Spoilage potential of Bacillus subtilis in a neutral-pH dairy dessert.

The objective of this study was the characterization of the microbiota associated with spoilage of vanilla cream pudding during storage at different temperatures. Commercial cream samples were stored aerobically at 4, 8, 12 and 15 °C for a maximum time period of 40 days. At appropriate time intervals, cream samples were subjected to: (i) microbiological analyses, and (ii) high-performance liquid chromatography (HPLC). Furthermore, the spoilage microbiota was identified through repetitive extragenic palindrome-PCR, while selected isolates were further characterized based on sequencing of the V1-V3 region of the 16S rRNA gene. Microbial growth was observed only during storage of cream samples at 12 and 15 °C, with the applied genotypic analysis demonstrating that Bacillus subtilis subsp. subtilis was the dominant spoilage microorganism of this product. Based on the HPLC analysis results, citric acid and sucrose were the most abundant organic acid and sugar, respectively throughout storage of cream pudding, whereas notable changes mainly included: (i) increase in the concentration of lactic acid and to a lesser extent of formic and acetic acids, and (ii) increase in the concentration of glucose and fructose at the expense of sucrose and lactose. The results of this study should be useful for the dairy industry in detecting and controlling microbiological spoilage in cream pudding and other chilled, neutral-pH dairy desserts.

Evaluation of Fourier transform infrared spectroscopy and multispectral imaging as means of estimating the microbiological spoilage of farmed sea bream.

The objective of the present study was the evaluation of Fourier transform infrared (FTIR) spectroscopy and multispectral imaging (MSI), in tandem with multivariate data analysis, as means of estimating the microbiological quality of sea bream. Farmed whole ungutted fish were stored aerobically at 0, 4 and 8 °C. At regular time intervals, fish samples (i.e. cut portions) were analysed microbiologically, while FTIR and MSI measurements also were acquired at both the skin and flesh sides of the samples. Partial least squares regression (PLSR) models were calibrated to provide quantitative estimations of the microbiological status of fish based on spectral data, in a temperature-independent manner. The PLSR model based on the FTIR data of fish skin exhibited good performance when externally validated, with the coefficient of determination (R2) and the root mean square error (RMSE) being 0.727 and 0.717, respectively. Hence, FTIR spectroscopy appears to be promising for the rapid and non-invasive monitoring of the microbiological spoilage of whole sea bream. Contrarily, the MSI models' performance was unsatisfactory, delimitating their potential exploitation in whole fish quality assessment. Model optimization results concerning fish flesh indicated that MSI may be propitious in skinned fish products, with its definite competence warranting further investigation.

Online Feature Selection for Robust Classification of the Microbiological Quality of Traditional Vanilla Cream by Means of Multispectral Imaging.

The performance of an Unsupervised Online feature Selection (UOS) algorithm was investigated for the selection of training features of multispectral images acquired from a dairy product (vanilla cream) stored under isothermal conditions. The selected features were further used as input in a support vector machine (SVM) model with linear kernel for the determination of the microbiological quality of vanilla cream. Model training (n = 65) was based on two batches of cream samples provided directly by the manufacturer and stored at different isothermal conditions (4, 8, 12, and 15 °C), whereas model testing (n = 132) and validation (n = 48) were based on real life conditions by analyzing samples from different retail outlets as well as expired samples from the market. Qualitative analysis was performed for the discrimination of cream samples in two microbiological quality classes based on the values of total viable counts [TVC ≤ 2.0 log CFU/g (fresh samples) and TVC ≥ 6.0 log CFU/g (spoiled samples)]. Results exhibited good performance with an overall accuracy of classification for the two classes of 91.7% for model validation. Further on, the model was extended to include the samples in the TVC range 2-6 log CFU/g, using 1 log step to define the microbiological quality of classes in order to assess the potential of the model to estimate increasing microbial populations. Results demonstrated that high rates of correct classification could be obtained in the range of 2-5 log CFU/g, whereas the percentage of erroneous classification increased in the TVC class (5,6) that was close to the spoilage level of the product. Overall, the results of this study demonstrated that the UOS algorithm in tandem with spectral data acquired from multispectral imaging could be a promising method for real-time assessment of the microbiological quality of vanilla cream samples.

