Cinco linhagens bacteriocinogênicas foram selecionadas quanto às suas propriedades sensoriais e tecnológicas em leite. Somente Lactococcus lactis ssp. lactis CTC204 apresentou as características (baixa acidificação e proteólise) adequadas para a fabricação de queijo Minas Frescal. Contudo, a baixa atividade da bacteriocina (3x102UA.mL-1) inviabilizou a sua utilização como bioconservante para a produção in situ no queijo. Estudos para obtenção de um bio-ingrediente foram conduzidos. A atividade da bacteriocina foi maior em leite adicionado com extrato de levedura e glicose (1,3x104UAmL-1) comparado com prebioticos (fruto-oligiossacarideo e inulina). A secagem em Spray-drier originou um bio-ingrediente com atividade de 1,3x105UAmL-1. Listeria monocytogenes ATCC7644 (4,0LogUFC.mL-1) foi inoculada nos queijos obtidos por acidificação do leite pasteurizado e microfiltrado. A contagem aumentou 6,2LogUFC.gL-1 e 2LogUFC.gL-1 no 15° e 21º dia de estocagem a 6±1°C, respectivamente, nos queijos sem e com adição de 10% do bio-ingrediente. Portanto, inibição de 4,2 LogUFC.g-1 do patógeno no 15° dia de estocagem. / Among the five bacteriocin-producing lactic bacteria strains tested, only Lactococcus lactis subsp. cremoris CTC 204 presented suitable sensory and technological properties for Minas Frescal cheesemaking process. However, because of the low yield of bacteriocin production in milk (3.0x102 AU.mL-1), its use as biopreservative for in situ production in cheese was impractical. We carried out experiments to obtain a bioingredient. Bacteriocin activity was higher in milk supplemented with glycose and yeast extract (1.3x104 AU.mL-1) compared to probiotics (fructooligosaccharides and inulin). Spray drying fermented milk originated a bioingredient presenting activity of 1.3x105 AU.mL-1. Population count of Listeria monocytogenes ATCC 7644, inoculated (4.0 Log CFU.mL-1) in cheese produced by direct acidification of pasteurized microfiltered milk, reached 6.2 Log CFU.g-1 and 2.0 Log CFU.g-1 at day 15 of storage at 6±1°C, in cheese without and with addition of 10% of bioingredient, respectively, therefore presenting pathogen inhibition of 4.2 Log CFU.g- 1 at day 15 of storage.

Apesar do sucesso do transplante na clínica, os importantes efeitos adversos dos imunossupressores, usados para prevenir e tratar a rejeição, apontam para a necessidade de novas terapias imunorreguladoras. A via oral tem sido efetiva na indução de imunorregulação, em diversos modelos experimentais, principalmente de doenças autoimunes. A Hsp60/65 é uma molécula com grande potencial imunoterapêutico, por sua capacidade de induzir respostas imunes pró-inflamatória e imunorreguladora. Testamos se a terapia oral com o probiótico Lactococcus lactis que expressa a Hsp65, combinada à administração de aloantígenos do doador (AloAg-doador), atua sinergicamente na indução de tolerância ao enxerto de pele semialogeneico murino, ou no aumento de sua sobrevida. Testamos diferentes combinações de terapia oral, assim como a influência da utilização de um anti-inflamatório, inibidor seletivo de COX-2 (celecoxibe). O transplante de pele foi realizado 10 dias após a última administração oral dos probióticos e aloantígenos do doador. Não observamos efeitos benéficos na sobrevida do enxerto no grupo de animais que receberam L.lactis que produz Hsp65, sozinho ou em combinação com AloAg-doador e/ou o anti-inflamatório. Em contraste, a terapia oral combinada com o probiótico L.lactis selvagem e AloAg-doador aumentou significativamente a sobrevida do enxerto (p=0,01), em comparação com o grupo não tratado. Nesse grupo que teve maior sobrevida do aloenxerto (L,lactis selvagem e AloAg-doador), também observamos maior quantidade de epitélio preservado (p=0,02) e maior expressão de TGF-beta (p=0,04), no enxerto, em comparação com o grupo sem tratamento. Não observamos diferenças significativas na expressão, in situ, de FOXP3 e IL-17, que foi baixa em todos os grupos experimentais. Concluímos que a Hsp65 não induziu efeito imunorregulador capaz de prolongar a sobrevida do enxerto. No entanto, a manipulação da microbiota com a terapia combinada com o L.lactis selvagem e a exposição a antígenos do doador, previamente, ao transplante, induz mecanismos imunorreguladores capazes de controlar, mesmo que parcialmente, as respostas inflamatórias dirigidas ao aloenxerto de