Occurrence, distribution and diversity of Listeria monocytogenes contamination on beef and pig carcasses after slaughter.

In this study we investigated the prevalence and location of Listeria monocytogenes and hygiene indicator bacteria on beef and pig carcasses. Carcasses were sampled after slaughter and before cooling at eight and nine sites on the carcass, respectively. For each sample, detection and enumeration of Listeria was performed, as well as the enumeration of Total Aerobic Counts (TAC) and Enterobacteriaceae. The L. monocytogenes isolates were also typed to determine pulsotypes and clonal complexes (CC). L. monocytogenes was detected on 46% [95% CI: 35-56%] of beef and 22% [95% CI: 11-32%] of pig carcasses. Contamination levels at the different carcass sites differed considerably between beef and pigs. Genetic typing of strains suggests that carcass contamination originates from both incoming animals with transmission during slaughter practices as well as persistent (CC9) contamination from the slaughterhouse environment. These findings can be used to understand the complexity of introduction and persistence of this pathogen in slaughter facilities. Accurate correlation of L. monocytogenes presence proved unfeasible with any of the tested hygiene indicator bacteria.

Digital microfluidics for time-resolved cytotoxicity studies on single non-adherent yeast cells.

Single cell analysis (SCA) has gained increased popularity for elucidating cellular heterogeneity at genomic, proteomic and cellular levels. Flow cytometry is considered as one of the most widely used techniques to characterize single cell responses; however, its inability to analyse cells with spatio-temporal resolution poses a major drawback. Here, we introduce a digital microfluidic (DMF) platform as a useful tool for conducting studies on isolated yeast cells in a high-throughput fashion. The reported system exhibits (i) a microwell array for trapping single non-adherent cells by shuttling a cell-containing droplet over the array, and allows (ii) implementation of high-throughput cytotoxicity assays with enhanced spatio-temporal resolution. The system was tested for five different concentrations of the antifungal drug Amphotericin B, and the cell responses were monitored over time by time lapse fluorescence microscopy. The DMF platform was validated by bulk experiments, which mimicked the DMF experimental design. A correlation analysis revealed that the results obtained on the DMF platform are not significantly different from those obtained in bulk; hence, the DMF platform can be used as a tool to perform SCA on non-adherent cells, with spatio-temporal resolution. In addition, no external forces, other than the physical forces generated by moving the droplet, were used to capture single cells, thereby avoiding cell damage. As such, the information on cellular behaviour during treatment could be obtained for every single cell over time making this platform noteworthy in the field of SCA.

Variability in growth/no growth boundaries of 188 different Escherichia coli strains reveals that approximately 75% have a higher growth probability under low pH conditions than E. coli O157:H7 strain ATCC 43888.

This study investigated the variation in growth/no growth boundaries of 188 Escherichia coli strains. Experiments were conducted in Luria-Bertani media under 36 combinations of lactic acid (LA) (0 and 25 mM), pH (3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 for 0 mM LA and 4.3, 4.4, 4.5, 4.6, 4.7 and 4.8 for 25 mM LA) and temperature (20, 25 and 30 °C). After 3 days of incubation, growth was monitored through optical density measurements. For each strain, a so-called purposeful selection approach was used to fit a logistic regression model that adequately predicted the likelihood for growth. Further, to assess the growth/no growth variability for all the strains at once, a generalized linear mixed model was fitted to the data. Strain was fitted as a fixed factor and replicate as a random blocking factor. E. coli O157:H7 strain ATCC 43888 was used as reference strain allowing a comparison with the other strains. Out of the 188 strains tested, 140 strains (∼75%) presented a significantly higher probability of growth under low pH conditions than the O157:H7 strain ATCC 43888, whereas 20 strains (∼11%) showed a significantly lower probability of growth under high pH conditions.

An educationally inspired illustration of two-dimensional Quantitative Microbiological Risk Assessment (QMRA) and sensitivity analysis.

