Development and validation of predictive models for the effect of storage temperature and pH on the growth boundaries and kinetics of Alicyclobacillus acidoterrestris ATCC 49025 in fruit drinks.

This study was undertaken to provide quantitative tools for predicting the behavior of the spoilage bacterium Alicyclobacillus acidoterrestris ATCC 49025 in fruit drinks. In the first part of the study, a growth/no growth interface model was developed, predicting the probability of growth as a function of temperature and pH. For this purpose, the growth ability of A. acidoterrestris was studied at different combinations of temperature (15-45 °C) and pH (2.02-5.05). The minimum pH and temperature where growth was observed was 2.52 (at 35 and 45 °C) and 25 °C (at pH ≥ 3.32), respectively. Then a logistic polynomial regression model was fitted to the binary data (0: no growth, 1: growth) and, based on the concordance index (98.8%) and the Hosmer-Lemeshow statistic (6.226, P = 0.622), a satisfactory goodness of fit was demonstrated. In the second part of the study, the effects of temperature (25-55 °C) and pH (3.03-5.53) on A. acidoterrestris growth rate were investigated and quantitatively described using the cardinal temperature model with inflection and the cardinal pH model, respectively. The estimated values for the cardinal parameters Tmin, Tmax, Topt and pHmin, pHmax, pHopt were 18.11, 55.68, 48.60 °C and 2.93, 5.90, 4.22, respectively. The developed models were validated against growth data of A. acidoterrestris obtained in eight commercial pasteurized fruit drinks. The validation results showed a good performance of both models. In all cases where the growth/no growth interface model predicted a probability lower than 0.5, A. acidoterrestris was, indeed, not able to grow in the tested fruit drinks; similarly, when the model predicted a probability above 0.9, growth was observed in all cases. A good agreement was also observed between growth predicted by the kinetic model and the observed kinetics of A. acidoterrestris in fruit drinks at both static and dynamic temperature conditions.

Risk assessment of fungal spoilage: A case study of Aspergillus niger on yogurt.

A quantitative risk assessment model of yogurt spoilage by Aspergillus niger was developed based on a stochastic modeling approach for mycelium growth by taking into account the important sources of variability such as time-temperature conditions during the different stages of chill chain and individual spore behavior. Input parameters were fitted to the appropriate distributions and A. niger colony's diameter at each stage of the chill chain was estimated using Monte Carlo simulation. By combining the output of the growth model with the fungus prevalence, that can be estimated by the industry using challenge tests, the risk of spoilage translated to number of yogurt cups in which a visible mycelium of A. niger is being formed at the time of consumption was assessed. The risk assessment output showed that for a batch of 100,000 cups in which the percentage of contaminated cups with A. niger was 1% the predicted numbers (median (5th, 95th percentiles)) of the cups with a visible mycelium at consumption time were 8 (5, 14). For higher percentages of 3, 5 and 10 the predicted numbers (median (5th, 95th percentiles)) of the spoiled cups at consumption time were estimated to be 24 (16, 35), 39 (29, 52) and 80 (64, 94), respectively. The developed model can lead to a more effective risk-based quality management of yogurt and support the decision making in yogurt production.

Variability in the adaptive acid tolerance response phenotype of Salmonella enterica strains.