pele, provavelmente, com a participação de TGF-beta / Despite the success of clinical transplantation, the significant side effects induced by immunosupressants used to prevent and treat rejection, indicate the need for novel immunoregulatory therapies. The oral route has been effective in inducing immunoregulation in several experimental models, mostly in pathological autoimmunity. Heat Shock protein 60/65 (Hsp) displays great immunotherapeutic potential due to its capacity to induce both pro-inflammatory and immunregulatory responses. We tested whether oral therapy with the probiotic Lactococcus lactis that expresses Hsp65, in combination with donor alloantigens (Donor-Allo-Ag), acted synergically, inducing immunotolerance or increasing graft survival, in a murine model of semiallogeneic skin transplantation. We tested different oral therapy combinations, as well as the association with a COX-2 selective nonsteroidal anti-inflammatory drug (celecoxib). Skin transplantation was performed 10 days after the last oral administration of probiotics and Donor-Allo-Ag. We observed no beneficial effect on graft survival in the group that received L.lactis that produce Hsp65, alone or in combination with Donor-Allo-Ag/and/or the anti-inflammatory drug. In contrast, combined oral therapy with wild type L.lactis and Donor-Allo-Ag significantly prolonged graft survival (p=0.01), in comparison to non-treated animals. In this prolonged-survival group (L.lactis and Donor-Allo-Ag), we also found higher extension of preserved epithelium (p=0.02) and higher expression of TGF-beta (p=0.04), within the graft, in comparison to non-treated animals. We found no significant differences in the intragraft expression of FOXP3 and IL-17, which was essentially absent or very low. We conclude that Hsp65 did not induce immunoregulatory effects capable of prolonging graft survival. However, the microbiota manipulation with the combined oral therapy with wild type L.lactis and Donor-Allo-Ag, prior to transplantation, induce immunoregulatory mechanisms capable of partially controlling the inflammatory responses to the graft, most likely involving the participation of TGF-beta

Cette thèse présente les résultats de travaux visant à étudier les interactions entre les conditions hydrodynamiques et le comportement bactérien en bioréacteur. En s'intéressant plus particulièrement aux phénomènes agissant aux petites échelles, nous avons cherché à identifier, caractériser et quantifier les couplages hydro-bio à l'échelle de la cellule. Ici, la souche Lactococcus lactis a été choisie comme modèle microbiologique, alors qu’un réacteur de type Couette a été préféré afin d’engendrer des contraintes hydrodynamiques connues et définies. Il a été démontré que dans des conditions spécifiques d’écoulement (Modulated Wavy Vortex Flow), les bactéries lactiques s’agrègent au sein d’une matrice riche en polysaccharides. Le lien entre ce phénotype atypique et les contraintes locales liées à l’écoulement a été étudié { l’aide de simulation numérique directe de l’écoulement combiné { un suivi de particule. Cette approche permet d’établir les profils temporels des contraintes subies et de comparer la nature des forces disruptives subies au mécanisme d’agrégation séparation des cellules bactériennes au sein de leur matrice. Ce travail de recherche donne ainsi d’autres exemples d’interactions cellule – environement en bioréacteurs, mettant en exergue des effets mécaniques / The aim of this work is to reach a better understanding of environmental effects on bacterial behaviour in bioreactors. Particular attention has been paid to hydrodynamically-induced stresses at the cell scale, with a view to characterizing and quantifying these local interactions. As a “model experiment”, Lactococcus lactis NCDO2118 has been cultivated in a CouetteBioreactor, a device generating a known and defined flow field. Under specific flow regime (Modulated Wavy Vortex Flow), the cells end up being entrapped in a polysaccharidic matrix. The phenotype of the cells has been demonstrated to be strongly affected by the flow conditions. The stress signal encountered by the cells has been characterized, through umerical simulation (Direct Numerical Simulation) and lagrangian particle tracking, and linked to the phenotypic expression. These studies provide further examples of bacterial response to local hydrodynamic conditions

