Growth and no-growth boundary of Clostridium perfringens in cooked cured meats - A logistic analysis and development of critical control surfaces using a solid growth medium.

This study was conducted to evaluate the effect of sodium nitrite (NaNO2, 100-200 ppm), sodium erythorbate (SE, 0-547 ppm), sodium tripolyphosphate (STPP, 0-0.5 %), and sodium chloride (NaCl, 2-3 %) on growth of C. perfringens using a solid growth medium and to develop a growth/no-growth boundary (critical control surface, or CCS) to prevent its growth in cooked cured meat under the optimal temperature condition. Melted Shahidi Ferguson Perfringens (SFP) agar, inoculated with a 3-strain spore cocktail and mixed with NaNO2, SE, STPP, and NaCl according to a Box-Behnken response surface experimental design, was dispersed in 96-well microplates and incubated anaerobically in an incubator programmed to remain at 4 °C for 24 h, heat to 80 °C in 1.75 h, quickly (0.5 h) cool to 46 °C (optimum temperature), and then maintain at 46 °C overnight. The plates were examined optically and visually for colony formation. Any well free of growth was designated as no-growth. Logistic regression was used to analyze the growth probability (P) as affected by NaNO2, SE, STPP, and NaCl and define a CSS as meeting the criterion of P < 1/96. The results showed that STPP and the interactions of SE with NaNO2 and NaCl could reduce the growth probability of C. perfringens in SFP agar. The validation of CCS with ground beef showed an accuracy of 96.3 % for no growth of C. perfringens in the inoculated samples. The results of this study proved that cured meat can be formulated with proper combinations of NaNO2, SE, STPP, and NaCl to prevent the growth of C. perfringens even under the optimum temperature condition, thus preventing food poisoning caused by the growth of this microorganism.

Thermal resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella in animal fat - Kinetic analysis and mathematical modeling.

The objective of this study was to evaluate the effect of fat on thermal resistance of L. monocytogenes, E. coli O157:H7, and Salmonella spp. A 4-strain cocktail of each microorganism was inoculated to beef tallow and heated isothermally at temperatures between 55 and 80℃. All survival curves did not follow the 1st-order inactivation kinetics but conformed to a two-stage linear pattern. The first stage was markedly less heat-resistant than the second, as manifested by significantly lower D values. The z values of E. coli O157 H7 and Salmonella spp. were 11.8 °C and 12.3 °C in the first stage (z1) but increased to 23.7 °C and 20.8 °C in the second stage (z2), respectively. For L. monocytogenes, while the z values were similar for both stages (z1 = 19.6 °C and z2 = 18.5 °C), the second stage D values are 3.6-5.9 times of those in the first stage. One-step analysis was used to fit the nonlinear curves to the Weibull model, yielding < 1 exponents for the model (0.495, 0.362, and 0.282, respectively, for L. monocytogenes, E. coli O157:H7, and Salmonella spp.), suggesting gradually increased thermal resistance during heating. The experimental results showed that these microorganisms could resist heating for longer time and at higher temperatures in tallow than they do in regular meats containing lower levels of fat. The kinetic models can be used to develop thermal processes to properly inactivate pathogens contaminated in the fat portions of meat products or other high fat products.

Numerical simulation of heat transfer during meat ball cooking and microbial food safety enhancement.

