La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l’absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L’expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l’est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l’expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l’expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodY / The variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon CodY

S. thermophilus est une bactérie lactique largement utilisé dans l'industrie laitière et, comme toute bactérie, doit s'adapter à des environnements variés tels que le lait, le yaourt et même le tractus digestif, après que le produit ait été ingéré. Les systèmes à deux composants (TCS) constituent un des mécanismes essentiels qu'utilisent les bactéries pour percevoir et s'adapter à des changements environnementaux. D'un point de vue structural, les TCS sont constitués de deux composants: un « senseur » ou protéine histidine kinase (HK) qui s'auto-phosphoryle en réponse à un stimulus puis transfère son groupement phosphate au « response regulator » (RR), le deuxième composant. Celui-ci se comporte alors le plus souvent comme un régulateur transcriptionnel permettant une réponse physiologique adaptée. Afin de mieux comprendre ces phénomènes de régulation impliqués dans la réponse aux changements environnementaux, nous avons étudié la contribution de chacun des 8 TCS de Streptococcus thermophilus LMD-9 à son adaptation dans le lait. Ainsi, des études transcriptionnelles effectuées sur des cultures en lait montrent que tous les RR sont exprimés, à des niveaux et profils d'expression différents. Nous avons noté en coculture avec Lactobacillus bulgaricus, le partenaire de Streptococcus thermophilus dans le yaourt, une induction de l'expression de 4 RR qui atteint, pour rr02 et rr09, un facteur 6. Nous avons construit par ailleurs des mutants négatifs pour 7 des 8 RR de S. thermophilus et montré l'essentialité de RR05, un orthologue de YycF chez B. subtilis ou de or de WalR chez S. aureus. Pour les 7 autres mutants RR, l'absence d'un seul gène rr n'impacte pas suffisamment la croissance du streptocoque en lait. Enfin, la détermination du régulon du TCS06 par des études post-génomiques a permis de montrer que ce système est impliqué dans la résistance à la bacitracine en modulant entre autres la voie de biosynthèse du polysaccharide à rhamnose (RGP). / The lactic acid bacterium Streptococcus thermophilus is widely used in the dairy industry and, as a food bacterium, has to cope with changing environments such as milk, yogurt as well as the digestive tract, after the product has been ingested. Two-component systems (TCS), typically composed of a sensor kinase (HK) that detects a stimulus and of a response regulator (RR) which acts as a transcriptional regulator, are among the most prevalent means for bacteria to adapt to changing environments via fine-tune gene expression. To get a more comprehensive view of the role of all two-component systems in S. thermophilus physiology, we have investigated the contribution of each S. thermophilus LMD-9 TCS to its fitness and adaptation to milk. Transcriptomic studies (RT-qPCR) and construction of negative mutants of the rr genes were performed for LMD-9 S. thermophilus strain. We have shown that all LMD-9 response regulators were expressed in milk, at different levels and with different profiles of expression during growth. In mixed culture with Lactobacillus bulgaricus, the S. thermophilus partner in yoghurt, the expression of four LMD-9 rr increased; for two of them, rr02 and rr09, the increase reached a factor 6. These results indicate that Lb. bulgaricus induces regulatory changes in S. thermophilus and that S. thermophilus is able to adapt to these changes by probable fine tuning regulations. We constructed negative mutants for 7 out of 8 LMD-9 RRs and we showed that RR05 -an ortholog of B. subtilis YycF or S. aureus WalR- was essential for the optimum growth of S. thermophilus. For the 7 other RR, the absence of a single rr gene was not sufficient to notably impact the growth of LMD-9 in milk. The determination of the TCS06 regulon by post-genomics shows that TCS06 is involved in bacitracin resistance through the modulation of the rhamnose polysaccharide pathway.

No description available.

L'amélioration de la digestion de lactose est une allégation "santé" liée aux ferments viviants du yaourt : Streptococcus thermophilus (S. thermophilus) et Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) validée par l'EFSA en 2010. La physiologie de S. thermophilus et de L. bulgaricus est connue dans le lait et particulièrement le yaourt, alors qu'elle n'a été que peu étudiée dans le tractus digestif (TD). Mon travail de thèse est basé sur l'hypothèse de travail suivante : l'utilisation de modèles animaux gnotobiotiques permet de mieux connaître la physiologie des bactéries lactiques et de proposer des mécanismes d'action de leurs effets "santé". La stratégie a donc été d'obtenir des animaux mono-associés avec chacune des deux bactéries du yaourt ou les deux en même temps. Les principaux résultats obtenus sont : 1/ S. thermophilus colonise le TD en s'adaptant progressivement à l'environnement colique et y induit une glycolyse massive et une production de lactate. La glycolyse est la signature majeure de S. thermophilus dans le TD et le lactate pourrait être est la molécule "signal" qui induit une réponse chez l'hôte par une augmentation des transporteurs de mono-carboxylates (SLC16A1 et SLC5A8) et d'une protéine impliquée dans l'arrêt du cycle cellulaire p27kip1. 