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).     

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.     

Inactivation kinetics of avirulent Bacillus anthracis spores in milk with a combination of heat and hydrogen peroxide

The combined effect of heat and hydrogen peroxide (HP) on the inactivation of avirulent Bacillus anthracis spores (Sterne strain 7702; strain ANR-1, an avirulent Ames derivative lacking the pXO2 plasmid; and strain 9131, a plasmid-less Sterne strain) was evaluated in milk. The study temperature ranged from 90 to 95 degrees C, and the concentration of added HP varied from 0.05 to 0.5%. Decimal reduction times (D-values) were determined using a sealed capillary tube technique. The mean D- and z-values of hydrated freeze-dried spores of all three strains in milk ranged from 550 s at 90 degrees C to 180 s at 94 degrees C and from 8.6 to 9.0 degrees C, respectively. When 0.05% HP was added to the milk, the D-values were decreased at least threefold, and at 0.5% HP the D-values ranged from 1 to 10 s. At 90 degrees C, all three strains had similar D-values when 0.05% HP was added. Increasing the concentration of HP to 0.5% had a greater reducing effect on the D-value for strain 7702 than on the values for strains ANR-1 and 9131. The rate of inactivation of each strain followed first-order reaction kinetics at each temperature-peroxide combination. Equations in the form of D = Constant x (HP concentration)n had R2 values greater than 0.97 for strains ANR-1 and 7702 and of at least 0.7 for strain 9131. This study suggests that a combination of high temperature (from 90 to 95 degrees C) and HP could be used for inactivation of B. anthracis spores in the event of deliberate contamination of milk such that the contaminated milk could be disposed of safely.     

Alicyclobacillus in orange juice: occurrence and heat resistance of spores.

Spore suspensions of a pure culture of Alicyclobacillus acidoterrestris DSM 2498 were submitted to different heat treatments (60 degrees C for 60 min, 60 degrees C for 30 min, 70 degrees C for 20 min, 80 degrees C for 5 min, 80 degrees C for 10 min, 80 degrees C for 30 min, and boiling for 5 min) to determine the best activation conditions in orange juice. The best treatment for spore activation was shown to be 70 degrees C/20 min. Seventy-five samples of concentrated orange juice from 11 different suppliers were examined for the presence of thermophilic acid-tolerant spore formers by the most probable number technique using Bacillus acidocaldarius medium (BAM broth) and incubation at 44 degrees C for 5 days after a prior spore activation. After incubation, isolation was carried out using BAM agar medium incubating at 44 degrees C for 5 days. Typical colonies were submitted to a microscopic examination, evaluation for the presence of spores, and various biochemical tests. Of the orange juice samples examined, 14.7% were found to be positive for Alicyclobacillus. The thermal death time open tube method was used to determine the heat resistance of the spores of strains confirmed as being Alicyclobacillus. The D-values determined were in the range from 60.8 to 94.5 min at 85 degrees C, 10.0 to 20.6 min at 90 degrees C, and 2.5 to 8.7 min at 95 degrees C. The z-values were between 7.2 degrees C and 11.3 degrees C. The results demonstrated the occurrence of Alicyclobacillus in orange juice and the high heat resistance of the spores that could survive the heat treatments normally applied in the processing of orange juice.     

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.     

Influence of recovery conditions on apparent heat resistance of Bacillus stearothermophilus spores

The effects of post-treatment environmental factors on the heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Nutrient Agar (NA), Antibiotic Assay Medium (AAM), Dextrose Tryptone Agar (DTA) and Tryptic Soy Agar (TSA) with Ca^{2+ added to a final concentration of 100 p.p.m. were used as recovery media. No significant differences were seen between D-values obtained except in the case of strain 12980 when comparing TSA with the other media and for strain 7953 comparing AAM and DTA. The optimum incubation temperature was slightly lower for heated than for unheated spores of} each strain, although, in general, 50 °C was adequate. Higher D-values were obtained at 50–55 °C. The effects of the pH of the medium in the range 5.0–7.0 and the addition of starch and phosphate on heat resistance have also been investigated. Maximum colony counts of heated spores were obtained at pH 7.0 and decreased as pH fell. D-values were significantly lower at pH ≤ 5.5. Increasing the concentration of phosphate in the recovery medium from 0 to 0.2% resulted in a progressive decrease in spore recovery and D-values. The addition of starch improved recoverability. The z-values obtained for the four strains studied under the different recovery conditions were similar with a mean value of 7.58 °C ± 0.28.     

