponding bases were recently reported (5,9). The synthesis of 5'-inosinic acid, ATP, nicotinamide adenine dinucleotide, coenzyme A, etc., are the examples of these methods. In a previous paper the production of FAD by a microorganism was reported; i.e., a large amount of FAD was produced by S. lutea from FMN and adenine in a medium containing sucrose and salts (7). But this previous method had some disadvantages. Among them are the high cost of FMN and the difficulty of separating FMN from FAD during the isolation procedure of FAD. To overcome these disadvantages, we attempted to use riboflavine instead of FMN as a precursor for FAD production. Riboflavine was an inferior precursor in comparison to FMN, because the ability to convert riboflavine into FMN was low in S. lutea. In general, fermentative production of nucleotides is markedly affected by the cellular permeability of the microorganism used. Therefore, studies were performed on the removal of the permeability barrier of S. lutea. The permeability barrier is removed by controlling the levels of trace nutrients or metals, or by the addition of the agents affecting cellular permeability such as surfactants, antibiotics, etc. As described above, FAD production from riboflavine was stimulated only by the addition of D-cycloserine. An example of using D-cycloserine was shown for fermentative production of 5'-inosinic acid by Nara et al. (6). They reported that the most important condition was to add the antibiotics at a very early stage of fermentation. A similar observation was made on FAD production. The addition of D-cycloserine at later than 24 h of incubation resulted in a marked reduction of FAD yields. It was estimated that the stimulation with the addition of D-cycloserine was caused by the improvement of the permeability barrier through the change of cell wall because D-cycloserine is known as a compound which affects the structure of microbial cell wall (4). This was confirmed by the experiments shown in Table 3. The change of cell permeability caused by D-cycloserine allowed for a more rapid conversion of riboflavine into FMN, the most immediate precursor of FAD. But it was desirable for production and isolation of FAD that the accumulation of FMN was not accelerated since accumulated FMN was easily converted to FAD by FAD pyrophosphorylase (ATP: FMN adenyltransferase; EC 2.7.7.2) in this strain. From a practical point of view, the present method has considerable advantages over any other microbial process previously reported (7,10). One of the advantages is that a high concentration of FAD is accumulated in culture fluid and another is that only traces of FMN are formed. These characteristics allow for the purification of product in a high yield without tedious procedure. Thus, the method presented here is considered to be a very advantageous one for FAD production.