Quantitative Microbiological Risk Assessment (QMRA) is a structured methodology used to assess the risk involved by ingestion of a pathogen. It applies mathematical models combined with an accurate exploitation of data sets, represented by distributions and - in the case of two-dimensional Monte Carlo simulations - their hyperparameters. This research aims to highlight background information, assumptions and truncations of a two-dimensional QMRA and advanced sensitivity analysis. We believe that such a detailed listing is not always clearly presented in actual risk assessment studies, while it is essential to ensure reliable and realistic simulations and interpretations. As a case-study, we are considering the occurrence of listeriosis in smoked fish products in Belgium during the period 2008-2009, using two-dimensional Monte Carlo and two sensitivity analysis methods (Spearman correlation and Sobol sensitivity indices) to estimate the most relevant factors of the final risk estimate. A risk estimate of 0.018% per consumption of contaminated smoked fish by an immunocompromised person was obtained. The final estimate of listeriosis cases (23) is within the actual reported result obtained for the same period and for the same population. Variability on the final risk estimate is determined by the variability regarding (i) consumer refrigerator temperatures, (ii) the reference growth rate of L. monocytogenes, (iii) the minimum growth temperature of L. monocytogenes and (iv) consumer portion size. Variability regarding the initial contamination level of L. monocytogenes tends to appear as a determinant of risk variability only when the minimum growth temperature is not included in the sensitivity analysis; when it is included the impact regarding the variability on the initial contamination level of L. monocytogenes is disappearing. Uncertainty determinants of the final risk indicated the need of gathering more information on the reference growth rate and the minimum growth temperature of L. monocytogenes. Uncertainty in the dose-response relationship was not included in the analysis, hence the level of its influence cannot be assessed in the present research. Finally, a baseline global workflow for QMRA and sensitivity analysis is proposed.

The metabolic response of cultured tomato cells to low oxygen stress.

The storage of fruits and vegetables under a controlled atmosphere can induce low oxygen stress, which can lead to post-harvest losses through the induction of disorders such as core breakdown and browning. To gain better understanding of the metabolic response of plant organs to low oxygen, cultured tomato cells (Lycopersicum esculentum) were used as a model system to study the metabolic stress response to low oxygen (0 and 1 kPa O2). By adding 13C labelled glucose, changes in the levels of polar metabolites and their 13C label accumulation were quantified. Low oxygen stress altered the metabolite profile of tomato cells, with the accumulation of the intermediates of glycolysis in addition to increases in lactate and sugar alcohols. 13C label data showed reduced label accumulation in almost all metabolites except lactate and some sugar alcohols. The results showed that low oxygen stress in tomato cell culture activated fermentative metabolism and sugar alcohol synthesis while inhibiting the activity of the TCA cycle and the biosynthesis of metabolites whose precursors are derived from central metabolism, including fluxes to most organic acids, amino acids and sugars.

Towards the quantification of the effect of acid treatment on the heat tolerance of Escherichia coli K12 at lethal temperatures.

The aim of this work is to investigate the effect of acid treatment -before and during heat inactivation- on the heat resistance of Escherichia coli K12 MG1655 cells at lethal temperatures. E. coli cells were grown in Brain Heart Infusion broth until they reached the stationary phase (≈10(9) cfu/mL). Approximately 30 min before thermal inactivation the early stationary phase cells were added in Brain Heart Infusion broth with a specific pH value, achieved with addition of either acetic (50% (v/v)), lactic (50% (v/v)) or hydrochloric acid (30% (v/v)), and inactivation experiments took place at 54 °C and 58 °C. The inactivation dynamics are analysed using the inactivation model of Geeraerd et al. (2000). This enables to define the induced thermotolerance of E. coli as a prolongation of the shoulder and/or a reduction of the inactivation rate. Generally, addition of acids increased the heat resistance of E. coli. The induced resistance depends on the type of acid and on the quantity added, i.e. different levels of acidification lead to a different level of heat resistance. This work provides additional knowledge on the reaction of bacterial cultures to heat after acid treatment -before and during heat treatment- and, therefore, it contributes to an improved understanding of the effect of acid exposure on the bacterial heat resistance.

Hierarchical Bayesian analysis of censored microbiological contamination data for use in risk assessment and mitigation.

