Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-12-16T13:48:33Z No. of bitstreams: 1 texto completo.pdf: 1250907 bytes, checksum: 2299d09b23deeb532f065839b105d4dc (MD5) / Made available in DSpace on 2015-12-16T13:48:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1250907 bytes, checksum: 2299d09b23deeb532f065839b105d4dc (MD5) Previous issue date: 2012-02-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Biossurfactantes - ou surfactantes microbianos - são moléculas com propriedades tensoativas produzidas por uma grande diversidade de micro-organismos, presentes em diversos habitats. Os biossurfactantes compõem uma classe de compostos que têm mostrado potencial de aplicação em diversas áreas industriais economicamente importantes. Nas aplicações relacionadas à cadeia do petróleo, a exemplo da recuperação avançada de petróleo, biorremediação de ambientes marinhos contaminados com óleo e tratamento de água de produção, é necessário que os biossurfactantes mantenham sua atividade em condições extremas, especialmente sob elevadas salinidade e temperatura. Nesse contexto, os biossurfactantes são os melhores substitutos de seus homólogos sintéticos, dada a sua tolerância a condições extremas. O objetivo deste estudo foi prospectar isolados produtores de biossurfactantes ativos em concentrações salinas elevadas. Amostras de solos, sem histórico de contaminação por hidrocarbonetos de petróleo, foram coletadas na ilha da Trindade, local de difícil acesso e com atividade antrópica reduzida ou inexistente. Das 12 amostras de solos coletadas, foram selecionados 14 isolados. Avaliaram-se a produção de biossurfactantes, a tensão interfacial (hexadecano) e superficial. Os isolados foram identificados por meio de perfil de ácidos graxos e sequenciamento da região rRNA 16S. Dentre os biossurfactantes obtidos, destacou- se o produzido por Brevibacterium halotolerans, o qual manteve sua atividade em solução contendo até 175 g.L-1 de NaCl. Esse é o primeiro relato de produção de um biossurfactante por essa espécie. Sua elevada tolerância à salinidade coloca o isolado como promissor para aplicações biotecnológicas na cadeia do petróleo. / Biosurfactants or microbial surfactants are biomolecules with tension active properties produced by a wide variety of microorganisms in various habitats, being a unique class of compounds which has shown potential application in various industrial fields economically important. In applications related to oil chain, such as the enhanced oil recovery, bioremediation of marine oil-contamined water treatment and production, it is necessary for maintaining its activity biosurfactants in extreme conditions, especially under high salinity and temperature. Biosurfactants are the best substitutes for their synthetic counter parts due to higher activity in extreme conditions, lower toxicity and better biodegradability. The objective of this study was to explore isolated producers of active biosurfactant in high salt concentrations, for application related to petroleum activities that require these features. n the present study were collected soil samples from a completely isolated region located at Trindade island, being a place of difficult access and little or no humam activity. From 12 soil samples collected 14 isolateds were selected, that had the biosurfactants production quantified, the surface and interfacial tension activity measured and sub sequently identified to species by the fatty acid profile and 16S rRNA region sequencing. Among the biosurfactants obtained, it was stood out produced by Brevibacterium halotolerans, which maintained its activity in a solution containing 175 g. L-1 NaCl. This is the first report of biosurfactant production by this species. Its high salt tolerance isolated places as promising for biotechnological applications in the oil chain.