The objective of this study was the assessment of the stationary-phase, low-pH-inducible acid tolerance response (ATR) of different Salmonella enterica strains. For this purpose, 30 strains of the pathogen were grown in tryptone soy broth in the absence (non-adapted cultures) and presence (1% w/v; acid-adapted cultures) of glucose, and then subjected to 4-h acid challenge trials at pH 3.0. Surviving populations of each strain were determined at 1-h intervals, and the Weibull model was fitted to the derived microbiological data. Extensive variability in the acid stress responses of the tested S. enterica strains was observed, with the total population reductions (log CFU/ml) attained in 4 h of acid challenge ranging from 0.9 to 5.5 and from 0.6 to 7.0 for the non-adapted and acid-adapted cultures, respectively. As demonstrated by the model scale parameter δ and shape parameter p, the effect of acid adaptation on the inactivation curves was strain-specific. Although acid adaptation resulted in enhanced acid survival for the majority of the tested strains, there were strains exhibiting similar or decreased acid resistance compared to their non-adapted counterparts. Moreover, acid adaptation appeared to decrease the strain variability of δ whereas increasing the strain variability of p: the coefficient of variation of δ among the tested strains was 97.2 and 54.9% for the non-adapted and acid-adapted cultures, respectively, while the corresponding values for p were 12.7 and 48.1%. The data of the present study, which is the first one to systematically evaluate the adaptive ATR of multiple S. enterica strains, clearly demonstrate that this phenotype (attempted to be induced by growing the pathogen in the presence of glucose) is strain-dependent.

Effect of storage temperature on the lag time of Geobacillus stearothermophilus individual spores.

The lag times (λ) of Geobacillus stearothermophilus single spores were studied at different storage temperatures ranging from 45 to 59 °C using the Bioscreen C method. A significant variability of λ was observed among individual spores at all temperatures tested. The storage temperature affected both the position and the spread of the λ distributions. The minimum mean value of λ (i.e. 10.87 h) was observed at 55 °C, while moving away from this temperature resulted in an increase for both the mean and standard deviation of λ. A Cardinal Model with Inflection (CMI) was fitted to the reverse mean λ, and the estimated values for the cardinal parameters Tmin, Tmax, Topt and the optimum mean λ of G. stearothermophilus were found to be 38.1, 64.2, 53.6 °C and 10.3 h, respectively. To interpret the observations, a probabilistic growth model for G. stearothermophilus individual spores, taking into account λ variability, was developed. The model describes the growth of a population, initially consisting of N0 spores, over time as the sum of cells in each of the N0 imminent subpopulations originating from a single spore. Growth simulations for different initial contamination levels showed that for low N0 the number of cells in the population at any time is highly variable. An increase in N0 to levels exceeding 100 spores results in a significant decrease of the above variability and a shorter λ of the population. Considering that the number of G. stearothermophilus surviving spores in the final product is usually very low, the data provided in this work can be used to evaluate the probability distribution of the time-to-spoilage and enable decision-making based on the "acceptable level of risk".

Development and application of Geobacillus stearothermophilus growth model for predicting spoilage of evaporated milk.

The presence of Geobacillus stearothermophilus spores in evaporated milk constitutes an important quality problem for the milk industry. This study was undertaken to provide an approach in modelling the effect of temperature on G. stearothermophilus ATCC 7953 growth and in predicting spoilage of evaporated milk. The growth of G. stearothermophilus was monitored in tryptone soy broth at isothermal conditions (35-67 °C). The data derived were used to model the effect of temperature on G. stearothermophilus growth with a cardinal type model. The cardinal values of the model for the maximum specific growth rate were Tmin = 33.76 °C, Tmax = 68.14 °C, Topt = 61.82 °C and µopt = 2.068/h. The growth of G. stearothermophilus was assessed in evaporated milk at Topt in order to adjust the model to milk. The efficiency of the model in predicting G. stearothermophilus growth at non-isothermal conditions was evaluated by comparing predictions with observed growth under dynamic conditions and the results showed a good performance of the model. The model was further used to predict the time-to-spoilage (tts) of evaporated milk. The spoilage of this product caused by acid coagulation when the pH approached a level around 5.2, eight generations after G. stearothermophilus reached the maximum population density (Nmax). Based on the above, the tts was predicted from the growth model as the sum of the time required for the microorganism to multiply from the initial to the maximum level ( [Formula: see text] ), plus the time required after the [Formula: see text] to complete eight generations. The observed tts was very close to the predicted one indicating that the model is able to describe satisfactorily the growth of G. stearothermophilus and to provide realistic predictions for evaporated milk spoilage.

Modelling biofilm formation of Salmonella enterica ser. Newport as a function of pH and water activity.

