A microbiota vaginal saudável é constituída, majoritariamente, por espécies de lactobacilos que representam uma barreira natural contra microrganismos causadores de doenças como a candidíase vulvovaginal (CVV), vaginose bacteriana (VB) e infecções do trato urinário (ITU), que juntas acometem cerca de um bilhão de mulheres no mundo anualmente. Um melhor entendimento da ecologia microbiana vaginal pode ser útil na otimização de tratamentos existentes para as infecções urogenitais, os quais podem destruir parcialmente a microbiota autóctone, predispor a novas infecções, contribuir para a seleção de microrganismos resistentes e causar efeitos colaterais indesejáveis. A utilização de microrganismos certificadamente probióticos, Lactobacillus rhamnosus GR-1 e Lactobacillus reuteri RC-14, representa uma alternativa terapêutica promissora na abordagem de VB e CVV, uma vez que são capazes de colonizar o trato vaginal, apresentam atividade inibitória frente a diversos patógenos do trato urogenital, exibem risco mínimo para a seleção de microrganismos resistentes e podem auxiliar na restauração da microbiota vaginal. Os objetivos deste trabalho foram: (i) avaliar a prevalência de espécies de lactobacilos na microbiota vaginal de mulheres saudáveis e diagnosticadas com infecções vaginais (CVV e VB) da cidade de Ribeirão Preto (São Paulo, Brasil), (ii) avaliar a capacidade de isolados de lactobacilos produzirem peróxido de hidrogênio (H2O2) e (iii) determinar a eficácia da utilização de L. rhamnosus GR-1 e L. reuteri RC-14 no tratamento de CVV e VB, quando co-administrados com medicamentos antimicrobianos tradicionais. Participaram deste estudo, 196 pacientes voluntárias, atendidas por médicos ginecologistas de centros de saúde ligados à Universidade de São Paulo, campus de Ribeirão Preto (64 saudáveis, 68 diagnosticadas com CVV e 64 diagnosticadas com VB) e duas amostras vaginais de cada paciente foram coletadas com o auxílio de zaragatoas esterilizadas. Uma zaragatoa foi utilizada para semeadura em ágar MRS (de Man, Rogosa & Sharpe), os isolados de bactérias láticas obtidos foram analisados através da técnica de PCR-ARDRA (Reação em cadeia da polimerase Análise de restrição do DNA ribossomal amplificado) e a habilidade de Lactobacillus spp. produzir H2O2 foi determinada semi-quantitativamente. A outra zaragatoa foi utilizada para a análise das espécies de lactobacilos da microbiota vaginal pela técnica independente de cultivo PCR-DGGE (Reação em cadeia da polimerase Eletroforese em gel de gradiente de desnaturação). As leveduras do gênero Candida foram obtidas através da semeadura das amostras vaginais provenientes de pacientes saudáveis e com CVV no meio Chromagar® Candida e identificadas através de provas bioquímicas de referência. As pacientes diagnosticadas com CVV participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de fluconazol (150mg) e suplementação diária durante 28 dias com (i) duas cápsulas contendo os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 (Urex-Cap-5®) ou (ii) duas cápsulas de placebo. As pacientes com VB também participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de tinidazol (2g) e suplementação diária com cápsulas do probiótico (Urex-Cap-5®) ou placebo, conforme descrito acima. Todas as pacientes foram reavaliadas ao final do tratamento, no 28º dia. Foram realizados experimentos para averiguar o possível efeito de L. rhamnosus GR-1 e L. reuteri RC-14 na modulação in vitro da infecção por Candida albicans em culturas de células epiteliais vaginais humanas (VK2/E6E7) Os resultados mostraram que, pela técnica de PCR-ADRA, L. crispatus foi a espécie mais prevalente nos grupos de pacientes saudáveis (37,0%) e com CVV (35,9%), enquanto que L. gasseri foi predominante no grupo de pacientes com VB (34,6%). De acordo com o método de PCR-DGGE, L. iners foi o microrganismo mais prevalente nos três grupos de pacientes avaliados: saudáveis, com CVV e VB (48,7%, 44,7% e 65,0%, respectivamente). A maioria dos isolados de Lactobacillus spp. obtidos nos grupos de pacientes saudáveis (98,6%) e diagnosticadas com CVV (97,4%) foram capazes de produzir H2O2 (1 a 100mg/L), em comparação a apenas 68,2%, determinado no grupo de pacientes com VB (p<0,05). L. crispatus e L. johnsonii produziram as maiores quantidades médias de H2O2 (30mg/L). A taxa de colonização por leveduras do gênero Candida foi de 26,6% no grupo de pacientes saudáveis (C. albicans correspondeu a 52,4% de todos os isolados), enquanto que no grupo de pacientes com CVV, 89,2% dos isolados leveduriformes foram identificados como C. albicans. Para as análises estatísticas dos dados obtidos com os testes clínicos, foram consideradas 55 pacientes diagnosticadas com CVV (pela presença de sinais e sintomas da infecção e cultura positiva para Candida sp.) e foi observado que a utilização de dose única de fluconazol e suplementação diária com o probiótico, resultou em taxa de cura mais elevada para a infecção (89,7%) em comparação com aquela verificada no grupo placebo (65,4%) (p<0,05). A utilização de tinidazol em associação com a ingestão diária de Urex-Cap-5® no tratamento de pacientes com VB também resultou em taxa de cura mais elevada para a condição (87,5%), em comparação àquela observada no grupo placebo (50,0%) (p<0,05). A atividade anti-Candida de L. rhamnosus GR-1 e L. reuteri RC-14 foi observada através do modelo in vitro para infecção vaginal. Em conclusão, quando as técnicas de PCR-ARDRA e PCR-DGGE foram comparadas entre si, foi observado que ambas apresentaram limitações, evidenciando a importância do emprego de diferentes metodologias para avaliação adequada das espécies de lactobacilos presentes na microbiota vaginal. As espécies de lactobacilos vaginais determinadas nas mulheres saudáveis do presente trabalho foram semelhantes àquelas verificadas em estudos prévios descritos na literatura com pacientes com dieta e localização geográfica notadamente distintas. Os dados do presente trabalho sugerem que a presença