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International audience; The apparent heat-resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis at 45°C and 20°C. B. weihenstephanensis spores were then heat-treated at 85°C, 90°C and 95°C and B. licheniformis spores at 95°C, 100°C and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis, 15°C to 60°C for B. licheniformis or a range of pH (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a low effect on the apparent heat resistance, except very near recovery boundaries. In contrast a decrease in the recovery pH had a progressive impact on apparent heat-resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions, was highly satisfactory.

National audience

Persistent bacteria in processing sites are of great concern in food industry, causing continuous recontamination and thus yielding compromised food quality and safety, lost productivity and environmental issues. The aim of this study was to develop a tool to reproduce bioadhesion under industrial conditions in order to investigate the impact of successive stress exposure on adhering microflora and further optimize food formulation, industrial process and surface sanitation.A pilot scale loop system was used to reproduce adhering flora after circulation of liquid egg in stainless steel pipeline at refrigerated temperature. Inoculation of the system was performed either by the circulation of non-pasteurized liquid egg or by the introduction of contaminated probe surface or insert. After 9 days of continuous flow, stainless steel inserts and sections were removed and analyzed with various conditions of incubation. The identification of culturable (ana)aerobe psychrotrophic and mesophilic isolates was performed by 16SrDNA sequencing. Moreover, the detection of Bacillus species was also performed using the PCR based GeneDisc® plateform.After loop circulation of non pasteurized liquid egg, bacterial isolates recovered from stainless steel surfaces were mainly composed of Gram-negative bacteria such as Pseudomonas, Myroides and Aeromonas. Enterobacteria and lactic acid bacteria were as well identified but with a lower extend, while Bacillus species were only detected by GeneDisc® plate. When the loop system was inoculated with an insert preliminary left for 2 months in an industrial pipeline, the proportion of recovered Gram-positive flora was higher. Indeed, Corynebacterium, Enterococcus and Staphylococcus were identified from industrial surfaces.Even though this system might favour fast growing colonizers, correlation was found between bacterial diversity recovered from pilot scale system and industrial pipeline surfaces. It is interesting to note a shift of diversity recovered from surfaces when industrial surfaces were used to inoculate the loop, suggesting the survival and persistence of given species after consecutive steps of production/sanitation. This system is promising to further study bacterial removal and inactivation upon exposure to acid or biocide taking into account protection due to embedded structure or food residues.

International audience; The genetic diversity of two major yeast species found in cheese, Debaryomyces hansenii and Kluyveromyces marxianus, was analyzed within the yeast flora in French traditional cheesemaking. Based on the amplification of sequences separating long terminal repeat (LTR) retrotransposon sequences, a molecular typing method was developed for D. hansenii and K. marxianus. This method was applied to a total of 56 D. hansenii strains and 61 K. marxianus strains, mostly isolated during fermentation and ripening of traditional cheese from Normandy and Haute-Savoie (French Alps) regions. A total of 32 and 43 robust profiles were obtained for D. hansenii and K. marxianus, respectively. Cluster analysis confirmed the large genetic diversity already shown for D. hansenii and revealed an even larger diversity for K. marxianus. After its use with Saccharomyces cerevisiae, the inter-LTR PCR proved to be efficient to discriminate between strains of the two species, D. hansenii and K. marxianus, isolated from the same ecological niches, confirming the high intra-specific variability of species found in cheese. This strain typing could not correlate the analyzed strains with their origin, would it be the cheese type, the cheese-making facility or the cheese batch, showing a high discrimination power. The method described here will provide a fast and reliable tool for the biodiversity study of these two major cheese yeasts.; La diversité génétique de deux levures majeures des fromages, Debaryomyces hansenii et Kluyveromyces marxianus, a été analysée dans la flore-levures de fromages français traditionnels. Basée sur l'amplification des séquences qui séparent les " Long Terminal Repeats " des rétrotransposons (LTR), une méthode de typage moléculaire a été développée pour D. hansenii et K. marxianus. La méthode a été appliquée à 56 souches de D. hansenii et 61 souches de K. marxianus, pour la plupart isolées pendant l'affinage de fromages traditionnels de Normandie et de Haute-Savoie. Un total de 32 et 43 profils robustes a été obtenu pour D. hansenii et K. marxianus, respectivement. Une analyse hiérarchique a confirmé la grande diversité génétique déjà observée pour D. hansenii et a révélé une grande diversité chez K. marxianus. Apres son utilisation chez Saccharomyces cerevisiae, la PCR inter-LTR s'est montrée très efficace pour discriminer entre isolats de D. hansenii et K. marxianus isolés des mêmes niches écologiques, confirmant ainsi la grande variabilité intra-spécifique des espèces présentes dans le fromage. Le typage de ces souches n'a pu être corrélé à la provenance des souches, que ce soit avec le type de fromage, la fromagerie ou le lot, indiquant un grand pouvoir de discrimination lors de ce typage. La méthode développée ici apporte un outil rapide et robuste pour étudier la biodiversité de ces deux espèces.

International audience; Several years ago, microbiologists developed a harmonized standard for the validation of alternative methods against the reference method, namely International Organization for Standardization (ISO) 16140:2003. Since then a new and better statistical approach known as the accuracy profile concept has been proposed for interpretation of data collected during a validation study. This approach defines acceptability limits that are used to verify that an alternative analytical method can produce results acceptable for a defined analytical objective. This paper demonstrates how the accuracy profile approach can be efficiently applied to any data set collected according to ISO guidelines and how to interpret the final calculations to decide if a method is valid or not. The procedure was applied to data obtained in the validation of a commercial kit for the enumeration of Enterobacteriaceae in foods. A comparison of several statistical outputs derived from application of the accuracy profile demonstrated that this simple numerical method can be applied despite the non-normal distribution of bacterial colony counts.

Dimensions du poster : * Collation : 1 p.

Détection et quantification des micro-organismes par amplification génique in vitro (PCR) en temps réel

International audience