ALBERT

Notes: SUMMARY— The inhibition of outgrowth of spores of Clostridium botulinum Type E by the addition of different concentrations of various solutes to a trypticase-peptone-sucrose-yeast extract medium (TPSY) was studied at different incubation temperatures. Solutes added to TPSY medium were sodium chloride (NaCl), sodium formate (NaCOOH), potassium chloride (KCI), sucrose and glucose. Incubation was at 30, 21.1, 15.6, 10 and 7.2°C, and growth was confirmed by mouse toxin assay. The effect of the solutes on the equilibrium relative humidities (ERHI of the various media was measured using the graphical interpolation method and expressed as water activity (aw). The possibility of an osmotic effect was also investigated. The molar (M) concentrations and osmotic pressures of the different solutes were calculated and related to the approximate inhibitory concentrations of 5.0% NaCl (aw 0.975), 6.0% KCI (aw 0.974), 5.5% NeCOOH (aw 0.9711, 38.5% sucrose (aw 0.976) and 22.5%. glucose (aw 0.970).The inhibitory salt concentrations were approximately 0.80 M and produced osmotic pressures of about 36.0 atmospheres. The approximate osmotic pressure of the inhibitory 1.12 and 1.24 M sucrose and glucose solutions were 25.08 and 27.97 atmospheres respectively.Growth response decreased as the incubation temperature decreased as measured by delayed outgrowth times and in decreased amount of cellular material produced by the Type E strains tested.

Notes: Efforts were continued in an attempt to assess quantitatively the behavior of certain cellular components while the spores were being exposed to moist heat, and to correlate such behavior with the thermal death reaction.Spores of Bacillus subtilis were preheated over prolonged intervals in an attempt to induce the release of cellular dipicolinic acid (DPA) without exercising serious loss of viability, and then to evaluate the subsequent effect of DPA release upon the thermal death rate of the preheated spores. At 45°C in 2.5mM PO4 buffer at pH 7, the net DPA released amounted only to 1.6 and 2.7% at the end of 20 and 30 hr. The treated spores exhibited no loss in viability and when heated at 98.5°C in 25mM PO4 buffer at pH 7, exhibited no differenee in their thermal death rates. Prolonging the heat treatment up to 9 days did not materially change the final results. To induce faster and greater DPA release, the above experiment was repeated at 80 and 90°C. At 80°C the net DPA released was 3.6 and 4.1% at the end of 3 and 8.5 hr, without loss in viability. However, the treated spores exhibited some reduction in their thermoresistance at 98.5°C. At 90°C greater amounts of DPA were released, but were associated with substantial loss in viability, and the surviving spores exhibited marked reduction in their thermoresistance at 98.5°C. These observations suggest the existence of DPA in more than one structural form; free or loosely bound to the spore structure and easily exuded upon mild heating or even standing under refrigeration; and a second more strongly bound form which required severe heating for its release. It is this latter form which might be associated with the mechanism of thermoresistance. Along with DPA, following the same pattern but at much higher rates, ninhydrin-positive material was exuded during sublethal heating at 80, 85, and 90°C. It was suggested that such material might be of similar nature to that exuded during germination of Bacillus species as described by Powell (1957).Spore suspensions of Bacillus coagulans (thermoacidurans) of varied thermostability were heated in 25mM phosphate buffer at pH 7, at 95°C and both the rate of death and DPA release were established. Results showed a difference in kinetics between the two reactions. Death progressed at higher rates than DPA release. Higher rates of death were associated with higher rates of DPA release. Spores of same strain were heated in both water and 10mM glyeylglycine, a death accelerating agent, at 100°C, sampled at intervals and analyzed for survival, dry weight, calcium, manganese, magnesium and DPA. Results showed the death of spores and their concurrent exudation of DPA and divalent cations were both markedly accelerated in the presence of glycylglyeine. The 25-min survival and exudate levels in glycylglyeine were near equivalent to those at the 65-min levels in water.The kinetics of death and the concurrent release of DPA and calcium were further assessed in more detail in 5mM phosphate buffer at pH 7 at 96 and 99°C using spores of B. coagulans (thermoacidurans). Death again progressed at a higher rate than that of either DPA or calcium release. Based on the molar ratio of Ca/DPA released in the supernataut, the initial ratio was always greater than 1, then rapidly dropped. and eventually plateaued at a value of less than 1. Similar patterns of death and calcium and DPA release were obtained for B. cereus when heated at 86°C.All of the above observations demonstrate the association of the thermal death reaction with the exudation of ninhydrin-positive material, DPA and divalent cations into the heating menstruum. The exact relationship between such cellular components and the spore mechanism of thermoresistance remains inconclusive.

