Validation and analysis of modeled predictions of growth of Bacillus cereus spores in boiled rice.

The growth of psychrotrophic Bacillus cereus 404 from spores in boiled rice was examined experimentally at 15, 20, and 30 degrees C. Using the Gompertz function, observed growth was modeled, and these kinetic values were compared with kinetic values for the growth of mesophilic vegetative cells as predicted by the U.S. Department of Agriculture's Pathogen Modeling Program, version 5.1. An analysis of variance indicated no statistically significant difference between observed and predicted values. A graphical comparison of kinetic values demonstrated that modeled predictions were "fail safe" for generation time and exponential growth rate at all temperatures. The model also was fail safe for lag-phase duration at 20 and 30 degrees C but not at 15 degrees C. Bias factors of 0.55, 0.82, and 1.82 for generation time, lag-phase duration, and exponential growth rate, respectively, indicated that the model generally was fail safe and hence provided a margin of safety in its growth predictions. Accuracy factors of 1.82, 1.60, and 1.82 for generation time, lag-phase duration, and exponential growth rate, respectively, quantitatively demonstrated the degree of difference between predicted and observed values. Although the Pathogen Modeling Program produced reasonably accurate predictions of the growth of psychrotrophic B. cereus from spores in boiled rice, the margin of safety provided by the model may be more conservative than desired for some applications. It is recommended that if microbial growth modeling is to be applied to any food safety or processing situation, it is best to validate the model before use. Once experimental data are gathered, graphical and quantitative methods of analysis can be useful tools for evaluating specific trends in model prediction and identifying important deviations between predicted and observed data.

Thermal resistance of bacterial spores in milk-based beverages supplemented with nisin.

The effect of nisin, added in the form of Nisaplin, on the thermal resistance of bacterial spores and the effects of medium composition, exposure time, and pH on nisin enhancement of heat sensitivity were evaluated. Nisin apparently required specific nutrients to sensitize spores to heat. For example, D130 degrees C values of approximately 10 s were observed in sodium phosphate buffer with and without 6% sucrose with no significant (P> or =0.05) differences detected as a result of increased nisin concentration. In a nutrient-rich chocolate milk model system (CMMS), increasing either the time of exposure to nisin (5, 15, or 24 h) before heating or nisin concentration (0, 2,000, or 4,000 IU/ml) increased the sensitivity of Bacillus stearothermophilus spores to heat. In the CMMS with 10 to 12% fat cocoa powder, increasing nisin concentration (at 5 h of exposure) significantly (P< or =0.05) reduced D130 degrees C values; D130 degrees C values were 21.7, 17.2, and 17.8 s, respectively, for the 0-, 2,000-, and 4,000-IU/ ml nisin treatments. Fifteen and 24 h of exposure further reduced D130 degrees C values in the nisin-containing treatments compared to the control (0 IU of nisin per ml). A lower-fat CMMS (0 to 1% fat cocoa powder) had lower D130 degrees C values (19.3, 15.8, and 14.7 s for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively). Nisin activity was enhanced by lowering pH in the CMMS (10 to 12% fat cocoa powder), with reductions in D130 degrees C values across all pH values (ranging from 18.0% at pH 6.4 to 41.9% at pH 5.0). zD values were 9.6, 9.0, and 8.4 degrees C for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively. Spores of B. licheniformis yielded results similar to those obtained with B. stearothermophilus. For example, decreasing CMMS (10 to 12% fat cocoa powder) pH values from 6.4 to 5.0 produced D100 degrees C values of 3.3, 2.8, and 2.8 min (pH 6.4) and 1.0, 0.8, and 0.8 min (pH 5.0) for the 0-, 2,000-, and 4,000-IU/ml nisin treatments. This study clearly verified that the addition of Nisaplin to dairy-based beverages, such as a chocolate milk drink, or other foods intended to be heated reduces the thermal resistance of selected bacterial spores. Increased spore sensitivity to heat may provide food processors with an opportunity to reduce their thermal processes and expenses while maintaining product quality, functionality, and shelf stability.

Nisin in milk sensitizes Bacillus spores to heat and prevents recovery of survivors.

