When cells of Escherichia coli ML30 were suspended in 2% gelatin and frozen at-40 C, no appreciable metabolic damage or death occurred. After freeze-drying for 8 hr at a platen temperature of 49 C and rehydration with a mineral salts medium, survival of the cells was 0.6%. Metabolic damage of the survivors was found to be 23%. Permeability alterations were detected by several criteria. Freeze-dried cells were susceptible to antibiotics normally ineffective against E. coli and leakage ' Contribution no.
Ground beef (GB) samples were maintained in atmospheres of 2 or 18% oxygen. Similar putrid spoilage occurred at both concentrations, although the microflora differed. Two of 10 isolates from fresh and spoiled GB, nonfluorescent pseudomonads, produced putrid aromas but only 4 volatile compounds in common when inoculated into sterile GB. AMorarelIa isolate produced a pronounced estery, decayed vegetable odor while others produced a decayed straw, ammoniacal or sour aroma. Differences in spoilage aromas apparently were not characterized by individual compounds but rather by the concentration of sulfur compounds and their ratio to other compound classes. Acetone, methyl ethyl ketone, dimethyl sulfide and dimethyl disulfide were indexes of microbial spoilage.
Volatile components present at spoilage of refrigerated chicken breasts were identified using high-vacuum-low-temperature distillation techniques followed by analysis with combined temperature-programmed gas chromatography and mass spectrometry. A comparison was made of the compounds detected from both irradiated and non-irradiated muscle stored at 2 and 10°C under both aerobic and anaerobic conditions. Isolates were randomly selected from the spoiled poultry, identified, and evaluated for their ability to produce volatile spoilage notes when grown on radiation-sterilized chicken. Several isolates that produced off-odors on sterile chicken breasts were examined. Twenty-two compounds were associated with spoilage. Some of the compounds found on both irradiated and unirradiated samples were considered to play only a minor role in the spoilage aroma or were present in low concentrations, since the aroma of spoiled irradiated chicken lacked the harsh odor notes typical of spoiled unirradiated chicken. Fifteen of the 22 compounds were considered to be unique to unirradiated, aerobically spoiled samples. Nine of these compounds, hydrogen sulfide, methyl mercaptan, dimethyl sulfide, dimethyl disulfide, methyl acetate, ethyl acetate, heptadiene, methanol, and ethanol, were found on chicken spoiled at both 2 and 10°C. Xylene, benzaldehyde, and 2,3-dithiahexane were detected only in samples stored at 20C and methyl thiolacetate, 2-butanone, and ethyl propionate were associated with 10°C spoilage. Fifty-eight isolates randomly selected from fresh, radiation-pasteurized, and unirradiated spoiled poultry were classified taxonomically, and 10 of them, which produced spoilage odors on sterilized chicken breasts, were selected for subsequent analysis of their volatiles. Isolates identified as Pseudomonas putrefaciens and Pseudomonas species that were members of groups I and II of Shewan's classification, as well as Flavobacterium and oxidative Moraxella, produced a number of the compounds found in the aroma of spoiled chicken. A total of 17 compounds were identified. Whereas no isolate produced all of the aroma compounds found in the aroma of spoiled chicken, together they did produce the nine found in unirradiated samples spoiled at either 2 or 10°C, as well as methyl thiolacetate and xylene. Six compounds were present in the volatiles produced by the isolates but were absent in the volatiles identified from spoiled chicken. These were hydrogen cyanide, methyl isopropyl sulfide, 2-propane thiol, methyl propionate, ethyl benzene, and an unidentified compound. The spoilage odor of meat, poultry, and fish at refrigeration temperatures is attributed mainly to microbial by-products (2, 10, 13, 15) and not to autolytic products from tissue (10, 13, 17). Whereas spoilage odors have been subjectively described (1, 14, 15), only recently have attempts been made to identify specific volatile compounds present at spoilage and relate them to the causative microorganism.
Although 95 % of the enterotoxin B produced by Staphylococcus aureus appears during the latter part of the exponential phase of growth, growth per se is not necessary for toxin synthesis. A procedure is described whereby a concentrated suspension (at least 6 x 1010 cells per ml) of a 16-hr culture of S. aureus was found to be capable of producing toxin, without replication, when air and glucose were present. This technique allows the growth requirement to be separated from toxin formation. Although higher (100 ,ug/ml) concentrations of toxin appeared in the medium when nitrogen was present, lower levels (30 ,ug/ml) were produced in the absence of N-Z-amine A. Toxin production proceeded without any net increase in deoxyribonucleic acid, ribonucleic acid, or protein. Chloramphenicol did not inhibit toxin formation in a nitrogen-free medium. The optimal pH for toxin production in a nitrogen-free medium was 8.0 to 8.5; for synthesis in a medium where nitrogen was available, the optimal pH was 7.0 to 7.5. Increasing the rate of aeration increased toxin release during growth, but decreased the amount of toxin subsequently produced when the bacteria were resuspended. These results suggest the presence of a precursor pool in the cells collected after 16 hr of growth. MATERIALS AND METHODS Media. Medium 1, as described by Rosenwald et al. (12), contained 4.0% N-Z-amine type A (Sheffield Chemical, Norwich, N.Y.), 0.4% yeast extract (Difco), and 0.1% K2HPO4 in distilled water. Medium 2 contained 4.0% N-Z-amine type A and 0.1% K2HPO4. Medium 3 contained 0.5% glucose and 3.5% K2HPO4. This medium was considered the nitrogenfree medium. Culture propagation. Stock cultures of S. aureus S-6 were maintained on slants of Trypticase Soy 506
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