Notes: The radiation resistance of spores of 6 strains of Type E Clostridium botulinum was determined in a beef stew substrate. The results are evaluated in terms of the minimum dose showing no spoilage for a 2 billion total inoculum level (20 cans each with 100 million spores per can) and in terms of a calculated radiation D value. The mean radiation D value for 6 strains is 0.132 megarad (range 0.125–0.138). A comparison with results for 6 strains of Type A and 5 strains of Type B shows that Type E strains possess about 45–5570 of the resistance to ionizing radiations of Types A and B. It is concluded that any radiation dose for food sterilization based upon the maximum resistance of Type A or B strains would provide ample protection against Type E strains. The unswelled cans at the lowest radiation level showing no spoilage were free of toxin and recoverable Type E spores.

Notes: SUMMARY— Aseptic samples from pig and rabbit muscles were inoculated with Achromobacter liquefaciens, Micrococcus luteus, Pediococcus cerevisiae, Pseudomonas fluorescens, Streptococcus faecal is end a mixed culture obtained from commercial hamburger. Some difficulty was encountered in getting the organisms to grow, and good growth was achieved only with A. liquefaciens end mixed culture from commercial meat. Both inoculated and uninoculated control samples were incubated at 3 and 10°C for 0, 8 and 20 days. The salt soluble proteins were extracted with Weber-Edsall solution and subjected to sucrose density gradient centrifugation, gel filtration and disc gel electrophoresis. The microorganisms utilized in this study had no measurable effect upon the myofibrillar proteins from either pig or rabbit muscle. However, bacterial growth decreased the amount of certain non-protein ultra-violet absorbing components in the ultracentrifugal pattern of Weber-Edsall extract. These components did not appear to be of myofibrillar origin. Disc gel patterns of Weber-Edsall extracts from pig muscle produced a more intensely staining band than those from rabbit muscle at Rm, = 0.59.

Notes: Mildly heat-shocked spores of four strains of Type E Clostridium botulinum (VH, Beluga, Iwanai, and 8E) were inoculated into a heat-sterilized beef stew substrate and incubated at 34, 36, and 38°F. Inoculum levels of approx 4-12 million spores per tube were used with different strains. At 38°F, toxin production and visible outgrowth as evidenced by gas formation beneath a vaspar seal occurred in one or more tubes of each of the strains within 31–45 days. No evidence of growth was visible after 14 days at 38°F, and at this time duplicate tubes of each strain were not toxic. After 31 days, strains Iwanai and Beluga showed 2 of 8 tubes with gas and toxin. None of 8 tubes of the VH and 8E strains showed gas after 31 days. Two tubes of each strain were not toxic. After 45 days, tubes of the VH and 8E strains showed visible gas production and toxin. Inoculated tubes incubated at 34 or 36°F showed no gas production during 104 days. Samples of all strains at both temperatures were toxin assayed at 54 days and 104 days, and toxin was absent in all cases. These results suggest a rather sharply defined critical temperature above and below which spores of Type E Cl. botulinum are or are not capable of germinating and initiating toxic outgrowth. The significance of these results in relation to extended refrigerated storage of food products is discussed.

Notes: SUMMARY– A comparison of starch gel patterns of sarcoplasmic proteins from aseptic and inoculated porcine and rabbit muscles after storage for 0, 8 and 20 days at 10°C indicated that different microorganisms preferentially utilized specific proteins. Pseudomonas fragi showed the greatest amount of proteolytic activity upon the sarcoplasmic fraction, causing extensive breakdown in both rabbit and porcine muscle. Leuconostoc mesenteroides caused extensive alteration in the sarcoplasmic proteins of rabbit muscle, but had less effect upon porcine muscle. Pediococcus cerevisiae exhibited similar action to L. mesenteroides upon rabbit muscle sarcoplasmic proteins, but had no effect upon pig muscle. Micrococcus luteus showed only minor breakdown of rabbit muscle sarcoplasmic proteins, and had no action upon porcine muscle. Both P. fragi and P. cerevisiae caused considerable breakdown of the urea-soluble proteins in pig muscle and to a lesser extent in rabbit muscle. Neither M. luteus nor L. mesenteroides exerted any measurable proteolytic effect upon the urea-soluble proteins. Possible implications concerning meat spoilage are discussed.

Notes: Spores of three strains of Clostridium botulinum Type E were inoculated into 202 × 202 cans of beef stew and irradiated to permit the survival of a small number (50–250) of spores per container. The cans containing survivors were incubated at 43 and 49°F to determine whether germination and outgrowth of surviving spores could take place. With all three strains at least some of the survivors were capable of developing to the point of producing swell spoilage and toxin at both temperatures. The significance. of these results in relation to extension of the refrigerated storage’ life of food by radiation “pasteurization” is discussed.