de lactobacilos produtores de H2O2, isoladamente, não confere proteção contra CVV, enquanto que a ausência desses microrganismos pode ser um fator contribuinte para VB. Além disso, foi demonstrado que a utilização de medicamentos antimicrobianos tradicionais e suplementação com os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 foi mais eficiente no tratamento de CVV e VB em comparação com medicamentos clássicos e placebo. Estes resultados podem contribuir para prolongar a vida útil de medicamentos cuja eficácia pode ser comprometida devido à seleção de microrganismos resistentes e também reduzir o tempo de tratamento para pacientes que necessitam de terapias clássicas por períodos prolongados. / The vaginal microbiota is mainly constituted by lactobacilli species, which represent a natural barrier against microorganisms that cause vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and urinary tract infections (UTI). Together, these conditions afflict each year an estimated one billion women worldwide. A better understanding of the vaginal microbial ecology may be useful to improve the current available treatments for urogenital infections, which can partially destroy the autochthonous microbiota, predispose to other infections, contribute for the selection of resistant microorganisms and cause undesirable collateral effects. The use of microorganisms with demonstrated probiotic properties, Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, represents a promising therapeutic alternative for BV and VVC, since they are able to colonize the vaginal tract, present inhibitory against several urogenital pathogens, pose minimal risk for the selection of resistant microorganisms and can help to restore the vaginal microbiota. The objectives of this work were: (i) to evaluate the prevalence of lactobacilli species in the vaginal microbiota of healthy women and those diagnosed with vaginal infections (VVC and BV) in the city of Ribeirão Preto (São Paulo, Brazil), (ii) to evaluate the ability of the lactobacilli isolates to produce hydrogen peroxide (H2O2) and (iii) to determine the efficacy of the use of L. rhamnosus GR-1 and L. reuteri RC-14 in the treatment of VVC and BV, in co-administration with traditional antimicrobials. 196 voluntary subjects were examined by the gynecologists team from health centers affiliated with Universidade de São Paulo, campus at Ribeirão Preto (64 healthy, 68 diagnosed with VVC and 64 diagnosed with BV) and two vaginal samples from each patient were collected by the use of two sterile swabs. One swab was cultured in MRS (de Man, Rogosa & Sharpe) agar, the isolates of lactic acid bacteria obtained were analyzed by PCR-ARDRA (Polymerase chain reaction - Amplified ribosomal DNA restriction analysis) and the ability of Lactobacillus spp. to produce hydrogen peroxide was determined semi-quantitatively. The other swab was used for the analysis of lactobacilli species from the vaginal microbiota using the culture-independent PCR-DGGE (Polymerase chain reaction Denaturating gradient gel electrophoresis). The yeasts belonging to Candida genus were obtained also by culturing the vaginal material from healthy and VVC patients in Chromagar® Candida and were identified by standard biochemical tests. VVC patients were enrolled in a randomized, double-blind, placebo-controlled trial and treated with a single dose fluconazole (150mg) and daily supplementation for 28 days with (i) two capsules containing the probiotic microorganisms L. rhamnosus GR-1 and L. reuteri RC-14 (Urex-Cap-5®) or (ii) two placebo capsules. BV patients were also enrolled in a randomized, double-blind, placebo controlled trial and treated with a single dose of tinidazole (2g) and supplementation with probiotic capsules (Urex-Cap-5®) or placebo, as described above. All patients were re-evaluated at the end of treatment, on the 28th day. Experiments were also conducted to assess the possible effect of L. rhamnosus GR-1 and L. reuteri RC-14 in the in vitro modulation of vaginal infection by Candida albicans on cultures of human vaginal epithelial cells (VK2/E6E7). The results revealed that according to PCR-ADRA, L.crispatus was the most prevalent species in the groups of healthy women (37.0%) and those with VVC (35.9%), while L. gasseri was dominant in BV patients (34.6%). By PCR-DGGE method, L. iners was the most prevalent Lactobacillus species in all the three groups evaluated: healthy, VVC and BV (48.7%, 44.7% and 65.0%, respectively). The majority of the isolates of Lactobacillus spp. from healthy women (98.6%) and those with VVC (97.4%) were able to produce H2O2 (1 to 100mg/L) in comparison with only 68.2% assessed for the BV group (p<0.05). L. crispatus and L. johnsonii produced the highest average levels of H2O2 (30mg/L). Colonization rate by yeasts belonging to Candida genus was 26.6% in the group of healthy patients (C. albicans represented 52.4% of all isolates), whereas in the VVC group, 89.2% of yeast isolates were identified as C. albicans. For the performance of statistical analysis of the results obtained with the clinical trials, 55 patients diagnosed with VVC (by the presence of symptoms and signals of the infection and positive culture for Candida sp.) were taken into consideration and it was observed that the use of a single dose of fluconazole and daily supplementation with probiotics, yielded a higher cure rate (89.7%), in comparison with the placebo group (65.4%) (p<0.05). The use of tinidazole plus probiotic also resulted in higher cure rate of the infection (87.5%), compared to placebo group (50.0%) (p<0.05). An anti-Candida activity of L. rhamnosus GR-1 and L. reuteri RC-14 was observed in the in vitro model of vaginal infection. In conclusion, when PCR-ARDRA and PCR-DGGE were compared, it was verified that both presented limitations, which evidences the need of using different techniques for a better knowledge of lactobacilli species present in the vaginal microbiota. The lactobacilli species found in healthy women in this work were similar to those reported in previous studies described in the literature for patients with distinctly different diet and geographic localization. The data of the present work indicate that solely the presence of H2O2-producing isolates does not render protection against VVC, whereas the absence of those microorganisms may be a contributing factor for BV. Moreover, it was demonstrated that the use of classical medicines supplemented with the probiotics L. rhamnosus GR-1 and L. reuteri RC-14 was more efficient to treat VVC and BV in comparison with classical medicines plus placebo. These results may contribute to extend the longevity of drugs whose efficacy is compromised due to the selection of resistant microorganisms and also to shorten the length of treatment courses for patients that require long regimens with standard therapy.