Microbiological contamination data often is censored because of the presence of non-detects or because measurement outcomes are known only to be smaller than, greater than, or between certain boundary values imposed by the laboratory procedures. Therefore, it is not straightforward to fit distributions that summarize contamination data for use in quantitative microbiological risk assessment, especially when variability and uncertainty are to be characterized separately. In this paper, distributions are fit using Bayesian analysis, and results are compared to results obtained with a methodology based on maximum likelihood estimation and the non-parametric bootstrap method. The Bayesian model is also extended hierarchically to estimate the effects of the individual elements of a covariate such as, for example, on a national level, the food processing company where the analyzed food samples were processed, or, on an international level, the geographical origin of contamination data. Including this extra information allows a risk assessor to differentiate between several scenario's and increase the specificity of the estimate of risk of illness, or compare different scenario's to each other. Furthermore, inference is made on the predictive importance of several different covariates while taking into account uncertainty, allowing to indicate which covariates are influential factors determining contamination.

On the critical evaluation of growth/no growth assessment of Zygosaccharomyces bailii with optical density measurements: liquid versus structured media.

Growth/no growth (G/NG) studies that include the effect of medium structure have typically been performed for (pathogenic) bacteria and on the basis of gelatin/agar as a gelling agent. In this study, the growth potential of the spoilage yeast Zygosaccharomyces bailii was investigated in two model systems that resemble the macroscopic physicochemical and rheological properties of acidic sauces. In a Carbopol model system, the effect of pH (3.5-4.5), glycerol concentration (17-32%), acetic acid concentration (1.5-2.0%) and medium structure (3 levels) was investigated. In xanthan gum, the behavior of the yeast was studied at different levels of pH (3.5-4.5), NaCl concentration (0.5-13.5%), acetic acid concentration (0-2.0%) and medium structure (2 levels). Rheologically, viscoelastic moduli failed to discriminate between different forms of microbial growth, whereas yield stress data appeared to provide a better indication. In general, G/NG results revealed an unexpected increase of growth probability as a function of medium structure, both at 22 and 30 °C. Whether this behavior is the result of an underlying growth-promoting mechanism could not be explained from a macroscopic point of view (e.g., macrorheology, a(w)), but may be more related to the local microscopic properties of the gels. In a second part of this study, the potential use and information content of optical density measurements for G/NG data collection in structured media were critically evaluated and confronted with their practical relevance to the food industry.

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure-amended soil under tropical climatic conditions in Sub-Saharan Africa.

AIMS: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure-amended agricultural soils under tropical conditions in Sub-Saharan Africa. METHODS AND RESULTS: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g(-1) in manure and manure-amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro-Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high-density inocula and enhanced the decline of low-density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure-amended soil than in manure. The 7 log CFU g(-1) E. coli O157:H7 and Salm. Typhimurium survived for 49-84 and 63-98 days, while at 4 log CFU g(-1) , persistence was 21-28 and 35-42 days, respectively. CONCLUSIONS: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.

Kinetic model-based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions.

AIM: Present a kinetic model-based approach for using isothermal data to predict the survival of manure-borne enteric bacteria under dynamic conditions in an agricultural environment. METHODS AND RESULTS: A model to predict the survival of Salmonella enterica serovar Typhimurium under dynamic temperature conditions in soil in the field was developed. The working hypothesis was that the inactivation phenomena associated with the survival kinetics of an organism in an agricultural matrix under dynamic temperature conditions is for a large part due to the cumulative effect of inactivation at various temperatures within the continuum registered in the matrix in the field. The modelling approach followed included (i) the recording of the temperature profile that the organism experiences in the field matrix, (ii) modelling the survival kinetics under isothermal conditions at a range of temperatures that were registered in the matrix in the field; and (iii) using the isothermal-based kinetic models to develop models for predicting survival under dynamic conditions. The time needed for 7 log CFU g(-1) Salmonella Typhimurium in manure and manure-amended soil to reach the detection limit of the enumeration method (2 log CFU g(-1) ) under tropical conditions in the Central Agro-Ecological Zone of Uganda was predicted to be 61-68 days and corresponded with observed CFU of about 2·2-3·0 log CFU g(-1) , respectively. The Bias and Accuracy factor of the prediction was 0·71-0·84 and 1·2-1·4, respectively. CONCLUSIONS: Survival of Salm. Typhimurium under dynamic field conditions could be for 71-84% determined by the developed modelling approach, hence substantiating the working hypothesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Survival kinetic models obtained under isothermal conditions can be used to develop models for predicting the persistence of manure-borne enteric bacteria under dynamic field conditions in an agricultural environment.

Transfer and internalisation of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cabbage cultivated on contaminated manure-amended soil under tropical field conditions in Sub-Saharan Africa.