This study was conducted to apply the finite volume method (FVM) to solve the partial differential equation (PDE) governing the heat transfer process during meat cooking with convective surface conditions. For a one-dimensional, round-shaped food, such as meat balls, the domain may be divided into shells of equal thickness, with energy balance established for each adjacent shell using in the finite difference scheme (FDS) to construct a set of finite difference equations, which were then solved simultaneously using the FORTRAN language and the IVPAG subroutine of the International Mathematics and Statistics Library. The FDS is flexible for temperature-dependent physical properties of foods, such as thermal conductivity (k), specific heat (Cp ), thermal diffusivity (α), and boundary conditions, for example, surface heat transfer coefficient (h), to predict the dynamic temperature profiles in beef and chicken meat balls cooked in an oven. Once the FVM model was established and validated, it was used to simulate the dynamic temperature profiles during cooking, which were then used in combination with the general method to evaluate the thermal lethality of Shiga toxin-producing Escherichia coli and Salmonella spp. using D and z values in ground meats during cooking. The method can be applied to design cooking processes that effectively inactivate foodborne pathogens while maintaining the quality of cooked meats and evaluate the adequacy of a cooking process. PRACTICAL APPLICATION: The temperature dependences of thermal conductivity (k) and thermal diffusivity (α) of raw ground beef and ground chicken meats were measured. These thermal properties were then used in numerical simulation to predict the dynamic heating temperature profile and thermal lethality of ground beef and chicken meat balls. The numerical simulation method may be used to optimize and evaluate thermal processes and ensure the inactivation of pathogens in meat products during cooking.

Shelf-life boundaries of Listeria monocytogenes in cold smoked salmon during refrigerated storage and temperature abuse.

Cold smoked salmon (CSS) is a high-value ready-to-eat product, but it generally has a short shelf-life even under refrigeration and can support the growth of Listeria monocytogenes. Therefore, the objective of this study was to examine the growth and survival of L. monocytogenes in CSS during refrigerated storage and temperature abuse. The growth and survival data of L. monocytogenes (116 records, 465 data points) were retrieved from ComBase (https://www.combase.cc). All records contained storage time and temperature, but other information (aw, pH, and salt) was not fully documented. Each data point, normalized with the initial population to calculate relative growth (RG, log CFU/g), was used to classify the probability of growth. Eighty percent (80%) of the data were randomly sampled for examining the effect of storage time and temperature on growth of L. monocytogenes, while the remaining 20% were set aside for model validation. Logistic regression was used to develop a model for classifying L. monocytogenes growth according to 7 different control thresholds (CT), ranging from 0 to 3 log CFU/g in RG. A probability threshold was set to judge if the bacterial growth has exceeded a CT. The validation showed > 89% of true negative rate for not exceeding the control thresholds. A dynamic method was then developed and demonstrated to predict the growth probabilities under fluctuating temperature conditions. The result of this study suggested that storage time and temperature could be used to predict the growth of L. monocytogenes in CSS and to control listeriosis using a risk-based strategy. It can be used by the retailers and consumers to determine if a packaged product is safe to consume based on its time and temperature history.

Growth competition between lactic acid bacteria and Listeria monocytogenes during simultaneous fermentation and drying of meat sausages - A mathematical modeling.

Listeria monocytogenes is a significant foodborne health hazard in many products and may survive and grow when making fermented meat sausages. The objective of this study was to investigate the competition between lactic acid bacteria (LAB) and L. monocytogenes during simultaneous fermentation and drying (SFD) of meat sausages. Sausages made from irradiated ground beef (90% lean), salt, sugar, and sodium nitrite were inoculated with a 4-stain cocktail of LAB (2 Lactobacillus plantarum and 2 Lb. brevis strains) and a 5-strain cocktail of L. monocytogenes, individually or in combination, and incubated (30 °C, relative humidity 76%) for 5 days to undergo SFD. The changes in the populations of LAB and L. monocytogenes were monitored to determine the growth kinetics and examine the competitive growth between the two. L. monocytogenes grew in the sausage samples unhindered without LAB but was suppressed by LAB during SFD. The interaction between LAB and L. monocytogenes could be described by a modified Lotka-Volterra equation. The decreases of pH and aw in sausages could be related to the SFD time using segmented linear models. The competition model could accurately predict the growth of LAB and L. monocytogenes during SFD and may be used to improve the safety of semi-dry and dry fermented meat sausages.