v3-fos

2018-04-03T00:53:27.506Z

1974-04-01T00:00:00.000Z

27083458

s2

Survival of Vibrio parahaemolyticus in cooked seafood at refrigeration temperatures. The growth and survival of two strains of Vibrio parahaemolyticus isolated during food-borne gastroenteritis outbreaks in Japan and surface inoculated on cooked shrimp, shrimp with sauce, or cooked crab were tested at various refrigeration temperatures during a 48-h holding period. On cooked shrimp and crab, the vibrios grew well at 18.3 C, but their numbers declined gradually at 10 C and below. At 12.8 C, vibrios remained static for the most part. Thus, it appeared that 12.8 C was the borderline temperature for growth of the organism on cooked seafood. When cocktail sauce was added to surface-inoculated shrimp at a ratio of 2:1, the vibrio die-off rate was accelerated. In the shrimp and sauce few cells remained after 48 h, but in the sauce alone die-off was complete at 6 h. The growth and survival of two strains of Vibrio parahaemolyticus isolated during food-borne gastroenteritis outbreaks in Japan and surface inoculated on cooked shrimp, shrimp with sauce, or cooked crab were tested at various refrigeration temperatures during a 48-h holding period. On cooked shrimp and crab, the vibrios grew well at 18.3 C, but their numbers declined gradually at 10 C and below. At 12.8 C, vibrios remained static for the most part. Thus, it appeared that 12.8 C was the borderline temperature for growth of the organism on cooked seafood. When cocktail sauce was added to surface-inoculated shrimp at a ratio of 2: 1, the vibrio die-off rate was accelerated. In the shrimp and sauce few cells remained after 48 h, but in the sauce alone die-off was complete at 6 h. The facultative halophile, Vibrio parahaemolyticus, is recognized as a public health hazard in seafoods of United States and foreign origin (13). Recent outbreaks of food poisoning in the United States were associated with the consumption of crab or shrimp contaminated with this organism (4)(5)(6)(7). Foods contaminated with V. parahaemolyticus through inadequate preparation or handling may be held under conditions that favor the growth of bacteria and increase the risk to the consumer. Generation times as low as 12 min were observed when these organisms were inoculated into sea fish (11). Minimal temperatures reported for V. parahaemolyticus multiplication were 5 C (3) and 8 C (1) in artificial media, and 10 C in oyster homogenate (15) and the marine environment (10). Under favorable growth temperatures, extensive multiplication may occur that probably increases the risk of food-borne illness. Restaurants and cafeterias commonly prepare seafood salads in advance of serving time and store them in the refrigerator until they are displayed on the serving line. Temperatures as high as 12.8 C in refrigerated showcases (2), 10 C in domestic refrigerators (19), and 15.6 C in coolers used in the blue crab industry (12) have been recorded. Such deviations from optimum refrigerator temperatures might well permit multiplication of contaminating V. parahaemolyticus. A few studies (8,9,15,18) of the survival of V. parahaemolyticus inoculated into the tissues or into homogenates of shrimp and oysters have been conducted. But such studies have limited applicability to the hazards of storing poorly refrigerated, contaminated seafood. This study was therefore undertaken to determine the effects of storage temperatures representative of both good and poor refrigeration on the growth and survival of V. parahaemolyticus on the surface of cooked seafoods such as shrimp and crab. MATERIALS AND METHODS Cultural methods. The two cultures of V. parahaemolyticus used in this study were Yanagisawa, 04: K9 (obtained from H. Zen-Yoji, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo) and 9382, 04: K11 (obtained from Y. Miyamoto, Kanagawa Prefectural Public Health Laboratory, Yokohama). Both cultures were isolated during foodborne gastroenteritis outbreaks in Japan. The media and culture conditions used for maintenance of stock cultures as well as for verification of strains have been previously described (16). Both strains exhibited properties that have been well established for identifying pathogenic V. parahaemolyticus (14). Kanagawa hemolysis was determined on Wagatsuma agar (Eiken) that contained 10% washed human red cells. Serotyping was accomplished by slide agglutination with 8 monovalent 0, 8 polyvalent K, and 52 monovalent K antisera (Toshiba Kazaku Co., Ltd., Tokyo). Inocula for a given experiment were prepared from strains grown on Trypticase soy agar (BBL) containing 3% NaCl at 35 C for 18 to 22 h. Cells were washed twice and resuspended in buffered physiological saline to a known optical density. Preparation of seafood. Frozen whole jumbo shrimp in shells and frozen pasteurized Alaskan king crab meat were used in this study. Samples were cooked at 100 C for 6 min; the cooking water was then poured off, and cold, sterile distilled water was added. Shells were aseptically removed from the shrimp. Twenty-five grams of either crab or shrimp was added to sterile, 3-oz (88.69-ml) plastic bottles. Samples were equilibrated at 35 i 1 C and surface inoculated with 1 ml of cell suspension adjusted to provide initial concentrations of approximately 104 organisms/g. Bottles were sealed in plastic bags and immersed in water baths set at the desired test temperatures. Duplicate plates