S. thermophilus et L. bulgaricus sont les bactéries lactiques utilisées dans la fabrication du yaourt. En milieu laitier, cette association est appelée proto-coopération lorsqu'elle est bénéfique au développement de chacune des espèces. Même si quelques facteurs nutritionnels impliqués dans cette coopération ont été identifiés, ce phénomène reste encore peu connu au niveau moléculaire. Notre objectif est d'identifier plus amplement les bases moléculaires de cette association, d'évaluer la nature et l'étendue des échanges bactériens et d'estimer si des phénomènes de régulation interviennent entre les deux espèces. Nous avons sélectionné le couple de souches, S. thermophilus LMG18311 et L. bulgaricus ATCC11842, dont les génomes sont séquencés, pour lequel nous avons observé un effet proto-coopératif. En utilisant des analyses protéomique et transcriptomique, nous avons comparé la croissance en lait de S. thermophilus en présence (co-culture) ou en absence (monoculture) de L. bulgaricus et à deux stades de croissance différents. Nous avons obtenu la variation de 88 différents gènes ou protéines (4.6% des CDS) de S. thermophilus qui variaient en présence de L. bulgaricus. Notamment des variations concernant les métabolismes azoté, des purines et du fer. De plus, la co-culture conduit à des changements de régulation chez S. thermophilus avec la variation d'un régulateur de réponse, homologue au régulateur de réponse CovR des streptocoques. Cette étude révèle des échanges nutritionnels inattendus et des relations de régulation entre St. thermophilus et Lb. bulgaricus offrant de nouvelles pistes pour la compréhension du comportement proto-coopératif entre ces deux bactéries.

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.

In questo studio abbiamo analizzato l'impatto dellethermophiluds Streptococcus sull'epitelio del colon di ratto. Dopo la generazione del modello di ratto axenico e inoculato con S. thermophilus abbiamo investigato l'interazione tra il batterio e epithelo del colon di ratto. Dopo questo studio integrativo abbiamo analizzato l’espressione genica del colon usando due diversi approcci: Ibridazione sottrattiva I trascritti ottenuti dopo il sequenziamento dei cloni ottenuti con la SSH sono stati raggruppati in diversi categorie funzionali: arresto del ciclo cellulare e induzione del differenziamento, comunicazione cellulare e binding . due geni candidati sono stati privilegiati, krupel like fattore 4 e 14-3-3σ. Questi geni candidati sono stati analizzati mediante RT-PCR, qRT-PCR e Western Blot in animali mono-associati e in animali germ-free. I test hanno confermato che l’espressione dei geni candidati aumenta in presenza di S. thermophilus. Microarray Analysis. L’spressione genica è stata misurata per due gruppi di animali: i) ratti privi di germi, ii) ratti mono-associati con il ceppo LMD9 di Streptococcus thermophilus. I risultati delle analisi dei dati di microarray indicano che Streptococcus thermophilus influenza notevolmente l'espressione genica nelle cellule epiteliali del colon. / In this study we have investigated the impact of Streptococcus thermophiluds on the rat colonic epithelium. After generation of the model axenic rat and inoculated with S. thermophilus we have investigated the interplay between bacteria and host colon. Colonic epithelium gene expression was investigated also, with two different approaches: Suppressive Subtractive Hybridization. The subtraction library was prepared subtracting mRNA between epithelial cells from colonic mono-associated rats and germ-free rats. The transcripts generated by SSH were grouped into divers Functional groups: cell-cycle arrest and induction of differentiation; cell-communication and binding. Tow candidates genes were privileged, krupel like factor 4 and 14-3-3σ. These candidate genes were tested by RT-PCR, qRT-PCR and Western blot in mono-associated animals and in germ-free animals. The tests confirmed that candidate genes increase their expression in the presence of S. thermophilus. Microarray analysis. Gene expression was measured in tow groups of animals: i) germ-free rats; ii) mono-associated rats inoculated with LMD9 strain of Streptococcus thermophilus. The results of microarray analysis data show that Streptococcus thermophilus remarkably affected gene expression in the colonic epithelial cells. Streptococcus thermophilus enhanced the expression of genes involved in different pathways in the host, compared to the gem free group.

