Predictive modeling of Salmonella spp. growth behavior in cooked and raw chicken samples: Real-time PCR quantification approach and model assessment in different handling scenarios.

The increasing prevalence of Salmonella contamination in poultry meat emphasizes the importance of suitable predictive microbiological models for estimating Salmonella growth behavior. This study was conducted to evaluate the potential of chicken juice as a model system to predict the behavior of Salmonella spp. in cooked and raw chicken products and to assess its ability to predict cross-contamination scenarios. A cocktail of four Salmonella serovars was inoculated into chicken juice, sliced chicken, ground chicken, and chicken patties, with subsequent incubation at 10, 15, 20, and 25°C for 39 h. The number of Salmonella spp. in each sample was determined using real-time polymerase chain reaction. Growth curves were fitted into the primary Baranyi and Roberts model to obtain growth parameters. Interactions between temperature and growth parameters were described using the secondary Ratkowsky's square root model. The predictive results generated by the chicken juice model were compared with those obtained from other chicken meat models. Furthermore, the parameters of the chicken juice model were used to predict Salmonella spp. numbers in six worst-case cross-contamination scenarios. Performance of the chicken juice model was evaluated using the acceptable prediction zone from -1.0 (fail-safe) to 0.5 (fail-dangerous) log. Chicken juice model accurately predicted all observed data points within the acceptable range, with the distribution of residuals being wider near the fail-safe zone (75%) than near the fail-dangerous zone (25%). This study offers valuable insights into a novel approach for modeling Salmonella growth in chicken meat products, with implications for food safety through the development of strategic interventions. PRACTICAL APPLICATION: The findings of this study have important implications in the food industry, as chicken juice could be a useful tool for predicting Salmonella behavior in different chicken products and thus reducing the risk of foodborne illnesses through the development of strategic interventions. However, it is important to recognize that some modifications to the chicken juice model will be necessary to accurately mimic all real-life conditions, as multiple factors particularly those related to food processing can vary between different products.

Modeling strain variability in Campylobacter jejuni thermal inactivation by quantifying the number of strains required.

There is a limited understanding of the survival responses of Campylobacter jejuni during thermal processing, which must be investigated for appropriate risk assessment and processing. Therefore, we aimed to elucidate the survival response of C. jejuni and develop a predictive model considering strain variability and uncertainty, which are important for quantitative microbial risk assessment (QMRA) or risk-based processing control measures. We employed the most probable curve (MPC) method to consider the uncertainty in cell concentrations. Further, the multivariate normal (MVN) distribution served as a model for strain variability in bacterial survival behavior. The prediction curves from the MVN successfully captured the parameter variability of the most probable curves of each strain. More than ten reference strains effectively described the strain variability in parameters using the MVN distribution. The findings indicated that, with sufficient strain data, the MVN could estimate the strain variability, including unknown strains. The multi-level model for strain variability can potentially become a specialized tool for QMRA and risk-based processing controls. The combined approach of MPC and MVN provides valuable insights into strain variability, emphasizing the importance of accounting for variability and uncertainty in predictive models for QMRA and risk-based processing control measures.

[Analysis of Maximum Growth Rate and Construction of Predictive Growth Model for Bacillus cereusin Mashed Potato by Calorimetric Method].

The maximum growth rate (µmax) of Bacillus cereus was estimated using a non-destructive isothermal calorimetric method, and a growth prediction model was constructed based on the measurement results. SCD medium and mashed potato were inoculated with serial-diluted inoculum of B. cereus. Heat generation curves were determined using an isothermal calorimeter at 35, 25, and 15℃. The µmax was determined from the relationship between the increase in B. cereus cell number and incubation time, which was detected through the heat generation of the B. cereus biological process. Moreover, the growth prediction model was constructed using Ratkowsky's square-root model. The results of the growth prediction model based on the data of the calorimetric and conventional culture methods for SCD were expressed as √µCalmax=0.0354 (T-4.9)[R2=0.99] and √µCCMmax=0.0335 (T-5.0)[R2=0.99]; a similar equation was provided by both methods. Conversely, the results of the growth prediction model based on the calorimetric method data for mashed potato were given as √µCalmax=0.0390 (T-8.5)[R2=0.99]; the maximum growth rates at 30 and 20℃ were predicted as 0.70 and 0.20 (1/hr), respectively. The maximum growth rates obtained using the conventional culture method were 0.63 and 0.29 (1/hr), respectively, similar to the calorimetric method results. The predictive microbiological analysis using the calorimetric method enabled the rapid provision of a growth prediction equation, and the number of samples could be substantially reduced compared with that for the conventional culture method.