were prepared from appropriate dilutions of duplicate samples in modified Twedt medium (17) at 0, 6, 12, 24, 36, and 48 h. Plates were incubated at 35 C for 42 to 44 h. V. parahaemolyticus were never detected in uninoculated control samples plated at 0 and 48 h. In certain assays, 50 g of a commercial sauce (Seafood Cocktail Sauce, Crosse & Blackwell Co., White Plains, N.Y.) was added to 25 g of inoculated shrimp to approximate a commercial seafood cocktail that is produced and then frozen for retail sale. In other assays, samples of sauce were inoculated and tested for survival. APPL. MICROBIOL. Verification. Five colonies from each of four countable plates at each incubation temperature in every experiment were picked to test in Trypticase soysalt broth for verification of V. parahaemolyticus. The biochemical tests performed on each included growth in 0, 1, 8, and 10% NaCl; production of acetoin, indole, and H2S; and fermentation of glucose and sucrose. Serotyping was performed by slide agglutination. RESULTS The growth and survival of two strains of V. parahaemolyticus that were surface inoculated on whole shrimp held at six refrigeration temperatures are shown in Table 1. The vibrios grew well at 18.3 C, but their numbers declined from 0.5 to 1 log at 10 C and below during the 48-h holding period. At 12.8 C, strain 9382 declined 1 log, and strain Yanagisawa remained relatively static. Table 2 illustrates the effect of added cocktail sauce on the growth and survival of vibrios on shrimp. In most cases counts fell 9382). In sauce alone, few viable cells were more than 1 log in 24 h at all temperatures. Few present at 6 h, and die-off was complete at 24 h organisms remained at 48 h. The rate of decline at all temperatures. The growth and survival of seemed less at 18.3 C than at the other tempera-V. parahaemolyticus on cooked crabmeat were tures. Indeed, multiplication occurred after 36 h similar to those observed on shrimp (Table 3). at this temperature in one experiment (strain The organisms grew well at 18.3 C, remained Downloaded from static (except for the small increase in experiment 1 at 48 h) at 12.8 C, and declined approximately 0.5 log at 7.2 C and 1 to 2 logs at 1.6 C during the 48-h holding period. DISCUSSION Our results indicate that V. parahaemolyticus inoculated onto the surface of cooked shrimp or crab gradually decline in numbers at incubation temperatures of 10 C and below and multiply if held at 18.3 C. Apparently, 12.8 C is the borderline temperature for growth of this organism on cooked seafood. The Yanagisawa strain remained nearly static, and the 9382 strain declined 1 log during the 48-h storage period. By utilizing different experimental menstrua and methods, other investigators have obtained analogous results. Vanderzant and Nickelson (18) observed a decrease of 2 logs during the first 2 days of storage at 3, 7, and 10 C of approximately 105 V. parahaemolyticus cells injected into whole uncooked shrimp. In shrimp homogenates inoculated with approximately 5 x 104 cells/ml, results were quite different. An initial slight increase in numbers over the first 12 h at all three temperatures was followed by a gradual decrease. Johnson and Liston (8) reported a slow decline in numbers of a V. parahaemolyticus strain after 2.5 days of storage at 11 C and below in depurated oysters naturally contaminated with 5.8 x 104 cells/g. Thomson and Thacker (15) inoculated V. parahaemolyticus strains into oyster homogenates at an initial level of approximately 5 x 103 cells/ml. They observed multiplication at 10 C and above and no change at 8 C, with a gradual decline of 1 to 2 logs at 4 C and below during 1 week of storage. Johnson et al. (9) reported little or no apparent decrease of V. parahaemolyticus in naturally contaminated oyster shellstock stored for 3 weeks at 4 C. Reports in the literature demonstrate that domestic and commercial refrigerators exhibiting fluctuating temperatures capable of supporting Vibrio survival or multiplication on cooked seafood are not uncommon. Bauman (2) reported refrigerator showcases to cycle between 4.4 and 12.8 C during a 12-h period. When van Walbeek et al. (19) tested domestic refrigerators in the early morning hours to avoid fluctuation caused by frequent opening, they recorded temperatures as high as 10 C. Commercial coolers exhibited temperatures up to 15.6 C during a survey of the blue crab industry by Phillips and Peeler (12). When commercial sauce was added to sur-face-inoculated shrimp, Vibrio decline was accelerated. In most cases, the die-off rate was greater than 1 log in 24 h. Only minimal numbers were present at 48 h. The explanation for this rapid decline may lie with the acidity of the sauce (pH 3.3 to 3.4). In sauce alone, Vibrio die-off was virtually complete in only 6 h. Since the shrimp was surface inoculated before admixing with sauce, it is very likely that the bacteriocidal effects of the acid pH were modified by buffering that would result from the association of vibrios with shrimp tissue. The gradual decline of V. parahaemolyticus on the surface of cooked seafood at refrigerator storage temperatures of 10 C and below can hardly be considered to eliminate the public health hazard inherent in a contaminated product. The danger of gastroenteritis is still present for the consumer, either from massive numbers of V. parahaemolyticus or from modest numbers of a highly infectious strain.