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.

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".

Growth interactions and antilisterial effects of the bacteriocinogenic Lactococcus lactis subsp. cremoris M104 and Enterococcus faecium KE82 strains in thermized milk in the presence or absence of a commercial starter culture.

Traditional Greek cheeses are often produced from thermized milk (TM) with the use of commercial starter cultures (CSCs), which may not inhibit growth of Listeria monocytogenes completely. Therefore, this study evaluated the behavior of an artificial L. monocytogenes contamination in commercially TM (63 °C; 30 s) inoculated with a CSC plus Lactococcus lactis subsp. lactis M104 and/or Enterococcus faecium KE82, two indigenous strains producing nisin A and enterocin A and B, respectively. Inoculation treatments included TM with the CSC only, and TM without the CSC but with strain M104 alone, or combined with strain KE82. All treatments were incubated at 37 °C for 6 h followed by 66 h at 18 °C. L. monocytogenes grew by 0.66-1.24 log cfu/ml at 37 °C, whereas its further growth at 18 °C was retarded, suppressed, or accompanied by different inactivation rates, depending on each TM treatment. Strain M104 caused the greatest inactivation, whereas the CSC per se was the least effective treatment. Strain KE82 assisted the CSC in controlling pathogen growth at 37 °C, whereas both reduced the nisin A-mediated antilisterial activity of strain M104. Overall, the most 'balanced' treatment against L. monocytogenes was CSC+M104+KE82. Hence, this starter/co-starter combination may be utilized in traditional Greek cheese technologies.

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.

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.

Biomarkers of cardiovascular disease risk in the neonatal population.

The consistently high prevalence of cardiovascular disease (CVD) has urged the need for punctual and effective prevention. Extended research on this specific area has demonstrated the influence of fetal and neonatal periods on the risk of developing CVD in adulthood. Thus, the role of traditional and novel biological markers to the effective screening of CVD among the neonatal population is widely investigated. The objective of the present narrative review is to examine those neonatal biomarkers that may play a role in the development of CVD, to exhibit scientific data that appertain to their association with various perinatal conditions leading to CVD predisposition, and their potential role on prediction and prevention strategies. Multiple biomarkers, traditional and novel, have been mined across the studied literature. Adiposity, insulin resistance, altered lipid profile, inflammation, and endothelial dysfunction seem among the headliners of CVD. Even though various novel molecules have been studied, their clinical utility remains controversial. Therefore, it is quite important for the scientific community to find elements with strong predictive value and practical clinical use.

Serum PCSK9 levels in infants with deviant birth weight: a biomarker of the lipoprotein metabolism.

OBJECTIVE: Proprotein Convertase Subtilisin/Kexin-Type 9 (PCSK9), a modulator of low-density lipoprotein (LDL) cholesterol metabolism, has been reported to be a promising biomarker for evaluating lipoprotein metabolism; however, evidence in infants is limited. In the current study, we sought to investigate potential differences in serum PCSK9 levels between infants with deviant birth weight and controls. METHODS: We enrolled 82 infants, classified into 33 small (SGA), 32 appropriate (AGA), and 17 large for gestation (LGA) infants. Serum PCSK9 was measured on routine blood analysis within the first postnatal 48 h. RESULTS: PCSK9 was significantly higher in SGA as compared to AGA and LGA infants [322 (236-431) as compared to 263 (217-302) and 218 (194-291) ng/ml respectively, p = .011]. In comparison to term AGA infants, PCSK9 was significantly elevated in preterm AGA and SGA infants. We also found a significantly higher level of PCSK9 in term female SGA infants as compared to term male SGA infants [325 (293-377) as compared to 174 (163-216) ng/ml, p = .011]. PCSK9 was significantly correlated with gestational age (R = -0.404, p < .001), birth weight (R = -0.419, p < .001), total cholesterol (R = 0.248, p = .028) and LDL cholesterol (R = 0.370, p = .001). SGA status (OR 2.56, p = .004, 95% CI 1.83-4.28) and prematurity (OR 3.10, p = .001, 95% CI 1.39-4.82) were strongly related to serum PCSK9 levels. CONCLUSION: PCSK9 levels were significantly associated with total and LDL cholesterol. Moreover, PCSK9 levels were higher in preterm and SGA infants, suggesting that PCSK9 might be a promising biomarker for evaluating infants with increased later cardiovascular risk.HighlightsWhat's already known? Proprotein Convertase Subtilisin/Kexin-Type 9 (PCSK9) is a promising biomarker for evaluating lipoprotein metabolism; however, evidence in infants is limited. Infants that were born with a deviant birth weight have a unique lipoprotein metabolism profile.What this study adds? Serum PCSK9 levels were significantly associated with total and LDL cholesterol. PCSK9 levels were higher in preterm and small for gestation infants, suggesting that PCSK9 might be a promising biomarker for evaluating infants with increased later cardiovascular risk.