Parmi les stratégies visant à contrôler la croissance de microorganismes pathogènes dans un aliment, la biopréservation qui s’appuie sur l’utilisation des capacités inhibitrices d’autres microorganismes offre une grande diversité d’opportunités. Il est cependant nécessaire de comprendre les mécanismes moléculaires et physiologiques régissant l’antagonisme du microorganisme protecteur vis-à-vis de la bactérie indésirable. L’objectif de cette thèse était de caractériser l’antagonisme de L. garvieae N201, isolé de fromage, vis-à-vis de souches de S. aureus par des approches in vitro : génomique, transcriptomique (ciblée concernant S. aureus, globale concernant L. garvieae) et phénotypique. Un acteur avait déjà été identifié : le peroxyde d’hydrogène (H2O2) produit par L. garvieae sous un niveau d’aération élevé. Lors de ces travaux de thèse, il a été montré que le peroxyde d’hydrogène serait également produit par L. garvieae sous une faible aération en quantité faible (indétectable par spectrophotométrie) mais suffisante pour induire une inhibition de S. aureus. Les gènes de production du H2O2 de L. garvieae (poxB, sodA) seraient exprimés constitutivement quel que soit le niveau d’aération. Les gènes de dégradation du H2O2 (katA, sodA, ahpC / ahpF) seraient plutôt surexprimés sous une faible aération, suggérant leur rôle dans un mode de contrôle de la concentration en H2O2 autogène par L. garvieae. En parallèle, trois autres mécanismes potentiellement impliqués dans l’antagonisme ont été mis en évidence : i) la répression de gènes de réponse au stress (clpC, ctsR, dnaK) de S. aureus par L. garvieae et l’aération, ii) la répression de gènes de division cellulaire de S. aureus (mraZ, mraW, potentiellement le cluster dcw) par L. garvieae, iii) la production d’un effecteur extracellulaire par L. garvieae dont la nature reste à caractériser. Ajouté à cela, la présence de L. garvieae modulerait l’expression des principaux gènes de virulence de S. aureus, réprimant ceux codant pour les entérotoxines sous une faible aération. Ainsi, la souche L. garvieae N201 s’est révélée être une candidate intéressante comme agent de biopréservation. Cependant, son innocuité pour l’Homme devra être vérifiée et son antagonisme sur S. aureus devra être évalué en matrice alimentaire. Les données générées ainsi que la démarche développée pourront être utilisées afin d’étudier des interactions entre d’autres espèces d’intérêt et dans des écosystèmes différents. / Among strategies aiming to control the growth of spoilage microorganisms in food, the biopreservation is based on the inhibitory capacities of other microorganisms and presents a considerable variety of opportunities. A good understanding of the molecular and physiologic mechanisms underlying the antagonism of the preservative microorganism against the spoilage bacterium is also required. This thesis aimed to characterize the antagonism of L. garvieae N201 dairy strain against S. aureus strains combining in vitro strategies: genomic, transcriptomic (targeted concerning S. aureus, global concerning L. garvieae) and phenotypic. The involvement of hydrogen peroxide (H2O2) produced by L. garvieae under high aeration was already known. Although H2O2 concentration was undetectable using spectrophotometry method, it was produced by L. garvieae under low aeration at sufficient concentration to induce S. aureus inhibition. L. garvieae H2O2 -synthesis genes (poxB, sodA) seemed constitutively expressed whatever the aeration level. L. garvieae H2O2-degradation (katA, sodA, ahpC / ahpF) genes were overexpressed under low aeration, suggesting their involvement in control of autogenous H2O2 level. In parallel, three other mechanisms may be involved in this antagonistic relationship: i) the repression of S. aureus stress-response genes (clpC, ctsR, dnaK) by L. garvieae and / or under high aeration, ii) the repression of S. aureus cell-division genes (mraZ, mraW and probably the dcw cluster) by L. garvieae, iii), the production by L. garvieae of an extracellular effector which has to be characterized. Additionally, L. garvieae can modulate the expression of S. aureus major virulence genes, repressing those coding for enterotoxins under low aeration. Thus, L. garvieae N201 turned out to be an interesting candidate for biopreservative applications. However, its safety for humans should be approved and its antagonism against S. aureus has to be investigated in food matrices. The data resulting from this work may be used to study other interactions between other valuable species and in other ecosystems.