2/ L'apport de lactose stimule la colonisation du TD, la glycolyse ainsi que la production de L-lactate par S. thermophilus in vivo. 3/ Contrairement à ce qui est observé pour S. thermophilus, L. bulgaricus ne s'implante pas en absence de lactose. Quand les deux bactéries sont en co-culture, S. thermophilus est toujours avantagé numériquement par rapport à L. bulgaricus aussi bien in vitro qu' in vivo. Au niveau nutritionnel, tous nos résultats sont cohérents avec les allégations "santé" du yaourt avec un effet prébiotique du lactose. L'étude d'animaux gnotobiotiques a permis de proposer des nouvelles voies de régulation du métabolisme des sucres de bactéries lactiques et de nouvelles voies moléculaires (via le lactate) par lesquelles des bactéries lactiques et de nouvelles voies moléculaires (via le lactate) par lesquelles des bactéries lactiques pourraient influencer la physiologie de l'hôte. / *

Alterações favoráveis na composição da microbiota intestinal podem ser observadas com o consumo regular de alimentos funcionais contendo probióticos. Os probióticos são micro-organismos vivos que conferem benefícios à saúde do hospedeiro, quando administrados em quantidades adequadas. Tradicionalmente, são incorporados aos leites fermentados e a outros produtos lácteos fermentados. Atualmente, a idéia de redução dos componentes lácteos como veículos para agentes probióticos tem sido promovida, em razão da alta proporção de indivíduos que apresentam intolerância à lactose, além da busca por alternativas vegetarianas. Nesse contexto, a soja aparece como um substituto ideal para o consumo, promovendo a saúde através de características nutricionais intrínsecas. O presente trabalho visou desenvolver um produto semelhante ao queijo petit-suisse, mas à base de soja, com polpa de fruta, e adicionado de micro-organismos probióticos, bem como avaliar a aceitabilidade do produto sob o ponto de vista sensorial e suas características físico-químicas, microbiológicas e de textura instrumental durante o seu armazenamento a 4±1 °C por até 28 dias. Três formulações foram produzidas (com três repetições cada): F1- formulação de queijo petit-suisse probiótico elaborado com massa-base de queijo quark, como controle (produto lácteo); F2 - formulação com \"queijo\" de soja e com creme de leite (produto misto à base de soja e de leite); F3 - formulação com \"queijo\" de soja e com creme de soja (produto de soja). Em todas as formulações, foi empregada a cultura probiótica ABT-4, constituída dos micro-organismos comprovadamente probióticos Bifidobacterium animalis subsp. lactis Bb-12, Lactobacillus acidophilus La-5 e da cultura starter Streptococcus thermophilus. Os produtos foram armazenados a 4±1 °C e avaliados sensorialmente (teste de aceitabilidade, utilizando escala hedônica estruturada), após 7, 14 e 21 dias, por 50 consumidores em cada período. Adicionalmente, foram analisados semanalmente durante o seu armazenamento por até 28 dias quanto à viabilidade dos probióticos e da cultura starter e quanto ao seu pH e o seu perfil instrumental de textura (teste de dupla compressão de amostras, em analisador de textura TA-XT2). Paralelamente, foi realizado um monitoramento microbiológico das amostras quanto à presença de contaminantes e a partir de amostras mantidas congeladas, foi determinada a composição centesimal dos produtos. A viabilidade de La-5 mostrou-se satisfatória até o 28º dia de armazenamento das formulações F1 e F2, com populações variando de 8,29 a 7,56 log ufc/g e de 8,17 a 6,49 log ufc/g, respectivamente. Entretanto, para F3, as populações de La-5 mostraram-se satisfatórias até o 21º dia (8,18 a 6,84 log ufc/g), não atingindo o mínimo recomendado na última semana. Por outro lado, a viabilidade de Bb-12 manteve-se acima de 8 log ufc/g até o 28º dia de armazenamento de F1, F2 e F3. A cultura starter apresentou populações sempre entre 9,73 e 8,86 log ufc/g. O pH manteve-se estável para todas as formulações, mas foi significativamente menor (p<0,05) para F2, em relação a F1 e F3. Nos parâmetros dureza e gomosidade, F1 apresentou comportamento antagônico em relação ao de F2 e F3. Todos os parâmetros de textura de F1 diferiram significativamente de F2 e F3 (p<0,05). Não houve diferença significativa (p>0,05) na análise sensorial de uma mesma formulação entre os períodos de armazenamento. No entanto, na comparação entre as formulações, foram observados escores médios superiores (p<0,05) para F3 aos 21 dias (6,4), quando comparada a F2 (4,6). Os alimentos à base de soja, F2 e F3, mostraram-se bons veículos para os micro-organismos probióticos, com populações adequadas para caracterizá-los como probióticos, sendo que F3 mostrou uma tendência a um melhor desempenho sensorial após 14 e 21 dias de armazenamento. / Favorable changes in the composition of the intestinal microbiota can be observed with the regular consumption of functional foods containing probiotics. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. They are traditionally incorporated into fermented milks and other fermented milk products. Currently, the idea of reducing milk components as vehicles for probiotic agents has been promoted due to the high proportion of people who have lactose intolerance, and the search for vegetarian alternatives. In this context, soy appears as an ideal replacement for consumption, promoting health through intrinsic nutritional characteristics. This work aimed to develop a product similar to petit-suisse cheese, but soy based, with fruit pulp, and with probiotic micro-organisms, as well as evaluating the acceptability of the product from the point of view of its sensory and physico-chemical characteristics, microbiological and instrumental texture profile during storage at 4 ± 1 °C for up to 28 days. Three formulations were produced (in triplicates): F1 - formulation of probiotic petit-suisse cheese prepared with quark cheese, as control (milk product) F2 - formulation with soy \"cheese\" and milk cream (mixed product with soy and milk), F3 - formulation with soy \"cheese\" and soy cream (soy product). The three formulations were produced with the probiotic ABT-4 culture, consisting of the probiotic microorganisms Bifidobacterium animalis subsp. lactis Bb-12, Lactobacillus acidophilus La-5 and the starter culture Streptococcus thermophilus. The products were stored at 4 ± 1 °C and subjected to sensory evaluation (acceptability test, using an hedonic scale), after 7, 14, and 21 days, by 50 consumers in each period. Additionally, the products were monitored weekly during storage for up to 28 days regarding the viability of the probiotics and starter culture and their pH profile and the instrumental texture (double compression of samples test, using a TA-XT2 texture analyzer). Also, samples were monitored for the presence of contaminants, and, the chemical composition of the products was determined from samples kept frozen. The viability of the La-5 was satisfactory until the 28th day of storage for F1 and F2, with populations ranging from 8.29 to 7.56 log cfu/g and from 8.17 to 6.49 log cfu/g respectively. However, for F3, the population of La-5 proved to be satisfactory until the 21st day (8.18 to 6.84 log cfu/g), not reaching the minimum recommended in the last week. On the other hand, Bb-12 viability remained above 8 log cfu/g throughout the 28 days of storage for F1, F2, and F3. The starter culture populations were always between 9.73 and 8.86 log cfu/g. The pH remained stable for all formulations, but was significantly lower (p<0.05) for F2, compared to F1 and F3. As for the texture parameters hardness and gumminess, F1 showed antagonistic behavior in relation to F2 and F3. All texture parameters evaluated differed significantly for F1, F2, and F3 (p<0.05). There was no significant difference (p>0.05) in the sensory scores obtained for the same formulation, when different storage periods were compared. However, when the 3 formulations were compared, higher average scores (p<0.05) were obtained on day 21 for F3 (6.4) than for F2 (4.6). The soy-based foods, F2 and F3, proved to be good vehicles for probiotic microorganisms, with appropriate populations to characterize them as probiotics, and F3 showed a trend towards a better performance in sensory evaluation on days 14 and 21 days of storage.

Les bactéries lactiques présentent un grand intérêt économique de par leur large utilisation dans l’industrie agroalimentaire. Parmi elles, Streptococcus thermophilus (ST) est d’une importance majeure puisqu’elle est la plus utilisée après Lactococcus lactis, pour la fabrication de produits laitiers fermentés et de fromages. De plus, cette bactérie est le seul streptocoque à bénéficier du statut GRAS (Generally Recognized As Safe). Outre son intérêt en industrie laitière, ST présente des effets bénéfiques sur la santé intestinale de l’Homme. Bien que ces effets soient largement documentés, le statut probiotique de ST reste encore à conforter. C’est pourquoi des études sont actuellement menées afin de sélectionner des souches de ST à fort potentiel probiotique. Parmi les critères importants figurent leur capacité à survivre aux conditions drastiques du tube digestif (TD) et leur capacité à adhérer aux cellules intestinales. Dans cette optique, les objectifs de cette thèse étaient d’étudier, dans un premier temps, la capacité d’adhésion in vitro de la souche ST LMD-9 à différentes lignées cellulaires intestinales d’origine humaine et d’évaluer la survie de cette souche au stress biliaire. Afin de mettre en évidence un rôle potentiel de certaines protéines de surface dans ces deux processus, trois mutants issus de cette souche et inactivés dans les gènes prtS (protéase pariétale), srtA (sortase A) et mucBP (protéine de liaison aux mucines), ont été inclus dans cette étude. Dans un second temps, l’impact de l’adhésion de LMD-9 a été analysé, d’une part sur l’expression de gènes codant certaines mucines dans les cellules eucaryotes, et d’autre part sur l’expression de gènes qui seraient spécifiquement induits durant le processus d’adhésion, ceci en utilisant la technologie R-IVET (Recombinase-based In Vivo Expression Technology). Les résultats obtenus ont permis de montrer que la souche LMD-9 était capable de survivre jusqu’à une concentration de 3 mM en sels biliaires et que les protéines de surface PrtS, SrtA et MucBP seraient impliquées dans la résistance à ce stress. Nos résultats ont également montré que LMD-9 adhérait aux trois différentes lignées cellulaires, suggérant ainsi que la souche pourrait interagir avec les différentes mucines qu’elle peut rencontrer dans le TD. De plus, l’implication de certaines protéines de surface dans l’adhésion de LMD-9 s’est avérée dépendante des caractéristiques de ces lignées, qu’il s’agisse de cellules entérocytaires (Caco-2) ou productrices de mucus (HT29-MTX et HT29-CL.16E). Concernant l’impact de l’adhésion de la souche LMD-9 sur l’expression des gènes MUC2 et MUC5AC, aucun effet sur le taux de transcrits n’a été observé dans nos conditions expérimentales. Par ailleurs, nos résultats ont permis, pour la première fois, d’identifier les gènes spécifiquement induits dans la souche