Predictive Growth Model of Listeria monocytogenes Under Fluctuating Temperature Conditions in Pasteurized Milk by Using Real-Time Polymerase Chain Reaction.

The aim of this study was to evaluate the application of real-time polymerase chain reaction (PCR)-based quantification as a rapid and accurate tool for the monitoring and prediction of Listeria monocytogenes growth in pasteurized milk under constant and fluctuating temperature conditions. The growth of L. monocytogenes was monitored under constant temperature conditions at 4°C, 10°C, 15°C, 20°C, and 35°C. High correlation was obtained between the bacterial growth rate and incubation temperature, where the R2 of the slope of the square root model was calculated to be 0.993 and 0.996 for real-time PCR and the conventional culture method, respectively. Moreover, the obtained maximum specific growth rate (µmax) data plots were correlated with 188 L. monocytogenes µmax data points from the existing model according to ComBase database, with an R2 of 0.961 for real-time PCR and of 0.931 for the conventional culture method. The growth models were examined under three different patterns of fluctuating temperature conditions ranging from 2°C to 30°C. The prediction results fell within ±20% of the relative error zone, showing that real-time PCR quantification could be used for fast, sensitive, and specific bacterial growth monitoring with high-throughput results. Real-time PCR should be considered a promising option and powerful tool for the construction of a bacterial growth prediction model for safety risk analysis in the dairy industry.

Allelic Mutations in the Ripening -Inhibitor Locus Generate Extensive Variation in Tomato Ripening.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

Inactivation of Escherichia coli O157 and Salmonella Enteritidis in raw beef liver by gamma irradiation.

Irradiation of ground beef and beef liver inoculated with Escherichia coli O157 466 and DT66 and Salmonella Enteritidis 3313 were performed with gamma rays from cobalt-60 at refrigerated and frozen temperatures under air- and vacuum-packaged conditions. Results showed that D10 values for all pathogens in frozen beef liver were higher than those in frozen ground beef samples, with significant differences observed between the D10 values of E. coli O157 466 and S. Enteritidis 3313 under air-packaged conditions, as well as in E. coli O157 DT66 and S. Enteritidis 3313 under vacuum-packaged conditions. To verify effective bacterial inactivation under high bacterial-contamination levels (105-107 CFU/g), survival/death interfaces of E. coli O157 DT66 and S. Enteritidis 3313 inoculated in beef liver under vacuum-packaged and frozen conditions were constructed, with results suggesting that doses from 5.3 kGy to 5.5 kGy and 8.2 kGy-8.5 kGy would be sufficient to kill 105 CFU/g of E. coli O157 and S. Enteritidis 3313, respectively, at a 95%-99% predicted confidence interval. These results suggested that food matrixes containing high amounts of antioxidants (such as beef liver) and treated under frozen and vacuum-packaged conditions require additional consideration and evaluation for applications of irradiation treatment.

Predictive Modeling for the Growth of Salmonella Enteritidis in Chicken Juice by Real-Time Polymerase Chain Reaction.

