Food safety objective approach for controlling Clostridium botulinum growth and toxin production in commercially sterile foods.

As existing technologies are refined and novel microbial inactivation technologies are developed, there is a growing need for a metric that can be used to judge equivalent levels of hazard control stringency to ensure food safety of commercially sterile foods. A food safety objective (FSO) is an output-oriented metric that designates the maximum level of a hazard (e.g., the pathogenic microorganism or toxin) tolerated in a food at the end of the food supply chain at the moment of consumption without specifying by which measures the hazard level is controlled. Using a risk-based approach, when the total outcome of controlling initial levels (H(0)), reducing levels (ΣR), and preventing an increase in levels (ΣI) is less than or equal to the target FSO, the product is considered safe. A cross-disciplinary international consortium of specialists from industry, academia, and government was organized with the objective of developing a document to illustrate the FSO approach for controlling Clostridium botulinum toxin in commercially sterile foods. This article outlines the general principles of an FSO risk management framework for controlling C. botulinum growth and toxin production in commercially sterile foods. Topics include historical approaches to establishing commercial sterility; a perspective on the establishment of an appropriate target FSO; a discussion of control of initial levels, reduction of levels, and prevention of an increase in levels of the hazard; and deterministic and stochastic examples that illustrate the impact that various control measure combinations have on the safety of well-established commercially sterile products and the ways in which variability all levels of control can heavily influence estimates in the FSO risk management framework. This risk-based framework should encourage development of innovative technologies that result in microbial safety levels equivalent to those achieved with traditional processing methods.

Significant shift in median guinea pig infectious dose shown by an outbreak-associated Listeria monocytogenes epidemic clone strain and a strain carrying a premature stop codon mutation in inlA.

Listeria monocytogenes contains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) in inlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of an L. monocytogenes infection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation in inlA. Recovery of L. monocytogenes from tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log(10) increase in the median infectious dose for the strain carrying a PMSC in inlA relative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with an inlA PMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that the L. monocytogenes dose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.

Development of a macrophage cell culture method to isolate and enrich Francisella tularensis from food matrices for subsequent detection by real-time PCR.

Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia in humans from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, there are no techniques currently available to detect this organism in specific food matrices. In this study, a macrophage cell culture system is combined with real-time PCR to identify F. tularensis in food matrices. The method utilizes a mouse macrophage cell line (RAW 264.7) as host for the isolation and intracellular replication of F. tularensis. Exposure of macrophages to F. tularensis-contaminated food matrices results in uptake and intracellular replication of the bacteria, which can be subsequently detected by real-time PCR analysis of the DNA released from infected macrophage cell lysates. Macrophage monolayers were exposed to infant formula, liquid egg whites, and lettuce contaminated with varying quantities of F. tularensis. As few as 10 CFU/ml (or CFU per gram) F. tularensis was detected in infant formula and lettuce after 5 h postinfection. As few as 10 CFU/ml F. tularensis was detected in liquid egg whites after 18 h postinfection. Intracellular F. tularensis could also be isolated on Mueller-Hinton medium from lysates of macrophages infected with the bacteria in infant formula, liquid egg whites, and lettuce for subsequent confirmatory identification. This method is the first to successfully identify F. tularensis from select food matrices.

Thermal resistance of Francisella tularensis in infant formula and fruit juices.

Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens.

Survival and growth of Listeria monocytogenes in broth as a function of temperature, pH, and potassium lactate and sodium diacetate concentrations.

The objective of this study was to determine the antimicrobial effect of a combination of potassium lactate and sodium diacetate (0, 1.8, 3, and 4.5%; PURASAL P Opti. Form 4, 60% solution) on the survival and growth of Listeria monocytogenes Scott A in pH-adjusted broth (5.5, 6.0, 6.5, and 7.0) stored at 4, 10, 17, 24, 30, and 37 degrees C. Appropriate dilutions of broth were enumerated by spiral plating on tryptose agar and counted with an automated colony counter. Growth data were iteratively fit, using nonlinear regression analysis to a three-phase linear model, using GraphPad PRISM. At pH 5.5, the combination of lactate-diacetate fully inhibited (P < 0.001) the growth of L. monocytogenes at all four levels and six temperatures. At pH 6.0, addition of 1.8% lactate-diacetate reduced (P < 0.001) the specific growth rate of L. monocytogenes and increased lag time; however, 3 and 4.5% completely inhibited the growth at the six temperatures studied. Efficacy of the lactate-diacetate mixture was decreased as pH increased and incubation temperature increased. Thus, at pH 6.5, at least 3% was required to retard (P < 0.001) the growth of L. monocytogenes in broth. There was a limited effect of the lactate-diacetate level on the specific growth rate of the pathogen at pH 7.0. However, 1.8 and 3% significantly lengthened the lag time at 4 and 10 degrees C. These results suggest that 1.8% of lactate-diacetate mixture can be used as a substantial hurdle to the growth of L. monocytogenes when refrigerated temperatures are maintained for products with pH less than 6.5.