Apesar do sucesso do transplante na clínica, os importantes efeitos adversos dos imunossupressores, usados para prevenir e tratar a rejeição, apontam para a necessidade de novas terapias imunorreguladoras. A via oral tem sido efetiva na indução de imunorregulação, em diversos modelos experimentais, principalmente de doenças autoimunes. A Hsp60/65 é uma molécula com grande potencial imunoterapêutico, por sua capacidade de induzir respostas imunes pró-inflamatória e imunorreguladora. Testamos se a terapia oral com o probiótico Lactococcus lactis que expressa a Hsp65, combinada à administração de aloantígenos do doador (AloAg-doador), atua sinergicamente na indução de tolerância ao enxerto de pele semialogeneico murino, ou no aumento de sua sobrevida. Testamos diferentes combinações de terapia oral, assim como a influência da utilização de um anti-inflamatório, inibidor seletivo de COX-2 (celecoxibe). O transplante de pele foi realizado 10 dias após a última administração oral dos probióticos e aloantígenos do doador. Não observamos efeitos benéficos na sobrevida do enxerto no grupo de animais que receberam L.lactis que produz Hsp65, sozinho ou em combinação com AloAg-doador e/ou o anti-inflamatório. Em contraste, a terapia oral combinada com o probiótico L.lactis selvagem e AloAg-doador aumentou significativamente a sobrevida do enxerto (p=0,01), em comparação com o grupo não tratado. Nesse grupo que teve maior sobrevida do aloenxerto (L,lactis selvagem e AloAg-doador), também observamos maior quantidade de epitélio preservado (p=0,02) e maior expressão de TGF-beta (p=0,04), no enxerto, em comparação com o grupo sem tratamento. Não observamos diferenças significativas na expressão, in situ, de FOXP3 e IL-17, que foi baixa em todos os grupos experimentais. Concluímos que a Hsp65 não induziu efeito imunorregulador capaz de prolongar a sobrevida do enxerto. No entanto, a manipulação da microbiota com a terapia combinada com o L.lactis selvagem e a exposição a antígenos do doador, previamente, ao transplante, induz mecanismos imunorreguladores capazes de controlar, mesmo que parcialmente, as respostas inflamatórias dirigidas ao aloenxerto de pele, provavelmente, com a participação de TGF-beta / Despite the success of clinical transplantation, the significant side effects induced by immunosupressants used to prevent and treat rejection, indicate the need for novel immunoregulatory therapies. The oral route has been effective in inducing immunoregulation in several experimental models, mostly in pathological autoimmunity. Heat Shock protein 60/65 (Hsp) displays great immunotherapeutic potential due to its capacity to induce both pro-inflammatory and immunregulatory responses. We tested whether oral therapy with the probiotic Lactococcus lactis that expresses Hsp65, in combination with donor alloantigens (Donor-Allo-Ag), acted synergically, inducing immunotolerance or increasing graft survival, in a murine model of semiallogeneic skin transplantation. We tested different oral therapy combinations, as well as the association with a COX-2 selective nonsteroidal anti-inflammatory drug (celecoxib). Skin transplantation was performed 10 days after the last oral administration of probiotics and Donor-Allo-Ag. We observed no beneficial effect on graft survival in the group that received L.lactis that produce Hsp65, alone or in combination with Donor-Allo-Ag/and/or the anti-inflammatory drug. In contrast, combined oral therapy with wild type L.lactis and Donor-Allo-Ag significantly prolonged graft survival (p=0.01), in comparison to non-treated animals. In this prolonged-survival group (L.lactis and Donor-Allo-Ag), we also found higher extension of preserved epithelium (p=0.02) and higher expression of TGF-beta (p=0.04), within the graft, in comparison to non-treated animals. We found no significant differences in the intragraft expression of FOXP3 and IL-17, which was essentially absent or very low. We conclude that Hsp65 did not induce immunoregulatory effects capable of prolonging graft survival. However, the microbiota manipulation with the combined oral therapy with wild type L.lactis and Donor-Allo-Ag, prior to transplantation, induce immunoregulatory mechanisms capable of partially controlling the inflammatory responses to the graft, most likely involving the participation of TGF-beta