The goals of this study were to monitor the growth kinetics of Salmonella Enteritidis in chicken juice using real-time polymerase chain reaction (PCR) and to evaluate its efficacy by comparing the results with an experimental database. Salmonella Enteritidis was inoculated in chicken juice samples at an initial inoculum of 104 CFU/mL with inoculated samples incubated at six different temperatures (10, 15, 20, 25, 30, and 35°C). Sampling was carried out for 36 h to observe the growth of Salmonella Enteritidis. The total DNA was extracted from the samples, and the copy number of the Salmonella invasion gene (invA) was quantified by real-time PCR and converted to Salmonella Enteritidis cell concentration. Growth kinetics data were analyzed by the Baranyi and Roberts model to obtain growth parameters, whereas the Ratkowsky's square-root model was used to describe the effect of the interactions between growth parameters and temperature on the growth of Salmonella Enteritidis. The growth parameters of Salmonella Enteritidis obtained from an experiment conducted at a constant temperature were validated with growth data from chicken juice samples that were incubated under fluctuating temperature conditions between 5°C and 30°C for 30-min periods. A high correlation was observed between maximum growth rate (µmax) and storage temperature, indicating that the real-time PCR-monitoring method provides a precise estimation of Salmonella Enteritidis growth in food material with a microbial flora. Moreover, the µmax data reflected data from microbial responses viewer database and ComBase. The results of this study suggested that real-time PCR monitoring provides a precise estimation of Salmonella Enteritidis growth in food materials with a background microbial flora.

Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening.

Tomato (Solanum lycopersicum) rin mutants completely fail to ripen: they do not produce red pigmentation, soften or induce an ethylene burst. Therefore, RIN has long been believed to function as a major regulator that is essential for the induction of ripening. Here, we provide evidence contradicting this concept of RIN function, showing induction of fruit ripening in the absence of RIN. A CRISPR/Cas9-mediated RIN-knockout mutation did not repress initiation of ripening and the mutant fruits showed moderate red colouring. Moreover, inactivation of the rin mutant allele partially restored the induction of ripening. Therefore, RIN is not required for the initiation of ripening and rin is not a null mutation, but rather is a gain-of-function mutation that produces a protein that actively represses ripening. Since the discovery of the rin mutant a half-century ago, many models have depicted RIN as indispensable for the induction of ripening; these models should be reconsidered in light of these results.

Microbioligical Hazard Contamination in Fermented Vegetables Sold in Local Markets in Cambodia.

　Fermented vegetables are common part of Cambodian diet. The food safety status for these foods has not been investigated. This study was conducted to evaluate the microbiological hazards that contaminated fermented vegetables. A total of 68 samples of fermented vegetables were purchased randomly from five wet markets in Phnom Penh. The conventional culture methods for microbiological analysis were used. Coliform bacteria (Escherichia coli, Cronobactersakazakii, and Enterobacter spp.), opportunistic non-Entrobacteriaceae, Enterococcus spp., Staphylococcus spp., and Listeria spp. were found in these fermented foods. The highest contamination rate of Enterococcus spp. was 34% of total fermented vegetable samples, followed by Bacillus spp. coliform bacteria and E. coli (31%, 24% and 10%, respectively). The potential foodborne pathogen, C. sakazakii, was identified in one sample. Fermented mixed vegetables showed higher contamination rate of coliform bacteria (50%) than fermented single-type vegetables (13%). The results showed that fermented vegetables sold in wet market are poor in hygiene. The stage in the processing chain where contamination occurred should be identified and basic sanitary practice should be enforced to improve the food safety of fermented vegetables in Cambodia.

Evaluation of TA10 Broth for Recovery of Listeria monocytogenes from Ground Beef.

In 2009, the enrichment broth TA10 was released for simultaneous recovery of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. This medium was compared with other Salmonella enrichment broths [lactose (LAC) broth, buffered peptone water (BPW), and universal pre-enrichment (UP) broth] for the recovery of heat- and freeze-injured Salmonella spp. in beef by the conventional culture method. There was a significant difference between TA10 and LAC enrichment broths for detecting injured Salmonella spp. In this study, the International Organization for Standardization Listeria pre-enrichment broth (Half-Fraser/Fraser) was compared with TA10 broth for the recovery of L. monocytogenes from ground beef. Ground beef samples were contaminated with single Listeria serovars at levels of 0.096 to 0.001 most probable number/g. Twenty 25 g test portions of the contaminated ground beef were pre-enriched in each broth, and the ISO-11290-1 Listeria official isolation protocol was used thereafter. There was a significant difference between TA10 broth (48 h) and Half-Fraser/Fraser broth (72 h) in the recovery of L. monocytogenes. In addition, the incubation time for TA10 broth was shorter than for Half-Fraser/Fraser broth. The results indicate that TA10 broth should be used instead of Half-Fraser/Fraser broth for analysis of beef that may be contaminated with very low levels of L. monocytogenes.