Surface contamination and internalisation of Escherichia coli O157:H7 and Salmonella Typhimurium in cabbage leaf tissues at harvest (120 days post-transplantation) following amendment of contaminated bovine manure to soil at different times during crop cultivation were investigated under tropical field conditions in the Central Agro-Ecological Zone of Uganda. Fresh bovine manure inoculated with rifampicin-resistant derivatives of non-virulent strains of E. coli O157:H7 and S. Typhimurium was incorporated into the soil to achieve inoculum concentrations of 4 and 7 log CFU/g at the point of transplantation, 56 or 105 days post-transplantation of cabbage seedlings. Frequent sampling of the soil enabled the accurate identification of the survival kinetics in soil, which could be described by the Double Weibull model in all but one of the cases. The persistence of 4 log CFU/g E. coli O157:H7 and S. Typhimurium in the soil was limited, i.e. only inocula applied 105 days post-transplantation were still present at harvest. Moreover, no internalisation in cabbage leaf tissues was observed. In contrast, at the 7 log CFU/g inoculum level, E. coli O157:H7 and S. Typhimurium survived in the soil throughout the cultivation period. All plants (18/18) examined for leaf contamination were positive for E. coli O157:H7 at harvest irrespective of the time of manure application. A similar incidence of leaf contamination was found for S. Typhimurium. On the other hand, only plants (18/18) cultivated on soil amended with contaminated manure at the point of transplantation showed internalised E. coli O157:H7 and S. Typhimurium at harvest. These results demonstrate that under tropical field conditions, the risk of surface contamination and internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest depend on the inoculum concentration and the time of manure application. Moreover, the internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest seems to be limited to the worst case situation, i.e., when highly contaminated manure is introduced into the soil at the time of transplantation of cabbage seedlings.

The importance of expressing antimicrobial agents on water basis in growth/no growth interface models: a case study for Zygosaccharomyces bailii.

In a previous study on Zygosaccharomyces bailii, three growth/no growth models have been developed, predicting growth probability of the yeast at different conditions typical for acidified foods (Dang, T.D.T., Mertens, L., Vermeulen, A., Geeraerd, A.H., Van Impe, J.F., Debevere, J., Devlieghere, F., 2010. Modeling the growth/no growth boundary of Z. bailii in acidic conditions: A contribution to the alternative method to preserve foods without using chemical preservatives. International Journal of Food Microbiology 137, 1-12). In these broth-based models, the variables were pH, water activity and acetic acid, with acetic acid concentration expressed in volume % on the total culture medium (i.e., broth). To continue the previous study, validation experiments were performed for 15 selected combinations of intrinsic factors to assess the performance of the model at 22°C (60days) in a real food product (ketchup). Although the majority of experimental results were consistent, some remarkable deviations between prediction and validation were observed, e.g., Z. bailii growth occurred in conditions where almost no growth had been predicted. A thorough investigation revealed that the difference between two ways of expressing acetic acid concentration (i.e., on broth basis and on water basis) is rather significant, particularly for media containing high amounts of dry matter. Consequently, the use of broth-based concentrations in the models was not appropriate. Three models with acetic acid concentration expressed on water basis were established and it was observed that predictions by these models well matched the validation results; therefore a "systematic error" in broth-based models was recognized. In practice, quantities of antimicrobial agents are often calculated based on the water content of food products. Hence, to assure reliable predictions and facilitate the application of models (developed from lab media with high dry matter contents), it is important to express antimicrobial agents' concentrations on a common basis-the water content. Reviews over other published growth/no growth models in literature are carried out and expressions of the stress factors' concentrations (on broth basis) found in these models confirm this finding.

Quantification of the influence of trimethylamine-N-oxide (TMAO) on the heat resistance of Escherichia coli K12 at lethal temperatures.

AIM: To quantify the influence of trimethylamine-N-oxide (TMAO) on the heat resistance of Escherichia coli K12 MG1655 cells at static temperatures. METHODS AND RESULTS: Stationary-phase E. coli cells were inactivated at 52, 54 and 58°C. The heat resistance is described as reduction in the inactivation rate, k(max) , and/or an increase in the time for one decimal reduction, D, and/or an increase in the time for the fourth decimal reduction, t(4D) . CONCLUSIONS: Resistance of E. coli changed - increased - at all temperatures under study. Generally, the addition of TMAO to the growth medium protected E. coli cells, leading to an increase in their heat resistance, i.e. reduced k(max) and increased D and t(4D) values are obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional knowledge on the reaction of E. coli to heat in the presence of the organic osmolyte TMAO at lethal temperatures is provided. This work contributes to an improved understanding of the level of the resistance of bacteria to heat in the presence of osmolytes.

