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These De Doctorat De THESE DE DOCTORAT DE L'UNIVERSITE DE BRETAGNE OCCIDENTALE COMUE UNIVERSITE BRETAGNE LOIRE ECOLE DOCTORALE N° 600 Ecole doctorale Ecologie, Géosciences, Agronomie et Alimentation Spécialité : Microbiologie Par Laura QUERO Développement de la spectrométrie de masse MALDI-TOF pour l’identification des champignons filamenteux d’intérêt alimentaire et étude de leur résistance aux molécules biocides Thèse présentée et soutenue à Plouzané, le 21 Décembre 2018 Unité de recherche : Laboratoire de Biodiversité et Ecologie Microbienne (EA 3882) Rapporteurs avant soutenance : Sabine GALINDO Professeur, UMR Quali’Sud, Polytech Montpellier Jean-Denis BAILLY Professeur, UMR INRA Toxalim, ENV de Toulouse Composition du Jury : Président : Emmanuel COTON Professeur, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, UBO Examinateurs : Sabine GALINDO Professeur, UMR Quali’Sud, Polytech Montpellier Anne-Gaëlle RANC Docteur, Institut des Agents Infectieux, Centre National de Référence des Légionelles, Centre National de Référence des Staphylocoques, CHU de Lyon Dir. de thèse : Jérôme MOUNIER Professeur, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, UBO Encadrants : Victoria GIRARD Docteur, bioMérieux, La Balme les Grottes Patrice NODET Docteur, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, UBO 2 Cette thèse CIFRE a été financée par bioMérieux et l’Association Nationale de la Recherche et de la Technologie (ANRT), en collaboration avec le Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (LUBEM) Convention #2015/0821 3 Remerciements Je tiens à adresser mes premier remerciements aux membres du jury pour avoir accepté de participer à l’évaluation de mon travail. Mes remerciements vont également aux organismes financeurs qui ont rendu ce projet possible : la société bioMérieux ainsi que l’ANRT. Je remercie le professeur Emmanuel Coton, directeur du LUBEM pour m’avoir permis de réaliser ma thèse au sein du laboratoire. Je remercie sincèrement mon directeur de thèse, le professeur Jérôme Mounier, pour m’avoir fait confiance, tout d’abord dans le cadre d’un stage mais surtout pour la réalisation de ce projet. Merci pour tes conseils et ton optimisme. Mes plus vifs remerciements à mon encadrante au sein de l’entreprise, le docteur Victoria Girard, pour ta disponibilité et pour la confiance que tu m’as accordée afin de mener ce projet à distance, j’espère en avoir été digne. Je remercie chaleureusement mes deux encadrants de thèse au sein du LUBEM, les docteurs Valérie Vasseur et Patrice Nodet, merci pour vos conseils toujours avisés et pour le temps que vous avez pu me consacrer. Mes remerciements vont également à toutes les personnes que j’ai pu cotoyer au sein du département R&D microbiologie de bioMérieux, et plus particulièrement à l’équipe VITEK MS : Valérie, Marie-Christine, Sophie, Delphine J., Delphine G., Laetitia et Nadine, merci de m’avoir si bien accueillie lors de mes séjour à La Balme et surtout de m’avoir transmis votre savoir, j’ai beaucoup appris à vos côtés, toujours dans la bonne humeur. Un grand merci à Sandrine, Béatrice et Priscillia pour tout le travail effectué sur l’analyse des données et pour votre disponibilité. Je souhaite aussi remercier tous les membres du LUBEM et de l’ESIAB, avec qui j’ai pu partager de bons moments, autour d’un repas, d’un café ou entre deux manips : Anne-Cécile, Christelle, Christophe, Dominique, Elisabeth, Florence, Franck, Gaetan B., Gaetan L.F., Jean-Luc, José, Karim, Laurence, Marie-Elisabeth, Marielle, Monika et Nolwenn. Un grand merci à Stella, pour ta disponibilité au quotidien qui facilite la vie des thésards. Une pensée spéciale à Audrey, merci 4 pour le travail que tu as effectué avec moi sur le projet et pour ta bonne humeur, et à Sylvie, merci de m’avoir laissée m’installer en soucho, j’espère que je ne t’ai pas trop envahie. Je souhaite aussi remercier Marc Le Romancer et Adeline Picot, pour m’avoir donné l’opportunité d’effectuer des missions d’enseignement. Enfin un grand merci à ma chère MHT, pour ta disponibilité et ton sens de l’humour, n’oublie pas de rester mauvaise mauvaise. Je remercie évidemment mes compagnons de fortune (ou d’infortune selon les jours), tous les doctorants que j’ai eu l’occasion de croiser lors de ces trois ans : Annie, Fabienne, Lucille, Marcia, Marie-Caroline, Nicolas. Guillaume, bon courage pour ta dernière année, j’ai aussi une pensée pour ceux qui commencent : Marion, Mélanie, Océane et Vincent. Un énorme merci à ceux qui sont devenus un peu plus que des collègues de travail, Amélie, merci pour tes conseils et ta confiance, je peux ouvrir ma propre collection de souches maintenant ! Merci aussi pour nos nombreuses discussions qui m’ont souvent aidée à relativiser et à décompresser. Marine et Maxence, merci pour cette dernière année riche en bons moments au labo comme en dehors, merci pour les fous-rires, les Maxime à peu près et les karaokés tard le soir. Une petite pensée pour Camille, qui a fait partie de la bande lors de son passage au LUBEM. Merci à ma famille, de m’avoir toujours laissée libre de choisir ma voie et de m’avoir soutenue dans tout ce que j’ai entrepris, je n’en serai jamais arrivée là sans vous. Enfin, merci à toi Jérémy d’avoir toujours été là pour me supporter, le meilleur est à venir. 