Fate of Escherichia coli O157 Cells Inoculated into Lightly Pickled Chinese Cabbage during Processing, Storage and Incubation in Artificial Gastric Juice.

Fate of Escherichia coli O157 cells was evaluated when inoculated into each step after production of lightly pickled Chinese cabbage. The efficacy of surface sterilization by 100 mg/L of chlorine water for 10 min on raw leaves (6.0 log CFU/g) was 2.2 log CFU/g reduction. No meaningful change of the population of E. coli O157 (3.5 log CFU/g to 1.5 log MPN/g) contaminated into 19 kinds of products was observed. These results indicated the difficulty of estimating the viable count of the cells between contaminated on farms and further processing and storage steps. The population of E. coli O157 (3 log CFU/g to 1 log MPN/g) inoculated into the Chinese cabbage products was reduced less than 0.6 log CFU/g after 2 h-incubation at 37℃ in artificial gastric juice. Prevention from initial contamination of E. coli O157 on the ingredients of Chinese cabbage products is important to reduce the risk of food poisoning because the reduction of the bacterial counts after processing and consumption are limited.

Survival of Inoculated Escherichia coli O157:H7 in Japanese Sweet Dumplings during Storage.

An outbreak of Escherichia coli O157:H7 occurred due to the consumption of sweet dumplings in Japan. We examined the survival of E. coli O157:H7 inoculated into several types of sweet dumplings to evaluate the progress of the residual contaminating pathogens after the production or packing processes. For all 4 types of tested typical sweet dumplings, no significant reduction in the viable cell counts of inoculated E. coli O157:H7 (3 log CFU/g) was observed during storage at -20 ℃ and 5 ℃ for 5 weeks. Approximately 1 log CFU/g of reduction was observed after storage for 5 weeks at 10 ℃ and 15 ℃, which corresponded to the growth of the naturally contaminating fungi. Similar results were obtained when we used several types of commercially distributed sweet dumplings. It is of vital importance to prevent the cross contamination of sweet dumplings after the heating process in order to reduce the risk of foodborne illness.

Prevalence of foodborne pathogens in retailed foods in Thailand.

The consumption of foodborne pathogens contaminated in food is one of the major causes of diarrheal diseases in Thailand. The objective of this study was to evaluate the prevalence and types of contaminating bacteria in retailed foodstuffs in Thailand. Food from four categories (137 samples total), including meat (51 samples), vegetables (38 samples), fish or seafood (37 samples), and fermented food (11 samples), was purchased randomly from seven different open-markets and seven supermarkets in Thailand from August 2010 to March 2011. Seven types of major foodborne pathogens were identified using conventional culture methods. Approximately 80% of meat samples tested was contaminated with Salmonella spp. In contrast, the Salmonella spp. contamination rate of vegetable (5%) or fermented food (9%) samples was comparatively low. Six strains of Cronobacter sakazakii and two strains of Yersinia enterocolitica were also isolated. A substantially higher rate of contamination by Bacillus cereus was observed in fermented food (82%) than in samples of meat (2%) and fish or seafood (5%). Seven Listeria spp. isolates were obtained from meat and fish or seafood samples. Approximately 39% of samples tested were found to be contaminated with Staphylococcus spp. (54 isolates). The rate of bacterial contamination of meat did not depend on the type of market. However, the contamination rate of Staphylococcus spp. in vegetables was higher in open markets than in supermarkets, and the contamination rate of Salmonella spp. and Staphylococcus spp. in fish or seafood samples purchased in open markets was likewise higher than in those purchased in supermarkets. Therefore, improvement of hygienic practices throughout the food chain may be required to reduce the risk of food poisoning.

A survey of iceberg lettuce for the presence of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Japan.