The effect of pH and water activity (aw) on the formation of biofilm by Salmonella enterica ser. Newport, previously identified as a strong biofilm producer, was assessed. Biofilm formation was evaluated in tryptone soy broth at 37 °C and at different combinations of pH (3.3-7.8) and aw (0.894-0.997). In total, 540 biofilm formation tests in 108 pH and aw combinations were carried out in polystyrene microtiter plates using crystal violet staining and optical density (OD; 580 nm) measurements. Since the individual effects of pH and aw on biofilm formation had a similar pattern to that observed for microbial growth rate, cardinal parameter models (CPMs) were used to describe these effects. CPMs described successfully the effects of these two environmental parameters, with the estimated cardinal values of pHmin, pHopt, pHmax, awmin and awopt being 3.58, 6.02, 9.71, 0.894 and 0.994, respectively. The CPMs assumption of the multiplicative inhibitory effect of environmental factors was validated in the case of biofilm formation using additional independent data (i.e. 430 OD data at 86 different combinations of pH and aw). The validation results showed a good agreement (r(2) = 0.938) between observed and predicted OD with no systematic error. In the second part of this study, a probabilistic model predicting the pathogen's biofilm formation boundaries was developed, and the degree of agreement between predicted probabilities and observations was as high as 99.8%. Hence, the effect of environmental parameters on biofilm formation can be quantitatively expressed using mathematical models, with the latter models, in turn, providing useful information for biofilm control in food industry environments.

Individual cell heterogeneity as variability source in population dynamics of microbial inactivation.

A statistical modeling approach was applied for describing and evaluating the individual cell heterogeneity as variability source in microbial inactivation. The inactivation data (Nt vs time) of Salmonella enterica serotype Agona, with initial concentration N0 = 10(9) CFU/ml in acidified tryptone soy broth (pH 3.5), were transformed to (N0 - Nt)/N0 vs time leading to the cumulative probability distribution of the individual cell inactivation times (ti), which was further fitted to a variety of continuous distributions using @Risk software. The best-fitted ti distribution (Gamma) was used to predict the inactivation of S. Agona populations of various N0 using Monte Carlo simulation, with the number of iterations in each simulation being equal to N0 and the number of simulations representing the variability of the population inactivation behavior. The Monte Carlo simulation results for a population with N0 = 10,000 CFU/ml showed that the variability in the predicted inactivation behavior is negligible for concentrations down to 100 cells. As the concentration decreases below 100 cells, however, the variability increases significantly. The results also indicated that the D-value used in deterministic first order kinetic models is valid only for large populations. For small populations, D-value shows a high variability, originating from individual cell heterogeneity, and, thus, can be better characterized by a probability distribution rather than a uniform value. Validation experiments with small populations confirmed the variability predicted by the statistical model. The use of the proposed approach to quantify the variability in the inactivation of mixed microbial populations, consisting of subpopulations with different probability distributions of ti, was also demonstrated.

Stochasticity in colonial growth dynamics of individual bacterial cells.

Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of Salmonella enterica serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (µmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N(0)). For bacterial populations with N(0) of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

Evaluation of the strain variability of Salmonella enterica acid and heat resistance.

The inherent acid and heat resistances of 60 Salmonella enterica strains were assessed in tryptone soy broth without dextrose acidified to pH 3.0 or heated at 57 °C. A total of 360 inactivation curves were generated. Regarding the acid challenge experiments, the inactivation rate (kacid), estimated using the log-linear model, ranged from 0.47 to 3.25 h(-1). A log-linear model with a "survival tail" was used to describe the thermal inactivation of the strains, and the estimated inactivation rate (kheat) ranged from 0.42 to 1.33 min(-1). The strain variability of kacid was considerably higher than that of kheat with the coefficient of variation of this kinetic parameter among the tested strains being 39.0% and 18.3%, respectively. No correlation was observed between the estimated kacid and kheat values of the 60 S. enterica strains. Furthermore, no trends among the tested strains related to origin, serotype or antibiotic resistance profile were evident. The present study is the first one to comparatively evaluate the inherent acid and heat resistance profiles of multiple S. enterica strains. Beyond their value in strain selection for use in food safety challenge studies, the collected data should be useful in describing and integrating the strain variability of S. enterica acid and heat resistance profiles in quantitative microbial risk assessment.