A formação de biofilmes é um fator preocupante para indústria de alimentos, pois pode comprometer a sanitização de superfícies de contato e aumentar o risco de contaminação por patógeno em alimentos processados. L. monocytogenes é uma bactéria de ampla distribuição no ambiente e com capacidade de formar biofilmes e sobreviver por longos períodos em condições adversas. Esta bactéria pode causar doenças em pessoas imunocomprometidas e mulheres grávidas manifestando-se por infecções do sistema nervoso central, abortos e nascimentos prematuros. Vários estudos têm demonstrado que algumas bactérias podem sofrer transição para o estado viável mas não cultivável em resposta ao estresse. Considerando-se a importância da garantia da segurança e qualidade dos alimentos, há necessidade de desenvolvimento e de padronização de técnicas rápidas para a quantificação de células viáveis de L. monocytogenes em alimentos e biofilmes. Neste estudo foi avaliada a formação de biofilmes e viabilidade celular de Listeria monocytogenes em condições de estresse. Foram utilizadas técnicas de semeadura em placa e quantificação direta por microscopia de fluorescência com os corantes cloreto de 5-ciano-2,3-di-(p-tolil) tetrazólio (CTC) e 4,6-diamino-2- fenilindol (DAPI). Foram também realizados ensaios preliminares para padronização da Reação da Polimerase em Cadeia em tempo real (PCR em tempo real) com amostras previamente tratadas com brometo de etídio monoazídico (EMA). Os resultados obtidos demonstraram que o método de semeadura em placa foi adequado somente para a enumeração de células viáveis de L. monocytogenes em biofilmes enquanto que o método de enumeração por microscopia de fluorescência com CTC-DAPI permitiu quantificar bactérias no estado viável e viável mas não cultivável. Também foi observado que a presença de bacteriocinas de L. sakei 1 e L. mensenteroides 11 causou a diminuição na viabilidade e formação de biofilmes por L. monocytogenes. Os resultados preliminares utilizando culturas puras de L. monocytogenes demonstraram que o tratamento com brometo de etídio monoazídico (EMA) antes da análise por PCR em tempo real reduziu a amplificação do DNA de células mortas em 1 log de UFC por mL. No entanto dependendo da concentração utilizada, pode ocorrer também a inibição da amplificação de células viáveis de L. monocytogenes. Os resultados do presente trabalho indicaram que a microscopia de fluorescência é comparável à placa para análise de células de L. monocytogenes não estressadas. Entretanto, para a quantificação de L. monocytogenes submetida a estresse, é desejável a utilização de corantes de viabilidade celular. O método de PCR em tempo real com pré-tratamento com EMA é promissor mas, ainda necessita de maior padronização para avaliação de sua aplicabilidade na determinação seletiva de células viáveis de L. monocytogenes. / Biofilm formation is of great concern for food industry because it may compromise sanitization of surfaces and increase contamination risk of processed foods by bacterial pathogens. L. monocytogenes is an ubiquitous bacterium which is able to form biofilms and to survive for long periods under adverse conditions. L. monocytogenes may cause disease in immunocomprommised people and pregnant women, manifesting as central nervous system infections, abortion and premature birth. Some bacteria can undergo transition to viable but non-cultivable state in response to stress and it is important to study techniques for rapid quantification of viable cells of L. monocytogenes in foods. In this study biofilm formation and cell viability of Listeria monocytogenes were studied in stress conditions by plate counting, direct quantification by fluorescence microscopy with double staining dyes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)/4\'-6 diamino-2 phenylindole (DAPI). Preliminary experiments with real time polymerase chain reaction and treatment with ethidium bromide monoazide (EMA) were also performed. The results showed that plate count method was suitable for enumeration only of viable cells of L. monocytogenes in biofilms since, fluorescence microscopy with CTC-DAPI yielded higher counts, probably due to the presence of viable but nonculturable cells. It was also observed that the presence of bacteriocins of L. sakei 1 and L. mensenteroides 11 decreased viability and formation of biofilm by L. monocytogenes. Results obtained with pure cultures of L. monocytogenes showed that the treatment with ethidium bromide monoazide (EMA) before real time PCR detection, reduced DNA amplification of dead cells in 1 log CFU per mL, but depending on the concentration used, EMA also inhibited amplification of viable cells of L. monocytogenes. The results indicated that plate counting and fluorescence microscopy are equivalent for enumeration of non-stressed L. monocytogenes cells. However, the use of double staining with fluorescence microscopy is a more suitable method if stressed cells are present. The EMA-real time PCR is a promissing tool for rapid evaluation of viable L. monocytogenes, but it needs further standartization.

