Richard C. Whiting

Modeling bacterial survival in unfavorable environments

SummaryThe long-term survival of pathogenic microorganisms was evaluated and modeled in simulated fermented and dried, uncooked sausages, such as salami and pepperoni.Listeria monocytogenes andSalmonella were inoculated in BHI broths with added lactic acid or lactate (0–1.5%), NaCl (0–19%) and NaNO2 (0–200 ppm) and then incubated at 4–42°C for up to 9 months. Enumerations of surviving cells showed several forms of declining curves, including classic first-order declines, shoulder or lag phases, and two-phase declines with shoulder. Two primary models were tested for their ability to depict the data. The effect of the environmental conditions on the parameters of the models were described with multiple regression equations (secondary models).

Inactivation of microorganisms with microwaves at reduced temperatures

We developed a pilot-plant nonthermal flow process using microwave energy to inactivate microorganisms. The process consists of multiple passes through the microwave generator. Each passed material goes to a receiving tank for subsequent passes. The flow rate was 0.96 to 1.26 kg/min and the dwell time per pass was 1.1 to 1.5 min. Five passes were used. The microwave energy is instantaneously and simultaneously applied to the system, and thermal energy is removed by a cooling tube within the process line in the microwave generator. The cooling tube maintains the temperature below 40 degrees C. There was significant reduction in microorganisms in water, 10% glucose solution, and apple juice, and in yeast in beer. There was a slight decrease in microorganisms in tomato juice, pineapple juice, apple cider, and beer; and no effect in skim milk.

Risk assessment: A means for linking HACCP plans and public health

HACCP plan adoption has greatly enhanced the food industrys ability to systematically design programs to ensure the microbiological safety of foods. Yet, this widening acceptance of the HACCP system has revealed several areas where its application is limited due to reliance on qualitative consideration of hazards and their control. In particular, HACCP planning is limited both conceptually and practically by its inability to quantify the potential combined influence of multiple control-point deviations and to relate the successful operation of a HACCP system to a measurable public-health impact. Recent advances in quantitative microbiological risk assessment appear to offer a means of overcoming these limitations. The integration of HACCP plans with the development of dynamic risk-assessment models offers a means for considering the entire farm-to-table continuum and for relating food-manufacturing operations to public health goals. Such capabilities may be critical to establishing equivalence among HACCP systems.

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.

Survival of Escherichia coli O157:H7 during the manufacture of pepperoni.

This study investigated the growth and survival of Escherichia coli O157:H7 during the manufacture of pepperoni to determine whether a 5-log10-unit decline in numbers, as recommended by the U.S. Food Safety and Inspection Service (FSIS), could be achieved. A range of pepperoni formulations with variations in salt (2.5 to 4.8%) and sodium nitrite (100 to 400 ppm) levels, and with pH (4.4 to 5.6) adjusted by manipulation of dextrose concentrations were prepared. The batters produced were inoculated with E. coli O157:H7 380-94 at a level of approximately 6.70 log10 CFU/g; changes in pathogen numbers, pH, titratable acidity, and sodium nitrite concentrations were monitored during fermentation and drying. With the standard commercial formulation (i.e., 2.5% salt, 100 ppm sodium nitrite, pH 4.8) E. coli O157:H7 numbers declined by approximately 0.41 log10 CFU/g during fermentation and a further 0.43 log10 CFU/g during subsequent drying (7 days). A regression equation was fitted to the data which showed significantly (P < 0.001) greater reductions in pathogen numbers in samples with increased salt and sodium nitrite contents and lowered pH. However declines were in all cases less than the target reduction of 5 log10 CFU/g.

Attributing risk to Listeria monocytogenes subgroups: dose response in relation to genetic lineages.

