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Fermentation Du Kivuguto, Lait Traditionnel Du Rwanda: Mise Au Point D’Un Starter Lactique

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Fermentation Du Kivuguto, Lait Traditionnel Du Rwanda: Mise Au Point D’Un Starter Lactique COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE GEMBLOUX AGRO-BIO TECH Fermentation du kivuguto, lait traditionnel du Rwanda: mise au point d’un starter lactique Eugène KARENZI Dissertation originale présentée en vue de l’obtention du grade de Docteur en Sciences agronomiques et Ingénierie biologique Promoteur: Pr Philippe JACQUES Co-promoteur: Pr Philippe THONART Gembloux, Janvier 2015 COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE GEMBLOUX AGRO-BIO TECH Fermentation du kivuguto, lait traditionnel du Rwanda: mise au point d’un starter lactique Eugène KARENZI Dissertation originale présentée en vue de l’obtention du grade de Docteur en Sciences agronomiques et Ingénierie biologique Promoteur: Pr Philippe JACQUES Co-promoteur: Pr Philippe THONART Gembloux, Janvier 2015 Copyright. Aux termes de la loi belge du 30 juin 1994, sur le droit d'auteur et les droits voisins, seul l'auteur a le droit de reproduire partiellement ou complètement cet ouvrage de quelque façon et forme que ce soit ou d'en autoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit. Toute photocopie ou reproduction sous autre forme est donc faite en violation de la dite loi et des modifications ultérieures. A Dieu Tout-Puissant Vous êtes et vous resterez toujours Grand. Amen A ma famille Merci pour la résistance et la tenue des babies hors des soucis A mon père Que cette thèse soit un prolongement de votre travail sur les fermentations traditionnelles pour la pérennisation d’une tradition familiale depuis 1400 et un couronnement de vos leçons d’Arithmétique & français sur un tableau de morceau de bois conçu par vos propres mains. Je ne peux être plus fier que de soutenir cette thèse de Doctorat en Bioindustries dans cette université de Liège Gembloux Agro-BioTech, Sorry for delaying, the things were so hard, the sky was so cloudy, the weather so bad, but people so nice and job so exciting Thanks for the good work you are doing now We love you forever, A ma mère,frères & soeurs Pour l’absence prolongée et la peine endurée. Karenzi E. ii Membres du jury M. Fréderic FRANCIS (président), ULG, Gembloux AGRO-BIO TECH M. Philippe JACQUES (promoteur), ULG, Gembloux AGRO-BIO TECH M. Philippe THONART (co-promoteur), ULG, Gembloux AGRO-BIO TECH M. Georges LOGNAY, ULG, Gembloux AGRO-BIO TECH Mme Marianne SINDIC (rapporteur interne), ULG, Gembloux AGRO-BIO TECH M. Georges SINNAEVE (rapporteur externe), Centre wallon de Recherches agronomiques CRA-W. iii SUMMARY/RESUME Eugène Karenzi (2015). Fermentation du kivuguto, lait traditionnel du Rwanda: mise au point d’un starter lactique. Thèse de Doctorat. Université de Liège Gembloux Agro-Bio Tech. Belgique. 191 pages. 16 tables. 28 figures. Résumé Une recherche pour la production industrielle du kivuguto, lait caillé traditionnel du Rwanda a été entreprise par la sélection de micro-organismes responsables de la fermentation de ce lait normalement produit artisanalement. Pour ce faire, il fallait une mise au point d’un starter lactique à partir des isolats issus des échantillons de lait artisanal kivuguto. Au terme de quatre échantillonnages, un dénombrement, un isolement et une purification ont abouti à conserver dans la Collection du CWBI 390 souches pures. Par des analyses phénotypiques (microscopiques, biochimiques), associées à des tests de résistance aux conditions extrêmes et à une analyse préliminaire des propriétés technologiques, 7 souches ont été pré-sélectionnées pour la poursuite du screening. Une caractérisation moléculaire par la méthode de 16S ADNr associé ou non à l’ITS 16S-23S ADNr a assimilé ces souches à deux Lactococcus lactis, deux Leuconostoc mesenteroides et trois Leuconostoc pseudomesenteroides. Des essais de formulation de laits fermentés par des mélanges de souches et leur conservation pendant 24 jours ont permis de formuler un lait fermenté semblable au lait artisanal kivuguto par l’association d’un Lactococcus lactis, d’un Leuconostoc mesenteroides et d’un Leuconostoc pseudomesenteroides. En effet, lors d’une analyse sensorielle discriminative, un jury de dégustation constitué de huit personnes est parvenu à identifier ce lait formulé parmi deux autres laits fermentés commercialisés aussi sur le marché à des différences significatives de p=0.05 dans une première série et p=0.01 dans une deuxième série. Des analyses technologiques proprement dites ont montré que ce lait formulé fermente après 14 heures avec une acidification de 73°D à pH4.6 et à 19°C, présente des caractéristiques d’un fluide visco-élastique. Son activité protéolytique est moyenne pour ne pas développer des peptides responsables de l’amertume en stockage. Son profil aromatique comporte cinq composés principaux 3-méthylbutan-1-ol, pentan-1-ol, acide acétique, furanméthan-2-ol et furan-2(5)H-one clairement identifiés par GC-MS. L’étude de la production et de la conservation de trois souches sélectionnées a montré une bonne stabilité sur trois mois à 4°C avec des viabilités cellulaires >90%, mais moins bonne à 20°C. iv Eugène Karenzi (2015). Fermentation of kivuguto, a rwandese traditional milk: making a starter culture (Ph.D. Thesis). University of Liège - Gembloux Agro-Bio Tech, Belgium. 