Thermal treatment of aseptically processed tomato paste

The heat resistance characteristics of Bacillus coagulans (NRRL B-1103) spores suspended in buffer (pH 7.0, 4.5), tomato serum and tomato paste were studied. It was found that the heat resistance of spores was reduced significantly when buffer of pH 4.5, tomato serum or tomato paste was used as suspension medium instead of buffer pH 7.0. This effect was more apparent at higher temperatures. It was concluded that a thermal process of F_105°C = 3 min is capable of causing at least 3D destruction of spores of the most heat resistant strains of B. coagulans     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Heat resistance of Alicyclobacillus acidocaldarius in water, various buffers, and orange juice

The effect of the pH or the composition of the heating medium and of the sporulation temperature on the heat resistance of spores of a thermoacidophilic spore-forming microorganism isolated from a dairy beverage containing orange fruit concentrate was investigated. The species was identified as Alicyclobacillus acidocaldarius. The spores showed the same heat resistance in citrate-phosphate buffers of pH 4 and 7, in distilled water, and in orange juice at any of the temperatures tested (D120 degrees C = 0.1 min and z = 7 degrees C). A raise in 20 degrees C in the sporulation temperature (from 45 to 65 degrees C) increased the heat resistance eightfold (from D110 degrees C = 0.48 min when sporulated at 45 degrees C to 3.9 min when sporulated at 65 degrees C). The z-values remained constant for all sporulation temperatures. The spores of this strain of A. acidocaldarius were very heat resistant and could easily survive any heat treatment currently applied to pasteurize fruit juices.     

Growth characteristics of virulent Bacillus anthracis and potential surrogate strains

The objectives of this study were to compare generation and lag times of virulent Bacillus anthracis strains with those of other Bacillus strains, to identify possible surrogates for growth studies, and to determine if the B. cereus module of the U.S. Department of Agriculture Pathogen Modeling Program (PMP) had predictive value for B. anthracis. Growth characteristics of B. anthracis, B. cereus, B. mycoides, and B. subtilis strains in brain heart infusion broth at pH 6.5, 6.0, and 5.5 were determined by absorbance measurements. Growth curves of B. anthracis Sterne and B. cereus strains appeared similar, and the generation times for strain Sterne fell within the PMP's 95% confidence interval for B. cereus. However, the virulent B. anthracis strains Vollum and Pasteur had shorter generation times than the avirulent Sterne strain and most other surrogates and were lower than the PMP's 95% confidence interval for B. cereus. Growth curves of B. cereus ATCC 9818 and B. subtilis ATCC 6633 were more similar to those of virulent B. anthracis strains, but all potential surrogates had significantly different generation times and lag times under some conditions.     

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.     

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.     

Heat resistance of juice spoilage microorganisms

The heat resistance of various yeasts (Saccharomyces cerevisiae, Rhodotorula mucilaginosa, Torulaspora delbrueckii, and Zygosaccharomyces rouxii), molds (Penicillium citrinum, Penicillium roquefortii, and Aspergillus niger), and lactic acid bacteria (Lactobacillus fermentum and Lactobacillus plantarum) obtained from spoiled acid or acidified food products was determined in 0.1 M citrate buffer at pH values of 3.0, 3.5, and 4.0. S. cerevisiae was the most heat resistant of the microorganisms in citrate buffer, and its heat resistance was further evaluated in apple, grapefruit, calcium-fortified apple, and tomato juices as well as in a juice base with high fructose corn syrup. Decimal reduction times (D-values) and changes in temperature required to change the D-value (z-values) for S. cerevisiae were higher in the juices than in citrate buffer at all pH values tested. The D57 degrees C(135 degrees F)-values varied from 9.4 min in the juice product with pH 2.8 to 32 min in a calcium-added apple juice with pH 3.9. The S. cerevisiae strain used in this study can be used in thermal-death-time experiments in acidic products to calculate process conditions and in challenge tests to validate the calculated temperatures and hold times during processing.     