Climate change threatens the microbiological stability of non-refrigerated foods.

Most studies on the impact of climate change on foods focus on the consequences to security and safety. In the present study we provide scientific evidence on an overlooked aspect of climate change related to the microbiological stability of foods. Most microbiologically stable processed foods are contaminated with spores of thermophilic spoilage bacteria which are highly heat-resistant and can survive thermal processing. Current temperatures during distribution and storage in temperate climates do not allow growth of thermophilic bacteria to levels that can cause spoilage, ensuring their microbiological stability. Our findings suggest that the latter limiting condition can be eliminated by global warming. By assessing different global warming scenarios for 38 European cities in a case study with canned milk, we show that failing to limit the increase of global mean surface temperature below 2 °C can lead to a very high risk of spoilage and subsequently cause a collapse of the shelf-stable food chain.

Effect of the growth environment on the strain variability of Salmonella enterica kinetic behavior.

Intra-species variability of microbial growth kinetic behavior is an event with important implications for food safety research. Aiming at the evaluation of the growth variability among Salmonella enterica strains as affected by the growth environment, the kinetic behavior of 60 isolates of the pathogen was assessed at 37 °C in tryptone soy broth of different pH values (4.3-7.0) and NaCl concentrations (0.5-6.0%). Maximum specific growth rate (µ(max)) values corresponding to each strain and growth condition were estimated by means of absorbance detection times of serially decimally diluted cultures using the automated turbidimetric system Bioscreen C. A total of 9600 optical density curves were generated for the strains and the growth conditions tested. The variability of µ(max) among the S. enterica strains was important and greater than that observed within the strains (i.e. among replicates). Moreover, strain variability increased as the growth conditions became more stressful both in terms of pH and NaCl. The coefficient of variation of µ(max) among the tested strains at pH 7.0-0.5% NaCl was 6.1%, while at pH 4.3-0.5% NaCl and pH 7.0-6.0% NaCl was 11.8% and 23.5%, respectively. Beyond the scientific interest in understanding strain variability, the findings of this study should be useful in strain selection for exploitation in food safety challenge studies as well as in incorporating strain variability in predictive microbiology and microbial risk assessment.

Development and application of predictive models for fungal growth as tools to improve quality control in yogurt production.

The effect of storage temperature (0-40 °C) and inoculum size (10¹-105 spores) on the mycelium growth kinetics of 12 fungal species on yogurt were monitored. A cardinal model with inflection (CMI) was used to describe the effect of temperature on the growth rate (µ) and the lag time (λ) of each isolate. Significant differences on the temperature dependence of the mycelium growth between the tested species were observed. Depending on the strain, the estimated minimum, optimum and maximum temperature parameters for µ (T(min), T(opt), T(max)) ranged from -7.6 to 9.6, 19.5 to 37.8 and 29.8 to 46.9 °C, respectively. Only λ was found to be affected by the inoculum size and a linear relation between Ln (λ) and Log (inoculum size) was revealed. The inoculum level did not influence the values of T(min), T(opt) and T(max) for λ. Based on the above observations, the combined effect of inoculum size and temperature on λ was modeled using a modified CMI. The parameter λ(opt) (λ at optimum conditions) was expressed as a function of the inoculum size. Validation studies showed a good performance of the developed models. The application scheme of the models for improving fungi control in yogurt productions is discussed.

Strain variability in biofilm formation: A food safety and quality perspective.