No information has been available on the prevalence of pathogens in fresh produce in Japan. In the present study, information was collected on the occurrence of contamination by Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in iceberg lettuce in a Japanese retail store. A total of 419 samples of lettuce that had been harvested in different districts and/or by different producers from July 2008 to March 2009 were examined. A multiplex PCR method was used to simultaneously identify the three bacterial pathogens. No pathogenic bacteria, including Salmonella, E. coli O157:H7, and L. monocytogenes, were detected from any of the samples with this highly sensitive and validated procedure. The aerobic bacteria plate counts and coliform bacteria counts in lettuce throughout the examination period did not show any seasonal trends, and the numbers were comparable to those reported by others from around the world. Based on the results of this study, we concluded that none of the three major pathogens were present in this limited survey of iceberg lettuce sold by a retailer in Japan.

Evaluation of TA10 broth for recovery of heat- and freeze-injured Salmonella from beef.

The Bacteriological Analytical Manual (BAM) Salmonella pre-enrichment broth [lactose (LAC) broth], buffered peptone water, and universal pre-enrichment (UP) broth were compared with TA10 broth, developed in our laboratory, for recovery of heat- and freeze-injured Salmonella (55 degrees C for 2-20 min and -20 degrees C for 2 months, respectively) from beef. Beef samples were contaminated with single Salmonella serovars, and contamination levels of 0.44 to <0.001 most probable number (MPN)/g and 0.74 to 0.14 MPN/g were used for heat- and freezing-induced injury studies, respectively. Twenty test portions (25 g) of the contaminated beef were pre-enriched in each broth, and the BAM Salmonella culture method was used thereafter. There was a significant difference (chi2 = 7.73) in recovery of heat-injured Salmonella between TA10 broth and LAC broth, 189 (67.5%) versus 156 (55.7%) positive samples, respectively, determined by plating onto selective agars and identification by biochemical tests. For the recovery of freeze-injured Salmonella, there was a significant difference (chi2 = 24.7) between TA10 and LAC broth, 189 (72.7%) versus 133 (51.2%) positive samples, respectively. TA10 broth was more effective than LAC broth and UP broth for recovery of freeze-injured Salmonella. The results indicate that TA10 broth should be used instead of LAC broth for testing of beef that may be contaminated with heat- and freeze-injured Salmonella spp.

Comparison of the effectiveness of acidified sodium chlorite and sodium hypochlorite in reducing Escherichia coli.

This study was designed to compare the effectiveness of acidified sodium chlorite (ASC) and sodium hypochlorite (NaClO) in reducing several Escherichia coli strains isolated from different retail meat and fresh produce. Forty nonpathogenic E. coli strains were isolated and used in this study. A type strain of E. coli (JCM 1649) and four O157:H7 serotypes of E. coli (CR-3, MN-28, MY-29, and DT-66) were used as reference. In vitro assay results revealed that the viable cell counts of each isolated E. coli strain and control strains exhibited a reduction of ∼ 4.3 ± 0.9 log and 7.8 ± 1.7 log CFU/mL after a 3-minute exposure to 100 mg/L NaClO and 20 mg/L ASC (pH 4.6), respectively, at 25°C, when compared with the viable bacterial counts obtained from phosphate-buffered saline. The one exception was the flocs-forming strain, which showed a reduction of only 1.0 log CFU/mL with both disinfectants. However, reductions of only 1.7 ± 0.3 log and 1.9 ± 0.4 log CFU/g were observed in lettuce after 5 minutes of washing with NaClO and ASC, respectively. On the other hand, reductions of 1.6 ± 0.2 log and 1.6 ± 0.4 log CFU/g were observed in spinach after 5 minutes of washing with NaClO and ASC, respectively. No reduction in the population was observed after washing the inoculated, fresh-cut vegetables with distilled water only. No significant difference in the reduction of E. coli was observed among all the tested strains with both sanitizers in the in vivo assay. These data suggest that the tested sanitizers exhibit a similar reduction of the surface-attached E. coli on leafy vegetables irrespective of the strain source.

Combined effect of low-dose irradiation and acidified sodium chlorite washing on Escherichia coli O157:H7 inoculated on mung bean seeds.