Involvement of a putative ATP-Binding Cassette (ABC) Involved in manganese transport in virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis, a disease associated with high fatality (20-30%) and hospitalization rates (>95%). ATP-Binding Cassette (ABC) transporters have been demonstrated to be involved in the general stress response. In previous studies, in-frame deletion mutants of the ABC transporter genes, LMOf2365_1875 and LMOf2365_1877, were constructed and analyzed; however, additional work is needed to investigate the virulence potential of these deletion mutants. In this study, two in vitro methods and one in vivo model were used to investigate the virulence potential of in-frame deletion mutants of ABC transporter genes. First, the invasion efficiency in host cells was measured using the HT-29 human cell line. Second, cell-to-cell spread activity was measured using a plaque forming assay. Lastly, virulence potential of the mutants was tested in the Galleria mellonella wax moth model. Our results demonstrated that the deletion mutant, ⊿LMOf2365_1875, displayed decreased invasion and cell-to-cell spread efficiency in comparison to the wild-type, LMOf2365, indicating that LMOf2365_1875 may be required for virulence. Furthermore, the reduced virulence of these mutants was confirmed using the Galleria mellonella wax moth model. In addition, the expression levels of 15 virulence and stress-related genes were analyzed by RT-PCR assays using stationary phase cells. Our results showed that virulence-related gene expression levels from the deletion mutants were elevated (15/15 genes from ⊿LMOf2365_1877 and 7/15 genes from ⊿LMOf2365_1875) compared to the wild type LMOf2365, suggesting that ABC transporters may negatively regulate virulence gene expression under specific conditions. The expression level of the stress-related gene, clpE, also was increased in both deletion mutants, indicating the involvement of ABC transporters in the stress response. Taken together, our findings suggest that ABC transporters may be used as potential targets to develop new therapeutic strategies to control L. monocytogenes.

Growth and No-Growth boundary of Listeria monocytogenes in beef - A logistic modeling.

Listeria monocytogenes is a potentially fatal foodborne pathogen. Its growth in ready-to-eat (RTE) foods must be strictly controlled to protect public food safety. This study was conducted to define the growth and no-growth boundary of L. monocytogenes with sodium tripolyphosphate (STPP), sodium lactate (NaL), sodium diacetate (NaDiAc), sodium chloride (NaCl), sodium nitrite (NaNO2), and pH as control factors. The growth of L. monocytogenes was first examined using a solid medium incubated under 37 °C for 48 h in 96-well microtiter plates. NaNO2 (1,800 ppm) and NaDiAc (2,500 ppm) were found not effective in preventing the growth when applied alone. STPP was shown highly effective in preventing the growth of L. monocytogenes. Its growth was unhindered at pH 6-7 but was increasingly inhibited beyond the neutral range. High concentrations of NaL and NaCl were needed to inhibit the growth of L. monocytogenes. A multifactor logistic regression model (LRM) was developed to calculate the growth probability (p) and then define the growth boundary using 2 thresholds. With Threshold 1 (p = 0.0104), the Accuracy of classification for growth events is 0.686, with a True positive rate (TPR) of 0.776 (or False negative rate (FNR) of 0.234), True negative rate (TNR) of 0.455 (or False positive rate (FPR) of 0.545), and Precision of 0.803, in PALCAM agar. However, with Threshold 2 (p = 0.04), the Accuracy becomes 0.826, with a TPR of 0.955 (or FNR of 0.045), a TNR of 0.690 (or FPR of 0.310), and Precision of 0.764. For validation in ground beef, the Accuracy of prediction of growth was 0.85, with a TPR of 0.9, TNR of 0.8, and Precision of 0.818 for Threshold 1. With Threshold 2, the Accuracy, TPR, TNR, and Precision are all 0.8, with both FNR and FPR of 0.2. Both thresholds and LRM may be used to formulate RTE products that may prevent the growth of L. monocytogenes even stored under the optimum temperature.