Lactococcus lactis est une bactérie lactique couramment utilisée dans l’industrie laitière. Elle assure en tant que ferment des fonctions technologiques multiples qui impactent la flaveur et la texture finale des produits. Cependant, la diversité fonctionnelle constatée au sein des levains de cette espèce impose de mettre en place un processus de sélections des souches. Ces travaux ont pour objectif d’identifier les déterminants de la spécificité organoleptique dite « crème » de la souche industrielle L. lactis ssp. lactis. Dans un premier temps, un diagnostic macro-cinétique de l’activité de ce ferment a été réalisé en lait pour évaluer l’impact sur la physiologie cellulaire (l’acidification, le stress oxydatif, et la thermisation différentielle du lait). Définir la singularité de notre souche d’intérêt nécessite d’évaluer la diversité fonctionnelle de levains laitiers de L. lactis ssp. lactis. Cette démarche s’est appuyée sur une approche de biologie intégrative du génotype au phénotype. Pour réduire le temps d’expérimentation, une sélection des variables discriminantes à été conduite. L’un de ses composés clef de cette typicité a fait l’objet d’une étude approfondie afin de tester les différents paramètres pouvant influencer sa synthèse. La dernière partie, plus applicative, s’est articulée sur la modélisation de la signature en fonction de quatre facteurs industriels (matière grasse, sèche, oxygénation et température) par utilisation de la méthodologie des surfaces de réponse. / The lactic acid bacterium Lactococcus lactis is widely used in dairy industry. Used as a starter, L. lactis subsp. lactis is in charge of many functional characteristics which determine the final texture and flavour of fermented products. However, the phenotypic diversity observed within the species requires strain selection development. This PhD aims at identifying the determinants of the creamy sensory specificity of the industrial strain L. lactis subsp. lactis. Firstly, a diagnosis of macro-kinetic activity of ND5F was carried out to assess the impact on cellular physiology of three environmental parameters (acidification, oxidative stress, and milk heat treatments). In order to assess the uniqueness of our strain of interest, a system biology approach from genotype to phenotype was implemented to study the functional diversity of L. lactis subsp. lactis starters. A group of nine strains representative of the genetic diversity of this subpopulation was made up. 82 variables selected as important dairy features; including physiological descriptors, production of metabolites and volatile organic compounds (VOCs); were monitored. This study demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination in dairy products. To reduce the time-consuming experimentation, we developed a method of variable selection. Twenty VOCs were therefore identified as major phenotypic determinants of this diversity. They define four robust stain clusters depending on their metabolic orientations: lipolysis, proteolysis, glycolysis and unexpressed. Inclusion of genotypic diversity in addition to phenotypic characters led to adjust the functional classification by integrating strain unexpressed genotypic potentialities. Comparative proteomics analysis in milk identified protein markers of this specificity. After selecting a subset of forty-six proteins the three levels (genotype, phenotype and proteomics) involved in this diversity expression were integrated to provide a predictive classification of starter diversity.The Integration of our strain to this model enabled the identification of fourteen phenotypic determinants and seven proteins to explain ND5F specificity. The pentane-2,3-dione, a key aromatic compounds of this typicity was subjected to a comprehensive analysis to point out the different factors that may influence its synthesis. On a more applied aspect, the last part was focused on the signature modelling based on four industrial factors (milk fat and total solid level, oxygen and temperature) using the response surface methodology. It enabled to enhance the “creamy” organoleptic characteristics of the fermented products.