The effect of low-dose irradiation (0.75 and 1.5 kGy) in combination with acidified sodium chlorite (ASC) on the reduction of Escherichia coli O157:H7 on mung bean seeds was examined. Washing with ASC (0.2, 0.5, 0.8, and 1.2 g/L sodium chlorite and 1.0 g/L citric acid) for 2 h reduced the E. coli O157:H7 population from 5.2 to 2.3-3.3 log CFU/g, depending on the concentrations of sodium chlorite. Gamma ray irradiation at 0.75 and 1.5 kGy resulted in reductions of about 1.8 and 2.8 log CFU/g, respectively. Therefore, a single treatment with ASC washing or gamma ray irradiation at 0.75 or 1.5 kGy could not achieve the complete elimination of E. coli O157:H7 on mung bean seeds. Conversely, low-dose irradiation (0.75 and 1.5 kGy) followed by washing with ASC (0.5-1.2 g/L) reduced the population of E. coli O157:H7 to below the detection limit (<1 log CFU/g). However, E. coli O157:H7 was detected in most samples in the enrichment and germination studies. When the treatment order was reversed (ASC washing followed by low-dose irradiation), the E. coli O157:H7 population was also observed to be below the detection limit. Under this treatment, fewer samples (16.7%) were shown to be positive in the enrichment and germination studies, and complete elimination was not achieved. The germination rates of mung bean seeds were not affected by ASC washing and gamma irradiation; however, the yield and length of sprouts were decreased by gamma irradiation.

Multiplex real-time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples.

Salmonella sp., Listeria monocytogenes, and Escherichia coli O157:H7 are foodborne pathogens capable of causing serious gastrointestinal illness. We previously described simultaneous detection of these pathogens by multiplex polymerase chain reaction (PCR) in 44 types of spiked food samples, including meat, produce, fish, and dairy products, targeting genes specific for each pathogen. Based on the previous work, a multiplex real-time PCR assay using fluorescent probes was developed to detect and accurately quantify Salmonella sp., L. monocytogenes, and E. coli O157:H7 in ground pork samples. The detection sensitivity for this method was 2.0 x 10(2) CFU/mL for each pathogen, and the quantification range was 10(2)-10(7) CFU/mL with a high correlation coefficient (R(2) > 0.99) and high PCR efficiency (84.2% to 99.2%). When this protocol was used for the detection of each of the pathogens in spiked pork samples, one cell per 25 g of inoculated sample after enrichment for 20 h could be detected within 24 h. As a result, this multiplex real-time PCR assay will be valuable as a screening method for foods contaminated with these pathogens.

Efficacy of chlorine and acidified sodium chlorite on microbial population and quality changes of spinach leaves.

Efficacy of washing with distilled water, chlorine solution, and acidified sodium chlorite (ASC) solution on populations of microorganisms on spinach leaves was evaluated. Washing with chlorine (100 mg/L) and ASC (sodium chlorite, 15 mg/L; citric acid, 200 mg/L) resulted in significant population reduction (1.1-1.9 log CFU/g) of aerobic microflora, coliform, and Escherichia coli O157:H7 (p < 0.05). There was no remarkable difference in decontamination efficacy between chlorine and ASC solution. In recent years, several sodium chlorite chemicals have been commercially available, and no difference in decontamination efficacy among the chemicals was observed when same concentration of sodium chlorite and citric acid were used. In addition, the reduction of E. coli O157:H7 population was influenced depending on the inoculation method and type of washing. It has been seen that dip-inoculated spinach leaves showed lower reduction than that of spot-inoculated spinach. After washing, populations of aerobic microflora, coliform, and E. coli O157:H7 were increased during storage at 10 degrees C, and washing condition before storage did not affect the subsequent increases in microbial population. Color of spinach leaves washed with ASC solution was not different from the color of those washed with water or chlorine solution, and washing with ASC solution was concluded to has no effect on appearance of spinach leaves.

Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing.

Conventional culture methods were compared to a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 from enrichment cultures of various types of artificially inoculated and naturally contaminated foods. The multiplex PCR assay was evaluated in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen for simultaneous detection. The sensitivity of the assay was