Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila.

The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48 degrees-65 degrees C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7 degrees C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30 degrees C) were twice as resistant as those in the early stage (5 h at 30 degrees C), and the maximum D-value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z-values significantly. The heat resistance of cells incubated for 48 h at 30 degrees C increased (twice) after holding at 7 degrees C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30 degrees C) did not influence their D-values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 to 4.0 reduced D-values by a factor of 5. Although the strain studied was heat-sensitive (D55 degrees C = 0.17 min; z = 5.11 degrees C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Thermal inactivation of Enterococcus faecium: effect of growth temperature and physiological state of microbial cells

AIMS: To provide data on the effects on culture temperature and physiological state of cells on heat resistance of Enterococcus faecium, which may be useful in establishing pasteurization procedures. METHODS AND RESULTS: The heat resistance of this Ent. faecium (ATCC 49624 strain) grown at different temperatures was monitored at various stages of growth. In all cases, the bacterial cells in the logarithmic phase of growth were more heat sensitive. For cells which had entered in the stationary phase, D70 values of 0.53 min at 5 degrees C, 0.74 min at 10 degrees C, 0.83 min at 20 degrees C, 0.79 min at 30 degrees C, 0.63 min at 37 degrees C, 0.48 min at 40 degrees C and 0.41 min at 45 degrees C were found. By extending the incubation times cells were more heat resistant as stationary phase progressed, although a different pattern was observed for cells grown at different temperatures. At the lower temperatures heat resistance increased progressively, reaching D70 values of 1.73 min for cells incubated at 5 degrees C for 50 days and 1.04 min for those grown at 10 degrees C for 16 days. At other temperatures assayed heat resistance became stable for late stationary phase cells, reaching D70 values of 1.05, 1.08 and 1.01 min for cultures incubated at 20, 30 and 37 degrees C. Heat resistance of cells obtained at higher temperatures, 40 and 45 degrees C, was significantly lower, with D70 values of 0.76 and 0.67 min, respectively. Neither the growth temperature nor the growth phase modified the z-values significantly. CONCLUSIONS: D70 values obtained for Ent. faecium (ATCC 49624) varies from 0.33 to 1.73 min as a function of culture temperature and physiological state of cells. However, z values calculated were not significantly influenced by these factors. A mean value of 4.50 +/- 0.39 degrees C was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall results strongly suggest that, to establish heat processing conditions of pasteurized foods ensuring elimination of Ent. faecium, it is advisable to take into account the complex interaction of growth temperature and growth phase of cells acting on bacterial thermal resistance.     

Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives

M. LÓPEZ, M. MAZAS, I. GONZÁLEZ, J. GONZALEZ AND A. BERNARDO. 1996. The effects of different heating systems on the heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were investigated. Spores were heated in distilled water, Sorensen buffer (0.18 moll⁻¹), McIlvaine buffer (0.0025-0.18 moll⁻¹), and several solutions containing sodium chloride (0.0612%), sodium nitrite (125 ppm), potassium sorbate (0.1%) and sodium benzoate (0.1%) over a wide range of temperatures (115-140°C). D-values obtained for McIlvaine and Sorensen buffers, at the same molarities, were not significantly different (P > 0.05), but decimal reduction times increased as phosphate concentrations in the solutions decreased. The concentrations, in which statistically significant differences (P < 0.05) were obtained, varied among strains. Among the additives assayed, only sodium chloride reduced heat resistance, being effective at concentrations as low as 0.06%. The z-values calculated in this study ranged from 6.99 to 8.40 with a mean value of 7.60±0.45. Although z-values observed for salt and buffers (180 moll⁻¹) were slightly higher than those obtained in the other conditions assayed, the difference was not statistically significant (P > 0.05).     

Thermal destruction of Clostridium botulinum spores suspended in tomato juice in aluminum thermal death time tubes.

The heat destruction characteristics of Clostridium botulinum spores suspended in tomato juice and phosphate buffer were determined by the survivor curve method with aluminum thermal death time tubes. Two type A strains of C. botulinum and a type B strain were evaluated. Strains A16037 and B15580 were implicated in outbreaks of botulism involving home-canned tomato products. Strain A16037 had a higher heat resistance than either 62A or B15580. The mean thermal resistance (D-values) for A16037 in tomato juice (pH 4.2) were: 115.6 degrees C, 0.4 min; 110.0 degrees C, 1.6 min; and 104.4 degrees C, 6.0 min. The mean D-values for A16037 in Sorensen 0.067 M phosphate buffer (pH 7) were: 115.6 degrees C, 1.3 min; 110.0 degrees C, 4.4 min; and 104.4 degrees C, 17.6 min. At each test temperature, the D-values were approximately three times higher in buffer than in tomato juice. The z-value for C. botulinum A16037 spores in tomato juice was 9.4 degrees C, and in buffer the z-value was 9.9 degrees C. The use of aluminum thermal death time tubes in a miniature retort system makes it possible to determine survivor curves for C. botulinum spores at 121.1 degrees C. This is possible because the lag correction factor for the aluminum tubes is only about 0.2 min, making possible heating times as short as 0.5 min.     