Lactococcus lactis est une espèce appartenant au groupe des bactéries lactiques, largement utilisées dans l’industrie pour leur capacité à produire de l’acide lactique au cours de la fabrication des produits laitiers fermentés. L’étude de la diversité globale de L. lactis ssp. lactis a été entreprise par l’intégration de données biologiques obtenues à partir d’analyses génétiques, génomiques, physiologiques, transcriptomiques et métaboliques. L’accès à la phylogénie de l’espèce par l’étude de la variabilité génétique du génome cœur par MLST (MultiLocus Sequence Typing) a permis de décrire deux groupes de souches : les souches environnementales, génétiquement très diverses, isolées de laits crus, de plantes ou d’animaux et les souches domestiquées, génétiquement très proches, isolées des levains utilisés dans l’industrie laitière. Malgré la perte de diversité génétique observée dans les souches domestiquées probablement associée à un processus de spécialisation à un environnement technologique, l’approche intégrative a permis de montrer que ce groupe présente une diversité génomique et fonctionnelle aussi importante que les souches environnementales. L’investigation des génomes de la sous-espèce lactis par la mesure de la taille des chromosomes et la caractérisation en nombre et en taille du contenu plasmidique, a révélé une variabilité de plus de 300 kb en capacité de codage génétique des souches domestiquées et environnementales. D’autre part, les souches domestiquées appartenant au biovar Diacetylactis ont montré des physiologies et des régulations métaboliques différentes, résultant en une production d’arômes de type diacetyl ou acétoïne variable selon la souche. Enfin, le séquençage du génome de la souche environnementale A12 isolée d’un levain de panification, et sa comparaison avec les 4 génomes actuellement séquencés de L. lactis a révélé un pangénome (ensemble des gènes de l’espèce) étendu, montrant que cette espèce offre un grand réservoir de diversité. Environ 20 % des gènes spécifiques de souches ont été mis en évidence témoignant des grandes capacités adaptatives de la sous-espèce. L’étude approfondie de la souche A12 par une analyse transcriptomique a permis de rendre compte des mécanismes impliqués dans l’adaptation d’une souche à un écosystème complexe / The Lactococcus lactis species belong to lactic acid bacteria group widely used for their ability to produce lactic acid in fermented dairy products. The study of the global diversity of L. lactis ssp. lactis was carry out by the integration of biological data obtained from genetic, genomic, physiological, transcriptomic and metabolic analyses. The genetic variability investigated by MLST (MultiLocus Sequence Typing) describe two strains groups according to their phylogeny : the “environmental” strains, displaying high genetic diversity and isolated from different natural environments such as raw milks, plants and animals and the “domesticated” strains, genetically closely related, isolated from starters in dairy industries. Despite the lost of genetic diversity in domesticated strains, probably associated to a specialisation process, the integrative approach showed a genomic and