One-step dynamic analysis of growth kinetics of Bacillus cereus from spores in simulated fried rice - Model development, validation, and Marko Chain Monte Carlo simulation.

Bacillus cereus is a spore-forming pathogen capable of producing an emetic toxin and several diarrheal enterotoxins that may cause outbreaks of foodborne illness often associated with rice-based and other farinaceous foods. Therefore, the objective of this study was to investigate the growth kinetics of B. cereus from spores in simulated egg fried rice. The growth of B. cereus was observed under dynamic conditions. Three independent growth curves were analyzed simultaneously using a one-step dynamic analysis (OSDA) to determine the kinetic parameters. The results showed that the minimum, optimum, and maximum growth temperatures were 11.8, 40.8, and 50.6 °C, respectively, with an optimum specific growth rate of 2.4 per h. The root-mean-square-error (RMSE) of model development was 0.4 log CFU/g. Deterministic validation with another 3 independent dynamic temperature profiles showed a RMSE of 0.5 log CFU/g. With Markov Chain Monte Carlo simulation, the RMSE of prediction was only 0.3 log CFU/g. This study proved that OSDA is an effective and efficient method for quickly developing integrated predictive models and estimating kinetic parameters. The resulting integrated model can be used to accurately predict the growth of B. cereus and for managing its risks associated with egg fried rice. The developed kinetic models also can be used to guide restaurant owners and catering establishments to properly prepare and store egg fried rice and other related products to prevent the growth of B. cereus. According to the model, the growth of mesophilic B. cereus is unlikely to occur if the food is stored below 10 °C.

Effect of water activity on inactivation of Listeria monocytogenes using gaseous chlorine dioxide - A kinetic analysis.

The aim of this study was to investigate the effect of water activity (aw) on inactivation of Listeria monocytogenes using gaseous chlorine dioxide (ClO2 (g)) under room temperature. Surface-inoculated tryptic soy agar (TSA) plates adjusted to 9 different water activity levels ranging from 0.994 to 0.429 were used as samples exposed to ClO2 (g) at 150, 250, and 350 ppm for different durations of treatment time. Results showed that the antimicrobial effect of ClO2 (g) significantly decreases as the aw level and ClO2 (g) concentration decrease. Nonlinear models, such as the modified Chick model and the Weibull model, were used to describe the inactivation kinetics of L. monocytogenes. The results showed that the modified Chick model, which is based on chemical reaction kinetics, was more suitable to describe the inactivation of L. monocytogenes (RMSE < 0.5 log CFU/g) than the Weibull model (RMSE < 1.0 log CFU/g). A multiple regression model was developed for the describing the effect of aw and ClO2 (g) concentration on bacterial inactivation. The results of this study may be used to design ClO2 (g) treatment processes to inactivate L. monocytogenes in low-moisture foods.

Effect of temperature on the growth of Staphylococcus aureus in ready-to-eat cooked rice with pork floss.

Cooked rice with pork floss (CRPF) wrapped in dried seaweed is one of the most popular ready-to-eat (RTE) foods in many Asian countries, particularly in Taiwan. The products are susceptible to Staphylococcus aureus contamination and temperature abuse during manufacturing, distribution, and storage. The objective of this study was to examine the effect of temperature on its growth in RTE CRPF for use in risk assessment and prevention of staphylococcal food poisoning (SFP). Inoculated CRPF samples were stored at 4, 12, 18, 25, and 35°C, and the change in the populations of S. aureus during storage were analyzed using three primary models to determine specific growth rate (µmax), lag-phase duration (λ), and maximum population density (ymax). The Ratkowsky square-root and Huang square-root (HSR) models were used as the secondary models to describe the effect of temperature on µmax, and a linear and an exponential regression models were used to describe the effect of temperature on λ and ymax, respectively. The model performance was evaluated by the root mean square error (RMSE), bias factor (Bf), and accuracy factor (Af) when appropriate. Results showed that three primary models were suitable for describing the growth curves, with RMSE ≤ 0.3 (log MPN/g). Using µmax obtained from the Huang model, the minimum growth temperature (Tmin) estimated by the HSR model was 7.0°C, well in agreement with the reported Tmin. The combination of primary and secondary models for predicting S. aureus growth was validated by additional growth curves at 30°C, which showed that the RMSE was 0.6 (log MPN/g). Therefore, the developed models were acceptable for predicting the growth of S. aureus in CRPF under likely temperature abuse conditions and can be applied to assess the risk of S. aureus in CRPF and design temperature controls to reduce the risk of SFP.