Orientador: Katia Sivieri / Resumo: Objetivo: desenvolver um leite fermentado com Lactobacillus acidophilus- LA-5, Bifidobacterium lactis- BB-12 e Streptococcus thermophilus com concentrações otimizadas de farinha de acerola e pectina. Verificar as características físico-químicas, sensoriais e microbiológicas da formulação otimizada. Avaliar a viabilidade dos microrganismos e a sobrevivência dos microrganismos probióticos frente à simulação das condições gastrointestinais in vitro. Métodos: a farinha de acerola foi avaliada com relação ao conteúdo fenólico, atividade antioxidante e teor de fibras totais. Foi utilizada a Metodologia de Superfície de Resposta (MSR) para obtenção das concentrações otimizadas de farinha de acerola e pectina no leite fermentado. O leite fermentado otimizado foi avaliado durante 28 dias de armazenamento refrigerado (5 ± 1 °C) quanto às características físico-químicas: composição centesimal, pH, acidez Dornic, atividade antioxidante, sinérese, perfil de textura; às características sensoriais: sabor, impressão global e textura e às características microbiológicas: viabilidade dos microrganismos e sobrevivência gastrointestinal a partir de simulação de condições gastrointestinais in vitro dos microrganismos probióticos. Resultados: a farinha de acerola apresentou 52,50 mg EAG g-1 de compostos fenólicos totais, 226,86 mmols ET g-1 de atividade antioxidante segundo método de DPPH e 51mmol ET g-1 por FRAP com 56,28 ± 0,19 % de fibras totais. Somente a farinha de acerola influenciou signifi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: to develop fermented milk with Lactobacillus acidophilus LA-5, Bifidobacterium lactis Bb-12 and Streptococcus thermophilus with optimized concentrations of acerola flour and pectin. The physical-chemical, sensorial and microbiological characteristics of the optimized formulation was verified. To assess the viability of microorganisms and the survival of probiotic microorganisms against the simulation of gastrointestinal conditions in vitro. Methods: acerola flour was evaluated for phenolic content, antioxidant activity and total fiber content. The Response Surface Methodology was used to obtain optimum concentrations of acerola flour and pectin in fermented milk. The optimized fermented milk was evaluated during 28 days of refrigerated storage (5 ± 1 °C) for the physico-chemical characteristics: centesimal composition, pH, Dornic acidity, antioxidant activity, syneresis, texture profile; sensory: taste, overall impression and texture and microbiological: viability of microorganisms and gastrointestinal survival from simulation of in vitro gastrointestinal conditions of probiotic microorganisms. Results: acerola flour presented 52.50 mg GAE g -1 of total phenolic compounds, 226.86 mmols TE g-1 of antioxidant activity according to DPPH method and 51 mmol TE g -1 by FRAP with 56.28 ± 0.19% of total fibers. Only acerola flour significantly influenced (p < 0.05) the sensorial attributes of symbiotic fermented milk. The developed mathematical model was effective in determining the optimized region of the independent variables. The optimized formulation showed 18.42 ± 0.75 g 100 g-1 dietary fiber. During the storage period there was no statistical difference (p <0.05) in the pH values comparing the treatment and control formulations. In relation... (Complete abstract click electronic access below) / Mestre

Orientador: Célia Maria de Sylos / Coorientador: Elizeu Antônio Rossi / Banca: Daise Aparecida Rossi / Banca: Daniela Cardoso Umbelino Cavallini / Banca: Daniele Peres Miguel / Banca: Sueli Ciabotti / Resumo: O objetivo deste trabalho foi desenvolver uma bebida láctea sabor morango carbonatada e fermentada com bactérias probióticas. Foram elaboradas quatro formulações de bebida láctea: Controle (BL); Fermentada (BLF); Carbonatada (BLC) e Fermentada Carbonatada (BLFC). Nas amostras submetidas à carbonatação, utilizou-se um carbonatador para injeção do gás dióxido de carbono (CO2) dissolvido em água potável, resultando em um produto com a proporção de 2: 1 (volume de gás/ volume de bebida láctea) e nas amostras fermentadas, adotou-se o cultivo constituído das bactérias lácticas Lactobacillus acidophilus-LA-5®, Bifidobacterium BB-12® e Streptococcus thermophilus. As amostras foram caracterizadas quanto a parâmetros físico-químicos (composição química, índice de proteólise e teor de carbonatação), parâmetros microbiológicos (contagens de coliformes totais, E. coli, bolores e leveduras e viabilidade das bactérias lácticas) e aspectos sensoriais através de testes de aceitação e intenção de compra, ao longo de 28 dias de armazenamento refrigerado das bebidas lácteas. As formulações de bebidas fermentadas BLF e BLFC apresentaram diferenças significativas (p < 0,05) em relação às amostras não fermentadas BL e BLC nos parâmetros glicídeos redutores em lactose e em sacarose, pH e índice de proteólise, com menores médias absolutas e acidez com maiores médias em todos os tempos estudados. As bebidas fermentadas BLF e BLFC não apresentaram presença de microrganismos contaminantes. A amostra BLC apresentou presença de leveduras e coliformes totais, mas as contagens indicaram que estava própria para o consumo no tempo 28 dias. Entretanto, a amostra controle (BL) apresentou contagens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The goal of this work was to develop a strawberry flavored dairy beverages carbonated and fermented with probiotic bacteria. Four formulas of dairy beverages were elaborated: Control (BL); Fermented (BLF); Carbonated (BLC) and Carbonated Fermented (BLFC). In samples submitted for carbonation, a carbonator was used for the carbon dioxide (CO2) gas injection dissolved in drinking water, resulting in a product with a ratio of 2:1 (volume of gas volume of dairy beverages) and the cultivation consisting of lactic bacteria Lactobacillus acidophilus-LA-5®, Bifidobacterium BB-12® and Streptococcus thermophilus was employed on the fermented samples. The samples were characterized by physical and chemical parameters (chemical composition, proteolysis index and carbonation proportion), microbiological parameters (total coliform counts, E. coli, yeasts and molds and viability of the lactic bacteria) and sensory aspects through acceptance testing and purchase intention, over the course of 28 days of refrigerated storage of the dairy beverages. The formulations of the BLF and BLFC fermented drink presented significant differences (p < 0,05) in relation to the non-fermented samples BL and BLC in glucides reducer of lactose and sucrose parameters, pH and proteolysis index, with lower absolute averages and acidity with higher averages at all studied times. The fermented beverages BLF and BLFC did not show any presence of contaminating microorganisms. The BLC sample showed the presence of yeasts and coliform counts, but the counts indicated that it was suitable for consumption in 28 days time. However, the sample... (Complete abstract click electronic access below) / Doutor

