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2018UBFCK013.Pdf UNIVERSITÉ DE BOURGOGNE-FRANCHE COMTÉ UNIVERSITÉ DE DIJON AGROSUP-DIJON INSTITUT NATIONAL SUPERIEUR DES SCIENCES AGRONOMIQUES, DE L’ALIMENTATION ET DE L’ENVIRONNEMENT UMR Procédés Alimentaires et Microbiologiques ÉCOLE DOCTORALE ENVIRONNEMENTS-SANTÉ THÈSE Présentée pour obtenir le grade de Docteur de l’Université de Bourgogne-Franche Comté Spécialité : Biotechnologie Agro-alimentaire Options : Biotechnologie, Biochimie Microbienne, Génie des Procédés, Microbiologie Par Sophal TRY Titre : PRODUCTION D’ARÔMES PAR FERMENTATION EN MILIEU SOLIDE Soutenance prévue le 25 Mai 2018 Andrée VOILLEY Pr. émérite, AgroSup Dijon Examinateur Christian LARROCHE Pr, Polytech Clermont-Ferrand, UCA Rapporteur Vincent PHALIP Pr, Université de Lille Rapporteur Yves WACHÉ Pr, AgroSup-Dijon, UBFC Directeur de thèse Joëlle DE-CONINCK Dr, Welience-PPB Dijon, UBFC Codirectrice de thèse Thavarith CHUNHIENG Dr, ITC, Cambodge Codirecteur de thèse Thèse financée par l’Ambassade de France au Cambodge. Production d’arômes par fermentation en milieu solide ©Sophal TRY 2018 RESUME La transposition d’un milieu liquide (FML) à un milieu solide (FMS) de la production de γ- décalactones par fermentation par Yarrowia lipolytica a été étudiée pour la première fois. Dans un premier temps, différentes matrices solides (rafles de maïs, éponges de cellulose, éponges de courgette et graines de ricin) ont été utilisées. L'éponge de courgette était le support sur lequel les lactones étaient produites aux concentrations les plus élevées. La production de lactones a donc été réalisée sur ce support et dans trois types de réacteurs FMS sous différentes conditions d’aérations (sans aération, avec aération statique et avec aération forcée). Une concentration élevée de 5 g/L d’hydroxy-lactone a été détectée dans la condition d’aération statique en fiole à col large. Une certaine concentration de lactones était entrainée lors de l’aération forcée au cours de la production en mini-réacteur. Pour calculer la production totale de lactones dans cette condition, un modèle mathématique a été utilisé pour modéliser les pertes. Par ailleurs, un nouveau procédé par injection d’air enrichi en oxygène (20%, 30%, 40% et 50%) a été mis en place pour étudier les effets de l’oxygène sur la β- oxydation chez Y. lipolytica. Dans cette partie, les résultats ont montré que l’oxygène est nécessaire pour la production d’hydroxy-lactone et la dégradation de γ-décalactone mais pas pour la dernière étape de production de cette dernière. Dans la dernière partie de ce travail, l’effet de l’activité de l’eau (Aw) a été étudié préliminairement sur la croissance en milieu gélosé, et pour la croissance et la production de lactone en FML. Mots clés : Aération ; Air enrichi en oxygène ; β-Oxydation ; Fermentation en milieu solide ; Métabolisme des acides gras hydroxylés ; Lactones ; Yarrowia lipolytica. i ABSTRACT The adaptation of the production of γ-decalactones from submerged fermentation (SmF) to solid state fermentation (SSF) by Yarrowia lipolytica was investigated in this work. First of all, different solid matrices (corncob, cellulose sponge, luffa sponge, and castor seeds) were used for the first adaptation of the production of γ-décalactones. Luffa sponge appeared to be the most interesting solid support on which lactones were produced in higher concentrations than in the other solid matrices used. Then, the production of lactones using luffa sponge as the solid support was carried out in three types of SSF reactors to monitor different aeration conditions (without aeration, with a static aeration and with a forced aeration). A 5 g/L-high concentration of hydroxy-lactone was detected in the condition of static aeration using wide- mouth Erlenmeyer flasks. Furthermore, a concentration of lactones was stripped when forced aeration was used during the production using mini-reactors. To calculate the total production of lactones in this case, a mathematical model was used to evaluate the losses. Moreover, a novel alternative process using oxygen-enriched air (20%, 30%, 40% and 50%) was employed to study the effect of oxygen on β-oxidation in Y. lipolytica. In this part, the results showed that oxygen is required for hydroxy-lactone production and γ-decalactone degradation but not for the last step of production of this latter lactone. In the last part of this work, the effect of water activity (Aw) was preliminary studied on growth in agar medium, and for growth and lactone production in SmF. Key words: Aeration ; Oxygen-enriched air ; β-Oxidation ; Solid state fermentation ; Hydroxylated fatty acid metabolism ; Lactones ; Yarrowia lipolytica. ii REMERCIEMENTS Avant de présenter ce travail, je voudrais remercier tous ceux qui, sous différentes formes, ont contribué à sa réalisation. Ce