v3-fos

2020-12-10T09:04:20.448Z

1974-11-01T00:00:00.000Z

237233346

s2

Fermentation of Feedlot Waste Filtrate by Fungi and Streptomycetes The soluble and dispersed nitrogen and carbon components in the filtrate fraction of cattle feedlot waste are a potential nutrient source from which single-cell protein could be produced for animal feeds. The ability of more than 200 fungi and streptomycetes to grow in this liquid was determined; these included isolates from the waste and associated sources, as well as organisms maintained in the Culture Collection of the Agricultural Research Service in Peoria, Ill. Utilization of waste nutrients was measured by changes in nitrogen content and chemical oxygen demand. Only 20% of the organisms were able to grow appreciably in the filtrate. Of these, dry-weight yields varied from 0.6 to 2.7 g of mycelium per liter; from 21 to 50% of the nitrogen in the filtrates was used during growth, whereas chemical oxygen demand levels diminished from 4 to 60%. In general, streptomycetes isolated from the feedlot used nutrients from the filtrates better than fungi did. Addition of readily available carbon sources such as glucose or whey significantly increased (as much as sixfold) cell yields of selected organisms and promoted better utilization of nitrogen (from two- to threefold); the effect on chemical oxygen demand varied (0 to 33% increase). The soluble and dispersed nitrogen and carbon components in the filtrate fraction of cattle feedlot waste are a potential nutrient source from which single-cell protein could be produced for animal feeds. The ability of more than 200 fungi and streptomycet es to grow in this liquid was determined; these included isolates from the waste and associated sources, as well as organisms maintained in the Culture Collection of the Agricultural Research Service in Peoria, Ill. Utilization of waste nutrients was measured by changes in nitrogen content and chemical oxygen demand. Only 20% of the organisms were able to grow appreciably in the filtrate. Of these, dry-weight yields varied from 0.6 to 2.7 g of mycelium per liter; from 21 to 50% of the nitrogen in the filtrates was used during growth, whereas chemical oxygen demand levels diminished from 4 to 60%. In general, streptomycetes isolated from the feedlot used nutrients from the filtrates better than fungi did. Addition of readily available carbon sources such as glucose or whey significantly increased (as much as sixfold) cell yields of selected organisms and promoted better utilization of nitrogen (from two-to threefold); the effect on chemical oxygen demand varied (0 to 33% increase). Livestock production centers must cope with huge quantities of waste which offer a pollution hazard and a disposal problem (6,7,13,15). Biological treatment by oxidation ditches and lagoons to stabilize the waste before land disposal is the most common system used (6,9). We have enumerated and identified the microbial groups in feedlot waste (FLW; 8, 12), but not the ability of individual aerobic organisms to grow on feedlot pollutants. Organisms grown on waste pollutants might provide a source of microbial protein for animal feed while decreasing the pollution potential of the material. In a survey of isolates from waste and others from our Culture Collection, we sought filamentous organisms that could reduce pollutants and filter easily for cell recovery. More than 200 fungi and streptomycetes were studied for their ability to use nitrogen and organic material in the waste, the latter being measured by chemical oxygen demand (COD). The production of cell mass and the effect of adding glucose and dairy whey to waste filtrates also were investigated. (This paper was presented at the 1974 Annual Meeting of the American Society for Microbiology in Chicago, Ill.) MATERIALS AND METHODS Source of microorganisms. Samples were taken from pens of a cattle feedlot located near Peoria, Ill. (12). Fungi were isolated from plates of Mycophil medium (pH 7.0; Bioquest, Div. of Becton, Dickinson and Co., Cockeysville, Md.) to which has been added 0.2 mg of dihydrostreptomycin sulfate and 330 U of penicillin G per ml. A salts-starch agar (11) amended with cycloheximide (0.5 mg/ml) was used to isolate streptomycetes. Preparation of liquid waste for fermentation. Feedlot manure (21 to 40% solids) was diluted with distilled water to a solids content of 15%. After it was mixed to break up lumps, 15% (wt/vol) diatomaceous earth was added to aid separation. Filtrate