Thermal resistance of spores from virulent strains of Bacillus anthracis and potential surrogates

The objective of this study was to determine the thermal resistance of spores of Bacillus anthracis and potential surrogates. The heat resistance of spores suspended in buffer (pH 7.0 or 4.5), milk, or orange juice was determined at 70, 80, and 90 degrees C. D-values for B. anthracis strains Sterne, Vollum, and Pasteur ranged from < 1 min at 90 degrees C to approximately 200 min at 70 degrees C and were lower under acidic than under neutral conditions. The D-values for B. anthracis spores fell within the range obtained for spores from eight strains of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis. However, there were significant differences (P < 0.001) among the D-values of the strains. The z-values in pH 7.0 buffer and milk averaged approximately 10.5 degrees C and were not significantly different among strains (P < 0.05). The z-values in pH 4.5 buffer and orange juice averaged 12.9 and 13.9 degrees C, respectively, significantly (P < 0.05) higher than those obtained in milk or in pH 7.0 buffer. The significance of this difference was driven by large differences among a few strains. The z-values for B. anthracis strain Pasteur were twice as high in the acid media than in the neutral media. This study confirms that B. anthracis spores are not unusually heat resistant and that spores from validated Bacillus species are appropriate surrogates for thermal resistance studies.     

The influence of nisin on the thermal resistance of Bacillus cereus

Decimal reduction times (D-values) at cooking and autoclaving temperatures (80 to 120 degrees C) of spores of Bacillus cereus ATCC 1479-8 in rice and milk (13% wt/vol) supplemented with nisin (25 microg/ml) were evaluated. The mean D-values at 97.8 degrees C in cooked white rice, phosphate buffer (pH 7.0), and rice water (pH 6.7) were 3.62, 1,99, and 1.34 min, respectively. From 80 to 100 degrees C, the mean reduction in D-values due to the addition of nisin to milk was 40%. The D-value at 110 degrees C was approximately 0.86 min for milk (control) and milk with nisin. The z-values ranged from 7.32 degrees C (phosphate buffer) to 10.37 degrees C (milk control).     

Incidence of Clostridium perfringens in commercially produced cured raw meat product mixtures and behavior in cooked products during chilling and refrigerated storage

A total of 445 whole-muscle and ground or emulsified raw pork, beef, and chicken product mixtures acquired from industry sources were monitored over a 10-month period for vegetative and spore forms of Clostridium perfringens. Black colonies that formed on Shahidi-Ferguson perfringens (SFP) agar after 24 h at 37 degrees C were considered presumptive positive. Samples that were positive after a 15-min heat shock at 75 degrees C were considered presumptive positive for spores. Of 194 cured whole-muscle samples, 1.6% were positive; spores were not detected from those samples. Populations of vegetative cells did not exceed 1.70 log10 CFU/g and averaged 1.56 log10 CFU/g. Of 152 cured ground or emulsified samples, 48.7% were positive, and 5.3% were positive for spores. Populations of vegetative cells did not exceed 2.72 log10 CFU/g and averaged 1.98 log10 CFU/g; spores did not exceed 2.00 log10 CFU/g and averaged 1.56 log10 CFU/g. Raw bologna (70% chicken), chunked ham with emulsion, and whole-muscle ham product mixtures were inoculated with C. perfringens spores (ATCC 12916, ATCC 3624, FD1041, and two product isolates) to ca. 3.0 log10 CFU/g before being subjected either to thermal processes mimicking cooking and chilling regimes determined by in-plant temperature probing or to cooking and extended chilling regimes. Populations of C. perfringens were recovered on SFP from each product at the peak cook temperatures, at 54.4, 26.7, and 7.2 degrees C, and after up to 14 days of storage under vacuum at 4.4 degrees C. In each product, populations remained relatively unchanged during chilling from 54.4 to 7.2 degrees C and declined slightly during refrigerated storage. These findings indicate processed meat products cured with sodium nitrite are not at risk for the growth of C. perfringens during extended chilling and cold storage.     