Effect of pH, NaCl content, and temperature on growth and survival of Arcobacter spp

Growth and survival of six human isolates of the pathogenic Arcobacter spp. in the presence of selected environmental factors were studied. Four strains of Arcobacter butzleri and two strains of Arcobacter cryaerophilus were exposed to pH levels of 3.5 to 8.0. Most strains grew between pH 5.5 and 8.0, with optimal growth of most A. butzleri and A. cryaerophilus strains at pH 6.0 to 7.0 and 7.0 to 7.5, respectively. The 24-h optimal growth range in the presence of NaCl was 0.5 to 1.0% for A. cryaerophilus. However, after 96 h, the optimum was between 0.5 and 2.0% NaCl. The optimum range for growth of A. butzleri strains was 0.09 to 0.5% NaCl after 96 h. The upper growth limits were 3.5 and 3.0% NaCl for A. butzleri and A. cryaerophilus, respectively. Survival at 25 degrees C in up to 5% NaCl was noted for A. butzleri 3556 and 3539 and A. cryaerophilus 3256. Decimal reduction times (D-values) at pH 7.3 in phosphate-buffered saline for three A. butzleri strains were 0.07 to 0.12 min at 60 degrees C, 0.38 to 0.76 min at 55 degrees C, and 5.12 to 5.81 min at 50 degrees C. At pH 5.5, decreased thermotolerance was observed, with D-values of 0.03 to 0.11 min at 60 degrees C, 0.30 to 0.42 min at 55 degrees C, and 1.97 to 4.42 min at 50 degrees C. Calculated z-values ranged from 5.20 to 6.28 degrees C. D-values of a three-strain mixture of A. butzleri in raw ground pork were 18.51 min at 50 degrees C and 2.18 min at 55 degrees C. Mild heat (50 degress C) followed by cold shock (4 or 8 degrees C exposure) had a synergistic lethal effect, reducing more cells than with an individual 50 degrees C treatment or with cold shock temperatures of 12 or 16 degrees C.     

Decontamination of fluid milk containing Bacillus spores using commercial household products

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.     

Immobilized bacterial spores for use as bioindicators in the validation of thermal sterilization processes

Spores of Bacillus subtilis ATCC 6051 and Bacillus stearothermophilus NCTC 10003 were immobilized in monodisperse alginate beads (diameter, 550 microm +/- 5%), and the capacity of the immobilized bioindicators to provide accurate and reliable F-values for sterilization processes was studied. The resistance of the beads to abrasion and heat was strong enough to ensure total retention of the bioindicators in the beads in a sterilization cycle. D- and z-values for free spores were identical to those for immobilized spores, which shows that immobilization does not modify the thermal resistance of the bioindicators. A D(100 degrees C) value of 1.5 min was found for free and immobilized B. subtilis spores heated in demineralized water, skimmed milk, and milk containing 4% fat, suggesting that a lipid concentration as low as 4% does not alter the thermal resistance of B. subtilis spores. Providing that the pH range is kept between 3.4 to 10 and that sufficiently low concentrations of Ca2+ competitors or complexants are present in the medium, immobilized bioindicators may serve as an efficient, accurate, and reliable tool with which to validate the efficiency of any sterilization process. The environmental factors (pH, media composition) affecting the thermoresistance of native contaminants are intrinsically reflected in the F-value, allowing for a sharper adjustment of the sterilization process. Immobilized spores of B. stearothermophilus were successfully used to validate a resonance and interference microwave system that is believed to offer a convenient alternative for the sterilization of temperature-sensitive products and medical wastes.     