5 Table des matières Liste des abréviations .............................................................................................................................. 7 Liste des figures ...................................................................................................................................... 8 Liste des tableaux .................................................................................................................................... 8 Introduction bibliographique .................................................................................................................. 9 I. Les champignons ...................................................................................................................... 10 1) Classification ......................................................................................................................... 10 2) Développement et reproduction ............................................................................................ 13 3) Rôle technologique ............................................................................................................... 14 II. Altérations fongiques des aliments ........................................................................................... 14 III. Identification des champignons filamenteux ........................................................................ 20 IV. Spectrométrie de masse MALDI-TOF .................................................................................. 24 V. Maîtrise des altérations fongiques ............................................................................................. 29 VI. Objectifs des travaux de thèse ............................................................................................... 35 Chapitre 1 : Développement et application de la spectrométrie de masse MALDI-TOF pour l’identification des champignons filamenteux d’altération ................................................................... 38 I. Résumé des travaux .................................................................................................................. 39 II. Article publié dans Food Microbiology .................................................................................... 43 III. Résultats supplémentaires: Evaluation des performances d’identification de champignons filamenteux par spectrométrie de masse MALDI-TOF après prélèvement directe de matériel biologique à partir d’aliments contaminés ........................................................................................ 61 Chapitre 2 : Application de la spectrométrie de masse MALDI-TOF à la différenciation de complexes d’espèces et au typage de champignons filamenteux d’altération – Application aux espèces appartenant à Aspergillus section Flavi et à Penicillium roqueforti ..................................................... 64 I. Résumé des travaux .................................................................................................................. 65 II. Article à soumettre dans International Journal of Food Microbiology .................................... 71 Chapitre 3 : Evaluation de la néphélométrie laser comme méthode haut-débit pour déterminer la résistance fongiques aux conservateurs – Etude préliminaire ............................................................. 100 I. Résumé des travaux ................................................................................................................ 101 II. Article à soumettre dans International Journal of Food Microbiology .................................. 107 Discussion générale et perspectives .................................................................................................... 147 Références bibliographiques ............................................................................................................... 158 Valorisations scientifiques .................................................................................................................. 178 6 Liste des abréviations ADN = Acide désoxyribonucléique ASC = Advanced Spectra Classifier CHCA = Acide α-cyano-4-hydroxycinnamique CMI = Concentration Minimale Inhibitrice
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