obtained by suction filtration through filter paper (Whatman 54) served as substrate for the fermentation studies. The pH ranged between 6.0 and 6.8. The filtrate presented a qualitatively predictable substrate without the particles which would make equivalent samples difficult to obtain. Survey of organisms. Initially the ability of organisms to grow in FLW filtrate was evaluated in two ways. (i) Streptomycetes isolated from feedlot sites and fungi from the Agricultural Research Service Culture Collection were first grown on agar prepared with FLW filtrate as the sole nutrient source. Those which visibly grew well were further tested in liquid fermentations. (ii) Fungi isolated from feedlot sites, 845 together with streptomycetes and fungi selected from (i), were grown in sterile FLW filtrate in single, shaken test tubes (10 ml in tubes 25 by 150 mm). Inoculum was provided by 1-week-old plate and slant cultures. Tubes were shaken on a rotary shaker (200 rpm, 5-cm displacement) at 28 C. After 7 days, fermentation samples were brought back to volume with distilled water, and the mycelium was recovered by vacuum filtration through Whatman 54 paper. Cell masses were dried at 103 C overnight. Filtrate was analyzed for nitrogen (2), COD (1), and total carbohydrate (4). Flask fermentations. Organisms selected from the preliminary survey were further evaluated in duplicate flask fermentations. Flasks containing FLW filtrate (50 ml in a 300-ml Erlenmeyer flask) were inoculated with 2% (vol/vol) washed and blended mycelium. The mycelial inoculum was grown in a medium of 2% glucose, 0.1% peptone, 0.1% yeast extract, and 2% malt extract. Flasks were incubated on a rotary shaker at 28 C; the contents were analyzed for nitrogen and COD as were the tube cultures. RESULTS Survey. Streptomycetes isolated from FLW were streaked on agar plates made with waste filtrates. Of 59 cultures, 35 grew well and were transferred to FLW filtrate in tubes. Eight isolates produced more than 2 g of cell mass per liter in the liquid. Filtrates of these were lower in nitrogen and COD content than initial levels by 37 to 50% and 53 to 60%, respectively. Mycelium weight ranged from 2.0 to 2.4 g/liter (Table 1). Only 17 of 170 fungi from the Agricultural Research Service Culture Collection, streaked on waste filtrate-agar, grew well; the test organisms represented 14 different genera. The 17 fungi were inoculated into tubes of FLW filtrate. Nitrogen and COD levels were diminished below initial values by 31 to 58% and 4 to 52%, respectively. Cell mass ranged from 0.7 to 1.9 g/liter. Isolates of fungi from feedlots were grown on FLW filtrates supplemented with 0.6% glucose ( Table 2). They produced more cell mass and lowered COD and nitrogen levels further than fungi grown in waste liquid without added glucose. Nutrient additions. Trichoderma viride Persoon ex S. F. Gray NRRL 3652 and Fusarium aquaeductuum (Radlkofer and Rabenhorst pro parte) Lagerheim NRRL 2503 grew poorly on FLW filtrate in shaken flasks and were ineffective in reducing COD and nitrogen ( Table 3). Addition of glucose increased cell mass and decreased nitrogen and COD levels more. Fusarium oxysporum grew twice as well as T. viride and F. aquaeductuum and was 2 to 3 times as effective in diminishing pollution potential. F. oxysporum, an isolate from FLW, reduced COD levels by one-half in 1 week of fermentation ( Table 4). Addition of glucose yielded more cell mass and greater use of nitrogen from the waste; however, residual COD was not lowered. Common nutrients that might be limiting were added to FLW filtrate; these mixtures were fermented for 1 week with F. oxysporum. Levels of COD were diminished by half in all flasks (Table 5). Nitrogen content was lowered by 37% with FLW filtrate without additives. Glucose alone and in combination with peptone and phosphate diminished nitrogen and COD levels by 44 and 47%, respectively, and produced more mycelium than in the control flasks. Addition of ammonium ion diminished nitrogen levels by 28% as compared with 37% for controls, but phosphate supplement allowed Filtrates obtained from heated FLW, as compared with unheated material (Table 5), contained more nitrogen (78%) and organic mate-rial (40%, measured as COD). F. oxysporum, grown 1 week on filtrate from heated FLW, took up one-third more nitrogen and one-tenth less COD substances than in unheated liquid. Mycelium production was the same with both filtrates. When a sterile mixture of dairy and FLW liquids (FLW