In Situ Generation of Chlorine Dioxide for Decontamination of Salmonella, Listeria monocytogenes, and Pathogenic Escherichia coli on Cantaloupes, Mung Beans, and Alfalfa Seeds.

ABSTRACT: In situ generation of chlorine dioxide to reduce microbial populations on produce surfaces has been shown to be effective on produce models. This study examined the treatment for decontamination of bacterial pathogens on whole cantaloupes and sprout seeds. Whole cantaloupes, mung beans, and alfalfa seeds were inoculated with Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli, sprayed with or dipped in 0.4 to 1.6% sodium chlorite (NaClO2) solutions, dried, and treated with 6 mM hydrochloric acid (HCl; sequential treatment). Controls were samples treated with NaClO2 or HCl (individual treatment). The pathogen populations on samples before and after treatments were enumerated to determine the reductions of pathogen populations by the treatments. The methods of applying NaClO2 and HCl (dipping for 30 min or spraying 0.2 g on cantaloupe rind [2 by 2 cm]), NaClO2 concentrations of 0.4 to 1.6% for cantaloupes, and treatment times of 5, 15, and 30 min for sprout seeds were evaluated to identify treatment parameters. For cantaloupes treated with spraying with 1.6% NaClO2, the sequential treatment caused significantly (P < 0.05) higher reductions (6.2 to 7.7 log CFU/cm2) than the combined reductions (3.2 to 5.2 log CFU/cm2) by the individual treatments. For cantaloupes treated by dipping in 1.6% NaClO2 and by spraying with 0.4 and 0.8% NaClO2, the reductions caused by the sequential treatment were not significantly (P > 0.05) different from those by the individual treatments. For mung beans, sequential 15- and 30-min treatments caused significantly (P < 0.05) higher reductions of 4.3 to 5.0 and 4.7 to 6.7 log CFU/g, respectively, than the individual treatments. The sequential 15-min treatment also caused high reductions of 5.1 to 7.3 log CFU/g on alfalfa seeds. The treatments did not bleach the color of cantaloupes and did not affect the germination rates of mung beans and alfalfa seeds. This study identified 1.6% NaClO2 and 6 mM HCl for sequential spraying treatment for cantaloupes and for sequential dipping (15-min) treatment for mung beans and alfalfa seeds that may be used for decontamination of whole cantaloupes and sprout seeds.

Growth/no growth boundary of Clostridium perfringens from spores in cooked meat: A logistic analysis.

Clostridium perfringens is a major foodborne health hazard that can cause acute gastroenteritis in consumers, and is often associated with cooked meat and poultry products. Improper cooling after cooking may allow this pathogen to grow in a product, producing an enterotoxin that causes food poisoning. This study was conducted to evaluate the effect of common ingredients, including sodium tripolyphosphate (STPP), sodium lactate (NaL), and sodium chloride (NaCl), on the germination and outgrowth of C. perfringens spores in meat products. The growth/no growth test was conducted in Shahidi Ferguson Perfringens agar mixed with STPP (0-2500ppm), NaL (0-4%), and NaCl (0-4%) in microplates. Turbidity measurements at 600nm were compared before and after anaerobic incubation at 46°C to evaluate growth and no growth conditions. The dichotomous responses were analyzed by logistic regression to develop a model for estimating the growth probability of C. perfringens. The probability model was used to define the threshold of growth (probability >0.1 or 0.2) of C. perfringens and validated using inoculated ground beef under optimum temperature. Inoculated ground beef was mixed with different combinations of STPP, NaL, and NaCl to observe growth or no growth of C. perfringens, and the probability was calculated from the formulation. If the threshold of growth was set to 0.2, the accuracy of the growth and no growth predictions was 95.7%, with 4.3% over-prediction of growth events (fail-safe). The results from this study suggested that proper combinations of STPP, NaL, and NaCl could be used to control the growth of C. perfringens in cooked beef under the optimum temperature. The results may also suggest that proper combinations of STPP, NaL, and NaCl in cooked meat and poultry products could be used to prevent the growth of C. perfringens during cooling.

