Development of a Cryptosporidium-arsenic multi-risk assessment model for infant formula prepared with tap water in France.

Tap water is used in France to reconstitute powder infant formula, although it is not sterile and possibly contaminated by microbiological and chemical hazards. The present study aims to quantify risks of using tap water in France for the preparation of infant formula, during the first six months of life. Cryptosporidium and arsenic were selected as hazards of greatest concern in microbiology and chemistry, respectively. A probabilistic model was developed using French (when available) and European (alternatively) data. Second order Monte Carlo simulation was used to separate uncertainty and variability of inputs. Outputs were expressed at the individual level as probability of illness and at the population level, using a common metric, the DALY (Disability Adjusted Life Year). Two scenarios of milk preparation were considered: with un-boiled or boiled tap water. Consuming infant formula rehydrated with un-boiled tap water during the first six months of life led to a total of 2250 DALYs per 100,000 infants (90% uncertainty interval [960; 7650]) for Cryptosporidium due to diarrhea, and 1 DALY [0.4; 2] for arsenic due to expected lifetime risk of lung and bladder cancer as a result of early exposure in life. For the entire population, boiling water would suppress the risk from Cryptosporidium. In contrast, the incremental cancer risk was low at the population level but elevated for 5% of the population exposed to high levels of arsenic. A stringent monitoring of tap water supply points should be continued. This multi-risk assessment model could help public health authorities and managers in evaluating both microbiological and chemical safety issues associated with using infant formula prepared with tap water.

Multi-criteria framework as an innovative tradeoff approach to determine the shelf-life of high pressure-treated poultry.

A multi-criteria framework combining safety, hygiene and sensorial quality was developed to investigate the possibility of extending the shelf-life and/or removing lactate by applying High Hydrostatic Pressure (HHP) in a ready-to-cook (RTC) poultry product. For this purpose, Salmonella and Listeria monocytogenes were considered as safety indicators and Escherichia coli as hygienic indicator. Predictive modeling was used to determine the influence of HHP and lactate concentration on microbial growth and survival of these indicators. To that end, probabilistic assessment exposure models developed in a previous study (Lerasle, M., Guillou, S., Simonin, H., Anthoine, V., Chéret, R., Federighi, M., Membré, J.M. 2014. Assessment of Salmonella and L. monocytogenes level in ready-to-cook poultry meat: Effect of various high pressure treatments and potassium lactate concentrations. International Journal of Food Microbiology 186, 74-83) were used for L. monocytogenes and Salmonella. Besides, for E. coli, an exposure assessment model was built by modeling data from challenge-test experiments. Finally, sensory tests and color measurements were performed to evaluate the effect of HHP on the organoleptic quality of an RTC product. Quantitative rules of decision based on safety, hygienic and organoleptic criteria were set. Hygienic and safety criteria were associated with probability to exceed maximum contamination levels of L. monocytogenes, Salmonella and E. coli at the end of the shelf-life whereas organoleptic criteria corresponded to absence of statistical difference between pressurized and unpressurized products. A tradeoff between safety and hygienic risk, color and taste, was then applied to define process and formulation enabling shelf-life extension. In the resulting operating window, one condition was experimentally assayed on naturally contaminated RTC products to validate the multi-criteria approach. As a conclusion, the framework was validated; it was possible to extend the shelf-life of an RTC poultry product containing 1.8% (w/w) lactate by one week, despite slight color alteration. This approach could be profitably implemented by food processors as a decision support tool for shelf-life determination.

Biodiversity of spoilage lactobacilli: phenotypic characterisation.

Preventing food spoilage is a challenge for the food industry, especially when applying mild preservation methods and when avoiding the use of preservatives. Therefore, it is essential to explore the boundaries of preservation by better understanding the causative microbes, their phenotypic behaviour and their genetic makeup. Traditionally in food microbiology, single strains or small sets of selected strains are studied. Here a collection of 120 strains of 6 different spoilage related Lactobacillus species and a multitude of sources was prepared and their growth characteristics determined in 384-well plates by optical density measurements (OD) over 20 days, for 20 carbon source-related phenotypic parameters and 25 preservation-related phenotypic parameters. Growth under all conditions was highly strain specific and there was no correlation of phenotypes at the species level. On average Lactobacillus brevis strains were amongst the most robust whereas Lactobacillus fructivorans strains had a much narrower growth range. The biodiversity data allowed the definition of preservation boundaries on the basis of the number of Lactobacillus strains that reached a threshold OD, which is different from current methods that are based on growth ability or growth rate of a few selected strains. Genetic information on these microbes and a correlation study will improve the mechanistic understanding of preservation resistance and this will support the future development of superior screening and preservation methods.