Determining the microbiological criteria for lot rejection from the performance objective or food safety objective.

The Microbiological Criteria (MC) is a set of parameters used to determine whether a specific lot of food is acceptable or not. These parameters are the microbial test protocol and its sensitivity, the confidence level that an unacceptable lot will be detected, the number of samples to be taken and the number of positive samples that are allowed before rejecting the lot. Determining the microbiological criteria begins with knowledge of the distribution of contamination from samples within a lot, particularly within a lot that is just at the unacceptable level of the microbial hazard. The just unacceptable lot can be defined by the Food Safety Objective (FSO) or Performance Objectives (PO), the small fraction of samples that can exceed these values and the standard deviation of the samples from the lot. With this information, a microbial test protocol is chosen to have a sensitivity level that would detect between approximately 15% and 45% of the samples. A confidence level for the MC and the number of positive samples that would be acceptable (c value which is usually zero) are also chosen. With this information the number of samples (n) required can be calculated. A critical factor in setting the microbiological criteria is the sensitivity of the microbiological test (m value). The sample size (weight) and sampling procedure can affect the standard deviation of the samples, particularly foods with non-homogeneous distribution and low numbers of microorganisms. Sampling, sample preparation and analytical procedures that reduce the variation between the samples will affect the choice of m value and maximum lot mean that meets the MC.

Control of growth and survival of Listeria monocytogenes on smoked salmon by combined potassium lactate and sodium diacetate and freezing stress during refrigeration and frozen storage.

In this study, we evaluated the antimicrobial effects of different levels of a potassium lactate (PL) plus sodium diacetate (SDA) mixture against the growth and survival of Listeria monocytogenes Scott A inoculated onto smoked salmon stored at 4, 10, and -20 degrees C. The effect of freezing stress on the growth kinetics of L. monocytogenes Scott A on smoked salmon at 4 and 10 degrees C was also investigated. The use of PL+SDA at all tested levels (1.5, 3.3, and 5% of a 60% commercial solution of PURASAL P Opti. Form 4) completely inhibited the growth of L. monocytogenes Scott A on smoked salmon stored at 4 degrees C during 32 days of storage. It also delayed the growth of L. monocytogenes Scott A on smoked salmon stored at 10 degrees C for up to 11 days, but a listeriostatic effect was observed only with 5% PURASAL P Opti. Form 4 at 10 degrees C after 11 days. Addition of PL+SDA at all tested levels decreased the surviving populations of L monocytogenes Scott A on smoked salmon during 10 months of frozen storage at -20 degrees C. Freezing stress significantly (P < 0.001) extended the lag time and delayed the growth of L. monocytogenes Scott A at both 4 and 10 degrees C. However, the effect of freezing stress was more significant at 4 degrees C than at 10 degrees C, indicating the importance of temperature control of smoked salmon during the retail storage period.

Effects of pH and agitation on the growth of Listeria monocytogenes Scott A in brain heart infusion broth containing combined potassium lactate and sodium diacetate during storage at 4 or 10 degrees C.

The objective of this study was to compare the effects of pH on the growth kinetics of Listeria monocytogenes Scott A in static and agitated broths stored at 4 and 10 degrees C with and without a combination of 1.85% potassium lactate (PL) and 0.13% sodium diacetate (SDA) (3.3% of a 60% commercial solution, PURASAL P Opti. Form 4). The pH of brain heart infusion broth without (control) or with 1.85% PL + 0.13% SDA was adjusted to 5.5, 6.0, 6.5, and 7.5. L. monocytogenes Scott A was inoculated (at 10(2) CFU/ml) into pH-adjusted broth, which was stored at 4 or 10 degrees C with or without agitation. At pH 5.5, a listeriostatic effect was observed for the broth containing 1.85% PL + 0.13% SDA at 4 and 10 degrees C both with and without agitation. At pH 6.0, 1.85% PL + 0.13% SDA fully controlled the growth of L. monocytogenes Scott A in static broth at 4 degrees C for up to 20 days and significantly slowed the growth of the pathogen in agitated broth. At 10 degrees C, the growth of L. monocytogenes Scott A was significantly reduced by 1.85% PL + 0.13% SDA in agitated and unagitated broths. At pH 6.5, 1.85% PL + 0.13% SDA significantly suppressed the growth of L. monocytogenes Scott A at both 4 degrees C (P < 0.001) and 10 degrees C (P < 0.01). At pH 7.5, 1.85% PL + 0.13% SDA had a limited effect on the growth of L. monocytogenes Scott A in broth stored at 4 and 10 degrees C. At 4 degrees C, agitation decreased the lag time and increased the growth rate of L. monocytogenes Scott A at all tested pHs. A similar but less obvious trend was observed for broths stored at 10 degrees C. These results indicate that lactate-diacetate combinations effectively acted with low pH and temperature to inhibit the growth of L. monocytogenes Scott A.