The inherent differences in microbial behavior among identically treated strains of the same microbial species, referred to as "strain variability", are regarded as an important source of variability in microbiological studies. Biofilms are defined as the structured multicellular communities with complex architecture that enable microorganisms to grow adhered to abiotic or living surfaces and constitute a fundamental aspect of microbial ecology. The research studies assessing the strain variability in biofilm formation are relatively few compared to the ones evaluating other aspects of microbial behavior such as virulence, growth and stress resistance. Among the available research data on intra-species variability in biofilm formation, compiled and discussed in the present review, most of them refer to foodborne pathogens as compared to spoilage microorganisms. Molecular and physiological aspects of biofilm formation potentially related to strain-specific responses, as well as information on the characterization and quantitative description of this type of biological variability are presented and discussed. Despite the considerable amount of available information on the strain variability in biofilm formation, there are certain data gaps and still-existing challenges that future research should cover and address. Current and future advances in systems biology and omics technologies are expected to aid significantly in the explanation of phenotypic strain variability, including biofilm formation variability, allowing for its integration in microbiological risk assessment.

Characterization of Fungal Surface Contaminants of the Small Maltese June Pear, Pyrus communis var. bambinella.

ABSTRACT: Fungal pathogens cause surface contamination and potential premature fruit spoilage of bambinella, a fruit endemic to the Maltese islands, leading to the loss of fruit during the postharvest phase. The objective of this study was to isolate, quantify, and characterize fungal contaminants of the small Maltese June Pear and describe their growth kinetics. In total, 284 fungicide-free fruits were collected over three consecutive summers (2014, 2015, 2016). The isolated fungi were identified by using forward and reverse colonial morphology. Species identification was determined using PCR-based methods. The number of CFU per square centimeter of bambinella outer skin was calculated. Mycelium diameter growth rate studies of the isolates were also carried out at seven different temperatures, ranging from 5 to 35°C. Fungi isolated from bambinella included Cladosporium ramotenellum, Alternaria arborescens, Penicillium lanosum, Penicillium expansum, and Aspergillus sydowii, listed from the most abundant to the least abundant. The Rosso model was fitted to the growth kinetic data and showed that the optimal temperatures for growth of all five fungi were in the range of 20 to 22°C, whereas growth was slower at temperatures below 10°C and above 30°C. As observed in the diameter studies, the order of highest to lowest germination rate was found to be P. expansum, A. sydowii, P. lanosum, C. ramotenellum, and A. arborescens. Germination studies showed that the highest germination rate was observed for P. lanosum, followed by A. arborescens, C. ramotenellum, P. expansum, and A. sydowii, in descending order. The highest germination lag time was observed for A. arborescens, followed by C. ramotenellum, P. expansum, P. lanosum, and A. sydowii, in ascending order.

Effect of treating lettuce surfaces with acidulants on the behaviour of Listeria monocytogenes during storage at 5 and 20 degrees C and subsequent exposure to simulated gastric fluid.

The effect of acid decontamination on the growth/survival of Listeria monocytogenes on fresh lettuce (Lactusa sativa) during storage and subsequent exposure to a simulated gastric fluid was studied. Fresh lettuce, inoculated with L. monocytogenes (3 log cfu/cm(2)), was immersed (20 degrees C, 90 s) in water (W), lactic acid (LA), acetic acid (AA), propionic acid (PA) and citric acid (CA) at concentrations 0.5 and 1.0%, and then stored aerobically at 5 and 20 degrees C for 20 and 10 days, respectively. The immediate post-treatment reduction of L. monocytogenes was less than 1 log cfu/cm(2) for all treatments tested. The level of L. monocytogenes on W treated lettuce did not change during storage at 5 degrees C but increased significantly (P<0.05) and reached levels of 10(6)-10(7) cfu/cm(2) at 20 degrees C. The residual effect of acid decontamination treatments during storage was found to be strongly dependent on the type and concentration of the acid solution. Decontamination with 0.5% AA, PA and CA did not have any antimicrobial effect and in some cases stimulated growth of the pathogen. The latter could be attributed to suppression of lettuce natural microflora. In contrast, decontamination with 0.5% LA resulted in a decrease of the pathogen during storage at 5 degrees C and in an extended lag phase at 20 degrees C. Increasing the concentration of acid solutions to 1% resulted in a rapid decline of L. monocytogenes for all acids tested except for CA, in which the behaviour of the pathogen was similar to that observed in W treated lettuce. The results from the sensory analysis showed that decontamination of lettuce with AA and PA led to a significant (P<0.05) negative effect on the overall appearance of lettuce while the sensory score of samples treated with LA and CA during storage was very close to that of W treated lettuce (P>0.05). The above results from the sensory analysis were confirmed by color analysis of lettuce. In order to evaluate the potential acid adaptation phenomena induced by acid decontamination interventions, the survival of L. monocytogenes during exposure to a simulated gastric fluid after prior storage of decontaminated lettuce was studied. The results showed that the tested decontamination treatments did not increase the acid tolerance of L. monocytogenes. In contrast, LA treatments sensitized the pathogen to the acidic simulated stomach conditions indicating a decreased virulence potential.