Assessment of Salmonella and Listeria monocytogenes level in ready-to-cook poultry meat: effect of various high pressure treatments and potassium lactate concentrations.

The objective of this study was to develop a probabilistic model in order to determine the contamination level of Salmonella and Listeria monocytogenes in ready-to-cook poultry meat, after a high pressure (HP) treatment. The model included four steps: i) Reception of raw meat materials, mincing and mixing meat, ii) Partitioning and packaging into 200-g modified atmosphere packs, iii) High pressure treatment of the meat, and iv) Storage in chilled conditions until the end of the shelf-life. The model excluded the cooking step and consumption at consumer's home as cooking practices and heating times are highly variable. The initial contamination level of Salmonella and L. monocytogenes was determined using data collected in meat primary processing plants. The effect of HP treatment and potassium lactate on microbial reduction was assessed in minced meat, using a full factorial design with three high pressure treatments (200, 350 and 500 MPa), three holding times (2, 8 and 14 min) and two potassium lactate concentrations (0 or 1.8% w/w). The inactivation curves fitted with a Weibull model highlighted that the inactivation rate was significantly dependent on the HP treatment. From the literature, it was established that Salmonella was not able to grow in the presence of lactate, under modified atmosphere and chilled conditions whereas the growth of L. monocytogenes was determined using an existing model validated in poultry (available in Seafood Spoilage and Safety Predictor software, V. 3.1). Once implemented in the Excel add-in @Risk, the model was run using Monte Carlo simulation. The probability distribution of contamination levels was determined for various scenarios. For an average scenario such as an HP treatment of 350 MPa for 8 min, of 200 g minced meat containing 1.8% lactate (pH 6.1; aw 0.96), conditioned under 50% CO2, the prevalence rate of Salmonella and L. monocytogenes, after a 20-day storage at 6 °C was estimated to be 4.1% and 7.1%, respectively. The contamination level was low considering that the product is going to be cooked by the consumer afterwards: the 99th percentile of the distribution was equal to -2.3log cfu/g for Salmonella and 0.5log cfu/g for L. monocytogenes. More generally, the model developed here from raw material reception up to the end of the shelf-life enables to recommend combinations of HP treatment and lactate formulation to guarantee an acceptable microbial concentration before cooking.

A risk assessment approach applied to the growth of Erwinia carotovora in vegetable juice for variable temperature conditions.

Risk assessment for food spoilage relies on probabilistic models of microbial growth to predict the likelihood that microbial populations will exceed predefined spoilage levels. To assist in the design and management of industrial food quality systems, predictive microbiological models have to incorporate major risk factors such as the variability in the microbial strain, environment and initial contamination levels. In addition, the application of results measured under laboratory conditions to the less controlled environment of an industrial process usually also involves uncertainty. Extra information regarding this uncertainty must be factored into industrial microbial risk assessment. In this paper, based on our previous analysis of the growth of Erwinia carotovora we show how different factors contribute to the risk of microbial spoilage of vegetable juice and we demonstrate an effective way of including these factors into risk assessment models. The association of risk components with different unavoidable and manageable factors is also valuable for the development of optimal strategies for reducing microbial risk.

A probabilistic modeling approach in thermal inactivation: estimation of postprocess Bacillus cereus spore prevalence and concentration.

The survival of spore-forming bacteria is linked to the safety and stability of refrigerated processed foods of extended durability (REPFEDs). A probabilistic modeling approach was used to assess the prevalence and concentration of Bacillus cereus spores surviving heat treatment for a semiliquid chilled food product. This product received heat treatment to inactivate nonproteolytic Clostridium botulinum during manufacture and was designed to be kept at refrigerator temperature postmanufacture. As key inputs for the modeling, the assessment took into consideration the following factors: (i) contamination frequency (prevalence) and level (concentration) of both psychrotrophic and mesophilic strains of B. cereus, (ii) heat resistance of both types (expressed as decimal reduction times at 90 degrees C), and (iii) intrapouch variability of thermal kinetics during heat processing (expressed as the time spent at 90 degrees C). These three inputs were established as statistical distributions using expert opinion, literature data, and specific modeling, respectively. They were analyzed in a probabilistic model in which the outputs, expressed as distributions as well, were the proportion of the contaminated pouches (the likely prevalence) and the number of spores in the contaminated pouches (the likely concentration). The prevalence after thermal processing was estimated to be 11 and 49% for psychrotrophic and mesophilic strains, respectively. In the positive pouches, the bacterial concentration (considering psychrotrophic and mesophilic strains combined) was estimated to be 30 CFU/g (95th percentile). Such a probabilistic approach seems promising to help in (i) optimizing heat processes, (ii) identifying which key factor(s) to control, and (iii) providing information for subsequent assessment of B. cereus resuscitation and growth.