A Model System for Testing the Microbiological Stability of Foods Processed in Laminated Flexible Pouches

A suitable time temperature process for packaging small potatoes in flexible pouches is described. Spores of Bacillus stearothermophilus were inoculated onto the surface of peeled potatoes in flexible pouches. These were evacuated, heat sealed and heated in a steam retort modified to allow a water cooking process with an air overpressure of ca. 68·95 kPa (10 lbf/in²). The D and z values determined in the retort were confirmed by parallel heat-resistance tests with spores in glass ampoules held in an oil bath. Heat treatment at 121·1 °C for 20 min in the retort satisfactorily killed test inocula of spores without overcooking the potatoes. Uninoculated peeled potatoes with a natural level of contamination ( ca. 4 x 10³ spores/sealed pouch) were treated in the retort for different times at 121·1 °C and then incubated at 30 °C for 6 months. No pouches heated for 17–25 min showed microbial growth. This procedure may be applied to any type of food if the numbers of naturally occurring heat-resistant contaminants can be related to the number and heat resistance of a suitable test micro-organism.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Thermal Activation and Dry-heat Inactivation of Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger

Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger were heat activated for different times at 60° and 80°C. Strain MD2 required considerable heat activation while B. subtilis var. niger did not. Maximum germination rates increased with heat activation dose and declined subsequently without loss of germinability. Germination rates and percentages were considerably greater in tryptone glucose extract (TGE) than in nutrient broth. The addition of 2°° dimethyl sulphoxide did not increase germination in nutrient broth. The spores of var. niger are more resistant to dry-heat than MD2 although they are less resistant to moist heat. Survivor curves in the dry-heat range 140°-170°C gave D-values from 4–123 to 0.106 min for MD2 and 5.679 to 0.233 min for var. niger recovered on TGE agar. D-values were lower on poorer media. The z-values for MD2 and var. niger on TGE were 18.7°C and 21.25C respectively.     

Repair and calcium dipicolinate release of bioindicators in propylene glycol and propylene glycol—water mixtures

B. PHILIPP AND H. SUCKER. 1992. The heat sterilization of spores of Bacillus stearothermophilus ATCC 7953 in propylene glycol (PG) and PG—water mixtures was investigated. Unusual non-logarithmic survival curves with a marked long shoulder, designated as the lag-time, were observed at the maximum of resistance. When the number of colony-forming units were determined after intervals of incubation, the growth curves of spores previously treated at 121°C in PG or PG-4% water varied considerably. Spores receiving a heat treatment that was longer than the lag-time showed no growth. When the heat treatment was shorter than the lag-time, spores showed a delayed growth phase. When spores received heat treatment that was long but which fell within the lag-time, there was a greater decrease in spore counts before the exponential growth phase began. In PG and PG with low water concentrations, a suppressed release of the specific spore substance calcium dipicolinate was observed during sterilization at 121°C of B. stearothermophilus ATCC 7953 and B. subtilis var. niger ATCC 9372. Calcium dipicolinate release, however, was observed in water.     

Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula

Enterobacter sakazakii may be related to outbreaks of meningitis, septicemia, and necrotizing enterocolitis, mainly in neonates. To reduce the risk of E. sakazakii in baby foods, thermal characteristics for Korean E. sakazakii isolates were determined at 52, 56, and 60 degrees C in saline solution, rehydrated powdered infant formula, and dried baby food. In saline solution, their D-values were 12-16, 3-5, and 0.9-1 min for each temperature. D-values increased to 16-20, 4-5, and 2-4 min in rehydrated infant formula and 14-17, 5-6, and 2-3 min in dried baby food. The overall calculated z-value was 6-8 for saline, 8-10 for powdered infant formula, and 9-11 for dried baby food. Thermal inactivation of E. sakazakii during rehydration of powdered infant formula was investigated by viable counts. Inactivation of cultured E. sakazakii in infant formula milk did not occur for 20 min at room temperature after rehydration with the water at 50 degrees C and their counts were reduced by about 1-2 log CFU/g at 60 degrees C and 4-6 log CFU/ml with the water at 65 and 70 degrees C. However, the thermostability of adapted E. sakazakii to the powdered infant formula increased more than two times. Considering that the levels of E. sakazakii observed in powdered infant formula have generally been 1 CFU/100 g of dry formula or less, contamination with E. sakazakii can be reduced or eliminated by rehydrating water with at least 10 degrees C higher temperature than the manufacturer-recommended 50 degrees C.     

Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure

Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44 degrees C, those sporulated at 30 degrees C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55 degrees C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca(2+) or Mg(2+), whereas the resistance did not change when spores were remineralized with Mn(2+) or K(+), suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.     

Heat and ozone resistance of Bacillus spores isolated from laboratory animals

Heat resistance of free-spores of 78 Bacillus strains isolated from laboratory animals was examined. Spores of 41 out of 78 strains survived for 320 minutes at 70 degrees C, 27 for 160 min, at 100 degrees C, only one for 20 min. at 110 degrees C by autoclaving, and none for 5 min. at 120 degrees C. D-values at 100 degrees C of 9 strains determined were between 5.03 and 30.06 min. Spores of 9 strains from stock cultures were exposed to ozone gas at various conditions. Ozone resistance of spores was closely dependent upon relative humidity. D-values of the spores tested by treatment with 200 ppm ozone at 60% RH were over 200 min., especially over 1,000 min. in 4 strains, indicating that exposure to ozone at a moderate humidity for 6 hours could not sterilize Bacillus spores. At 90% RH, however, treatment with 200 ppm ozone for 6 hr. might be effective for a routine sterilization in laboratory animal facilities.     

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.     

Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.