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis.

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5°C for 15h), (ii) defrosting at room temperature (25°C for 12h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25°C, microwave defrosting, defrosting at 5°C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.

Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef.

Antimicrobial films were prepared by incorporating different levels of oregano oil (0.5%, 1.0%, and 1.5% w/w in the film forming solution) into sorbitol-plasticized whey protein isolate (WPI) films. The moisture uptake behavior and the water vapor permeability (WVP) were not affected by the addition of oregano oil at any of the concentrations used. A reduction of the glass transition temperature (∼10-20°C), as determined by dynamic mechanical thermal analysis (DMTA), was caused by addition of oil into the protein matrix. A decrease of Young modulus (E) and maximum tensile strength (σ(max)) accompanied with an increase in elongation at break (%EB) was observed with increasing oil concentration up to a level of 1.0% (w/w). Wrapping of beef cuts with the antimicrobial films resulted in smaller changes in total color difference (ΔΕ) and saturation difference (Δ(chroma)) during refrigeration (5°C, 12days). The maximum specific growth rate (µ(max)) of total flora (total viable count, TVC) and pseudomonads were significantly reduced (P<0.05) by a factor of two with the use of antimicrobial films (1.5% w/w oil in the film forming solution), while the growth of lactic acid bacteria was completely inhibited. These results pointed to the effectiveness of oregano oil containing whey protein films to increase the shelf life of fresh beef.

Assessment of Escherichia coli O157:H7 growth in ground beef in the Greek chill chain.

A predictive mathematical model of the effect of temperature (10-47 °C) on the growth of Escherichia coli O157:H7 in natural contaminated ground beef was developed. The estimated values for the cardinal parameters Tmin, Tmax, Topt and the optimum maximum specific growth rate (µopt) of E. coli O157:H7 were found to be 3.36, 46.87, 43.16 °C and 1.385/h, respectively. The developed model was further validated against observed growth of E. coli O157:H7 in ground beef at non - isothermal chilling conditions by using two periodically changing temperature profiles with temperature ranging from 0 to 15 °C. Overall the model predicted satisfactorily the growth of E. coli O157:H7 in ground beef at dynamic temperature conditions. The model was combined with temperature data collected from ground beef chill chain in Greece in order to assess the growth of the pathogen from purchase of the product at retail to consumption. Retail storage average temperature from 50 retail cabinets in Greek super markets ranged from 0.1 to 7.4 °C with a mean of 3.2 °C and a mean standard deviation of 1.7 °C. The predicted growth of the pathogen after 7 days of storage at retail ranged between 0 and 2.03 log10 CFU/g, with an average growth to 0.31 log10 CFU/g. The growth of the pathogen during transportation from retail to domestic refrigerators ranged between 0.03 and 0.45 log10 CFU/g, with an average growth to 0.16 log10 CFU/g. The average temperature of 160 domestic refrigerators ranged from -2.7 to 18.1 °C. Differences in the temperature among the shelves of the refrigerators were observed. The predicted growth of E. coli O157:H7 in ground beef stored in domestic refrigerators for 1 day ranged between 0.00 and 2.3 log10 CFU/g. For a scenario storage of ground beef in retail for 3 days, transportation from retail domestic refrigerators over a period of 6 h and storage in domestic refrigerators for 3 days the 99th percentile of the total growth was 4.83 log10 CFU/g for storage at the upper self of the domestic refrigerator. The data and models provided in the present work can be further used in a quantitative risk assessment model of E. coli O157:H7 in ground beef consumed in Greece.