The magnitude of heterogeneity in individual-cell growth dynamics is an inherent characteristic of Salmonella enterica ser. Typhimurium strains.

Individual-cell heterogeneity is a major source of variability in biological systems affecting importantly, among others, microbial behavior. Characterization of cell populations of pathogenic bacterial strains in their entirety, ignoring the phenotypic variability of single cells, may result in erroneous safety risk estimates. The objective of the present study was the evaluation and comparison of the heterogeneity in the individual-cell growth dynamics of different strains of Salmonella enterica. The stochasticity in the growth of single cells of five S. enterica ser. Typhimurium strains was quantitatively described using time-lapse microscopy, and the existence of a strain effect was statistically assessed. In total, 831 growing microcolonies originating from single cells were monitored and analyzed, and the growth kinetic parameters of lag time (λ) and maximum specific growth rate (µmax) for each one of them were estimated. An extensive heterogeneity in individual-cell growth kinetics was recorded, while significant inter-strain differences in their heterogeneity were evident based on simultaneous Bonferroni confidence intervals and Levene's tests. The Logistic and LogLogistic probability distribution provided the best fitting for µmax and λ data, respectively for all the tested strains. The strain effect on the above distributions was also demonstrated with pairwise comparisons of the decile differences. The impact of strain-dependent heterogeneity on microbial growth was visualized by comparing stochastic growth curves of different strains using Monte Carlo simulation. In conclusion, the individual-cell growth dynamics of S. enterica are heterogeneous, with the magnitude of the observed heterogeneity appearing to be an inherent characteristic of bacterial strains.

Spectroscopy and imaging technologies coupled with machine learning for the assessment of the microbiological spoilage associated to ready-to-eat leafy vegetables.

Based on both new and previously utilized experimental data, the present study provides a comparative assessment of sensors and machine learning approaches for evaluating the microbiological spoilage of ready-to-eat leafy vegetables (baby spinach and rocket). Fourier-transform infrared (FTIR), near-infrared (NIR), visible (VIS) spectroscopy and multispectral imaging (MSI) were used. Two data partitioning approaches and two algorithms, namely partial least squares regression and support vector regression (SVR), were evaluated. Concerning baby spinach, when model testing was performed on samples randomly selected, the performance was better than or similar to the one attained when testing was performed based on dynamic temperatures data, depending on the applied analytical technology. The two applied algorithms yielded similar model performances for the majority of baby spinach cases. Regarding rocket, the random data partitioning approach performed considerably better results in almost all cases of sensor/algorithm combination. Furthermore, SVR algorithm resulted in considerably or slightly better model performances for the FTIR, VIS and NIR sensors, depending on the data partitioning approach. However, PLSR algorithm provided better models for the MSI sensor. Overall, the microbiological spoilage of baby spinach was better assessed by models derived mainly from the VIS sensor, while FTIR and MSI were more suitable in rocket. According to the findings of this study, a distinct sensor and computational analysis application is needed for each vegetable type, suggesting that there is not a single combination of analytical approach/algorithm that could be applied successfully in all food products and throughout the food supply 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.

Growth of Listeria monocytogenes in Partially Cooked Battered Chicken Nuggets as a Function of Storage Temperature.