La conception de nouveaux produits laitiers fermentés fonctionnalisés avec les peptides bioactifs est l'une des stratégies prometteuses pour le développement d'aliments fonctionnels. L'objectif de cette thèse s'inscrit ainsi dans une démarche de production d'un lait fermenté contenant un peptide anxiolytique, l'alpha-casozépine (alpha-CZP ; f91-100 de la CN-alpha s1 bovine) ou du fragment plus court (f91-97). Dans un premier temps, nous avons étudié la stabilité des peptides bioactifs vis-à-vis des souches ayant le phénotype PrtS+ (LMD-9) et PrtS- (CNRZ1066) de S. thermophilus. Il s'est avéré que les deux souches sont capables d'hydrolyser le peptide en fragments plus courts par l'activité peptidasique de surface. Il a été vérifié que l'hydrolyse n'est due ni aux peptidases intracellulaires issues d'une éventuelle lyse cellulaire ni à la protéase chaperonne HtrA. Les activités de type amino-, X-prolyl dipeptidyl-, carboxy-peptidases et peptidyl dipeptidase de surface ont été mises en évidence. L'ajout de peptides bioactifs dans des laits fermentés nécessite donc la caractérisation préalable du potentiel protéolytique des bactéries lactiques. Afin de n'ajouter qu'un type de peptides et non pas une fraction peptidique, nous avons exprimé de manière hétérologue l'alpha-CZP sous forme multimérique chez L. lactis en utilisant le système d'induction NICE. Il a été révélé par le test ELISA qu'une petite quantité d'alpha-CZP multimérique a été produite par les cellules induites mais également non induites par la nisine. Le ralentissement important de la croissance des cellules de L. lactis recombinante induites par la nisine suggère que le multimère peptidique a des effets toxiques sur les cellules / The designing of novel fermented milk products functionalized with bioactive peptides is one of the promising strategies to develop functional foods. The objective of the thesis is thus the development of fermented milks comprising of an anxiolytic peptide, alpha-casozepine (f91-100 of bovine alpha s1 CN) or its shorter fragment (f91 97). Firstly, we studied the stability of different bioactive peptides towards two strains, with PrtS+ (LMD¬9) and PrtS- (CNRZ1066) phenotypes, of S. thermophilus, a starter bacterium widely used for the manufacture of yogurt. It was revealed that both strains hydrolysed the peptides into shorter fragments by surface peptidases. The hydrolysis was neither due to intracellular peptidases eventually released from cell lysis nor to extracellular chaperone protease HtrA, since mutant LMD-9-deltahtrA also showed the same type of hydrolysis. Aminopeptidase, X-prolyl dipeptidyl peptidase, peptidyl dipeptidase and carboxypeptidase activities associated to the cell-surface of both strains were observed. Therefore, addition of bioactive peptides in fermented milks requires well prior characterization of the proteolytic potential of lactic acid bacteria. Finally, in order to add only one type of peptides instead of a peptide fraction, we expressed heterologously alpha-CZP in multimeric form in L. lactis using NICE system. It was revealed by ELISA test that a small quantity of recombinant alpha-CZP multimer was produced by both nisin-induced as well as non-induced cells. The significant decrease in the growth of nisin-induced recombinant L. lactis suggests that the peptide multimer has toxic effects on the cells

No description available.