Les ferments lactiques, d’une importance industrielle considérable, sont très largement commercialisés sous forme concentrée, congelée ou lyophilisée en vue de leur utilisation ultérieure dans les procédés industriels tels que les procédés de production de fromage. Cependant, les procédés de stabilisation (congélation et lyophilisation) engendrent différents stress qui peuvent conduire à de faibles taux de survie et des pertes de fonctionnalités des microorganismes. Dans ce contexte, ce travail de thèse vise à mieux comprendre l’incidence de l’état physiologique des cellules de L. lactis TOMSC161 au moment de la récolte sur leur tolérance à la lyophilisation et au stockage, et de développer des outils simples mais efficaces d’évaluation de l’état physiologique des cellules au cours de la fermentation pour les industriels. Dans une première partie, l’influence de différents paramètres de fermentation (température, pH et phase de croissance) sur la croissance et la résistance de L. lactis TOMSC161 à chaque étape du procédé de lyophilisation et au stockage a été étudiée. Alors que les performances de la souche ne sont pas dégradées après congélation, L. lactis s’est avéré sensible au séchage et au stockage à température ambiante. De plus, la température de fermentation et l’instant de récolte influencent la résistance au séchage de cette bactérie. Ainsi, les cellules de L. lactis TOMSC161 cultivées à 32 °C, pH 6,2 et récoltées tardivement (en phase stationnaire avancée) présentent une croissance optimale et la meilleure résistance à la lyophilisation et au stockage à 4 °C. Dans une seconde partie, une caractérisation approfondie de la membrane de L. lactis TOMSC161 aux niveaux biochimique et biophysique a été réalisée au cours de fermentations à différentes températures (22 et 30 °C) et a été mise en lien avec la résistance des ferments à la lyophilisation et au stockage. La cyclopropanation des acides gras insaturés de L. lactis TOMSC161 au cours de la fermentation est reliée à une rigidification de la membrane et permet d’améliorer la tolérance des cellules à la lyophilisation et au stockage. A l’inverse, la culture des cellules à une température inférieure à la température optimale de croissance induit une adaptation homéovisqueuse de la membrane, mise en évidence par la température de transition lipidique, mais n’a pas induite une amélioration de la résistance à ce procédé de préservation. Dans une troisième partie, la caractérisation de l’état physiologique des cellules de L. lactis TOMSC161 a été complétée au niveau du transcriptome, du protéome et de l’état d’oxydation cellulaire. Le procédé de lyophilisation provoque la formation d’espèces réactives de l’oxygène (ROS) intracellulaires qui dégradent la performance des ferments au cours du stockage. Par ailleurs, l’état d’oxydation des cellules diminue au cours de la fermentation et est expliqué, en phase stationnaire, par un ralentissement du métabolisme énergétique aérobie et une induction des réponses au stress oxydatif. Cette « pré-adaptation » initiale des cellules de L. lactis TOMSC161 au cours de la fermentation permet, là encore, une amélioration de leur tolérance à la lyophilisation et au stockage par une limitation de l’accumulation de ROS lors de ce procédé de préservation. Ce travail se conclut par une vérification des fonctionnalités du ferment lyophilisé, dans les conditions de production optimisées lors de cette thèse, en fabrication fromagère. Malgré l’étape de lyophilisation, les propriétés technologiques de L. lactis TOMSC161 sont conservées, validant ainsi le travail d’optimisation réalisé. / Lactic acid bacteria, which have a significant industrial importance, are widely distributed in frozen or freeze-dried state for further use in industrial processes such as cheesemaking. However, stabilization processes (freezing or freeze-drying) causes different stresses which can lead to low survival rates and functionality losses of microorganisms. In this context, this thesis aimed at better understanding the impact of the physiological state of L. lactis TOMSC161 cells during fermentation on their freeze-drying and storage resistance, and at developing simple but efficient tools to evaluate the cells physiological state during fermentation for industrials.In the first part of this work, the influence of fermentation parameters (temperature, pH and harvesting time) on the growth and resistance of L. lactis TOMSC161 to each step of the freeze-drying process and storage has been investigated While the strain performance was not deteriorated after freezing, L. lactis was sensitive to the drying step and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of this bacterium. L. lactis TOMSC161 cells grown at 32 °C, pH 6.2 and harvested late (at late stationary phase) exhibited therefore both an optimal growth and the highest resistance to freeze-drying and storage at 4 °C. In the second part, a deep characterization of the L. lactis TOMSC161 membrane at a biochemical and biophysical level was analyzed during fermentation at different temperatures and was linked to the freeze-drying and storage resistance of starters. The cyclopropanation of unsaturated fatty acids of L. lactis TOMSC161 during fermentation was correlated with a membrane rigidification and allowed an improvement of the cell tolerance to freeze-drying and storage. Conversely, cultivating cells at lower fermentation temperature than the optimum growth temperature induced as expected a homeoviscous adaptation as evidenced