Decimal reduction times (D values) were determined for Bacillus cereus T spores and B. stearothermophilus ATCC 12980 spores in skim milk supplemented with various concentrations (0, 2,000, and 4,000 IU/ml) of the bacteriocin nisin by using an immersed, sealed capillary tube procedure. For both organisms, the addition of nisin lowered the apparent D values. For B. cereus, the addition of 2,000 IU of nisin per ml to skim milk before heating significantly (P< or =0.05) lowered the apparent D value compared to the control treatment. The D values at 97 degrees C were 7.0, 4.8, and 4.7 min for the control and 2,000- and 4,000-IU/ml nisin treatments, respectively. At 103 degrees C, the D values were 1.5, 0.85, and 0.88 min for the control and 2,000-and 4,000-IU/ml nisin treatments. When calculated across both nisin treatments, the mean reductions in apparent D values at 97, 100, and 103 degrees C due to addition of nisin in comparison to the controls were 32, 20, and 42%, respectively. The zD values for B. cereus ranged from 8.0 to 8.9 degrees C. With B. stearothermophilus, the apparent D values at 130 degrees C were reduced by 13 and 21% respectively, because of the presence of 2,000 or 4,000 IU of nisin per ml. The D values were 16.0, 13.8, and 12.5 s for the control and 2,000- and 4,000-IU/ml nisin treatments, respectively. There was a significant (P< or =0.05) decrease in the apparent D value between the control and 4,000-IU/ml treatment. Overall, log populations of survivors for B. stearothermophilus compared to the control were lower at any given sampling time due to the presence of nisin. The results of these studies suggest that spore control is likely due to enhanced sensitivity of spores to heat and the presence of residual nisin in the recovery medium that could prevent outgrowth of survivors.

Heat resistance of Alicyclobacillus acidoterrestris spores as affected by various pH values and organic acids.

Alicyclobacillus acidoterrestris, a thermoacidophilic sporeformer, has caused spoilage of fruit juices which had been treated with thermal processes intended to commercially sterilize the juice. The objective of this research was to document the effect of pH, acid, and temperature on the heat resistance of spores of three fruit-juice isolates of A. acidoterrestris. The thermal resistance of spores of A. acidoterrestris strains VF, WAC, and IP were studied in a model fruit-juice system composed of 12% glucose and 30 mM of either citric, malic, or tartaric acid, adjusted to selected pH values ranging from 2.8 to 4.0. Decimal reduction times (D values) and inactivation rates were determined. Spores of strains VF and WAC were similarly resistant to heat under acidic conditions, while strain IP spores were less resistant. In the range of pH 2.8 to 4.0, a statistically effect of hydrogen ion concentration on heat resistance was observed at lower temperatures, but not at the higher temperatures, but not at the higher temperatures. For examples, at 91 degrees C and pH 3.1 and 3.7, D values were 31.3 and 54.3 min, respectively, while at 97 degrees C D values at pH 3.1 and 3.7 were 7.9 and 8.8 min, respectively. The type of acid did not significantly affect the heat resistance. The zd values ranged from 5.9 to 10 degrees C, depending on the acid, pH, and the strain. The models generated from this research can be used to determine adequate thermal processes, accounting for the acid type, pH, and temperature, to destroy A. acidoterrestris spores in beverages, since this organism is able to survive the typical hot-fill and hold process (2 min at 88 to 96 degrees C) currently used to process fruit juice.

Construction and expression of a bifunctional single-chain antibody against Bacillus cereus p6ores.

The variable-region genes of monoclonal antibody against Bacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coli under the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) had B. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

Development of a streptavidin-conjugated single-chain antibody that binds Bacillus cereus spores.

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 x 10(4) B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.

Driving predictive modelling on a risk assessment path for enhanced food safety.

How do we best protect our citizens to allow the highest quality of life? Where do we put our food safety resources so that we gain the greatest positive impact? Risk assessment provides the critical scientific basis for these types of important risk management decisions. Increasingly, risk assessment is used to guide legislated and voluntary changes intended to improve safety, yet its formal application for enhanced food safety is in its infancy. Risk assessment includes disease characterization. dose-response assessment, exposure assessment, and risk characterization. Quantitative data is critical for risk assessment to realize its full value, yet much of our knowledge about the incidence of pathogens or toxins in foods, dose-response knowledge, incidence of acute food-borne illness, incidence of chronic sequelae, and cost of food-borne illness is qualitative or estimates are controversial. Predictive modelling should help to improve estimates and thereby allow quantitation of food safety risks. Predictive modelling will also find application for assessing prevention strategies in risk management.

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.

Cold Temperature Growth of Clinical and Food Isolates of Bacillus cereus ‡.

A collection of 27 Bacillus cereus food and clinical isolates were screened for the ability to grow at cold temperatures. Growth was examined using fluid or solid nutrient media or milk incubated at 10, 7, or 5°C. Fourteen isolates were capable of visible colony formation on brain heart infusion (BHI) agar by day 7 at 10°C; two isolates formed visible colonies by day 10 at 7°C on Trypticase soy agar. Nineteen of the isolates could grow in BHI at 7°C if previously adapted to 7°C over a five-week period. Both food and clinical isolates demonstrated a cold adaptation response. This should be considered when modeling B. cereus growth in foods or in assessing shelf life or safety relative to B. cereus .

Assessment of Risks Associated with Foodborne Pathogens: An Overview of a Council for Agricultural Science and Technology Report.

A review is presented of a report published by the Council for Agricultural Science and Technology and entitled Foodborne Pathogens: Risks and Consequences. The risk assessment approach provided the framework to define the hazard and occurrence of foodborne disease as well as the estimated economic consequences. Fifteen recommendations are detailed, including that food safety policy should be based on risk assessment, that control practices should be applied from food source to consumption, that and fundamental and applied food safety research is needed.