functional diversity as huge as in environmental strains. The characterization of chromosome size and plasmidic content of the lactis subspecies revealed a variation higher than 300 kb in genetic coding capacity for domesticated and environmental strains. Moreover, the domesticated strains belonging to the biovar Diacetylactis showed different physiologies and metabolic regulations resulting in variable amount of aroma produced according to the strains. Finally, the genome sequencing of the A12 strain isolated from sourdough bread and its comparison with 4 other L. lactis genomes already sequenced revealed a spread pangenome (all the genes of a species). Approximately 20 % of each genome correspond to strain specific genes, showing large adaptive capacities of the subspecies. The in-depth study of the A12 strain by transcriptomic analysis allows to highlight mechanisms involved in the adaptation of a strain to a complex ecosystem

Cinco linhagens bacteriocinogênicas foram selecionadas quanto às suas propriedades sensoriais e tecnológicas em leite. Somente Lactococcus lactis ssp. lactis CTC204 apresentou as características (baixa acidificação e proteólise) adequadas para a fabricação de queijo Minas Frescal. Contudo, a baixa atividade da bacteriocina (3x102UA.mL-1) inviabilizou a sua utilização como bioconservante para a produção in situ no queijo. Estudos para obtenção de um bio-ingrediente foram conduzidos. A atividade da bacteriocina foi maior em leite adicionado com extrato de levedura e glicose (1,3x104UAmL-1) comparado com prebioticos (fruto-oligiossacarideo e inulina). A secagem em Spray-drier originou um bio-ingrediente com atividade de 1,3x105UAmL-1. Listeria monocytogenes ATCC7644 (4,0LogUFC.mL-1) foi inoculada nos queijos obtidos por acidificação do leite pasteurizado e microfiltrado. A contagem aumentou 6,2LogUFC.gL-1 e 2LogUFC.gL-1 no 15° e 21º dia de estocagem a 6±1°C, respectivamente, nos queijos sem e com adição de 10% do bio-ingrediente. Portanto, inibição de 4,2 LogUFC.g-1 do patógeno no 15° dia de estocagem. / Among the five bacteriocin-producing lactic bacteria strains tested, only Lactococcus lactis subsp. cremoris CTC 204 presented suitable sensory and technological properties for Minas Frescal cheesemaking process. However, because of the low yield of bacteriocin production in milk (3.0x102 AU.mL-1), its use as biopreservative for in situ production in cheese was impractical. We carried out experiments to obtain a bioingredient. Bacteriocin activity was higher in milk supplemented with glycose and yeast extract (1.3x104 AU.mL-1) compared to probiotics (fructooligosaccharides and inulin). Spray drying fermented milk originated a bioingredient presenting activity of 1.3x105 AU.mL-1. Population count of Listeria monocytogenes ATCC 7644, inoculated (4.0 Log CFU.mL-1) in cheese produced by direct acidification of pasteurized microfiltered milk, reached 6.2 Log CFU.g-1 and 2.0 Log CFU.g-1 at day 15 of storage at 6±1°C, in cheese without and with addition of 10% of bioingredient, respectively, therefore presenting pathogen inhibition of 4.2 Log CFU.g- 1 at day 15 of storage.

Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.