by lowered lipid phase transition temperature but did not induce any improvement of resistance to this preservation process.In the third part, the physiological state characterization of L. lactis TOMSC161 cells was completed by investigating at the transcriptomic and proteomic levels as well as the cellular oxidation state. The results proved that the freeze-drying process caused intracellular reactive oxygen species (ROS) formation, responsible of degradation of the starter performance during storage at 25 °C. Furthermore, the cellular oxidation state decreased during fermentation and was explained, in the stationary phase, by a slowdown of the aerobic energy metabolism and the induction of oxidative stress responses. This initial “pre-adaptation” of L. lactis TOMSC161 during fermentation allowed improving their tolerance to freeze-drying and storage by a limitation of ROS accumulation through the whole preservation process.Finally, this work has been concluded by verifying the functionalities of starter freeze-dried in the optimized production conditions defined in this thesis during cheesemaking. Despite the freezedrying step, the technological properties of L. lactis TOMSC161 were preserved, thus validating the performed optimization.

Les produits laitiers fermentés tels que les yaourts, les laits fermentés et les fromages frais, occupent une place importante dans l’économie française. La stabilité de ces produits et leurs chances d’exportation sont cependant limitées par de fréquentes altérations fongiques. De plus, l’augmentation des résistances de certains champignons aux conservateurs chimiques ainsi que la forte demande des consommateurs pour des produits dépourvus d’additifs poussent les industriels à en réduire l’ajout. Dans ce contexte, il est nécessaire de développer des alternatives innovantes, telle que la bio-conservation. Les bactéries lactiques, utilisées depuis des millénaires dans la fermentation de nombreux aliments, et généralement reconnues comme inoffensives pour la santé, sont potentiellement de bonnes candidates pour la bio-conservation. Dans le but d’obtenir une ou deux souches de bactéries lactiques antifongiques, capables d’être compétitives au sein de produits laitiers fermentés tels que le yaourt, une collection de bactéries lactiques antifongiques a d’abord été constituée. Pour cela, un criblage de colonies isolées de laits crus de vache, de chèvre et de brebis a été effectué sur une période d’une année. Ce criblage, ciblé contre 4 champignons communément retrouvés dans les produits laitiers contaminés, a abouti à l’isolement de 1235 colonies de bactéries lactiques antifongiques. Ceci a permis d’évaluer la biodiversité des bactéries lactiques antifongiques d’une part, et celle des champignons, d’autre part, dans ces échantillons de lait, afin d’observer une éventuelle corrélation entre la présence de champignons et l’expression des activités antifongiques. L’influence de l’origine du lait, de la période d’échantillonnage, du milieu d’isolement et du champignon ciblé, sur le pourcentage de colonies actives isolées a été mise en évidence. Parmi les champignons ciblés, Pénicillium expansum était le plus facilement inhibé, et la majorité des colonies ont été isolées durant la troisième période d’échantillonnage, majoritairement sur les milieux à base de MRS. Les laits de vache et de chèvre se sont révélés être des réservoirs de bactéries lactiques antifongiques, contrairement aux laits de brebis. La majorité des bactéries lactiques antifongiques isolées et identifiées appartenait au genre Lactobacillus, majoritairement du groupe Lb. casei. Onze isolats, dont 10 appartenant au groupe Lb. casei, ont été sélectionnés selon l’intensité de leur activité antifongique et leur spectre d’action sur milieu MRS. Certaines de leurs propriétés technologiques ont été caractérisées, en vue de leur utilisation comme cultures protectrices dans des produits laitiers fermentés. Leur activité antifongique a également été testée dans le lait et dans le yaourt, contre 6 contaminants fongiques communément responsables d’altérations dans les yaourts. Une souche, Lb. harbinensis K.V9.3.1Np, a révélé de fortes activités antifongiques dans le yaourt contre 6 cibles fongiques. Les études complémentaires effectuées ont permis de montrer que la variation de certains paramètres technologiques (présence ou absence de saccharose, temps de fermentation) n’avait pas d’influence sur l’activité antifongique de cette souche. La découverte du potentiel antifongique de Lb. harbinensis K.V9.3.1Np est innovante. Cette espèce, isolée pour la première fois en 2005 d’un produit fermenté végétal, n’avait encore jamais été décrite comme présentant des activités antimicrobiennes. Sa forte activité antifongique dans le yaourt fait de Lb. harbinensis K.V9.3.1Np un bon candidat pour la bioconservation de produit(s) laitier(s) fermenté(s). / Fermented dairy products such as yogurt, fermented milks and fresh cheeses are of great importance in the French economy. The stability of these products and export opportunities are however limited by frequent fungal spoilages. In addition, the increase of fungal resistances to Chemical preservatives and the strong consumer demand for products deprived of Chemical additives are pushing manufacturers to reduce the quantities of added Chemical preservatives in these products. In this context, it is necessary to develop alternatives to classical food preservation methods, such as biopreservation. Lactic acid bacteria (LAB), used for