LMOf2365_0442 Encoding for a Fructose Specific PTS Permease IIA May Be Required for Virulence in L. monocytogenes Strain F2365.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, which is a major public health concern due to the high fatality rate. LMOf2365_0442, 0443, and 0444 encode for fructose-specific EIIABC components of phosphotransferase transport system (PTS) permease that is responsible for sugar transport. In previous studies, in-frame deletion mutants of a putative fructose-specific PTS permease (LMOf2365_0442, 0443, and 0444) were constructed and analyzed. However, the virulence potential of these deletion mutants has not been studied. In this study, two in vitro methods were used to analyze the virulence potential of these L. monocytogenes deletion mutants. First, invasion assays were used to measure the invasion efficiencies to host cells using the human HT-29 cell line. Second, plaque forming assays were used to measure cell-to-cell spread in host cells. Our results showed that the deletion mutant ΔLMOf2365_0442 had reduced invasion and cell-to-cell spread efficiencies in human cell line compared to the parental strain LMOf2365, indicating that LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes strain F2365. In addition, the gene expression levels of 15 virulence and stress-related genes were analyzed in the stationary phase cells of the deletion mutants using RT-PCR assays. Virulence-related gene expression levels were elevated in the deletion mutants ΔLMOf2365_0442-0444 compared to the wild type parental strain LMOf2365, indicating the down-regulation of virulence genes by this PTS permease in L. monocytogenes. Finally, stress-related gene clpC expression levels were also increased in all of the deletion mutants, suggesting the involvement of this PTS permease in stress response. Furthermore, these deletion mutants displayed the same pressure tolerance and the same capacity for biofilm formation compared to the wild-type parental strain LMOf2365. In summary, our findings suggest that the LMOf2365_0442 gene can be used as a potential target to develop inhibitors for new therapeutic and pathogen control strategies for public health.

In Situ Generation of Chlorine Dioxide for Surface Decontamination of Produce.

Fresh fruits and vegetables are frequently contaminated with bacterial pathogens and implicated in foodborne illnesses. The objective of this study was to develop a unique surface decontamination method for produce using sodium chlorite and an acid in a sequential treatment. The surfaces of cantaloupe rinds, peels of cucumbers, stem scars of grape tomatoes, and leaves of baby spinach were inoculated with Salmonella or Listeria monocytogenes at 5 to 6 log CFU/g, submerged in 1.6 to 4% sodium chlorite solutions for 10 or 30 min, dried for 20 min, and then soaked in 6 mM hydrogen chloride (HCl) for 10 or 30 min and dried for 20 min. Control samples were treated with deionized water, sodium chlorite, HCl, or a premixed solution of sodium chlorite and HCl for comparison. The control treatments reduced the levels of both pathogens on the samples by only 0.3 to 2.9 log CFU/g, whereas the sequential treatment caused significantly higher reductions (P < 0.05) of 5.1 to 5.6 log CFU/g, effectively eliminating the inoculated pathogens. The more effective decontamination resulting from the sequential treatment was attributed to the in situ formation of chlorine dioxide within the plant tissues under the surface by the reaction between sodium chlorite absorbed by the produce and HCl. These results suggest that the sequential use of sodium chlorite and acid is a potentially effective treatment for elimination of foodborne pathogens on produce.