LMD-9 durant l’adhésion aux cellules épithéliales. Nous avons ainsi montré que l’adhésion de la souche LMD-9 ne dépend pas uniquement des protéines de surface, mais d’autres fonctions et voies métaboliques seraient également impliquées. Ce travail de thèse contribue ainsi à apporter de nouvelles connaissances liées (i) au choix du modèle cellulaire dans les études d’adhésion bactérienne in vitro, (ii) à l’aptitude de la souche LMD-9 à survivre au stress biliaire en faisant intervenir certaines protéines de surface et (iii) à la compréhension des mécanismes moléculaires de l’adhésion de LMD-9 aux cellules épithéliales intestinales / Lactic acid bacteria are of great economic interest because of their use in the food industry. Among them, Streptococcus thermophilus (ST) is of major interest since it is the most used after Lactococcus lactis, for the manufacture of fermented dairy products and cheese. In addition, this bacterium is the only streptococcus to benefit from GRAS status (Generally Recognized As Safe). Beside its interest in the dairy industry, ST has beneficial effects on human intestinal health. Although these effects are widely documented, the probiotic status of ST remains to be consolidated. Therefore, studies are currently being conducted in order to select strains of ST with a high probiotic potential. Among criteria that are important to select ST strains include their ability to survive stress the drastic conditions of the digestive tract (DT) and their ability to adhere to intestinal cells. In this context, the aim of this thesis was to investigate firstly the in vitro adhesion capacity of the ST LMD-9 strain to different human intestinal cell lines and to evaluate the survival of this strain to bile salt stress. In order to highlight the potential role of some surface proteins in these two processes, three mutants derived from this strain and inactivated in the genes prtS (parietal protease), srtA (sortase A) and mucBP (Mucin Binding-protein) were also included in this study. Secondly, the impact of LMD-9 adhesion was analyzed, on one hand on the expression of some mucin-encoding genes in eukaryotic cells, and on the other hand on the expression of genes that would be specifically induced during adhesion process using the R-IVET (Recombinase-based In Vivo Expression Technology) approach. The results obtained demonstrated the ability of LMD-9 to survive up to the concentration of 3 mM of bile salts and that the PrtS, SrtA and MucBP surface proteins would be involved in the resistance to this stress. Our results also showed that the LMD-9 strain was capable of adhering to three cell lines used suggesting that this strain could interact with different mucins that may encounter in the DT. Moreover, the involvement of some surface proteins in the adhesion of LMD-9 has been found to be dependent on the surface characteristics of these cell lines, whether they are enterocytic (Caco-2) or mucus-secreting cells (HT29-MTX and HT29-CL.16E). Regarding the impact of LMD-9 adhesion on MUC2 and MUC5AC gene expression, no effect has been observed on the transcript level under our experimental conditions. Furthermore, for the first time, our results allowed us to identify genes specifically induced in the LMD-9 strain during adhesion process to epithelial cells. We have thus shown that the LMD-9 adhesion does not depend solely on surface proteins, but other functions and metabolic pathways are also involved. This thesis work contributes thus to new knowledge related to (i) the choice of the cellular model in in vitro bacterial adhesion studies, (ii) the ability of LMD-9 to survive bile salt stress by involving some surface proteins and (iii) understanding the molecular mechanisms of LMD-9 adhesion to epithelial cells

Alterações favoráveis na composição da microbiota intestinal podem ser observadas com o consumo regular de alimentos funcionais contendo probióticos. Os probióticos são micro-organismos vivos que conferem benefícios à saúde do hospedeiro, quando administrados em quantidades adequadas. Tradicionalmente, são incorporados aos leites fermentados e a outros produtos lácteos fermentados. Atualmente, a idéia de redução dos componentes lácteos como veículos para agentes probióticos tem sido promovida, em razão da alta proporção de indivíduos que apresentam intolerância à lactose, além da busca por alternativas vegetarianas. Nesse contexto, a soja aparece como um substituto ideal para o consumo, promovendo a saúde através de características nutricionais intrínsecas. O presente trabalho visou desenvolver um produto semelhante ao queijo petit-suisse, mas à base de soja, com polpa de fruta, e adicionado de micro-organismos probióticos, bem como avaliar a aceitabilidade do produto sob o ponto de vista sensorial e suas características físico-químicas, microbiológicas e de textura instrumental durante o seu armazenamento a 4±1 °C por até 28 dias. Três formulações foram produzidas (com três repetições cada): F1- formulação de queijo petit-suisse probiótico elaborado com massa-base de queijo quark, como controle (produto lácteo); F2 - formulação com \"queijo\" de soja e com creme de leite (produto misto à base de soja e de leite); F3 - formulação com \"queijo\" de soja e com creme de soja (produto de soja). Em todas as formulações, foi empregada a cultura probiótica ABT-4, constituída dos micro-organismos comprovadamente probióticos Bifidobacterium animalis subsp. lactis Bb-12, Lactobacillus acidophilus La-5 e da cultura starter Streptococcus thermophilus. Os produtos foram armazenados a 4±1 °C e