191 pages. 16 tables. 28 figures. Summary A research for the industrial production of kivuguto traditional curd of Rwanda was undertaken by conducting a selection of microorganisms involved in the fermentation of that milk normally produced artisanally by householders. For that purpose, the objective of this work was the development of a starter culture using strains isolated in the curd milk kivuguto. After four samplings, an enumeration, isolation and purification led to preserve in the CWBI Collection 390 pure strains. By phenotypic analyses (microscopic, biochemical) associated with resistance tests under extreme conditions, as well as a preliminary analysis of technological properties, 7 strains were pre-selected for a further screening. Molecular characterization by 16S rDNA method with or without the 16S-23S rDNA ITS assimilated these strains to two Lactococcus lactis, two Leuconostoc mesenteroides and three Leuconostoc pseudomesenteroides. Thereafter, these strains were used in mixed assays for milk formulation of fermented milks and their preservation over a 24 days period helped to make a fermented milk similar to the traditional kivuguto. An association of one Lactococcus lactis, one Leuconostoc mesenteroides and one Leuconostoc pseudomesenteroides developed a much closer kivuguto artisanal milk. Indeed, in a discriminative sensory analysis, a tasting panel of eight people reached its identification among two other fermented milks sold also on the local market with significant differences of p=0.05 in the first series and p=0.01 milk in a second series. Technological analyzes showed that this formulated milk ferments within 14 hours after acidification of 73 °D at pH4.6 and 19 °C, has the characteristics of a viscoelastic fluid. It presents a mean value of proteolytic activity, so that it can’t develop bitter peptides in milk during storage. Its flavor profile includes five main compounds: 3-methylbutan-1-ol, pentan-1-ol, acetic acid, furanmethan-2-ol and furan-2(5)H-one clearly identified by GC-MS. The study of production and viability in storage of the three selected strains showed good stability over three months at 4 °C with cell viabilities >90% and less at 20°C. v REMERCIEMENTS Cette thèse vient consacrer plusieurs années d’études et de recherche qui s’inscrivent dans le cadre d’une formation universitaire pour l’obtention d’un doctorat (PhD) en Sciences agronomiques, Option Bioindustries. Bien que cet ouvrage représente une petite contribution personnelle à la science, il demeure en réalité une œuvre de plusieurs personnes. A tout un chacun, il est très important pour moi d’exprimer mes plus sincères remerciements. A Pr Philippe Jacques, promoteur de cette thèse après le départ de Pr Thonart, recevez toute ma gratitude pour avoir accepté la poursuite et la finalisation de cette thèse dans votre unité, et à travers vous, merci à tous les responsables de l’université de Liège et surtout ceux de Gembloux Agro-Bio Tech. A Professeur Philippe Thonart, pour votre accueil dans le Centre Wallon de Biologie Industrielle (CWBI), pour votre encadrement scientifique, pour votre confiance et surtout pour votre patience, pour vos encouragements à continuer à croire à ce jour, pour votre soutien moral et matériel, merci et merci encore. I will not forget! Je remercie également Pr Munyanganizi, pour ses encouragements et ses conseils judicieux. Et "Pole"! Il sied également de remercier les membres de mon jury et de mon comité de thèse: Pr Francis, Pr Wathelet, Pr Lognay, Pr Sindic, pour leur participation active et leur orientation de ce travail, leur disponibilité et leurs précieux conseils. Je remercie vivement Pr Sindic et Dr Sinnaeve de m’avoir fait l'honneur d'être rapporteurs de cette thèse. Les fondements de cette thèse sont les cours de Biologie moléculaire, de Génie des Procédés biotechnologiques, de Génie chimique, de Génie des Industries agro-alimentaires et d’Ingénierie des Formulations des aliments. Toute ma gratitude va à tous les professeurs qui ont assuré cette formation de base, car vous êtes et resterez mon inspiration, l'étincelle de ma créativité et, sans aucun doute, serez toujours mes professeurs préférés. Et de citer Pr Thonart, Pr Deroanne, Pr Blecker, Pr Sindic, Pr Béra, Pr Kettman, Pr Delvigne, Dr Destain, Dr Pochet, et Dr Aldrich. Très sincèrement, je remercie Dr Destain, pour sa rigueur scientifique, pour la lecture de tous les articles scientifiques de cette thèse et pour m’avoir donné des conseils scientifiques de ce travail. Je tiens aussi à adresser ma gratitude à Dr Robin Du Bois-Dauphin pour l’initiation à l’identification moléculaire et l’enregistrement de mes séquences dans la base de données de Genbank. Ce travail a été réalisé grâce au soutien matériel et financier du CUD, du CWBI, du Service social de Gembloux Agro-Bio Tech ULG, de ma famille et des amitiés personnelles ici présentes ou empêchées.
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