The "Sym'Previus" software, a tool to support decisions to the foodstuff safety.

Describing the Sym'Previus project, the software and its deliverable facilities is the aim of this present paper. This software concerns all the partners of the food industry who are involved in the management of food safety and allows food-borne pathogen behaviour in food to be predicted, as function of the environment (nature of the food, manufacturing process, conditions of conservation). This analysis of microbial behaviour has been possible thanks to the progress made in predictive microbiology since the 1980s. Sym'Previus offers to food industry professionals and their partners the possibility of applying this progress, by giving access to a database, to simulation systems and expertise.

Partitioning of the variance in the growth parameters of Erwinia carotovora on vegetable products.

The objective of this paper was to estimate and partition the variability in the microbial growth model parameters describing the growth of Erwinia carotovora on pasteurised and non-pasteurised vegetable juice from laboratory experiments performed under different temperature-varying conditions. We partitioned the model parameter variance and covariance components into effects due to temperature profile and replicate using a maximum likelihood technique. Temperature profile and replicate were treated as random effects and the food substrate was treated as a fixed effect. The replicate variance component was small indicating a high level of control in this experiment. Our analysis of the combined E. carotovora growth data sets used the Baranyi primary microbial growth model along with the Ratkowsky secondary growth model. The variability in the microbial growth parameters estimated from these microbial growth experiments is essential for predicting the mean and variance through time of the E. carotovora population size in a product supply chain and is the basis for microbiological risk assessment and food product shelf-life estimation. The variance partitioning made here also assists in the management of optimal product distribution networks by identifying elements of the supply chain contributing most to product variability.

Metabolic characteristics of the faecal microflora in humans from three age groups.

BACKGROUND: Little is known about the influence of the ageing process on the bacterial metabolism. The aim of this work was to study the inter-individual variation and the influence of age on metabolic characteristics of the faecal microflora from persons of three age groups. METHODS: Nine enzyme activities and 11 metabolites were analysed in faecal samples of 21 elderly persons (69-89 years of age), 15 adults (30-46 years of age) and 14 children (3-15 years of age). RESULTS: They provide a pattern of the faecal microflora metabolism in a French population aged between 3 and 89 years. They showed large inter-individual differences in the three groups. Although the enzyme activities were not significantly different between groups, glycolytic activities appeared less variable in adults than in elderly persons and children. The major metabolites (acetate, propionate, butyrate and L-lactate) resulting from carbohydrate fermentation were similar in the three groups. Samples from elderly persons were characterized by higher D/L lactate ratio and higher concentrations of metabolites resulting from protein fermentation (ammonia, valerate, iso-butyrate and iso-valerate). CONCLUSION: The data showed significant differences between elderly persons and younger adults and children, but the major metabolic characteristics of the faecal microflora were not greatly altered by the ageing process.

Predictive modelling approach applied to spoilage fungi: growth of Penicillium brevicompactum on solid media.

Growth of Penicillium brevicompactum was examined on five solid media. Fungal growth was established by diameter measurements up to 50 days. Seventy experimental curves were fitted by Baranyi's primary predictive model. The growth rates were then analysed by non-parametric statistical methods. Penicillium brevicompactum could colonize the surface of solid media containing up to 700 g l-1 of sugar (50% glucose-50% fructose) with a growth rate of 0.9 mm day-1 (median values). Fitting curves by non-linear models followed by a non-parametric multiple comparison seems to be a convenient method for detecting differences in fungal growth on solid media. These two methods would be useful for studying fungal spoilage of bakery products with intermediate water activity.

Combined effects of pH and sugar on growth rate of Zygosaccharomyces rouxii, a bakery product spoilage yeast.

The effects of citric acid-modified pH (pH 2.5, 2.75, 3, 3.5, 4, 4.5, 5, and 5.5) and a 30% glucose-70% sucrose mixture (300, 400, 500, 600, 700, 800, 875, and 900 g/liter) on an osmophilic yeast, Zygosaccharomyces rouxii, were determined by using synthetic medium. One hundred experiments were carried out; 50-ml culture flasks were inoculated with 10(3) CFU ml(-1) by using a collection strain and a wild-type strain cocktail. The biomass was measured by counting cell colonies, and growth curves were fitted by using a Baranyi equation. The growth rate decreased linearly with sugar concentration, while the effect of pH was nonlinear. Indeed, the optimal pH range was found to be pH 3.5 to 5, and pH 2.5 resulted in a 30% reduction in the growth rate. Finally, we evaluated the performance of two nonlinear predictive models developed previously to describe bacterial contamination. Equations derived from the Rosso and Ratkowsky models gave similar results; however, the model that included dimensionless terms based on the Ratkowsky equation was preferred because it contained fewer estimated parameters and also because biological interpretation of the results was easier.