Development of a predictive model for Salmonella spp. reduction in meat jerky product with temperature, potassium sorbate, pH, and water activity as controlling factors.

The objective of this study was to develop a predictive model for the inactivation of Salmonella spp. in ground beef jerky as a function of temperature (T), pH, potassium sorbate (PS), and final water activity (aw). Following a central composite design, ground beef was combined with PS (0 to 0.3%, w/w), pH adjusted from 5 to 7, inoculated with a cocktail of 6 serotypes of Salmonella spp. and heat processed at temperatures between 65 and 85°C until the final aw ranging from 0.65 to 0.85 was achieved. Surviving Salmonella cells were enumerated on tryptic soy agar overlaid with xylose lysine deoxycholate agar (pre-tempered to 47°C) after incubation for 48h at 30°C. Bacterial inactivation was quantified in terms of logarithmic reductions of Salmonella counts (log10CFU/g) and inactivation rate (log10(CFU/g)/h). The results indicated that pH, PS and T significantly (p<0.05) interacted to inactivate Salmonella in beef jerky. Decreasing meat pH significantly (p<0.05) increased the efficacy of PS and T to reduce the levels of Salmonella spp. Beef jerky processed at 82°C, pH5.5, with 0.25% PS to a final aw of 0.7 resulted in a maximum Salmonella logarithmic reduction of 5.0log10CFU/g and an inactivation rate of 1.3log10(CFU/g)/h. The predictive model developed can be used to effectively design drying processes for beef jerky under low humidity conditions and thereby, ensuring an adequate degree of protection against risks associated with Salmonella spp.

Modeling the Effect of Water Activity, pH, and Temperature on the Probability of Enterotoxin A Production by Staphylococcus aureus.

The objectives of this study were to develop a probability model of Staphylococcus aureus enterotoxin A (SEA) production as affected by water activity (a(w)), pH, and temperature in broth and assess its applicability for milk. The probability of SEA production was assessed in tryptic soy broth using 24 combinations of a(w) (0.86 to 0.99), pH (5.0 to 7.0), and storage temperature (10 to 30°C). The observed probabilities were fitted with a logistic regression to develop a probability model. The model had a concordant value of 97.5% and concordant index of 0.98, indicating that the model satisfactorily describes the probability of SEA production. The model showed that a(w), pH, and temperature were significant factors affecting the probability of toxin production. The model predictions were in good agreement with the observed values obtained from milk. The model may help manufacturers in selecting product pH and a(w) and storage temperatures to prevent SEA production.

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

Effect of Acidified Sorbate Solutions on the Lag-Phase Durations and Growth Rates of Listeria monocytogenes on Meat Surfaces.

The surfaces of ready-to-eat meats are susceptible to postprocessing contamination by Listeria monocytogenes. This study quantified the lag-phase durations (LPD) and growth rates (GR) of L. monocytogenes on the surfaces of cooked ham as affected by sorbate solutions of different concentrations and pH levels. Slices of cooked ham inoculated with a four-strain mixture of L. monocytogenes (ca. 10(3) CFU/g) were surface treated with sorbate solutions of 0 to 4% (wt/vol) at pH 4.0 to 6.5, vacuum packaged, and stored at 4 to 12 °C for up to 45 days. The LPD and GR of L. monocytogenes were used to develop response surface models. The models estimated that the LPD of L. monocytogenes in samples treated with solutions of pH 4.0 to 5.5 (no sorbate) were 0 to 11 days and the GR were 0.25 to 0.36 log CFU/day, respectively, at 4 °C. With the treatments of 2 and 4% (wt/vol) sorbate solutions, the LPD were estimated to be extended to 2 to 26 days and 34 to >45 days, and the GR were reduced to 0.15 to 0.30 and 0 to 0.19 log CFU/day, respectively. At 4 °C, increasing sorbate concentrations by 1% (wt/vol) to 2, 3, and 4% (wt/vol) at pH 5.5 to 4.0 led to an extension of LPD by 2 to 11, 10 to 19, and 18 to 27 days, whereas the GR were reduced by 0.037 to 0.055, 0.048 to 0.066, and 0.060 to 0.078 log CFU/day, respectively. Sorbate also extended the LPD and reduced the GR of L. monocytogenes at 8 and 12 °C. Results indicated that sorbate concentration and pH level were significant factors affecting the LPD and GR of L. monocytogenes and that the combination of sorbate and low pH has potential for use as a surface treatment to control L. monocytogenes on meat surfaces.