The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers — fragments of the invading nucleic acids — into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving no time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, I show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. I also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. While the acquisition of CRISPR spacers is not random and while the supplementation of eDNA influences that bias, eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection. / Thesis / Doctor of Philosophy (PhD) / Phages are viruses that can infect and kill bacteria with a 99.9999% success rate. To defend themselves, bacteria have evolved an adaptive immune system called the CRISPR-Cas system. This system uses a piece of DNA, called a spacer, that matches the phage to destroy it. However, in order to use their CRISPR-Cas system, they need to obtain this spacer. Given how dangerous phages are, how bacteria acquire this spacer is a mystery. My project investigates the possibility that bacteria use DNA floating in the environment to vaccinate themselves against phages before ever encountering them. In this thesis I show that DNA floating in the environment helps bacteria acquire these spacers. I also show that it is specific sections of phage DNA that helps bacteria. This shows that bacteria can use their environment to defend themselves against threats before they even happen.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-05T12:37:57Z No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) / Made available in DSpace on 2016-10-05T12:37:57Z (GMT). No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) Previous issue date: 2011-05-27 / Yogurt is a fermented milk resulting from a mutual interaction between the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. For yoghurt quality assurance, the cell numbers of each microorganism, should not be less than 1x107per gram, therefore a relative ratio lactobacilli and streptococci should be 1:1, although a 1:2 ratio is also accepted. During storage of yoghurt exposed for sale, post-acidification may occur, changing the ratio of the two microorganisms. The objectives of this research were to evaluate yoghurts of four different manufacturers named A, B, C and D, in relation to the number and balance between streptococii and lactobacilli during the storage and its relation with acidity and pH. In this way, yoghurts with up to 20 days of manufacturing (band I) and more than 20 days (band II) were evaluated. Better recovery of L. delbruckii subsp. bulgaricus and S. thermophilus was observed with LB and M17 media respectively, which were used to determine the relative ratio of the two microorganisms. An imbalance in the lactobacilli numbers which were lower than that of streptococci was verified and considered inadequate, in two out of four commercial brands. In yoghurts from the manufacturer A, there was a significant reduction in the number of lactobacilli from band I to band II leading to an increase in the relative ratio of streptococii to lactobacilli. There was no significant difference in the counts of streptococci or bacilli from one band to another with the other three brands of yoghurts. The acidity of yoghurts from manufacturer D revealed significantly higher (P< 0.05) than the others, although it did not result in an increased pH reduction. All samples attended the legislation in relation to total lactic acid bacteria counts, acidity, pH and mould and yeast count / Iogurte ? um leite fermentado resultante de uma intera??o microbiana mutualista entre as bact?rias l?cticas Streptococcus thermophilus e Lactobacillus delbrueckii subsp. bulgaricus. Para que a qualidade do iogurte seja garantida o n?mero de c?lulas destes dois microrganismos individualmente, n?o deve ser inferior a 1x107 por grama, portanto uma propor??o relativa de lactobacilos e estreptococos de 1:1 sendo a propor??o 1:2 tamb?m aceita. Durante o armazenamento dos iogurtes expostos para venda podem ocorrer p?s-acidifica??o e modifica??o na propor??o dos dois microrganismos. Os objetivos desta pesquisa foram avaliar iogurtes de quatro diferentes fabricantes as quais foram denominados A, B, C, D quanto ao n?mero e equil?brio entre as duas bact?rias l?cticas durante o armazenamento, e ao mesmo tempo determinar as caracter?sticas f?sico-quimicas (pH e acidez) e contagem de bolores e leveduras. Para tanto, adotou-se duas faixas de avalia??o, faixa I (at? 20 dias de fabrica??o) e faixa II (ap?s 20 dias). Foi observada melhor recupera??o L. delbruckii subsp. bulgaricus e S. thermophilus, respectivamente com os meios LB e M17 que foram usados para determina??o da propor??o relativa dos dois microrganismos. Foi verificado um desequil?brio no n?mero de lactobacilos que foi inferior ao de cocos e, considerado inadequado, em duas das quatro marcas comerciais. Nos iogurtes da marca A houve redu??o significativa no n?mero de lactobacilos da faixa I para faixa II levando a um aumento na propor??o de cocos relativa ao de bacilos. Nos iogurtes dos tr?s outros fabricantes n?o houve diferen?a significativa nas contagens de cocos ou de lactobacilos de uma faixa para outra. Tamb?m n?o foi observada diferen?a significativa de acidez e pH relacionados ao tempo de prazo comercial nas faixas I e II nas quatro marcas de iogurte analisadas. A acidez dos iogurtes do fabricante D foi significativamente mais elevada (P<0,05) que a dos demais, embora n?o tenha resultado em maior redu??o de pH nestes produtos. Todas as amostras analisadas estavam em conformidade com a legisla??o vigente no que se refere ao m?nimo exigido de bact?rias l?ticas totais que ? de 1x107 UFC/mL. Tamb?m estavam em conformidade com rela??o ? acidez, pH e contagem de bolores e leveduras. Palavras-chave: leite

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).