EFFECTS OF THE HEATING MEDIUM pH ON HEAT RESISTANCE OF BACILLUS CEREUS SPORES

The influence of the pH of the heating medium (which included several foods and buffers) on the thermal resistance (D and z-values) of spores of three Bacillus cereus strains was studied. Acidification from pH 7.0 to 4.0 produced a 5-fold decrease in D-values. Plots of log D vs pH gave straight lines, which made it possible to develop an equation to approximately predict the changes in heat sensitivity of B. cereus spores which occurred with changing pH. z-Values for two of the strains studied were not affected by acidification. On the other hand, with the strain ATCC 9818, a clear and statistically significant increase in z-value was observed as the pH decreased.     

Validation of bacon processing conditions to verify control of Clostridium perfringens and Staphylococcus aureus

It is unclear how rapidly meat products, such as bacon, that have been heat treated but not fully cooked should be cooled to prevent the outgrowth of spore-forming bacterial pathogens and limit the growth of vegetative cells. Clostridium perfringens spores and vegetative cells and Staphylococcus aureus cells were inoculated into ground cured pork bellies with and without 1.25% liquid smoke. Bellies were subjected to the thermal profiles of industrial smoking to 48.9 degrees C (120 degrees F) and normal cooling of bacon (3 h) as well as a cooling phase of 15 h until the meat reached 7.2 degrees C (45 degrees F). A laboratory-scale bacon smoking and cooling operation was also performed. Under normal smoking and cooling thermal conditions, growth of C. perfringens in ground pork bellies was <1 log regardless of smoke. Increase of S. aureus was 2.38 log CFU/g but only 0.68 log CFU/g with smoke. When cooling spanned 15 h, both C. perfringens and S. aureus grew by a total of about 4 log. The addition of liquid smoke inhibited C. perfringens, but S. aureus still achieved a 3.97-log increase. Staphylococcal enterotoxins were detected in five of six samples cooled for 15 h without smoke but in none of the six samples of smoked bellies. In laboratory-scale smoking of whole belly pieces, initial C. perfringens populations of 2.23 +/- 0.25 log CFU/g were reduced during smoking to 0.99 +/- 0.50 log CFU/g and were 0.65 +/- 0.21 log CFU/g after 15 h of cooling. Populations of S. aureus were reduced from 2.00 +/- 0.74 to a final concentration of 0.74 +/- 0.53 log CFU/g after cooling. Contrary to findings in the ground pork belly system, the 15-h cooling of whole belly pieces did not permit growth of either pathogen. This study demonstrates that if smoked bacon is cooled from 48.9 to 7.2 degrees C (120 to 45 degrees F) within 15 h, a food safety hazard from either C. perfringens or S. aureus is not likely to occur.     

Thermal inactivation of Bacillus cereus spores affected by the solutes used to control water activity of the heating medium

The heat resistance of B. cereus spores (ATCC 7004, 4342 and 9818) over a wide temperature range (92-125 degrees C) in aqueous solutions of NaCl, LiCl, sucrose and glycerol at different water activities (1.00-0.71) was investigated. Sodium chloride in the heating medium tended to protect the spores of B. cereus against heat. The z-values increased significantly (P < 0.05) at and above a concentration of 4.0 M. The effects of LiCl were lower than those caused by the NaCl at the same a(w) values. An increase in z-values was observed. but the differences were only statistically significant (P<0.05) at the highest concentration tested (5.0 M). A concentration of sucrose 0.87 M caused in all cases a reduction in D-values, which was most pronounced for strains 4342 and 9818. With increasing concentration of sucrose ( > 0.87 M), the D-values showed an increase, although only those obtained for strain 4342 in sucrose solutions 2.22 M were higher than those found in pure water. The z-values were significantly higher (P < 0.05) when sucrose was added at concentrations above 1.42 M, except for strain 4342. When a(w) was lowered from 0.96 to 0.71 with glycerol, D-values obtained gradually increased, about 30, 50 and 60 fold for 4342, 7004 and 9818 strains, respectively. No significant effect on z-values were detected.     