Variation among Escherichia coli O157:H7 strains relative to their growth, survival, thermal inactivation, and toxin production in broth.

To estimate the potential outcomes of food processing on the fate of foodborne pathogens. variations in microbial parameters such as growth rate, survival time, thermal inactivation time, and toxin production must be known. Previous microbial studies using single strains or cocktails provide error estimates for the uncertainty of the experimental and statistical procedures, but not for variations among strains. In this study, the behavior of 17 strains of Escherichia coli O157:H7 were followed when placed in synthetic media that permitted growth, survival, or thermal inactivation. The parameter values were not rejected as being normal, lognormal, gamma, or Weibull distributions. The ratio of the standard deviation to mean (normal distribution) for the exponential growth rate was 0.16 and for the lag phase duration, it was 0.38. The ratios of times to achieve a 4-log10 reduction at two survival conditions were 0.39 and 0.46; ratios of thermal D values at 55 and 60 degrees C were 0.42 and 0.33, respectively. The ratio of the negative log10 of toxin production was 0.24. These distributions are larger than the coefficient of variations observed for experimental errors in single strain and cocktail experiments. This indicates the limitations in precision that predictions of future population numbers can have when the potential presence of all strains needs to be considered. This variation among strains is applicable whether predictions are made by traditional subjective and point estimates or by using models and risk assessments.

Modeling the lag phase of Listeria monocytogenes.

An estimate of the lag phase duration is an important component for predicting the growth of a bacterium and for creating process models and risk assessments. Most current research and data for predictive modeling programs initiated growth studies with cells grown to the stationary phase in a favorable pH, nutrient and temperature environment. In this work, Listeria monocytogenes Scott A cells were grown in brain heart infusion (BHI) broth at different temperatures from 4 to 37 degrees C to the exponential growth or stationary phases. Additional cells were suspended in a dilute broth, desiccated or frozen. These cells were then transferred to BHI broth at various temperatures from 4 to 37 degrees C and the lag phase durations were determined by enumerating cells at appropriate time intervals. Long lag phases were observed for cells initially grown at high temperatures and transferred to low temperatures. In general, exponential growth cells had the shortest lag phases, stationary phase and starved cells had longer, frozen cells had slightly longer and desiccated cells had the longest lag phases. These data were from immediate temperature transitions. When a computer-controlled water bath linearly changed the temperature from 37 to 5 degrees C over a 3.0- or 6.0-h period, the cells had short lags and grew continuously with declining growth rates. Transitions of 0.75 or 1.0 h had 20-h lag phases, essentially that of immediate transitions. When the transition was 1.5 h, an intermediate pattern of less than 1 log of growth followed by no additional growth for 20 h occurred.

Food-borne Listeria monocytogenes risk assessment.

Listeria monocytogenes is ubiquitous in the environment and in food processing plants. Consequently, foods are frequently contaminated. However, the occurrence rate of listeriosis is only about five cases per million people per year. Listeriosis primarily strikes immunocompromised individuals, pregnant women and the elderly with a fatality rate of 20-25%. The FDA is in the process of finishing a risk assessment that is being conducted as an initial step in reviewing its approach to maximizing the public protection from foodborne L. monocytogenes. The risk assessment evaluated the presence and quantitative levels of L. monocytogenes in 21 groups of ready-to-eat foods. The potential growth of L. monocytogenes between retail point-of-sale, where contamination data originated, and consumption was modelled. The frequency and amount of consumption of these foods completed the data for the exposure assessment. For the hazard characterization or dose response part of the risk assessment, data from animal studies, virulence assays and epidemiological investigations were used to estimate the likelihood of illness for different human groups from consuming different numbers of L. monocytogenes. This risk assessment is a virtual review of current scientific knowledge. Quantitative modelling provides greater insight than a qualitative review and also indicates the uncertainty about our knowledge. The risk assessment does not attempt to define an acceptable or tolerable level of L. monocytogenes consumption or propose changes in regulations. These decisions are the responsibility of risk managers who consider additional factors such as food preferences, technical feasibility and societal values when evaluating regulatory policies.