avaliados sensorialmente (teste de aceitabilidade, utilizando escala hedônica estruturada), após 7, 14 e 21 dias, por 50 consumidores em cada período. Adicionalmente, foram analisados semanalmente durante o seu armazenamento por até 28 dias quanto à viabilidade dos probióticos e da cultura starter e quanto ao seu pH e o seu perfil instrumental de textura (teste de dupla compressão de amostras, em analisador de textura TA-XT2). Paralelamente, foi realizado um monitoramento microbiológico das amostras quanto à presença de contaminantes e a partir de amostras mantidas congeladas, foi determinada a composição centesimal dos produtos. A viabilidade de La-5 mostrou-se satisfatória até o 28º dia de armazenamento das formulações F1 e F2, com populações variando de 8,29 a 7,56 log ufc/g e de 8,17 a 6,49 log ufc/g, respectivamente. Entretanto, para F3, as populações de La-5 mostraram-se satisfatórias até o 21º dia (8,18 a 6,84 log ufc/g), não atingindo o mínimo recomendado na última semana. Por outro lado, a viabilidade de Bb-12 manteve-se acima de 8 log ufc/g até o 28º dia de armazenamento de F1, F2 e F3. A cultura starter apresentou populações sempre entre 9,73 e 8,86 log ufc/g. O pH manteve-se estável para todas as formulações, mas foi significativamente menor (p<0,05) para F2, em relação a F1 e F3. Nos parâmetros dureza e gomosidade, F1 apresentou comportamento antagônico em relação ao de F2 e F3. Todos os parâmetros de textura de F1 diferiram significativamente de F2 e F3 (p<0,05). Não houve diferença significativa (p>0,05) na análise sensorial de uma mesma formulação entre os períodos de armazenamento. No entanto, na comparação entre as formulações, foram observados escores médios superiores (p<0,05) para F3 aos 21 dias (6,4), quando comparada a F2 (4,6). Os alimentos à base de soja, F2 e F3, mostraram-se bons veículos para os micro-organismos probióticos, com populações adequadas para caracterizá-los como probióticos, sendo que F3 mostrou uma tendência a um melhor desempenho sensorial após 14 e 21 dias de armazenamento. / Favorable changes in the composition of the intestinal microbiota can be observed with the regular consumption of functional foods containing probiotics. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. They are traditionally incorporated into fermented milks and other fermented milk products. Currently, the idea of reducing milk components as vehicles for probiotic agents has been promoted due to the high proportion of people who have lactose intolerance, and the search for vegetarian alternatives. In this context, soy appears as an ideal replacement for consumption, promoting health through intrinsic nutritional characteristics. This work aimed to develop a product similar to petit-suisse cheese, but soy based, with fruit pulp, and with probiotic micro-organisms, as well as evaluating the acceptability of the product from the point of view of its sensory and physico-chemical characteristics, microbiological and instrumental texture profile during storage at 4 ± 1 °C for up to 28 days. Three formulations were produced (in triplicates): F1 - formulation of probiotic petit-suisse cheese prepared with quark cheese, as control (milk product) F2 - formulation with soy \"cheese\" and milk cream (mixed product with soy and milk), F3 - formulation with soy \"cheese\" and soy cream (soy product). The three formulations were produced with the probiotic ABT-4 culture, consisting of the probiotic microorganisms Bifidobacterium animalis subsp. lactis Bb-12, Lactobacillus acidophilus La-5 and the starter culture Streptococcus thermophilus. The products were stored at 4 ± 1 °C and subjected to sensory evaluation (acceptability test, using an hedonic scale), after 7, 14, and 21 days, by 50 consumers in each period. Additionally, the products were monitored weekly during storage for up to 28 days regarding the viability of the probiotics and starter culture and their pH profile and the instrumental texture (double compression of samples test, using a TA-XT2 texture analyzer). Also, samples were monitored for the presence of contaminants, and, the chemical composition of the products was determined from samples kept frozen. The viability of the La-5 was satisfactory until the 28th day of storage for F1 and F2, with populations ranging from 8.29 to 7.56 log cfu/g and from 8.17 to 6.49 log cfu/g respectively. However, for F3, the population of La-5 proved to be satisfactory until the 21st day (8.18 to 6.84 log cfu/g), not reaching the minimum recommended in the last week. On the other hand, Bb-12 viability remained above 8 log cfu/g throughout the 28 days of storage for F1, F2, and F3. The starter culture populations were always between 9.73 and 8.86 log cfu/g. The pH remained stable for all formulations, but was significantly lower (p<0.05) for F2, compared to F1 and F3. As for the texture parameters hardness and gumminess, F1 showed antagonistic behavior in relation to F2 and F3. All texture parameters evaluated differed significantly for F1, F2, and F3 (p<0.05). There was no significant difference (p>0.05) in the sensory scores obtained for the same formulation, when different storage periods were compared. However, when the 3 formulations were compared, higher average scores (p<0.05) were obtained on day 21 for F3 (6.4) than for F2 (4.6). The soy-based foods, F2 and F3, proved to be good vehicles for probiotic microorganisms, with appropriate populations to characterize them as probiotics, and F3 showed a trend towards a better performance in sensory evaluation on days 14 and 21 days of storage.