Acetic, lactic and citric acids and pH inhibition of Listeria monocytogenes Scott A and the effect on intracellular pH.

The effects of acetic, lactic and citric acids and pH on the growth and intracellular pH (pHin) of Listeria monocytogenes Scott A were documented and compared for total acid concentrations ranging from 50 mmol ml-1 to 250 mmol ml-1 for acetic and lactic acids and from 25 mmol ml-1 to 100 mmol ml-1 for citric acid. Initial pH values ranged from 4.7 to 6.0. Although the growth rate of L. monocytogenes Scott A was slower when incubated at 25 degrees C than at 37 degrees C, the relative acid and pH inhibition was identical at both temperatures. As the initial pH values decreased and/or the total acid concentrations increased, the growth rates of L. monocytogenes Scott A decreased. Compared at equal initial pH values and on an equimolar total acid basis, the relative inhibition effect was generally acetic > lactic > citric. When based on initial undissociated acid concentrations, the inhibition effect was citric > lactic > acetic. The effect of differing acid and pH environments on pHin was determined. At equimolar total acid concentrations, the pHin of the cell was changed the least by citric acid and the most by acetic acid. Growth rates were influenced by the pHin and the acid used to adjust the system.

A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes.

Listeria innocua M1 was developed as a thermal processing indicator organism for L. monocytogenes by selection of a rifampin- and streptomycin-resistant mutant. zetaD values were 5.6 and 5.8 degrees C, and D (68 degrees C) values were 3.8 and 4.9 s for L. monocytogenes and L. innocua, respectively, in skim milk. The advantages of easy selection, similar heat resistance, and nonpathogenicity make L. innocua M1 appropriate for challenge studies designed to evaluate process lethality with respect to L. monocytogenes.

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.

Purification and some characteristics of a calcium-binding protein from Bacillus cereus spores.

A novel calcium-binding protein has been purified from the dormant spores of Bacillus cereus T. Purity of this protein was verified by SDS-PAGE and reversed-phase HPLC. Its calcium-binding ability was verified by a competitive calcium-binding assay using Chelex-100 resin and 45Ca autoradiography. The protein is heat-stable and is retained by hydrophobic matrices (phenyl-Sepharose) in a calcium-dependent manner. SDS-PAGE and amino acid composition indicate the molecular mass of the protein to be 24 kDa.

Listeria innocua Transformed with an Antibiotic Resistance Plasmid as a Thermal-resistance Indicator for Listeria monocytogenes a.

Listeria innocua PFEI is a chloramphenicol- and erythromycin-resistant organism obtained by electotransforming L. innocua ATCC 33091 with the plasmid pGK12. L. innocua ATCC 33091 and L. innocua PFEI were more heat resistant between 56 and 66°C than Listeria monocytogenes F5069 and Scott A when evaluated in sterile phosphate buffer or milk. Decimal reduction times of each L. innocua strain were 1.5 to 3 times longer in either heating menstruum than were D-values of the most heat resistant L. monocytogenes strain studied (F5069). L. innocua PFEI retained the plasmid during heating so that, of 300 survivors evaluated, 100% were resistant to chloramphenicol and 98% were resistant to erythromycin. Thus, L. innocua ATCC 33091 or PFEI would be useful indicator organisms to evaluate the lethality of thermal processes with respect to L. monocytogenes . L. innocua PFEI has the advantages that it could easily be selected and enumerated among a large, complex, background microflora, but it may not be appropriate for application in a food processing environment.

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores.

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.

Listeria monocytogenes F5069 Thermal Death Times in Liquid Whole Egg 1, 2.

Thermal death times (F-values) for L. monocytogenes F5069 inoculated into sterile liquid whole egg were determined between 62 and 73°C by a submerged capillary tube procedure. The initial population was 5 × 106 to 2 × 107 CFU/tube (0.05 ml). High populations intentionally were selected to build in a safety factor. At each temperature, F-values were determined to be the shortest heating time which did not permit recovery of L. monocytogenes from six or more replicate tubes. L. monocytogenes were recovered by incubating the entire contents of the capillary tube in brain heart infusion broth at 25°C for 2 weeks. At 62°C, F = 16 min and at 69°C, F = 1.6 min. The zF-value was 7.1°C. Minimal pasteurization of egg would not result in product free from L. monocytogenes if initial populations were large. Ultrapasteurization processes may be designed to produce product free from L. monocytogenes and appropriate for prolonged refrigeration.

Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.

Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores.

Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM), the lactate germination requirement was overcome. The data suggest that the L-alanine germination sites were modified by hypochlorite so that a higher concentration of alanine was required for activity. Lactate appeared to be an activator of modified or non-hypochlorite-modified L-alanine germination sites.