Effects of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni.

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log(10) units.

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.

Development and validation of a dynamic growth model for Listeria monocytogenes in fluid whole milk.

Listeria monocytogenes, a psychrotrophic microorganism, has been the cause of several food-borne illness outbreaks, including those traced back to pasteurized fluid milk and milk products. This microorganism is especially important because it can grow at storage temperatures recommended for milk (< or =7 degrees C). Growth of L. monocytogenes in fluid milk depends to a large extent on the varying temperatures it is exposed to in the postpasteurization phase, i.e., during in-plant storage, transportation, and storage at retail stores. Growth data for L. monocytogenes in sterilized whole milk were collected at 4, 6, 8, 10, 15, 20, 25, 30, and 35 degrees C. Specific growth rate and maximum population density were calculated at each temperature using these data. The data for growth rates versus temperature were fitted to the Zwietering square root model. This equation was used to develop a dynamic growth model (i.e., the Baranyi dynamic growth model or BDGM) for L. monocytogenes based on a system of equations which had an intrinsic parameter for simulating the lag phase. Results from validation of the BDGM for a rapidly fluctuating temperature profile showed that although the exponential growth phase of the culture under dynamic temperature conditions was modeled accurately, the lag phase duration was overestimated. For an alpha0 (initial physiological state parameter) value of 0.137, which corresponded to the mean temperature of 15 degrees C, the population densities were underpredicted, although the experimental data fell within the narrow band calculated for extreme values of alpha0. The maximum relative error between the experimental data and the curve based on an average alpha0 value was 10.42%, and the root mean square error was 0.28 log CFU/ml.

Risk assessment: a means for linking HACCP plans and public health.

HACCP plan adoption has greatly enhanced the food industry's 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.

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.

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.

Development of a quantitative risk assessment model for Salmonella enteritidis in pasteurized liquid eggs.

The performance of hazard analyses and the establishment of critical limits by the food industry are both hampered by the inability to directly relate food processing operations from farm-to-table with their public health impact. Using a 'unit operations' and stochastic simulation approach, data on the frequency of pathogens in raw ingredients, predictive microbiology models for growth and inactivation (thermal and non-thermal), and dose-response models for infectivity were integrated to create a quantitative risk assessment model for a Salmonella enteritidis infection from thermally processed liquid whole eggs made into mayonnaise in the home. The risk assessment indicated pasteurization provides sufficient consumer protection from a high incidence of infected birds and from temperature abuse between the farm and the egg breakers. However scenarios showed how inadequate pasteurization temperatures and/or temperature abuse during storage leads to a hazardous product. This dynamic approach to modeling risk should aid in identification and setting critical control points and assessing the impact of altering food formulations or processes.

Time-to-turbidity model for non-protective type B Clostridium botulinum.

A model to predict the time for growth to turbidity from spores of non-proteolytic type B strains of Clostridium botulinum was developed in broth media with varying temperatures (4-28 degrees C), pH values (5-7), NaCl additions (0-4%) and total spores (10(1)-10(5)). The model estimates the probability that a sample will have growth on a given day for up to 90 days of storage. The parameters of the model include the probability of growth after 90 days (Pmax) and the mean time of growth (tau) for those that showed growth. The 95% confidence interval (CI95%) for tau was also determined. The tau decreased with increasing temperature and pH, but NaCl levels below 3% had little effect. Decreasing the number of spores in a sample increased both tau and the confidence intervals about tau, reflecting the increasing uncertainty about the estimation of growth times for low spore numbers.

Model for the survival of Staphylococcus aureus in nongrowth environments.

A model was developed to estimate the survival times of Staphylococcus aureus in nongrowth environments. A four strain mixture of S. aureus was inoculated into BHI broth that had a lactate buffer with various combinations of pH (3-7) and lactate (0-1%), NaCl (0.5-20%) and NaNO2 (0-200 ppm) and stored at different temperatures (4-42 degrees C). At appropriate times the survivors were enumerated by sampling and spreading on TSA plates. The survival curves were modeled with two forms of a logistic equation and the D values were determined. Polynomial regression equations were then calculated to predict the effect of the environmental factors on the D values. Survival times were increased with higher pH values, lower temperatures, and lower nitrite and lactate concentrations. Added salt increased survival times until the salt concentrations exceeded that of most foods.