millennia for diverse food fermentations, and generally recognized as safe for human health, can potentially be used for biopreservation. In order to select 1 or 2 LAB strains with antifungal activity capable to compete in fermented dairy products such as yogurt, a collection of antifungal LAB was first created. This was done by screening colonies isolated from cow, goat and ewe raw milk samples over a one-year period. This screening step, targeted against 4 fungi found in contaminated dairy products, resulted in the isolation of 1235 antifungal colonies. The biodiversity of the isolated antifungal LAB was then determined along with that of the fungi found in the different raw milks, in order to observe a possible correlation between the presence of fungi and the expression of antifungal activities. The influence of milk origin, sampling period, isolation medium and targeted fungus on the percentage of isolated active colonies was highlighted and clearly showed that both cow and goat milks were reservoirs of antifungal LAB. Among the targeted fungi, P. expansum was more easily inhibited, and the majority of colonies were isolated during the third sampling period, mainly on MRS-based media. The majority of identified antifungal LAB belonged to the genus Lactobacillus, mainly to the Lb. casei group. Eleven isolates, including 10 belonging to the Lb. casei group, were selected from these lactobacilli, according to their activity level and action spectrum in MRS medium. Some of their technological properties were characterized for their potential use as protective cultures in fermented dairy products and their antifungal activity was tested in milk and yogurt. To do this, 6 fungal contaminants commonly encountered in yogurt spoilage were used. A particular strain, Lb. harbinensis K.V9.3.1Np, showed a strong antifungal activity in yogurt. Additional experiments showed that the variation of technological parameters (presence or absence of sugar, fermentation time) had no influence on the antifungal activity of this strain. This is the first time that an antifungal potential has been observed for Lb. harbinensis, a species isolated for the first time in 2005 from a fermented vegetable product. Because of its effectiveness in yogurt, Lb. harbinensis K.V9.3.1Np is a promising strain for biopreservation of fermented dairy product(s).

Oenococcus oeni est la principale bactérie lactique retrouvée dans les fermentations malolactiques (FML) spontanées du vin. Pendant la FML, l’acide malique est converti en acide lactique, modulant l’acidité du vin et améliorant son goût. L’activité métabolique d’O. oeni produit aussi des changements dans la composition du vin, modifiant son profil aromatique. Des études précédentes ont suggéré que l’espèce est divisée en deux principaux groupes génétiques, désignés A et B. Nous avons examiné les souches d’O. oeni sous des approches de génomique comparative à l’aide d’outils bioinformatiques développés sur place, dévoilant l’existence de nouveaux de groupes et sous-groupes de souches. En outre, nos résultats suggèrent que certains groupes contiennent des souches qui sont adaptées à des produits spécifiques tels que le vin rouge, vin blanc, champagne et cidre. Ce phénomène est visible à différents niveaux des génomes des souches : l’identité de séquence, les signatures génomiques, et les caractéristiques génomiques spécifiques de groupes telles que la présence/absence de gènes et les mutations uniques. Afin de comprendre l’impact des caractéristiques génomiques dans l’adaptation de l’espèce à différents produits, nous avons sélectionné une collection de souches isolées de la même région, mais appartenant à deux groupes génétiques différents et adaptées soit au vin rouge, soit au vin blanc. Une analyse de données génomiques et métabolomiques intégrées révèle que les caractéristiques génomiques des souches de chaque groupe ont un impact sur l’adaptation des bactéries à leurs niches respectives et sur la composition de la fraction volatile du vin. / Oenococcus oeni is the main lactic acid bacteria found in spontaneous malolactic fermentation (MLF) of wine. During MLF, malic acid is converted into lactic acid, modulating wine’s acidity and improving its taste. The metabolic activity of O. oeni also produces changes in the composition of wine, modifying its aromatic profile. Previous studies have suggested that the species is divided in two major phylogenetic groups, namely A and B. We have examined O. oeni under comparative genomics approaches by the aid of bioinformatics tools developed in-place, unveiling the existence of more phylogenetic groups of O. oeni than previously thought. Moreover, our results suggest that certain groups are domesticated to specific products such as red wine, white wine, champagne and cider. This phenomenon is visible at different levels of the strains’ genomes: sequence identity, genomic signatures, and group-specific features such as presence/absence of genes and unique mutations. With the aim of understanding the impact of group-specific genomic features on the species adaptation to different products, we have selected a set of strains isolated from the same region, but belonging to two different genetic groups and adapted either to red wine, either to white wine. An integrated analysis of genomic and metabolomic data reveals that the genomic features of each genetic group have an impact on the strains adaptation to their respective niches, affecting the composition of the volatile fraction of wine.