Battered poultry products may be wrongly regarded and treated by consumers as ready-to-eat and, as such, be implicated in foodborne disease outbreaks. This study aimed at the quantitative description of the growth behavior of Listeria monocytogenes in fresh, partially cooked (non-ready-to-eat) battered chicken nuggets as function of temperature. Commercially prepared chicken breast nuggets were inoculated with L. monocytogenes and stored at different isothermal conditions (4, 8, 12, and 16 °C). The pathogen's growth behavior was characterized via a two-step predictive modelling approach: estimation of growth kinetic parameters using a primary model, and description of the effect of temperature on the estimated maximum specific growth rate (µmax) using a secondary model. Model evaluation was undertaken using independent growth data under both constant and dynamic temperature conditions. According to the findings of this study, L. monocytogenes may proliferate in battered chicken nuggets in the course of their shelf life to levels potentially hazardous for susceptible population groups, even under well-controlled refrigerated storage conditions. Model evaluation demonstrated a satisfactory performance, where the estimated bias factor (Bf) was 0.92 and 1.08 under constant and dynamic temperature conditions, respectively, while the accuracy factor (Af) value was 1.08, in both cases. The collected data should be useful in model development and quantitative microbiological risk assessment in battered poultry products.

Detection of Meat Adulteration Using Spectroscopy-Based Sensors.

Minced meat is a vulnerable to adulteration food commodity because species- and/or tissue-specific morphological characteristics cannot be easily identified. Hence, the economically motivated adulteration of minced meat is rather likely to be practiced. The objective of this work was to assess the potential of spectroscopy-based sensors in detecting fraudulent minced meat substitution, specifically of (i) beef with bovine offal and (ii) pork with chicken (and vice versa) both in fresh and frozen-thawed samples. For each case, meat pieces were minced and mixed so that different levels of adulteration with a 25% increment were achieved while two categories of pure meat also were considered. From each level of adulteration, six different samples were prepared. In total, 120 samples were subjected to visible (Vis) and fluorescence (Fluo) spectra and multispectral image (MSI) acquisition. Support Vector Machine classification models were developed and evaluated. The MSI-based models outperformed the ones based on the other sensors with accuracy scores varying from 87% to 100%. The Vis-based models followed in terms of accuracy with attained scores varying from 57% to 97% while the lowest performance was demonstrated by the Fluo-based models. Overall, spectroscopic data hold a considerable potential for the detection and quantification of minced meat adulteration, which, however, appears to be sensor-specific.

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.

Assuring growth inhibition of listerial contamination during processing and storage of traditional Greek Graviera cheese: compliance with the new European Union regulatory criteria for Listeria monocytogenes.

The current microbiological regulatory criteria in the European Union specify a maximum Listeria monocytogenes population of 100 CFU/g allowable in ready-to-eat foods provided the product will not exceed this limit throughout its shelf life. The aim of this study was to validate the manufacturing method for traditional Greek Graviera cheese produced from thermized milk. Initial challenge experiments evaluated the fate of inoculated L. monocytogenes (ca. 4 log CFU/ml, three-strain cocktail) in thermized Graviera cheese milk (TGCM; 63 degrees C for 30 s) in the presence and absence of a product-specific starter culture (SC) in vitro. Milk samples were incubated for 6 h at 37 degrees C and then for 66 h at 18 degrees C. Experiments were conducted to evaluate the fate of a cocktail of three nonpathogenic L. monocytogenes and L. innocua indicator strains inoculated (ca. 3 log CFU/g) in Graviera cheeses commercially manufactured from TGCM+SC. Cheeses were brined, ripened at 18 degrees C and 90% relative humidity for 20 days, and stored at 4 degrees C for up to day 60 under vacuum. In TGCM, L. monocytogenes increased by ca. 2 log units, whereas in TGCM+SC L. monocytogenes growth was retarded (P < 0.05) after a ca. 1-log increase within 6 h at 37 degrees C. Populations of Listeria indicator strains did not grow in TGCM+SC cheeses at any stage; they declined 10-fold in fresh cheeses within 5 days and then survived with little death thereafter. Thus, growth inhibition but not inactivation of potent natural Listeria contaminants at levels below 100 CFU/g occurs in the core of traditional Greek Graviera cheese during fermentation, ripening, and storage.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.