Streptococcus thermophilus est la bactérie lactique la plus utilisée après Lactococcus lactis dans l’industrie laitière pour la fabrication de yaourts et de fromages. Il s’agit du seul streptocoque à avoir le statut de bactérie GRAS (Generally Recognized As Safe). Malgré de récentes études montrant sa capacité à survivre dans le tractus digestif humain et des effets santé intéressants, le statut probiotique de S. thermophilus reste l’objet d’interrogations. Ainsi, les objectifs de cette thèse ont été (i) d’approfondir les connaissances sur la capacité de survie de S. thermophilus en conditions digestives humaines simulées, grâce, en particulier, au système dynamique multi-compartimenté TIM (TNO gastro-intestinal model) et (ii) d’identifier des gènes de S. thermophilus spécifiquement activés dans des conditions complexes comme l’environnement digestif, à l’aide de la technologie R-IVET (Recombinase-based In Vivo Expression Technology) basée sur l’excision d’un gène rapporteur. Le système R-IVET est composé d’un vecteur plasmidique portant la recombinase cre démunie de son promoteur et d’une cassette chromosomique composée d’un gène marqueur entouré de sites loxP reconnus par Cre. Ainsi, dans un premier temps, nous avons implanté la technologie R-IVET chez S. thermophilus LMD-9. Sa fonctionnalité a été testée et validée in vitro et dans le tractus digestif de la souris. Puis, l’étude de la survie de quatre souches de S. thermophilus dans le système TIM a montré que trois d’entre elles étaient plus résistantes que la quatrième, très sensible aux stress gastro-intestinaux. Ces résultats confirment donc que la survie de S. thermophilus dans l’environnement digestif est souche-dépendante. Ils montrent également que la survie de S. thermophilus est influencée par la matrice alimentaire, celle-ci étant plus importante en lait fermenté qu’en lait liquide. Enfin, dans un troisième temps, nous avons construit une première banque génomique R-IVET, en clonant en amont de cre des fragments d’ADN génomiques provenant de LMD-9. Cette banque a été testée uniquement en conditions gastriques simulées dans le TIM. Puis, après avoir optimisé notre outil chez S. thermophilus en améliorant la méthode d’identification des gènes activés, une seconde banque R-IVET a été testée dans l’ensemble du tractus gastro-intestinal (TIM) et en système batch en présence du microbiote intestinal. Ces expériences nous ont permis de mettre en évidence, pour la première fois, des gènes de S. thermophilus spécifiquement activés dans les différents compartiments digestifs de l’homme. Ce travail de thèse contribue ainsi à approfondir les connaissances sur le comportement de cette bactérie dans le tractus gastro-intestinal humain. A moyen terme, ces travaux devraient permettre d’identifier des marqueurs de survie de S. thermophilus et de mieux comprendre son activité métabolique dans l’environnement digestif, facilitant la sélection de souches dans la perspective de développement d’aliments fonctionnels. / Streptococcus thermophilus is the lactic acid bacterium most commonly used after Lactococcus lactis in the dairy industry for the production of yogurt and cheese. It is the only streptococcus strain to have the GRAS status (Generally Recognized As Safe). Despite recent studies showing its ability to survive through the human digestive tract and valuable health effects, the probiotic status of S. thermophilus remains questioned. Thus, the objectives of this pHD work were (i) to increase knowledge on the survival of S. thermophilus in human digestive environment, by using the dynamic multi-compartmental TIM system (TNO gastro-intestinal model) and (ii) to identify the genes from S. thermophilus that are specifically activated in complex digestive environment using the R-IVET technology (Recombinase-based In Vivo Expression Technology). R-IVET is based on the excision of a reporter gene and consists of a plasmid vector carrying the promoterless recombinase cre and a chromosomal cassette composed of a marker gene flanked by loxP recognized by Cre. First, we introduced the R-IVET technology in S. thermophilus LMD-9. Its functionality was tested and validated in vitro and in the mice digestive tract. Then, the survival of four S. thermophilus strains was investigated in the TIM system and we showed that 3 of these strains were more resistant than the other one, very sensitive to gastrointestinal stresses. These results strengthen the idea that the survival of S. thermophilus is strain-dependent. We also highlighted that the survival of S. thermophilus was influenced by the food matrix, being higher in fermented compared to liquid milk. Lastly, we constructed a first genomic R-IVET library, by cloning upstream of cre genomic DNA fragments from LMD-9. This library was tested only in gastric condition (TIM). After optimization of our tool in S. thermophilus (improvement of the method allowing identification of the activated genes), a second R-IVET library was tested throughout the gastrointestinal system (TIM) and in batch system including intestinal microbiota. By identifying bacterial genes specifically activated in human digestive conditions, this work contributes to extend our knowledge on the behavior of S. thermophilus in the human gastrointestinal tract. This could open up opportunities in determining survival markers for S. thermophilus and better describing its metabolic activity in the human gut, then facilitating the selection of strains that can be included in functional foods.