The objective of this study was to evaluate the hypothesis that the dose-response relationship for Listeria monocytogenes in humans varies with genotypic lineage or subtype. The linkages between molecular subtyping data and enumeration data for L. monocytogenes subtypes in foods consumed by the at-risk population were examined to test this hypothesis. We applied a conditional probability model to conduct a subtype-specific dose-response analysis, with the focus on invasive listeriosis. L. monocytogenes differed not only in the molecular subtype and lineage but also in the contamination level when isolates of the pathogen occurred in retail samples of ready-to-eat foods. Using the exponential model parameter r-value as a measure (essentially the probability of a single cell causing illness), we found that the virulence varied among L. monocytogenes lineages by several orders of magnitude. Under the assumptions made, for L. monocytogenes lineages I and II the consumption of a single cell would result in listeriosis with log average probabilities of -7.88 (equivalent to once in 10(7.78) times) and -10.3, respectively, as compared with -9.72 for L. monocytogenes independent of subtype. A greater difference in r-values was found for selected ribotypes. The uncertainty about the r-value estimates was small compared with the large differences in the virulence parameters themselves. Thus, for L. monocytogenes both subtype and the number of cells consumed matter, highlighting the usefulness of considering both exposure concentration and subtype prevalence in dose-response analysis. As advances are made in molecular subtyping and quantitative tools for dose-response analysis, further studies integrating genomic data into quantitative risk assessments will enable better attribution of disease risk to L. monocytogenes subtypes.

Methods for detection of Clostridium botulinum toxin in foods

Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.

Risk Assessment and Predictive Microbiology

The need and desirability of being able to perform quantitative microbial risk assessments for food processing and preparation operations has been discussed extensively for the past several years. However, there has been little application of this approach in large part due to the need to account for the changes in bacterial populations as a result of food environments and processing. The use of predictive food microbiology models has the potential for overcoming these limitations. Through integration of predictive modeling with risk assessment, it is possible to estimate how changes in unit operations are likely to effect the overall safety of a food. Hypothetical examples of how these techniques could be applied to both single-step and multiple-step food-processing and preparation operations are provided.

Effect of pH on survival, thermotolerance, and verotoxin production of Escherichia coli O157:H7 during simulated fermentation and storage

Heat treatment is increasingly being introduced to fermented meat processing, since the acid tolerance properties of Escherichia coli O157:H7 can permit this organism to survive traditional processing procedures. This study investigated the effect of growth pH and fermentation on the thermotolerance at 55 degrees C of E. coli O157:H7 in a model fermented meat system. E. coli O157:H7 (strain 380-94) was grown at pH 5.6 or 7.4 (18 h at 37 degrees C), fermented to pH 4.8 or 4.4 in brain heart infusion broth, and stored for 96 h. Cells grown at pH 5.6 had higher D values at 55 degrees C (D55 degrees C) than cells grown at pH 7.4 (P < 0.001). Cells fermented to pH 4.8 had higher D55 degrees C than those fermented to pH 4.4 (P < 0.001). Cells fermented to pH 4.8 demonstrated an increase in D55 degrees C during storage (P < 0.001), whereas cells fermented to pH 4.4 showed a decrease in D55 degrees C during the same period (P < 0.001). The effect of growth pH on verotoxin production by E. coli O157:H7 was assessed using the verotoxin assay. Cells grown at pH 5.6 had lower verotoxin production then cells grown at pH 7.4. This effect was not sustained over storage. These results indicate that a lower growth pH can confer cross-protection against heat. This has implications for the production of acidic foods, such as fermented meat, during which a heating step may be used to improve product safety.

Comparison of organic acid salts for Clostridium botulinum control in an uncured turkey product.

Health concerns have led consumers toward purchasing nitrite-free, low salt meat and poultry products. Lacking these barriers to control growth of bacterial pathogens, such products carry heightened risks for botulism, especially if temperature abused. To address this threat, five organic acid salts were evaluated as potential antibotulinal agents. Ground turkey breast was formulated with 1.4% NaCl, 0.3% sodium pyrophosphate, 0-6% organic acid salts, 10% ice, and 500 spores per g of a 6-strain mixture of proteolytic Clostridium botulinum . Vacuum-packaged product (10 g) was heated in 75°C water for 20 min, cooled, and incubated for up to 18 d at 28°C. Botulinal neurotoxin was detected by mouse bioassay at 2 d in samples which lacked any of the test compounds. Samples containing 2% acid salt developed neurotoxin, which was detected at 2, 2, 4, 5, and 5 d for pyruvate, citrate, lactate, acetate, and propionate, respectively. With 6% acid salt additions, samples remained neurotoxin free until 7 d with pyruvate, 18 d with citrate, and >18 d for the remaining compounds. Monocarboxylic acid salts exhibited antibotulinal activity related to their dissociation constants (pKa) Citrate did not fit this pattern, however, suggesting a different mechanism of action. This study reveals that a variety of organic acid salts possess activity that can be used alone or possibly in combination to enhance the safety of nitrite-free turkey products.