filtrate-whey, 2:1, vol/vol) in flasks was inoculated with F. oxysporum, 10.3 g of cell mass per liter was made in 1 week. Initial COD and nitrogen levels were lowered by 85 and 73%, respectively (Table 5). F. oxysporum inoculated into the mixed culture of unsterile dairy liquid and filtrate from FLW overgrew other organisms. After 7 days of growth, nitrogen and COD materials were diminished 62 and 96%, respectively (Table 5). Sterile mixtures, similarly fermented by this organism, had an equivalent amount of nitrogen but less organic material. Dairy whey was fractionated by dialysis in an Amicon apparatus and combined with waste filtrate in ratios of 1:2 (Table 6). F. oxysporum produced 6.4-fold greater cell mass on this mixture (10.3 g/liter) than on FLW filtrate alone (1.6 g/liter). Although nitrogen uptake was improved, comparable final COD values indicate that admixture with whey did not increase utilization of FLW filtrates. Dialysates of whey gave similar results. Thus, increased yields of mycelium from combined waste are attributable to usable nutrients in whey. Amendment of FLW filtrate with 1.7% lactose gave two-thirds the cell mass and showed decreases of nitrogen and COD material comparable to that for combined waste liquids. DISCUSSION As expected, a large proportion of streptomycetes isolated from FLW grew well as streaks on FLW filtrate-agar. Such growth may reflect survival and adaptation in a limiting environment. However, limited microbial growth in FLW in situ was indicated by the relatively constant number of organisms found in waste at a feedlot regardless of season (12). Streptomycetes which were isolated from the feedlot reduced pollutants in waste filtrates with modest yields of cells (Table 1). Fungi which were similarly isolated and grown on FLW filtrate with glucose also reduced nitrogen and COD levels, but formed more mycelium ( Table 2). Fungi that were not adapted to FLW nor supplemented with glucose reduced pollut- ants less well and produced less cell mass (Table 1). FLW filtrate contains 3 mg of carbohydrate and 0.5 mg of N per ml. Assuming that carbohydrate was the major nonprotein carbonaceous material in FLW, the calculated carbon-tonitrogen ratio of FLW filtrate is 2.4. This ratio was compared with 6.6 and 7.6, which values are based on the elemental composition of mycelium (10). Nitrogen levels of Aspergillus niger mycelium have been shown to be a function of initial nitrogen content of medium (14). Foster (5), reporting elemental data by Porges, gave a carbon-to-nitrogen ratio of 18 for fungi. A suitable carbohydrate source is often crucial for growth of molds (3). Consequently, carbohydrate supplements were supplied to microorganisms isolated from a feed lot and to others selected from the Agricultural Research Service Culture Collection. Selected fungi exhibited varied ability to take up pollutants from waste filtrates as a function of glucose amendent. Although T. viride, grown in FLW filtrate with glucose, reduced COD levels below those found without supplement, utilization of COD by F. aquaeductuum and F. oxysporum was either unaffected or impaired by glucose addition. Less uptake of organic material with increasing glucose content suggested that diminished utilization occurred because of a sparing action. In contrast, uptake of nitrogen was invariably increased by addition of glucose to the waste. Addition to FLW filtrate of nutrients common to microbial media did not greatly affect uptake of nitrogen or COD substances ( Table 5). Liberation of waste nutrients by heating FLW (before filtration) affected subsequent pollutant utilization even less. Evidently, F. oxysporum is so acclimated to waste substrates that common supplements give little benefit. Two dissimilar wastes that complemented deficiencies in nutrient composition were combined. For example, fermentation of dairy whey in combination with FLW filtrate resulted in a high uptake of nitrogenous and organic materials (Table 6). It is likely that dairy whey complemented feedlot filtrate by providing lactose because waste was deficient in total carbohydrates. Liquids resulting from fermentation of supplemented waste filtrate are not suitable for release to surface waters because of residual nitrogen and COD levels. However, stabilized fermentation liquids could be used for flushing feedlot surfaces and for irrigation. In conditions of protein shortages, the fungal mycelium of those fermentations might prove useful in animal feeds.

v3-fos