Orientador: Marcelo Cristianini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T10:26:08Z (GMT). No. of bitstreams: 1 Oliveira_MiguelMeirellesde_M.pdf: 1688064 bytes, checksum: 7da6b05069a8467a053c06082ae009f5 (MD5) Previous issue date: 2013 / Resumo: Leites fermentados probióticos são considerados alimentos funcionais, devendo conferir proteção às culturas. A alta pressão dinâmica (APD) vem sendo investigada como alternativa na produção de leite fermentado, pois altera a funcionalidade de alguns constituintes. Este trabalho objetivou avaliar o uso de leite submetido à APD na cinética de fermentação de S. thermophilus e L. acidophilus, bem como a viabilidade das culturas, as características físico-químicas e reológicas durante a estocagem refrigerada do leite fermentado probiótico.Leite (3% de gordura) foi submetido ao processo de APD (100MPa e 200MPa) e comparados com controle (15/5MPa), e avaliada a cinética de fermentação (pH), atividade proteolítica e a viabilidade durante a fermentação do L. acidophilus (Cap.2). Outro experimento foi realizado com leite (2% de gordura) submetido à APD (50/5MPa, 100/5MPa, 150/5MPa e 180/5MPa) e comparado com o controle (15/5MPa), no qual foi avaliada a cinética de fermentação de S. thermophilus em co-cultura com L. acidophilus (Cap.3), bem como o comportamento reológico (Cap.3) e as características físico-químicas (Cap.4) do leite fermentado probiótico durante a estocagem refrigerada. A cinética de fermentação de Lactobacillus acidophilus foi alterada pela APD (100MPa e 200MPa) em comparação com o controle (15/5MPa), aumentando consideravelmente o tempo de fermentação com o aumento da pressão, sendo que após 24 horas de fermentação as amostras apresentaram valores de pH 4,49 (15/5MPa), pH 4,72 (100MPa) e 4,93 (200MPa). A atividade proteolítica foi alterada, sendo que pressões elevadas reduziram a atividade, atingindo no final da fermentação uma redução de 21,39% (100MPa) e 35,89% (200MPa), quando comparadas com o controle. Além disso, as amostras submetidas à APD apresentaram menor número de células viáveis de L. acidophilus no fim da fermentação em relação ao controle (0,28 ciclos log) (p<0,05). A cinética de fermentação do Streptococcus thermophilus e Lactobacillus acidophilus não foi alterada pela APD. O comportamento reológico durante a fermentação de leite processado à 100/5MPa e 180/5MPa apresentou correlação positiva entre a pressão usada com a consistência do leite fermentado, gerando um produto com maior consistência. Além disso, o processo de APD aumentou a capacidade de retenção de água (CRA) em 9,15% e reduziu a sinérese espontânea do produto em 31%, entre o controle e a amostras processada a 180/5MPa. A viabilidade do S. thermophilus não foi (p<0,05) influenciada pela APD (50/5MPa, 100/5MPa, 50/5MPa e 180/5MPa) em comparação com a homogeneização convencional (15/5MPa) durante a estocagem refrigerada, reduzindo a contagem em aproximadamente 0,2 ciclos log em todos os tratamentos estudados. A viabilidade do L. acidophilus foi maior na amostra processada a 180/5MPa a partir do 14° dia, em comparação ao controle (15/5MPa). O pH apresentou uma pequena redução (p<0,05) somente na amostra processada a 180/5MPa no ultimo dia. A atividade proteolítica das culturas não foi influenciada pela ADP. Os resultados demonstraram que a APD pode melhorar a textura de leite fermentado, aumentar a capacidade de retenção de água, reduzir a sinérese e alterar a cinética de fermentação, dependendo da cultura utilizada. No entanto, a viabilidade das culturas, a redução do pH e a atividade proteolítica não sofreram alterações expressivas pela APD, apesar de apresentar diferenças significativas / Abstract: Probiotics fermented milk are considered functional food, should provide protection to cultures. Dynamic high pressure (DHP) is being investigated as an alternative in the production of fermented milk, because changing the functionality of some constituents. This study evaluated the use of milk submitted to DHP in fermentation kinetics of S. thermophilus and L. acidophilus, as well as the viability of the cultures, the physico-chemical and rheological during refrigerated storage of fermented milk probiotic. Milk (3% fat) was subjected to the process of DHP (100MPa and 200MPa) and compared with control (15/5MPa), and evaluated the kinetics of fermentation (pH), proteolytic activity and viability during fermentation of L. acidophilus (Cap.2). Another experiment was performed with milk (2% fat) subject to DHP (50/5MPa, 100/5MPa, and 150/5MPa 180/5MPa) and compared with the control (15/5MPa), in which was evaluated the kinetics fermentation of S. thermophilus in co-cultured with L. acidophilus (Cap.3), and rheological behavior (Cap.3) and the physico-chemical (Cap.4) of probiotic fermented milk during refrigerated storage. The fermentation kinetics of Lactobacillus acidophilus was modified by DHP (100MPa and 200MPa), comparated to control (15/5 MPa) considerably increasing the fermentation time the higher the pressure used, and that after 24 hours of fermentation samples showed pH 4.49 (15/5MPa), pH 4.72 (100MPa) 4.93 (200MPa). The proteolytic activity was changed, with elevated pressures reducing proteolytic activity at the end of the fermentation, reaching a reduction of 21.39% (100MPa) and 35.89% (200MPa) compared with the control. In addition, the samples submitted to APD had lower number of viable probiotics in the end of fermentation compared to control (0,28 log cycles) (p<0.05). The fermentation kinetics of Streptococcus thermophilus and Lactobacillus acidophilus was not altered by DHP. The rheological behavior during fermentation in milk processed by DHP (100/5 MPa and 180/5 MPa) demonstrated a positive correlation between the pressure and consistency of the fermented milk. Moreover, the process of DHP favored water-holding capacity (WHC) in 10.13% and reduced syneresis spontaneous in 31% between the control and 180/5 MPa. The viability of S. thermophilus was not influenced by DHP (50/5MPa, 100/5MPa, 150/5MPa and 180/5MPa), comparated to conventional homogenization (15/5 MPa) during refrigerated storage, reducing the count in approximately 0.2 log cycles in all treatments. The viability of L. acidophilus was higher in the processed sample 180/5MPa from day 14, compared to the control (15/5 MPa). The pH showed a small reduction in sample subject to 180/5 MPa at the final day of storage. The proteolytic activity of the culture was not influenced by DHP. The results showed that the DHP may improve the texture of fermented milk, increase water-holding capacity, reduce syneresis and alter the kinetics of the fermentation. However, viability of cultures, the decrease in pH and proteolytic activity did not change by DHP, despite showing significant differences / Mestrado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