Recent studies suggest aromatic amino acid catabolism by starter lactococci and flavor adjunct bacteria have a significant impact on off-flavor development during Cheddar cheese ripening. We hypothesized that a flavor adjunct bacterium, Brevibacterium linens BL2, produces off-flavor compounds from aromatic amino acid metabolism that will have a detrimental impact on cheese flavor. The mechanism of tryptophan (Trp) catabolism in Brevibacterium linens BL2, was investigated in a chemically defined medium during incubation in laboratory conditions (no carbohydrate, pH 6.50, 220 rpm, 25°C) and cheese-like conditions (no carbohydrate, 4% NaCl, static incubation, l5°C). In laboratory conditions, metabolic studies and enzyme assays confirmed that Trp was converted to kynurenine and anthranilic acid. However, cells incubated in cheese-like conditions did not utilize Trp, indicating that these enzymes are not likely to be involved in formation of Trp compounds associated with off-flavors in Cheddar cheese. In an attempt to verify the metabolic activity of the cells during incubation by monitoring the amino acid metabolism in chemically defined medium inoculated with B. linens BL2, a capillary electrophoresis-laser-induced fluorescence method was developed that could separate, detect, and quantitate 18 amino acids within 38 min. The data indicated that B. linens BL2 was metabolically active. Presumably, the cells will be metabolically active and metabolize amino acids in cheese as well. The ability to determine the Trp metabolic activity of B. linens BL2 in cheese, and to quantify Trp catabolic compounds in cheese during ripening, requires a quantitative extraction procedure. An analytical method was developed to extract and quantify aromatic amino acids and Trp catabolites from cheese using capillary electrophoresis. Methanol was used to extract Cheddar cheese made with Lactococcus lactis S3 alone and in combination with B. linens BL2 to quantitatively determine the influence of BL2 on the occurrence of aromatic catabolites. All cheeses contained aromatic amino acids, indole acetic acid, and indole. The concentration and time taken for development of these compounds were significantly decreased or delayed by the addition of B. linens BL2. After 6 months of aging, the concentrations of Trp catabolites were significantly lower in cheese made with B. linens BL2. Addition of BL2 did not directly contribute to off-flavors derived from Trp catabolism in Cheddar cheese. Therefore, the hypothesis was rejected.

L'objectif de ce travail était d'étudier une bactérie d'affinage de cet écosystème, Brevibacterium aurantiacum (BA). La mise en place d'outils moléculaires pour l'étude de la biodiversité du genre Brevibacterium. L'approche par MLST avec 9 gènes de ménage est un nouvel outil prometteur pour l'identification des Brevibacteriaceae, nous avons identifié une nouvelle espèce appartenant aux souches d'intérêt technologique, B. antiquum. L'approche par puce ADN a permis d'étudier la biodiversité au sein de l'espèce de BA, les résultats montrent que 13% et 15% du génome de la souche séquencée sont absents et/ou divergents dans les souches BL2 et ATCC 9175. Nous avons ensuite réalisé une reconstruction du métabolisme du soufre chez BA ATCC 9175 et étudié sa régulation en fonction de différentes sources de soufre en utilisant des approches omics. Les résultats montrent une répression des voies d'assimilation du sulfate et de la biosynthèse de la cystéine en présence de cystine et une répression des voies de biosynthèse de la méthionine via l'homocystéine et la voie de transulfuration par la méthionine. Enfin, lors d'un ajout de méthionine dans le milieu, nous observons une induction coordonnée des gènes codant pour la méthionine gamma-lyase et un transporteur de la méthionine suggérant la présence d'un régulateur spécifique pour cette voie. Enfin, nous avons étudié le comportement de BA en présence ou en absence d'une levure d'affinage de fromage Kluyveromyces lactis (KL) par des approches biochimiques et transcriptomiques. Chez BA, on observe une modification du métabolisme carboné et de l'azote, de la voie de biosynthèse des pigments.

Le métabolisme du soufre, qui occupe une place centrale au sein de la cellule, est aussi important lors de la fabrication des fromages à pâte molle à croûte lavée. L'écosystème fromager assimile les acides aminés soufrés et peut ainsi produire des composés soufrés volatils (CSVs) indispensables à la flaveur de ces produits. Nous avons étudié le métabolisme du soufre chez deux micro-organismes d'affinage, les levures hémiascomycètes Kluyveromyces lactis et Yarrowia lipolytica. L'analyse in silico du phylum des hémiascomycètes nous a donné pour la première fois une vision évolutive de ce métabolisme. Nous avons relevé des différences fondamentales au niveau de la synthèse de la cystéine mais aussi au niveau des enzymes impliquées dans la production de CSVs. Cette analyse constitue une base solide pour l'étude du métabolisme du soufre. Nous avons combiné plusieurs approches exploratoires (transcriptome, métabolome, dosage des CSVs) afin d'avoir une vision globale de ce métabolisme chez K. lactis et Y.lipolytica. Les différences observées se situent notamment au niveau des voies de synthèse de la cystéine et de la taurine. La production de CSVs semble liée à la surexpression de transaminases spécifiques à chaque espèce combinée à l'accumulation de méthionine intracellulaire. L'affinage du fromage dépendant de tout un écosystème, nous avons également étudié l'interaction entre deux micro-organismes d'affinage, K. lactis et Brevibacterium aurantiacum via une approche transcriptomique, en comparant l'expression d'une co-culture à celle des cultures pures. Nous avons observé de profondes modifications métaboliques touchant notamment le métabolisme du carbone et celui de la biotine.