Interactive effects of temperature, pH, and water activity on the growth kinetics of Shiga toxin-producing Escherichia coli O104:H4 3.

The risk of non-O157 Shiga toxin-producing Escherichia coli strains has become a growing public health concern. Several studies characterized the behavior of E. coli O157:H7; however, no reports on the influence of multiple factors on E. coli O104:H4 are available. This study examined the effects and interactions of temperature (7 to 46°C), pH (4.5 to 8.5), and water activity (aw ; 0.95 to 0.99) on the growth kinetics of E. coli O104:H4 and developed predictive models to estimate its growth potential in foods. Growth kinetics studies for each of the 23 variable combinations from a central composite design were performed. Growth data were used to obtain the lag phase duration (LPD), exponential growth rate, generation time, and maximum population density (MPD). These growth parameters as a function of temperature, pH, and aw as controlling factors were analyzed to generate second-order response surface models. The results indicate that the observed MPD was dependent on the pH, aw, and temperature of the growth medium. Increasing temperature resulted in a concomitant decrease in LPD. Regression analysis suggests that temperature, pH, and aw significantly affect the LPD, exponential growth rate, generation time, and MPD of E. coli O104:H4. A comparison between the observed values and those of E. coli O157:H7 predictions obtained by using the U. S. Department of Agriculture Pathogen Modeling Program indicated that E. coli O104:H4 grows faster than E. coli O157:H7. The developed models were validated with alfalfa and broccoli sprouts. These models will provide risk assessors and food safety managers a rapid means of estimating the likelihood that the pathogen, if present, would grow in response to the interaction of the three variables assessed.

Predictive model for the reduction of heat resistance of Listeria monocytogenes in ground beef by the combined effect of sodium chloride and apple polyphenols.

We investigated the combined effect of three internal temperatures (57.5, 60, and 62.5°C) and different concentrations (0 to 3.0 wt/wt.%) of sodium chloride (NaCl) and apple polyphenols (APP), individually and in combination, on the heat-resistance of a five-strain cocktail of Listeria monocytogenes in ground beef. A complete factorial design (3×4×4) was used to assess the effects and interactions of heating temperature, NaCl, and APP. All 48 combinations were tested twice, to yield 96 survival curves. Mathematical models were then used to quantitate the combined effect of these parameters on heat resistance of the pathogen. The theoretical analysis shows that compared with heat alone, the addition of NaCl enhanced and that of APP reduced the heat resistance of L. monocytogenes measured as D-values. By contrast, the protective effect of NaCl against thermal inactivation of the pathogen was reduced when both additives were present in combination, as evidenced by reduction of up to ~68% in D-values at 57.5°C; 65% at 60°C; and 25% at 62.5°C. The observed high antimicrobial activity of the combination of APP and low salt levels (e.g., 2.5% APP and 0.5% salt) suggests that commercial and home processors of meat could reduce the salt concentration by adding APP to the ground meat. The influence of the combined effect allows a reduction of the temperature of heat treatments as well as the salt content of the meat. Meat processors can use the predictive model to design processing times and temperatures that can protect against adverse effects of contaminated meat products. Additional benefits include reduced energy use in cooking, and the addition of antioxidative apple polyphenols may provide beneficial health affects to consumers.