Les bactéries lactiques sont largement utilisées en tant que ferments dans l'industrie laitière. Leur production s’effectue généralement dans des milieux semi-définis ou complexes dans lesquels certains nutriments peuvent être apportés par des extraits de levure (EXLs). Ce projet de thèse, qui associe deux partenaires industriels, les groupes Lesaffre et Sacco, ainsi que l’INRA, s’est focalisé sur l’effet des peptides de deux EXLs (EXL1 et EXL2) sur une souche industrielle de Streptococcus thermophilus, un levain lactique d’intérêt économique majeur. L’hypothèse sous-jacente était que ces peptides pourraient avoir un double rôle de nutrition et de régulation de fonctions cellulaires pouvant présenter un intérêt technologique. Afin d’explorer cette question, une stratégie expérimentale à deux niveaux a été élaborée : i) caractérisation et suivi cinétique de la fraction peptidique des deux EXLs par spectrométrie de masse (peptidomique) durant la fermentation de S. thermophilus en bioréacteurs, et ii) suivi cinétique parallèle du transcriptome et du protéome de la bactérie. L’objectif final était de croiser ces deux niveaux d’information afin de corréler des différences de contenu peptidique avec des différences d’activation de systèmes participant aux performances globales du levain.La caractérisation et le suivi du peptidome des EXLs en cours de fermentation a nécessité un important travail de développement méthodologique ayant abouti in fine à l’élaboration d’un outil analytique complet, combinant analyse peptidomique à haut-débit des échantillons et traitement bioinformatique et statistique des données. Cet outil a permis d’identifier environ 4000 peptides différents composant les deux EXLs. Le suivi cinétique a notamment permis de préciser la spécificité du transporteur d’oligopeptides de la bactérie (Ami). En particulier, il s’est avéré qu’une charge nette positive était le facteur prévalent pour le transport des peptides chez S. thermophilus. En complément de cette approche semi-quantitative, des analyses quantitatives ont été réalisées sur des fractions peptidiques des EXLs (dosages différentiels par HPLC des acides aminés avant et après hydrolyse). Elles ont notamment permis de révéler d’importantes différences de teneurs en oligopeptides entre les deux EXLs.En parallèle, le suivi transcriptomique et protéomique réalisé durant la croissance de la bactérie a révélé deux faits marquants. Le premier fait a trait à la surexpression dans l’EXL1 d’un locus génétique régulé par un mécanisme de quorum sensing utilisant un peptide phéromone comme signal moléculaire. Le deuxième fait marquant concerne diverses voies de biosynthèse (acides aminés et purines) différentiellement affectées par les deux EXLs. L’origine de ces dynamiques pourrait être au moins pour partie le fait de différences de contenu peptidique entre les deux substrats. Notamment, certaines voies de biosynthèse pourraient avoir été modulées différentiellement sous l’action de régulateurs centraux tels que CodY, dont l’activité est corrélée au contenu peptidique du milieu, ou encore YebC, un régulateur CodY-like dont le lien fonctionnel avec CodY reste encore inconnu chez S. thermophilus. Tous ces résultats ouvrent d’intéressantes perspectives pour mieux explorer le lien entre peptides et métabolisme bactérien. A terme, cette démarche pourrait se traduire par l’identification de biomarqueurs de performances dans les EXLs, et l’élaboration à façon de produits permettant de maximiser le potentiel technologique des ferments lactiques. / Lactic acid bacteria are widely used as starters in dairy industry. They are generally produced in complex fermentation media containing a wide array of nutrients that can be provided by yeast extracts (YEs). The main goal of this thesis project, involving two industrial partners, Lesaffre and Sacco, as well as INRA, was to investigate the effect of the peptide fraction of two YEs (YE1 and YE2) on an industrial Streptococcus thermophilus strain, a major lactic acid starter. The underlying hypothesis of this whole project was that YE peptides could have a role in nutrition but also regulate cellular functions of technological relevance. In order to explore this question, a two-step strategy was elaborated: i) mass spectrometry characterization (peptidomics) of both YE peptide fractions and time course analysis of their relative abundance during the growth in bioreactors of S. thermophilus, and ii) parallel time course analysis of the strain transcriptome and proteome. The final objective was to cross these two levels of information in order to correlate differences of peptide content with differentially activated systems related to technological performances.YE peptidome characterization and kinetic analysis first required an important methodological development. It eventually resulted in a complete analytical tool that combines high throughput peptidomic analysis as well as bioinformatic and statistical data processing. This powerful tool was able to identify around 4,000 different peptides in both YEs. Then, the time course analysis also clarified the in vivo substrate specificities of the oligopeptide transport system of the bacterium (Ami). A peptide positive net charge notably turned out to be the leading factor governing peptide transport. In addition to this semi-quantitative approach, quantitative analyses were carried out on YE peptide fractions (differential HPLC analyses of amino acids before and after sample hydrolysis). They notably revealed significant differences in oligopeptides content between both YEs.Meanwhile, genome scale transcriptomic and proteomic analyses performed during the strain growth highlighted two significant events. The first one concerns the overexpression in YE1 of a quorum sensing-based genetic locus that uses a pheromone peptide as molecular signal. The second event relates to several biosynthesis pathways (amino acids and purines) that were differentially affected by both YEs. These dynamics could result from differences in peptide content between both substrates. In particular, some pathways could have been differentially modulated by central regulators such as CodY, whose activity is correlated to the medium peptide richness, or YebC, a CodY-like regulator whose functional link with CodY is still unknown in S. thermophilus. All these results open avenues for a better understanding of the interplay between peptides and bacterial metabolism. In the future, this whole approach could lead to the identification of performance biomarkers in YEs, which in turns may eventually translate into the conception of new customized products granting high technological performances to dairy starters.