Application of gaseous ozone to control populations of Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs

The effect of ozonation as a method to reduce Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs was investigated. Dried figs were sprinkle inoculated with E. coli, B. cereus and B. cereus spores in sterile bags at a level of 10(7)microorganism g(-1), mixed and allowed to dry for 1h at 25 degrees C prior to ozonation. Inoculated samples were exposed to gaseous ozone in a chamber at 20 degrees C and 70% relative humidity. Ozone concentrations of 0.1, 0.5 and 1.0 ppm up to 360 min were used to inactivate E. coli and B. cereus while 1.0, 5.0, 7.0 and 9.0 ppm ozone concentrations for 360 min were used to treat B. cereus spores. E. coli and B. cereus counts were decreased by 3.5 log numbers at 1.0 ppm ozone concentration for 360 min ozone treatment. Up to 2 log reductions in the number of B. cereus spores were observed above 1.0 ppm ozone concentration at the end of 360 min of ozonation. No significant changes in color, pH and moisture content values of dried figs were observed after the ozonation treatments. No significant changes were found between sweetness, rancidity, flavor, appearance and overall palatability of ozonated and non-ozonated dried figs. Ozonation was found to be effective especially in reduction of vegetative cells in dried figs and a promising method for the decontamination of dried figs.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.     

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.     

Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas

Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas at different concentrations (0.5, 1, 1.5, 3 and 5 mgl(-1)) for 10 min were studied. A cocktail of three strains of each targeted organism (100 microl) was spotted onto the surface of the strawberries (approximately 8-9 log ml(-1)) separately followed by air drying, and then treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity. Approximately a 4.3-4.7 logCFU reduction per strawberry of all examined bacteria was achieved by treatment with 5 mgl(-1) ClO(2) for 10 min. The inactivation kinetics of E. coli O157:H7, L. monocytogenes and S. enterica were determined using first-order kinetic models to establish D-values and z-values. The D-values of E. coli, L. monocytogenes and S. enterica were 2.6+/-0.2, 2.3+/-0.2 and 2.7+/-0.7 min, respectively, at 5 mgl(-1) ClO(2). The z-values of E. coli, L. monocytogenes and S. enterica were 16.8+/-3.5, 15.8+/-3.5 and 23.3+/-3.3 mgl(-1), respectively. Furthermore, treatment with ClO(2) gas significantly (p < or = 0.05) reduced the initial microflora (mesophilic, psychrotrophic bacteria, yeasts and molds) on strawberries. Treatment with ClO(2) gas did not affect the color of strawberries and extended the shelf-life to 16 days compared to 8 days for the untreated control.     

Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas

The purpose of this investigation was to study inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce leaves by ClO(2) gas at different concentrations (0.5, 1.0, 1.5, 3.0, and 5.0 mg l(-1)) for 10 min and to determine the effect of ClO(2) gas on the quality and shelf life of lettuce during storage at 4 degrees C for 7 days. One hundred microliters of each targeted organism was separately spot-inoculated onto the surface (5 cm(2)) of lettuce (approximately 8-9 log CFU ml(-1)), air-dried, and treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity for 10 min. Surviving bacterial populations on lettuce were determined using a membrane transferring method, which included a non-selective medium followed by a selective medium. The inactivation kinetics of E. coli O157:H7 and S. enterica was determined using first-order kinetics to establish D-values and z-values. The D-values of E. coli and S. enterica were 2.9+/-0.1 and 3.8+/-0.5 min, respectively, at 5.0 mg l(-1) ClO(2) gas. The z-values of E. coli and S. enterica were 16.2+/-2.4 and 21.4+/-0.5 mg l(-1), respectively. A 5 log CFU reduction (recommended by the United States Food and Drug Administration) for E. coli and S. enterica could be achieved with 5.0 mg l(-1) ClO(2) gas for 14.5 and 19.0 min, respectively. Treatment with ClO(2) gas significantly reduced inherent microflora on lettuce and microbial counts remained significantly (p<0.05) lower than the uninoculated control during storage at 4 degrees C for 7 days. However, treatment with ClO(2) gas had a significantly (p<0.05) negative impact on visual leaf quality. These results showed that treatment with ClO(2) gas significantly reduced selected pathogens and inherent microorganisms on lettuce; however, the processing conditions would likely need to be altered for consumer acceptance.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.     