Les effets anti-allergiques des bactéries lactiques sont suggérés par plusieurs études épidémiologiques, des essais cliniques et des modèles expérimentaux d’allergie. Cependant, les propriétés immunomodulatrices des bactéries lactiques sont sous-exploitées par les stratégies vaccinales développées pour combattre l’allergie et les mécanismes empruntés par ces bactéries pour moduler l’allergie restent peu caractérisés. Dès lors, nous avons caractérisé les propriétés immunomodulatrices qu’exerce Lactobacillus plantarum NCIMB8826, une bactérie lactique modèle, sur la cellule dendritique étant donné le rôle déterminant de cette cellule sur la réponse allergique. Nous montrons que L. plantarum induit une forte sécrétion d’IL-12 p40, d’IL-12 p70, de TNF-a mais une faible production d’IL-10. Cette faculté à induire la sécrétion de cytokines polarisantes dépend de TLR2, de TLR9, de MyD88, de NF-kB, des MAPKs (en particulier JNK, p38 et ERK 1/2), de la composition de l’acide lipotéichoïque de L. plantarum et de CD14. Nous montrons aussi que l’ADN génomique de L. plantarum est un agoniste de TLR9 et que CD14 et CD36 facilitent la liaison de la cellule dendritique avec L. plantarum. Ensuite, nous avons évalué le potentiel vaccinal d’une coadministration L. plantarum + Der p 1 dans un modèle murin d’allergie à Der p 1. Cette formulation vaccinale prévient la production d’IgE Der p 1-spécifique et atténue l’éosinophilie pulmonaire tout en stimulant une forte production d’anticorps IgG2a Der p 1-spécifiques et d’IFN-g par les cellules spléniques. Ces effets bénéfiques nous ont conduit à élaborer une bactérie lactique recombinante dérivée de L. plantarum produisant Der p 1 pour la vaccination contre l’allergie aux acariens. La forme antigénique que nous avons réussi à faire produire par L. plantarum correspond à une protéine de fusion entre la Maltose Binding Protein de E. coli et ProDer p 1 (le zymogène de Der p 1), la présence de ce partenaire de fusion étant indispensable à la production de ProDer p 1. En prophylaxie, la vaccination par cette bactérie recombinante prévient la production d’anticorps IgE-Der p 1-spécifiques et stimule la production d’anticorps IgG2a spécifiques, reproduisant les effets de la coadministration L. plantarum + Der p 1. Elle réduit de manière drastique la production d’IL-5 des cellules spléniques et des cellules ganglionnaires médiastinales et prévient l’éosinophilie pulmonaire mais n’a pas d’effet sur l’hyperréactivité bronchique. Der p 1 étant un des allergènes d’acarien les plus immunodominants, cet ensemble de données montre donc que cette bactérie recombinante constitue un vaccin prophylactique prometteur pour la prévention de l’allergie aux acariens. Des résultats préliminaires obtenus à partir de cellules dendritiques humaines et lymphocytes T autologues montrent la forte capacité de cette bactérie recombinante à induire le développement d’une réponse Th1 fortement polarisée (production d’IFN-g en l’absence de production d’IL-4 et d’IL-5), ce qui suggère que l’utilisation de cette bactérie recombinante pourrait être envisagée pour le traitement de l’allergie chez l’homme.

La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana. / The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 6 uppsatser.

In der Milchindustrie spielt die Auswahl der Starterkulturen eine wichtige Rolle bei der Herstellung fermentierter Produkte mit gewünschter Textur und entsprechenden sensorischen Eigenschaften. Milchsäurebakterien mit der Fähigkeit extrazelluläre Polysaccharide (EPS) zu synthetisieren sind von besonderem Interesse, da auf Grund der in situ gebildeten Hydrokolloide der Einsatz von Zusatzstoffen vermieden werden kann. Die Wirkung von EPS auf die Produkt-eigenschaften ist in der Literatur bereits mehrfach beschrieben, wird jedoch auf Grund der Vielzahl an unterschiedlichen Stämmen und Fermentationsparametern bzw. einer fehlenden Systematisierung immer noch sehr kontrovers diskutiert. Des Weiteren stellt die wissenschaftliche Aufklärung der komplexen Struktur-Funktionsbeziehungen und der Wechselwirkungen mit anderen Produktkomponenten eine große Herausforderung dar. Um die Zusammenhänge besser verstehen zu können, wurde in dieser Arbeit ein neuer Ansatz gewählt: isolierte und aufgereinigte EPS wurden der Milch vor der Säuerung zugesetzt und die daraus hergestellten Milchgele mit jenen mit in situ produzierten EPS verglichen. In Milchgelen aus Einzelstammkulturen von Streptococcus thermophilus oder Lactobacillus delbrueckii ssp. bulgaricus wurden EPS‑Gehalte von 40 - 150 mg/kg ermittelt. Die Gele unterschieden sich hinsichtlich ihrer Viskosität und ihres fadenziehenden Charakters, was erste Hinweise auf die Art der gebildeten EPS liefert. Die Synthese größerer Mengen an EPS zur Charakterisierung und Untersuchung ihrer Funktionalität erfolgte entkoppelt von der Produkt­herstellung mit ausgewählten Stämmen in Batch-Fermentationen mit konstantem pH in komplexen oder semidefinierten Medien. S. thermophilus ST‑143 produzierte ~ 300 mg/L fadenziehende EPS, die durch entsprechende Aufreinigungsschritte als drei EPS‑Fraktionen gewonnen werden konnten: freie EPS (EPSf), kapsuläre EPS (EPSk) und ein Gemisch aus beiden (EPSf+k). EPSf haben eine höhere Molekülmasse (M = 2,6 x 10^6 Da) und eine höhere intrinsische Viskosität (1,14 mL/mg) im Vergleich zu EPSk (M = 7,4 x 10^3 Da, 1,4 x 10^5 Da; intrinsische Viskosität = 0,06 mL/mg) und führten bereits in geringen Mengen