Behaviour of Listeria monocytogenes under combined chilling processes.

The behaviour of Listeria monocytogenes under chilling processes was investigated. Growth kinetics were measured at 7 degrees C in TSBYE culture medium as a function of pH (7.2 and 6.2), pre-incubation temperatures (4 or 7 degrees C), cooling (0.05 or 0.1 degree C min-1) and freezing (0 and -5 degrees C) treatments. Growth curves generated were fitted by Gompertz and Baranyi functions. The Baranyi function gave better parameter estimation values than the Gompertz equation which over-estimated the specific growth rate values. Listeria monocytogenes grew at 7 degrees C without a lag phase, except when the sub-culture was performed at 37 degrees C, whereas the specific growth rate was affected by the chilling processes. In fact, L. monocytogenes grew slightly faster at 7 degrees C when a 4 degrees C pre-incubation treatment was applied than with a 7 degrees C pre-incubation treatment. These results suggest that to mimic the processes of contamination in industry, predictive microbiology studies with L. monocytogenes should be performed with organisms cultured at low temperatures.

Behavior of Listeria spp. in smoked fish products affected by liquid smoke, NaCl concentration, and temperature.

The growth, survival, and death of Listeria monocytogenes were studied in a synthetic medium as a function of temperature, NaCl content, and amount of liquid smoke, and the findings were validated in smoked fish products. The smoke preservative compound was simulated by adding liquid smoke, and the concentration was expressed as phenol concentration. The growth of L. monocytogenes was limited at a temperature as low as 4 degrees C or at a phenol concentration as high as 20 ppm. The predicted values were obtained using a mathematical model established in liquid medium in a previous study. They accurately fit values observed in L. monocytogenes challenge tests on smoked fish. After 21 days of storage the deviation between the predicted and experimental values was within 0.5 log for 60% of the data. This model may be useful in predicting Listeria contamination in smoked fish. Moreover, this study emphasizes the importance of phenol concentration to control the growth of Listeria spp. in smoked food products.

Degradation of pectic compounds during pasteurised vegetable juice spoilage by Chryseomonas luteola: a predictive microbiology approach.

Predictive modelling consists in describing effects of environmental factors on microbial growth parameters. With food spoilage bacteria, this approach must be extended to both growth and food damage characterisation. In order to study the incidence of storage temperature on vegetable damage, using predictive microbiology tools, kinetics of pectic compound degradation were studied. Chryseomonas luteola has been chosen because of its ability to grow on post-harvested vegetables. Experiments were performed at refrigerated temperatures (0-10 degrees C) with low initial bacterial charges (10(1)-10(3.5) cfu/ml). Microbial specific growth rate (mu), stability phase before pectic degradation (Sp) and alteration percentage (Ap) were chosen as reference parameters. Then, sub-optimal temperature effects on these three parameters were estimated using modified Ratkowsky model. Results obtained in synthetic medium were compared with data observed in endive juice to appreciate the alteration of vegetable during post-harvest storage.

Physiology and development of Pectinatus cerevisiiphilus and Pectinatus frisingensis, two strict anaerobic beer spoilage bacteria.

The genus Pectinatus was isolated recently and the deposited strains were classified as beer spoilage bacteria producing propionate as a major fermentation product. A recent investigation of this genus demonstrated the existence of two species: Pectinatus cerevisiiphilus, the type strain and Pectinatus frisingensis, a new species with a different pattern of growth substrates. Different culture media tested for both species demonstrated a higher specific growth rate for P. cerevisiiphilus. However, final biomass production was in every case around 20% higher in P. frisingensis. A 400% decrease of final biomass production was measured when the species were cultivated on poor culture medium; this decrease was found to be broadly proportional to amounts of acetate excreted in the medium. Both species produced CO2 from glucose; however, no significant modifications of biomass and volatile fatty acid production were demonstrated when varying head space composition with regard to CO2 levels. Growth experiments on glucose with increasing amounts of ethanol added in the culture, revealed a higher sensitivity of P. cerevisiiphilus to ethanol inhibition. Ethanol concentrations over 1.7 M resulted in a complete inhibition of growth for both Pectinatus species. Combined effects of culture medium pH, lactate, and glucose concentrations, demonstrated the prevailing role of glucose in the development of the bacteria. However, these three parameters had a different influence on growth characteristics of both Pectinatus species. P cerevisiiphilus grew very poorly with a glucose concentration of 5 mM for pH values below 4.1. This species had optimal pH for growth between 6 and 6.2 and it excreted increasing amounts of acetate with increase of pH, glucose or lactate in the culture medium. P. frisingensis showed a wide range of pH allowing a good growth. For glucose concentrations below 20 mM, highest final biomass productions were measured in the culture for pH values around 4.9; this also corresponded to a minimum in acetate excretion. The above results pointed at P. frisingensis as the prevailing species of Pectinatus in beer spoilage.