High Pressure Inactivation Kinetics of Saccharomyces cerevisiae Ascospores in Orange and Apple Juices

High pressure inactivation kinetics (D and z values) of Saccharomyces cerevisiae ascospores were determined in fruit juices and a model juice buffer at pH 3.5 to 5.0. Approximately 0.5 to 1.0 × 10⁶ ascospores/mL were pressurized at 300 to 500 MPa in juice or buffer. D-values ranged from 8 sec to 10.8 min at 500 and 300 MPa, respectively. The range for z-values was 115 to 121 MPa. No differences (P≥0.05) in D (at constant pressure) or z-values among buffers or juices at any pH were determined, indicating little influence of pH in this range and absence of protective or detrimental effects of juice constituents.     

THERMAL RESISTANCE OF BACILLUS CEREUS SPORES AS AFFECTED BY ADDITIVES IN THE RECOVERY MEDIUM

The effects of the addition of starch, glucose, sodium chloride, sodium citrate, monopotassium phosphate and disodium phosphate to the recovery medium on apparent heat resistance of Bacillus cereus spores (ATCC 4342, 7004 and 9818) were investigated. Sodium citrate, monopotassium and disodium phosphate at concentrations of 0.1% were effective inhibitory agents for heat injured B. cereus spores especially for strain 9818, although only monopotassium and disodium phosphate caused a significant reduction (p < 0.05) in D-values obtained for strain 9818. Sodium chloride also had a marked effect on the recovery of heat injured spores. Concentration as low as 0.5% caused a significant reduction in the recovery rates for strains 9818 and 7004. In all cases, increasing the salt levels from 0.5 to 4% resulted in a progressive decrease in spore recovery. D-values gradually decreased as the salt content increased, although the concentrations which produced statistically significant differences (p < 0.05) varied among strains. The addition of starch at 0.1% resulted in a significant increase in the counts for strains 9818 and 7004. In contrast, glucose (0.1%), did not significantly modify the counts obtained Neither of these compounds affected decimal reduction times. No statistical significance (p>0.05) differences were detected among z-values for the spores of the three strains recovered in the presence of different additives assayed. z-Values ranged from 6.67 to 8.32, with a mean value of 7.56 ± 0.46C .     

Sucrose, sodium dodecyl sulfate, urea, and 2-mercaptoethanol affect the thermal inactivation of R-phycoerythrin.

Thermal inactivation kinetics (D- and z-values) of the algal protein, R-phycoerythrin (R-PE), were studied under different buffer conditions (pH 4.0, 7.0, and 10.0) and concentrations of sucrose, sodium dodecyl sulfate (SDS), urea, and 2-mercaptoethanol (ME). R-PE solutions were heated in capillary tubes at temperatures between 40 and 90 degrees C depending on buffer conditions. Thermal inactivation parameters for R-PE, calculated on the basis of fluorescence loss, were modified by addition of chemicals. Overall, sucrose and ME had a thermostabilizing effect, while SDS and urea decreased thermal stability of R-PE. The z-values ranged from 5.9 degrees C in 50 mM NaCl, 20 mM glycine buffer, pH 10.0, to 37.8 degrees C in 60% sucrose, 50 mM NaCl, 20 mM phosphate buffer, pH 7.0. The z-values obtained for R-PE closely matched the z-values of some target microorganisms in food processes, suggesting R-PE might be used as a time-temperature integrator to verify thermal processing adequacy.     

Heat Resistance of Eurotium herbariorum, a Xerophilic Mold

Eurotium haerbariorum, isolated from a spoilage outbreak involving grape preserves, was a true osmophile in that optimal growth was obtained in media containing 40-60% sucrose. Survival curves showed logarithmic death of heated spores followed by a tailing. In 5° Brix grape juice D-values at 70°C and z-values were 2.5 min and 9.1°C compared to 5.2 min and 7.1°C in 65°C Brix juice. Low concentrations of sorbic acid and fumaric acid in the heating menstruum had little effect on heat resistance.