The development of Escherichia coli and Listeria monocytogenes variants resistant to high-pressure carbon dioxide inactivation.

AIMS: The objective of this study was to investigate whether bacterial cells could develop resistance (as a part of their adaptation strategy) to high-pressure CO(2) (HPCD) inactivation. METHODS AND RESULTS: Alternating cycles of exposure to pressurized CO(2) (10.5 MPa, 35 degrees C, 400 min(-1), 70% working volume ratio during 10 min) and re-growth of the surviving subpopulation were used to investigate possible increases in the resistance of Escherichia coli and Listeria monocytogenes to HPCD. The results show an increased resistance of both pathogens tested after seven cycles of inactivation. Increase in the resistance after 15 cycles resulted in a difference of 2.4 log CFU ml(-1) in log N(0)/N(i) when parental (N(0)) and treated cultures (N(i)) of E. coli and L. monocytogenes were compared. CONCLUSIONS: Current findings indicate the ability of micro-organisms to adapt to HPCD preservation technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence of HPCD-resistant micro-organisms could pose a new hazard to the safety and stability of HPCD-processed foods.

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment.

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35-45 degrees C), pressure (10.5-21.0 MPa) and treatment time (0-60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.

Estimating distributions out of qualitative and (semi)quantitative microbiological contamination data for use in risk assessment.

A framework using maximum likelihood estimation (MLE) is used to fit a probability distribution to a set of qualitative (e.g., absence in 25 g), semi-quantitative (e.g., presence in 25 g and absence in 1g) and/or quantitative test results (e.g., 10 CFU/g). Uncertainty about the parameters of the variability distribution is characterized through a non-parametric bootstrapping method. The resulting distribution function can be used as an input for a second order Monte Carlo simulation in quantitative risk assessment. As an illustration, the method is applied to two sets of in silico generated data. It is demonstrated that correct interpretation of data results in an accurate representation of the contamination level distribution. Subsequently, two case studies are analyzed, namely (i) quantitative analyses of Campylobacter spp. in food samples with nondetects, and (ii) combined quantitative, qualitative, semiquantitative analyses and nondetects of Listeria monocytogenes in smoked fish samples. The first of these case studies is also used to illustrate what the influence is of the limit of quantification, measurement error, and the number of samples included in the data set. Application of these techniques offers a way for meta-analysis of the many relevant yet diverse data sets that are available in literature and (inter)national reports of surveillance or baseline surveys, therefore increases the information input of a risk assessment and, by consequence, the correctness of the outcome of the risk assessment.

On the selection of relevant environmental factors to predict microbial dynamics in solidified media.

Several studies have shown that food structure causes slower growth rates and narrower growth boundaries of bacteria compared to laboratory media. In predictive microbiology, both a(w) or corresponding solute concentration (mainly NaCl) have been used as a growth influencing factor for kinetic models or growth/no growth interface models. The majority of these models have been based on data generated in liquid broth media with NaCl as the predominant a(w) influencing solute. However, in complex food systems, other a(w) influencing components might be present, next to NaCl. In this study, the growth rate of Salmonella typhimurium was studied in the growth region and the growth/no growth response was tested in Tryptic Soy Broth at 20 degrees C at varying gelatin concentration (0, 10, 50 g L(-1) gelatin), pH (3.25-5.5) and water activity (a(w)) (0.929-0.996). From the viewpoint of water activity, the results suggest that NaCl is the main a(w) affecting compound. However, gelatin seemed to have an effect on medium a(w) too. Moreover, there is also an interaction effect between NaCl and gelatin. From the microbial viewpoint, the results confirmed that the a(w) decreasing effect of gelatin is less harmful to cells than the effect of Na(+) ions. The unexpected shift of the growth/no growth interface to more severe conditions when going from a liquid medium to a medium with 10 g L(-1) gelatin is more pronounced when formulating the models in terms of a(w) than in terms of NaCl concentrations. At 50 g L(-1) gelatin, the model factored with NaCl concentration shifts to milder conditions (concordant to literature results) while the model with a(w) indicates a further shift to more severe conditions, which is due to the water activity lowering effect of gelatin and the interaction between gelatin and NaCl. The results suggest that solute concentration should be used instead of a(w), both for kinetic models in the growth region and for growth/no growth interface models, if the transferability of models to solid foods is to be increased.