Spore formation by Bacillus cereus in broth as affected by temperature, nutrient availability, and manganese

A study was done to determine the effect of interacting factors on sporulation of Bacillus cereus in broth. Vegetative cells (1.4 to 2.2 log CFU/ml) of B. cereus strain 038-2 (capable of growing at 12 degrees C) and strain F3812/84 (capable of growing at 8 degrees C) were inoculated into 30 ml of tryptic soy broth (TSB), TSB supplemented with manganese (50 microg/ml), diluted (10%) TSB (dTSB), and dTSB supplemented with manganese (50 microg/ml) and incubated at 8, 12, or 22 degrees C for up to 30, 30, or 10 days, respectively. Unheated and heated (80 degrees C for 10 min) cultures were plated on brain heart infusion agar to determine total cell counts (vegetative cells plus spores) and the number of spores produced, respectively. Both strains of B. cereus survived in TSB and dTSB for 30 days at 8 degrees C but did not sporulate. At 12 degrees C, cells grew in TSB to a population of 6.0 +/- 0.8 log CFU/ml, which was maintained for 30 days. Neither strain grew in dTSB at 12 degrees C and survived for at least 30 days. Spores were not produced in any of the test broths at 12 degrees C. At 22 degrees C, cells reached a stationary growth phase between 12 and 24 h in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese, and approximately 1% of the CFU were spores. In dTSB, cell growth and spore formation were retarded at 22 degrees C and a significantly lower number of spores was produced compared with the number of spores produced in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese. The addition of manganese to TSB did not affect cell growth or spore formation, but manganese did enhance sporulation in dTSB. This study provides useful information on spore formation by B. cereus as affected by conditions that may be imposed in liquid milieus on the surface of foods and on food contact surfaces in processing environments.     

Synergistic Effect of Electrolyzed Water and Citric Acid Against Bacillus Cereus Cells and Spores on Cereal Grains

ABSTRACT:  The effects of acidic electrolyzed water (AcEW), alkaline electrolyzed water (AlEW), 100 ppm sodium hypochlorite (NaClO), and 1% citric acid (CA) alone, and combinations of AcEW with 1% CA (AcEW + CA) and AlEW with 1% CA (AlEW + CA) against Bacillus cereus vegetative cells and spores was evaluated as a function of temperature (25, 30, 40, 50, or 60 °C) and dipping time (3 or 6 h). A 3-strain cocktail of Bacillus cereus cells or spores of approximately 10⁷ CFU/g was inoculated in various cereal grains (brown rice, Job's tear rice, glutinous rice, and barley rice). B . cereus vegetative cells and spores were more rapidly inactivated at 40 °C than at 25 °C. Regardless of the dipping time, all treatments reduced the numbers of B . cereus vegetative cells and spore by more than 1 log CFU/g, except the deionized water (DIW), which showed approximately 0.7 log reduction. The reductions of B . cereus cells increased with increasing dipping temperature (25 to 60 °C). B . cereus vegetative cells were much more sensitive to the combined treatments than spores. The effectiveness of the combined electrolyzed water (EW) and 1% CA was considerable in inhibiting B . cereus on cereal grains. The application of combined EW and CA for controlling B . cereus cells and spores on cereal grains has not been previously reported. Therefore, the synergistic effect of EW and CA may provide a valuable insight on reducing foodborne pathogens on fruits, vegetables, and cereal grains.     

The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens in a 'sous-vide' beef goulash under temperature abuse

The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens at three different concentrations (0, 1.5 and 3% w/w) and at different temperatures (10, 15 and 20 degrees C for B. cereus and 15, 20 and 25 degrees C for C. perfringens) was investigated, using beef goulash as a model system for pasteurised vacuum-packaged convenience foods. Calcium lactate at a level of 3% reduced the pH values of the samples from 6.0 to 5.5. No B. cereus growth was observed at 10 degrees C, but after 7 days at an incubation temperature of 15 degrees C, cell number increased by 1 log cfu/g in the control samples. At this temperature, lactates were seen to be effective at inhibiting growth. Calcium lactate was more inhibitory than sodium lactate as the growth of B. cereus was inhibited at 1.5 and 3% concentrations at 20 degrees C, respectively. Growth of C. perfringens was arrested in the presence of 1.5% calcium lactate at all storage temperatures, whereas growth was inhibited by 3% sodium lactate only at 15 degrees C.     