zu rheologischen Veränderungen. Allerdings scheinen die EPSk Wechselwirkungen zwischen EPSf Molekülen zu unterstützen. In chemisch gesäuerten Milchgelen konnte durch den definierten Zusatz aufgereinigter Fraktionen von EPSf und EPSf+k vor der Säuerung (c = 0 - 0,35 mg/g) erstmals eine konzentrationsabhängige Wirkung aufgezeigt werden. Mit EPSf stieg der maximale Speichermodul der Milchgele als Maß für die Gelsteifigkeit linear an (457 - 722 Pa). EPSk zeigten hingegen keinen Einfluss. Als Modellpolysaccharid wurde vergleichend das gut beschriebene, ebenfalls ungeladene und nicht gelbildende Homopoly­saccharid Dextran herangezogen (c = 0 - 300 mg/g). EPSf und Dextran veränderten die Gelbildung, erhöhten die Steifigkeit stichfester Gele und die Viskosität gerührter Gele in ähnlichem Maße, es waren jedoch deutlich unterschiedliche Konzentrationen notwendig. Die in den Milchgelen beschriebenen Einflüsse können unter anderem auf Depletionseffekte zwischen gleichgeladenen Polymeren (hier Proteine und Polysaccharide) zurückgeführt werden. / The selection of suitable starter cultures for the production of fermented milk with a desired texture and corresponding sensory attributes is of great importance for the dairy industry. Lactic acid bacteria with the ability to synthesise extracellular polysaccharides (EPS) are of particular interest, because these in situ produced hydrocolloids may allow to omit the use of additives. Many effects of EPS on product properties are already described in the scientific literature, but are still discussed controversially because of the multitude of different strains and fermentation parameters and, hence, a lack of systematisation. Furthermore, research on the mechanisms behind the structure-function relationship and interactions with other product components is a challenging area. To obtain a deeper understanding of this complex system, a new approach was chosen for the present study: EPS were isolated, purified and added to the milk prior to acidification, and the respective milk gels were compared with those with in situ produced EPS. In milk gels acidified by single strains of Streptococcus thermophilus or Lactobacillus delbrueckii ssp. bulgaricus, EPS contents of 40 - 150 g/kg were determined. The gels differed in viscosity and their ropy character, which is a first indicator for the type of the EPS. To allow for their chemical and technofunctional characterisation, the synthesis of higher amounts of EPS was performed by batch-fermentation at constant pH and decoupled from the product manufacturing with selected strains in complex or semidefined media. S. thermophilus ST‑143 synthesised ~ 300 mg/L ropy EPS, which were isolated as three different EPS fractions by applying particular purification steps: free EPS (EPSf), capsular derived EPS (EPSk) and a mixture of both EPS (EPSf+k). EPSf had a higher molecular mass (M = 2.6 x 10^6 Da) and a higher intrinsic viscosity (1.14 mL/mg) compared to EPSk (M = 7.4 x 10^3 Da, 1.4 x 10^5 Da; intrinsic viscosity = 0.06 mL/mg) and affected the rheological properties of aqueous solutions already at low concentration. However, EPSk appear to support interactions between the EPSf molecules. In chemically acidified milk gels a concentration dependent impact of EPSf and EPSf+k, which were added to the milk prior to acidification (c = 0 - 0,35 mg/g), was described for the first time. The maximum of the storage modulus as a measure for stiffness of the milk gels linearly increased with EPSf content (457 - 722 Pa). With EPSk no effects were observed. For the purpose of comparison dextran, a well described also uncharged and non gelling homopolysaccharide, was used as a model polysaccharide (0 - 300 mg/g). EPSf and dextran affected the gelation, increased gel stiffness of set gels and viscosity of stirred gels to a similar way, but the concentrations needed for that found to be completely different. The effects described for milk gels can be ascribed among others to depletion interactions between similar charged polymers (here proteins and polysaccharides).

Work size - 60 pages, including 35 pictures, 1 table. List of literature - 44 sources. The beginning of the work -2004 09 01, the end of the work - 2006 05 15. Purpose of work: To explore, what influence the additive lysozyme on technological properties of milk which are important in manufacture of fermental cheeses and sour - milk products has. In work presents the analysis of lysozyme influence on the technological properties of. The results show that lysozyme prevent to develop of undesirable microorganisms and positively influences on the quality of fermented milks. It was established that the additive of lysozyme prolongs the duration of the bactericidal phase. The investigation of the rennet formation time has shown that the clothing of the milk in samples with lysozyme formed 12 - 15  faster as in compared with the control sample without lysozyme. Besides, it is established, that the additive of lysozyme intensifies the process removal of the whey. The research investigation show that in samples with lysozyme, whey distinguish in smaller optical density as compared with control samples. The development of lactic bacteria during fermentation process was examined too. It was found that lysozyme influence on this process is very insignificant. It was established that the additive of lysozyme insignificant reduces viscosity and acidity of fermented milk gels.