travail de thèse a été réalisé en codirection au sein de l’équipe des Procédés Microbiologiques et Biotechnologiques (PMB) de l’Unité Mixte de Recherche des Procédés Alimentaires et Microbiologiques (UMR-PAM), en partenariat avec le département de Génie Chimique et Alimentaire (GCA) de l’Institut de Technologie du Cambodge (ITC). Je tiens tout d'abord à remercier mon directeur de thèse, Yves WACHÉ, pour m'avoir accueilli au sein de son équipe de recherche. Je le remercie également pour sa patience, son encouragement et sa confiance tout au long de ses années. Je le remercie particulièrement pour son écoute, son ouverture d’esprit, ses précieux conseils et ses connaissances scientifiques. Mes remerciements vont également à ma codirectrice de thèse, Joëlle DE-CONINCK, pour m’avoir accueilli à son équipe PPB, ainsi que pour ses conseils et son aide précieuse tout au long de mes études doctorales. Je souhaite remercier Dr. Thavarith CHUNHIENG, mon codirecteur de thèse pour ses conseils tout au long de mes études supérieures, pas seulement pour mes études doctorales, mais aussi depuis mon master recherche à l’INP-ENSAT et ma formation d’ingénieur et mon travail à l’ITC où il m’a prodigué de précieux conseils. Je tiens à remercier sincèrement Madame Andrée VOILLEY, Professeur émérite à AgroSup- Dijon, pour sa gentillesse et son dévouement. Je la remercie énormément pour avoir partagé ses connaissances scientifiques avec moi, spécifiquement celles concernant les phénomènes physicochimiques des arômes et les systèmes de récupération d’arômes. Mes remerciements s’adressent à tous les membres du comité de thèse qui se compose de : Dr. Florence HUSSON, Dr. Céline LAROCHE, et Pr. Andrée VOILLEY pour leurs conseils scientifiques qui ont contribué à l’avancement de ce travail. Je voudrais remercier également les Pr. Christian LARROCHE et Pr. Vincent PHALIP pour avoir bien voulu juger la qualité de ce travail. iii Je souhaite remercier le Dr. Thi Hanh PHAN, le Dr. Bao Ngoc PHAM-HOANG et le Dr. Pascale WINCKLER pour leur soutien technique concernant les analyses en CPG-FID, CPG- MS et en microscope à fluorescence et confocale. Je tiens à remercier Madame Marion ALLIET pour avoir partagé avec moi ses connaissances concernant la modélisation lors de mon stage à l’INP-ENSIACET. Merci également à Madame Anne-Marie SEUVRE pour ses conseils scientifiques concernant les interactions eau-produit et l’activité de l’eau. Je remercie le Dr. Sergey KLYKOV pour la discussion concernant le modèle de croissance. J’adresse également mes remerciements au Pr. Rémy CACHON, Pr. Jean-François CAVIN et Dr. Hélène LICANDRO pour leurs conseils scientifiques. Un grand merci à Christine et Sylvie de l’UMR-PAM pour leur soutien technique et administratif pour toutes les commandes de produits. Merci également à Guillaume pour son soutien technique au laboratoire de la PPB. Mes remerciements s’adressent également à Florence BOFFY et Alexandre GAUDRY pour leur soutien administratif et informatique. Un grand merci à mes chers amis Bao, Dat, Antonio et Chi, pour le partage de leurs connaissances et de leurs expériences. Merci également pour les discussions scientifiques et tous les moments sympathiques de la vie au laboratoire au long de ces années. Merci aussi à mes chers amis Thu, Da, Adeline, Mai, Aurore, Maxime pour ces moments sympathiques de la vie doctorale au laboratoire. Merci à mes stagiaires, Sotheary et Alison pour leurs résultats et pour avoir eu le courage de travailler avec moi. Ce travail n'aurait pu être mené à bien sans l'aide de différentes subventions. Je souhaite remercier l’Ambassade de France au Cambodge pour le financement des 29 mois de bourse pour la période écoulée en France. Je n’oublie pas de remercier l’AUF pour son financement de mobilité de recherche lors de la période de travail au Cambodge. Je dédicace ce travail à mes parents, mes frères/sœurs qui m’ont soutenu et encouragé au cours de ces années de thèse. Enfin, j’aimerais exprimer mes sincères remerciements à ceux dont le nom n’apparait pas dans cette page et qui m’ont aidé durant ces trois années à Dijon. iv ABREVIATIONS Aw : Activité de l’eau ATP : Adénosine triphosphate CoA : Co-enzyme A FML : Fermentation en milieu liquide (en anglais SmF : Submerged fermentation) FMS : Fermentation en milieu solide (en anglais SSF : Solid state fermentation) G3P : Glycérol-3-phosphate -1 KLa : Coefficient de transfert d’oxygène (h ) NAD+ : Nicotinamide adénine dinucléotide sous une forme oxydée NADH, H+ : Nicotinamide adénine dinucléotide sous une forme réduite pH : Potentiel hydrogène Pa : Pascal rpm : Rotations par minute v LISTE DES ILLUSTRATIONS Figures (hors publications) Figure 1 : Structure générale de lactones .................................................................................
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