Increased inactivation of ozone-treated Clostridium perfringens vegetative cells and spores on fabricated beef surfaces using mild heat

Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75 degrees C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59 +/- 0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09 +/- 0.72 log CFU/g and 3.50 +/- 0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55 degrees C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94 +/- 0.37 log spores per g to 2.07 +/- 0.38 log spores per g and 1.70 +/- 0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75 degrees C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.     

Influence of nisin on the resistance of Bacillus anthracis sterne spores to heat and hydrostatic pressure

The influence of nisin on the heat and pressure resistance of Bacillus anthracis Sterne spores was examined. The decimal reduction times (D-value) of spores in milk (2% fat) at 80, 85, and 90 degrees C were determined. In the absence of nisin, the D-values were 30.09, 9.30, and 3.86 min, respectively. The D-values of spores heated in the presence of nisin (1 mg/ml) were not significantly different (P = 0.05). However, spores heated in the presence of nisin had a 1- to 2-log reduction in viability, after which the death kinetics became similar to those of spores in the absence of nisin. The z-values all were 11.2 degrees C regardless of the presence or absence of nisin. The pressure sensitivity of B. anthracis Sterne spores in the presence and absence of nisin also was determined. Spores treated with nisin were 10 times more pressure sensitive than were spores subjected to pressure in the absence of nisin under the conditions used in this study.     

Effect of dissolved carbon dioxide on thermal inactivation of microorganisms in milk

Postpasteurization addition of CO2 inhibits growth of certain microorganisms in dairy products, but few workers have investigated the effect of CO2 on the thermal inactivation of microorganisms during pasteurization. Concentrations of CO2 ranging from 44 to 58 mM added to raw whole milk significantly (P < 0.05) reduced the number of surviving standard plate count (SPC) organisms in milk heated over the range of 67 to 93 degrees C. A decrease in thermal survival rates (D-values) for Pseudomonas fluorescens R1-232 and Bacillus cereus ATCC 14579 spores in milk was positively correlated with CO2 concentrations (1 to 36 mM). D(50 degrees C)-values for P. fluorescens significantly decreased (P < 0.05) in a linear fashion from 14.4 to 7.2 min. D(89 degrees C)-values for B. cereus spores were significantly (P < 0.05) decreased from 5.56 min in control milk to 5.29 min in milk containing 33 mM CO2. The Weibull function was used as a model to describe the thermal inactivation of P. fluorescens, B. cereus spores, and SPC organisms in raw milk. Nonlinear parameters for the Weibull function were estimated, and survival data fitted to this model had higher R2 values than when fitted to the linear model, further providing support that the thermal inactivation of bacteria does not always follow first-order reaction rate kinetics. These results suggest that CO2 could be used as a processing aid to enhance microbial inactivation during pasteurization.     

Effects of chilling rate on outgrowth of Clostridium perfringens spores in vacuum-packaged cooked beef and pork

Cooked, chilled beef and cooked, chilled pork were inoculated with three strains of Clostridium perfringens (NCTC 8238 [Hobbs serotype 2], NCTC 8239 [Hobbs serotype 3], and NCTC 10240). Inoculated products were heated to 75 degrees C, held for 10 min in a circulating water bath to heat activate the spores, and then chilled by circulating chilled brine through the water bath. Samples were chilled from 54.4 to 26.6 degrees C in 2 h and from 26.6 to 4.4 degrees C in 5 h. Differences in initial C. perfringens log counts and log counts after chilling were determined and compared with the U.S. Department of Agriculture (USDA) stabilization guidelines requiring that the chilling process allow no more than 1 log total growth of C. perfringens in the finished product. This chilling method resulted in average C. perfringens increases of 0.52 and 0.68 log units in cooked beef and cooked pork, respectively. These log increases were well within the maximum 1-log increase permitted by the USDA, thus meeting the USDA compliance guidelines for the cooling of heat-treated meat and poultry products.