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.

Diss. med. dent. Zürich. / Literaturverz.

Universiẗat, Diss., 2006--Frankfurt (Main).

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

Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.

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.

Streptococcus thermophilus est la bactérie lactique la plus utilisée après Lactococcus lactis dans l'industrie laitière pour la fabrication de yaourts et de fromages à pâte cuite (Emmental, gruyère), filée (Mozarella) ou pressée (Cheddar). Il s'agit du seul streptocoque à avoir le statut de bactérie GRAS (Generally Recognized As Safe). Dans un premier temps le potentiel probiotique de 30 souches de S. thermophilus de différentes origines a été testé par l'étude de leurs capacités à résister aux différentes conditions de stress rencontrées pendant leur passage dans le tractus gastro-intestinal (bas pH, sels biliaires et stress oxydant), de leur capacité à adhérer aux cellules épithéliales intestinales et de leurs propriétés immunomodulatrices. La majorité des souches réduit la production d'interleukine IL-8 (pro-inflammatoire) alors qu'elles induisent la production d'interleukine IL-10 (anti-inflammatoire) et l'IL-12 (pro-inflammatoire). Sur la base du rapport IL-10/IL-12, qui permet d'apprécier le potentiel anti-inflammatoire d'une souche, nous avons observé que certaines d'entre-elles pourraient avoir un fort potentiel anti-inflammatoire. L'Analyse en Composantes Principales (ACP) nous a permis de séparer les souches en 6 catégories différentes présentant des propriétés distinctes. A l'intérieur de chaque classe, une variabilité entre les souches a été observée et des caractéristiques intéressantes identifiées. Cependant, aucune des classes ne peut être considérée comme contenant le probiotique « parfait ». Suite à cette étude et sur la base de sa résistance aux stress gastriques et de ses capacités d'adhésion, et sachant que la séquence de son génome est disponible, la souche LMD-9 a été sélectionnée pour la deuxième partie de ce travail, à savoir la construction d'un outil basé sur la technologie R-IVET pour étudier l'état physiologique de S. thermophilus dans le tractus digestif. L'outil R-IVET mis au point se compose de deux éléments: un vecteur plasmidique portant le gène cre codant une recombinase spécifique dépourvu de son promoteur et une cassette chromosomique composée d'un gène de résistance à la spectinomycine flanqué par des sites loxP, reconnus par la recombinase Cre. La fonctionnalité de l'outil R-IVET a ensuite été testée par le clonage de trois promoteurs différents de S. thermophilus (PprtS, Pshsp and Plac) en amont de cre. Le système a été valide in vitro avec les trois promoteurs et in vivo avec le promoteur Plac / Streptococcus thermophilus is a lactic acid bacterium used after Lactococcus lactis in the dairy industry for the production of yogurt and cheeses like Emmental, Gruyere, Mozarella and Cheddar. It is the only streptococcus to have the GRAS (Generally Recognized As Safe) status. In this work, the probiotic potential of 30 S. thermophilus strains from different origins was tested by studying their ability to resist different stress conditions encountered during their passage through the GIT (low pH, bile salts and oxidative stress), their ability to adhere to intestinal epithelial cells and their immunomodulatory properties. Majority of the strains reduced the production of interleukin IL-8 (pro-inflammatory) and induced the production of interleukin IL-10 (anti-inflammatory) and IL-12 (pro-inflammatory). On the basis of the ratio IL-10/IL-12 which allows to evaluate the anti-inflammatory potential of a probiotic, several strains appeared to have a high the anti-inflammatory potential. Principal Component Analysis (PCA) allowed us to classify strains in 6 different categories with different properties. Within each class, variability and interesting features were observed, but none of the classes could be considered as containing the perfect probiotic. Following this study and on the basis of its resistance to gastric stress and its adhesion capacity and knowing that its genome sequence is available, the strain LMD-9 was selected for the second part of this work, namely the construction of a tool based on R-IVET to study the physiological state of S. thermophilus in the digestive tract. This tool is composed of two elements: a plasmid vector (pULNcreB), carrying the gene cre encoding a site-specific recombinase without its promoter and a chromosomal cassette composed of a gene of resistance to spectinomycin flanked by loxP sites which are recognized by the recombinase Cre. The functionality of the tool R-IVET was then tested by cloning three different promoters of S. thermophilus (PprtS, Pshsp and Plac) upstream of cre. System was valid in vitro with all the three promoters and in vivo by using the lactose operon promoter Plac