Após a constatação da escassez de estudos realizados com vegetais crus na busca por novas estirpes de bactérias láticas (BAL) produtoras de bacteriocinas e diante do potencial tecnológico da aplicação destas cepas tanto como agentes de conservação em alimento, bem como cultura probiótica em alimentos funcionais, este estudo objetivou isolar e identificar cepas de bactérias láticas potencialmente bacteriocinogênicas de amostras de rúcula obtidas no comércio local de São Paulo, SP - Brasil, identificar e caracterizar as bacteriocinas produzidas pelos isolados e avaliar o potencial probiótico dos isolados testando sua sobrevivência no modelo dinâmico do trato gastrointestinal TNO gastro-Intestinal Model - TIM-1 disponível no TNO (The Netherlands Organization for Applied Scientific Research) divisão Quality of Life (Zeist, Holanda). A produção de bacteriocinas neste modelo também foi avaliada, comparando-se com L. sakei 2a, também produtora de bacteriocinas e ainda avaliou-se a interferência na viabilidade de E. faecium LMA1. A cepa Lactococcus lactis subsp. lactis MK02R de rúcula produziu uma bacteriocina sensível à enzimas proteolíticas, termoestável e não influenciada pelo pH, sendo capaz de inibir Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii e Listeria Monocytogenes de diferentes grupos sorológicos. Os ensaios genéticos utilizando primers Nisf e Nisr confirmaram que a bacteriocina MK02R é uma nisina, apresentando uma alteração dos aminoácidos no peptídeo líder em relação às nisinas A, Z, Q, F e U, porém com a estrutura do peptídeo maduro idêntica ao da nisina F. Estes resultados foram confirmados por espectrometria de massas de amostras purificadas por HPLC. L. lactis MK02R resistiu à passagem no modelo dinâmico TIM-1, apresentando uma alta capacidade de sobreviver nas condições simuladas do trato gastrointestinal humano. Entretanto, não foi capaz de causar a redução no número de E. faecium LMA1. Em contrapartida, L. sakei 2a, mesmo apresentando uma sobrevivência menor, foi capaz de causar uma redução de 70% na população de E. faecium LMA1 no ambiente simulado do TGI. Não foi detectada atividade residual da ação antimicrobiana das bacteriocinas produzidas por L. lactis MK02R ou L. sakei 2a após a passagem pelo modelo dinâmico TIM-1. Estes resultados evidenciam a possível aplicação de L. lactis MK02R como um agente de controle biológico na conservação de alimentos e também como uma cultura potencialmente probiótica. / Given the scarcity of studies performed with raw vegetables addressing the search for new bacteriocinogenic strains of lactic acid bacteria (LAB) and considering the technological application of these strains as food preservatives and probiotic cultures in functional foods, this study was aimed at isolation and identification of bacteriocinogenic LAB strains from samples of rocket salad obtained in the local market of São Paulo, SP - Brazil, subsequent characterization of the bacteriocins produced by these LABs and evaluation of their probiotic potential by testing their survival in the dynamic gastrointestinal model TNO gastro- Intestinal-Model - TIM-1, available at the TNO (Netherlands Organization for Applied Scientific Research) Quality of Life division (Zeist, Netherlands). The studies in the TIM-1 model were also done with another bacteriocinogenic strain L. sakei 2a for comparison, evaluating their interference on the viability of E. faecium LMA1. The bacteriocin produced by strain Lactococcus lactis subsp. lactis MK02R isolated from rocket salad was sensitive to proteolytic enzymes, heat-stable and not influenced by the pH. The bacteriocin inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii the primers Nisf and Nisr indicated that the bacteriocin produced by the strain MK02R is a nisin, with a change in the amino acid sequence of the leader peptide when compared to nisin A, Z, Q, U and F, but with the structure of the mature peptide homologous to that of nisin F. These results were confirmed by mass spectrometry of purified samples obtained by HPLC. L. lactis MK02R withstood the test in the dynamic model TIM-1, presenting capability to survive in the simulated conditions of the human gastrointestinal tract. However, the strain was not able to cause a reduction in the number of E. faecium LMA1. On the other hand, L. sakei 2a, even presenting lower survival, was able to cause 70% reduction in the population of E. faecium LMA1 in the gut simulated environment. No residual antimicrobial activity of bacteriocin produced by L. lactis MK02R or L. sakei 2a was detected after the transit through the dynamic model TIM-1. These results demonstrate the possible application of L. lactis MK02R both as a biocontrol agent in food preservation and as a potentially probiotic culture.