Effect of chlorine stress on the subsequent growth behavior of individual Salmonella cells.

The present work investigates the effect of chlorine stress on the subsequent growth behavior of individual Salmonella cells. A time-lapse microscopy method was used which allowed to evaluate the effect of chlorine on the division times of Salmonella individual cells and the percentage of cells able to divide after the treatment. The results showed that the percentage of cells able to divide after the chlorine treatment decreased from 92.7% for untreated cells to 43.12% and 22% for cell exposed to 127 and 150 mg/l chlorine for 3 min, respectively. The first division time of Salmonella cells was not affected by the chlorine stress at the lower tested concentration of 11 mg/l. Exposure at higher chlorine concentrations however, resulted in significantly longer and more variable division times. The mean first division times were 1.46 ±â€¯0.61, 1.41 ±â€¯0.53, 1.69 ±â€¯0.59, 5.34 ±â€¯4.03 and 19.2 ±â€¯8.71 h after 3 min treatments with 0, 11, 61, 127 and 150 mg/l chlorine, respectively. The effect of chlorine on the second division time of the cells was milder compared to the first division. Exposure of cells to chlorine concentrations up to 61 mg/l did not affect the second division. These results indicate that the daughter cells have no "memory" of the chlorine treatment at these concentrations. For cells exposed to the highest tested chlorine concentration of 150 mg/l the mean second division time was almost 3.5 times longer compared to untreated cells indicating that potential damages of the cells caused by the chlorine treatment are not fully repaired in the second generation. The quantitative data provided by this study at the level of individual cell may lead to a better understanding of microbial resistance to chlorine and improve sanitation and decontamination procedures in the food industry.

Development of a microbial time/temperature indicator prototype for monitoring the microbiological quality of chilled foods.

A time/temperature indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain was developed for monitoring food quality throughout the chilled-food chain. In the designed system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline that results from microbial growth and metabolism in a selected medium. The relation of the TTI response (color change) to the growth and metabolic activity (glucose consumption, lactic acid production, pH decrease) of L. sakei was studied. In addition, the temperature dependence of the TTI kinetics was investigated isothermally in the range of 0 to 16 degrees C and modeled with a system of differential equations. At all temperatures tested, the pH and color changes of the TTI system followed closely the growth of L. sakei, with the endpoint (the time at which a distinct visual color change to the final yellow was observed) of the TTI coinciding with a population level of 10(7) to 10(8) CFU/ml. The endpoint decreased from 27 days at 0 degrees C to 2.5 days at 16 degrees C, yielding an activation energy of 97.7 kJ/mol, which was very close to the activation energy of the L. sakei growth rate in the TTI substrate (103.2 kJ/mol). Furthermore, experiments conducted on the effect of the inoculum level showed a negative linear relationship between the level of L. sakei inoculated in the system medium and the endpoint of the TTI. For example, the endpoint at 8 degrees C ranged from 6 to 2 days for inoculum levels of 10(1) and 10(6) CFU/ml, respectively. This relationship allows the easy adjustment of the TTI endpoint at a certain temperature according to the shelf life of the food product of concern by using an appropriate inoculum level of L. sakei. The microbial TTI prototype developed in the present study could be used as an effective tool for monitoring shelf life during the distribution and storage of food products that are spoiled primarily by lactic acid bacteria or other bacteria exhibiting similar kinetic responses and spoilage potentials. Apart from the low cost, the main advantage of the proposed TTI is that its response closely matches the loss of the quality of a food product by simulating the microbial spoilage process in particular environments.