Modelling the growth/no growth boundary of Zygosaccharomyces bailii in acidic conditions: a contribution to the alternative method to preserve foods without using chemical preservatives.

The aim of the study was to develop mathematical models describing growth/no growth (G/NG) boundaries of the highly resistant food spoilage yeast-Zygosaccharomyces bailii-in different environmental conditions, taking acidified sauces as the target product. By applying these models, the stability of products with characteristics within the investigated pH, a(w) and acetic acid ranges can be evaluated. Besides, the well-defined no growth regions can be used in the development of guidelines regarding formulation of new shelf-stable foods without using chemical preservatives, which would facilitate the innovation of additive-free products. Experiments were performed at different temperatures and periods (22 degrees C for 45 and 60days, 30 degrees C for 45days) in 150 modified Sabouraud media characterized by high amount of sugars (glucose and fructose, 15% (w/v)), acetic acid (0.0-2.5% (v/v), 6 levels), pH (3.0-5.0, 5 levels) and a(w) (0.93-0.97, 5 levels). These time and temperature combinations were chosen as they are commonly applied for shelf-stable foods. The media were inoculated with ca. 4.5 log CFU/ml and yeast growth was monitored daily using optical density measurements. Every condition was examined in 20 replicates in order to yield accurate growth probabilities. Three separate ordinary logistic regression models were developed for different tested temperatures and incubation time. The total acetic acid concentration was considered as variable for all models. In general, when one intrinsic inhibitory factor became more stringent, the G/NG boundary shifted to less stressful conditions of the other two factors, resulting in enlarged no growth zones. Abrupt changes of growth probability often occurred around the transition zones (between growth and no growth regions), which indicates that minor variations in environmental conditions near the G/NG boundaries can cause a significant impact on the growth probability. When comparing growth after 45days between the two tested temperatures, an unexpected phenomenon was observed: the no growth region at 30 degrees C was larger than the one at 22 degrees C, though it is known that 30 degrees C is the optimal growth temperature for Z. bailii. These results show that lowering temperature does not always lead to a reduced growth of the yeast (i.e. more stable foods) and storing shelf-stable products at the higher temperature (30 degrees C) is not always the worst case. In addition, at 22 degrees C, there was no significant difference in no growth zones between the two incubation periods (45 and 60days), implying that the no growth zones remain unchanged if the experimental time is sufficiently long.

Design of an experimental viscoelastic food model system for studying Zygosaccharomyces bailii spoilage in acidic sauces.

Within the field of predictive microbiology, the number of studies that quantify the effect of food structure on microbial behavior is very limited. This is mainly due to impracticalities related to the use of a nonliquid growth medium. In this study, an experimental food model system for studying yeast spoilage in acid sauces was developed by selecting a suitable thickening/gelling agent. In a first step, a variety of thickening/gelling agents was screened, with respect to the main physicochemical (pH, water activity, and acetic acid and sugar concentrations) and rheological (weak gel viscoelastic behavior and presence of a yield stress) characteristics of acid sauces. Second, the rheological behavior of the selected thickening/gelling agent, Carbopol 980, was extensively studied within the following range of conditions: pH 4.0 to 5.0, acetic acid concentration of 0 to 1.0% (vol/vol), glycerol concentration of 0 to 15% (wt/vol), and Carbopol concentration of 1.0 to 1.5% (wt/vol). Finally, the applicability of the model system was illustrated by performing growth experiments in microtiter plates for Zygosaccharomyces bailii at 0, 0.5, 1.0, and 1.5% (wt/vol) Carbopol, 5% (wt/vol) glycerol, 0% (vol/vol) acetic acid, and pH 5.0. A shift from planktonic growth to growth in colonies was observed when the Carbopol concentration increased from 0.5 to 1.0%. The applicability of the model system was illustrated by estimating mu(max) at 0.5% Carbopol from absorbance detection times.