Modelling the growth, survival and death of Listeria monocytogenes.

In this paper, the predictive microbiology approach has been generalized to the study of growth, survival and death of Listeria monocytogenes. As this micro-organism is involved in food poisoning, its growth, survival and death were studied as functions of low temperatures, NaCl and phenol compounds, in a synthetic medium, by a factorially designed experiment. A significant inactivation of L. monocytogenes was obtained with 20 ppm of phenol and 4% (w/v) NaCl at temperatures from 4 to 12 degrees C. An empirical model is proposed to describe, in a single step, the biomass profile vs studied factors. Thereby, the influence of temperature, NaCl and phenol concentration on L. monocytogenes biomass quantity (0.5-8 log cfu ml(-1)) are presented as a function of storage duration. The comparisons of the proposed model with existing models (Gompertz for growth, vitalistic for survival and death) were performed. The use of a single equation allows the prediction of contamination levels in all experimental conditions without knowledge a priori. The model offers considerable prospects for its use in food microbiology.

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach.

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N2, H2 or 20% CO2-containing atmosphere. Combinations of culture medium pH values from pH 3 x 9 to pH 7 x 2, of glucose levels between 5 and 55 mmol l-1, and of lactate concentrations varied from 4 to 40 mmol l-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6 x 2 for P. cerevisiiphilus, and between pH 4 x 5 and pH 4 x 9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1 x 3 mol l-1 ethanol for P. cerevisiiphilus, and at 1 x 8 mol l-1 for P. frisingensis.

Statistical approach for comparison of the growth rates of five strains of Staphylococcus aureus.

The interaction of temperature (10, 14, 25, 31, and 37 degrees C), pH (pH 5, 5.6, 6.5, 7.4, and 8), and NaCl (0, 2, 5, 8, and 10%) in a laboratory medium affects the specific growth of Staphylococcus aureus. From growth curves obtained by the turbidimetric technique, a nonlinear model in which the specific growth rate (mu) is fitted directly, without data transformation and with the residual error variations taken into account, is proposed. This model correctly fits experimental data and gives more biological information than the quadratic polynomial model. Moreover, the comparison of five strains of S. aureus was performed by a principal-component analysis in which the specific growth rate was the identifying characteristic for S. aureus strains. The results obtained from model coefficient comparison among the five strains and from multivariate data analysis allow the same classification of strains to be performed. Two of them have similar behaviors during food spoilage, two others could be distinguished by their capacity to grow at a low temperature, whereas the last one was markedly different from the others.

Effects of Temperature, pH, and NaCl on Growth and Pectinolytic Activity of Pseudomonas marginalis.

The interaction of temperature (4, 10, 18, and 30 degrees C), pH (6, 7, and 8), and NaCl (0, 2.5, and 5%) and their effects on specific growth rate, lag phase, and pectinolytic enzymes of Pseudomonas marginalis were evaluated. Response surface methodology was adapted to describe the response of growth parameters to environmental changes. To obtain good conditions of storage, the combined action of salt and temperature is necessary. At 4 degrees C with an NaCl concentration of 5% and a pH of 7, the lag time was 8 days and no growth was observed at 4 degrees C with 5% NaCl and a pH of 6. In the absence of salt, P. marginalis could grow regardless of temperature and pH. Pectate lyase and pectin lyase were produced by P. marginalis, while pectin methyl esterase activity was not observed in our culture conditions. The enzyme production depended on temperature, pH, and salt concentration but also on the age of the culture. Pectinolytic enzymes were abundantly excreted during the stationary phase, and even at 4 degrees C, after 2 weeks of storage, enzyme activities in supernatant culture were sufficient to damage vegetables. Both bacterial growth and enzymatic production have to be taken into account in order to estimate correctly the shelf life of vegetables.