Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-11Bitstream added on 2014-06-13T19:03:40Z : No. of bitstreams: 1 coelho_lf_dr_rcla.pdf: 3999428 bytes, checksum: b2a1c737e7c2bb5f6e89843add264993 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste trabalho foi isolar micro-organismos produtores de D-(-) e L-(+) ácido lático, os quais são utilizados na síntese de polímeros empregados na produção de diversos materiais resistentes e biodegradáveis, além de otimizar a produção de ácido lático, a partir da utilização de diversos resíduos agro-industriais. Os micro-organismos mais promissores para produção de L-(+) ácido lático foram os isolados de Keffir (Ke6, Ke11, Ke8 e Ke24) e o Lactobacillus rhamnosus B103, já para a produção de D-(-) ácido lático, os mais promissores foram os isolados de iogurte (Y15C e Y15A) e o Leuconostoc mesenteroides B512. Pode-se afirmar que os micro-organismos selecionados apresentaram grande potencial para utilização na indústria de biopolímeros e indústria de alimentos. O soro de queijo e a manipueira foram os melhores resíduos para produção de L-(+) ácido lático por Lactobacillus rhamnosus B103. Quando se utilizou 160 g/L de lactose de soro de queijo, 60 mL/L de água de maceração de milho (AMM), 2 mL/L de Tween 80 e 0,10 g/L de MnSO4, observou-se alta produção de L- (+) ácido lático (142 g/L) e baixo residual de lactose (3,2 g/L). Para a otimização com manipueira, foi obtido 41,58 g/L de L-(+) ácido lático, a partir de 50 g/L de açúcar redutor total (ART), 65,40 mL/L de AMM e 1,27 mL/L de Tween 80. Nas otimizações com Leuconostoc mesenteroides B512 foi observado produção de 60,20 g/L de D-(-) ácido lático, utilizando 116,90 g/L de ART de caldo de cana e 44,25 g/L de autolisado de levedura. Nas otimizações com L. plantarum Lmism6 observou-se uma produção de 63,40 g/L de ácido lático, 0,40 g/L de ART residual e alta conversão de substrato (99,40%), quando se utilizou 70 g/L de ART de melaço, 30,00 mL/L de AMM, 2 g/L de K2HPO4 e 1 mL/L de Tween 80 / The aim of this study was to isolate D-(-) and L-(+) lactic acid producers micro-organisms, which are used in the synthesis of polymers used in the production of many resistant and biodegradable materials and optimize the lactic acid production, from agro-industrial residues. The most promising micro-organisms for L-(+) lactic acid production were Lactobacillus rhamnosus B103, as well as, the isolated from Keffir (Ke6, Ke11, Ke8 Ke24) and the most promising D-(-) lactic acid producers were strains of yogurt (Y15C and Y15A) and Leuconostoc mesenteroides B512. Cheese whey and cassava wastewater (CW) were the best residues for L-(+) lactic acid production by Lactobacillus rhamnosus B103. Using 160 g/L of lactose from whey, 60 mL/L of CSL, 2 mL/L of Tween 80 and 0.10 g/L of MnSO4, there was higher production of L-(+) lactic acid (142 g/L) and low lactose residual (3.20 g/L). For optimizations with CW, it was obtained 41.58 g/L of L-(+) lactic acid from 50 g/L of reducing sugar, 65.40 mL/L and 1.27 mL of corn steep liquor (CSL) and Tween 80 respectively. Leuconostoc mesenteroides B512 produced 60.20 g/L of D-(-) lactic acid, using 116.90 g/L of sugarcane juice and 44.25 g/L of yeast autolysate. L. plantarum Lmism6 produced 63.40 g/L of lactic acid, with less residual reducing sugar (0.41 g/L) and higher substrate conversion (99.41%), by using 70 g/L of sugar reducing from molasses, 30 mL/L of CSL, 2 g/L of K2HPO4, and 1 mL/L of Tween 80

Le principal objectif de ce projet était d'étudier un système d'inoculation et préfermentation en continu du lait par une culture mixte avec cellules immobilisées et contenant une souche forte productrice de nisine Z, Lc. diacetylactis UL719, et deux souches sensibles à la bactériocine, un Lb. rhamnosus, producteur d’exopolysaccharides, et un Lc. cremoris, sélectionné pour ses capacités d’acidification. Des fermentations du lait inoculé de façon traditionnelle par la culture mixte en cellules libres ont également été réalisées. Durant ces fermentations en batch, la croissance de Lc. diacetylactis est stimulée par la présence des deux autres souches, entraînant une augmentation de la production de nisine. Le système d'inoculation et préfermentation en continu du lait avec cellules immobilisées, conduit sur une période de trois semaines, a permis d'inoculer massivement le lait par Lc. diacetylactis et Lb. rhamnosus. Le lait fermenté obtenu après une étape d'incubation additionnelle contenait également une population élevée de ces deux souches conduisant à une production élevée de nisine et une augmentation de la concentration d'exopolysaccharides comparativement à des fermentations en batch du lait par la culture mixte en cellules libres et inoculé de façon traditionnelle. La méthode de PCR en temps réel développée durant ce projet a permis de quantifier les faibles niveaux de populations de Lc. cremoris dans les laits fermentés et préfermentés. Une augmentation avec le temps de préfermentation de la tolérance à la nisine des cellules de Lb. rhamnosus ainsi que de la capacité acidifiante pour les deux souches majoritairement retrouvées dans le lait préfermenté fut également observée. / The objective of this work was to study an immobilized-cell system for continuous inoculation and prefermentation of milk by a mixed culture containing a nisin Z producer (Lc. diacetylactis UL719) and two nisin-sensitive strains for acidification (Lc. cremoris) and exopolysaccharide production (Lb. rhamnosus RW-9595M). Batch fermentations of milk traditionally inoculated with free-cell mixed culture of the three strains were also performed at different temperatures. In these conditions, we showed that growth of Lc. diacetylactis was stimulated by commensalism, leading to an increase of nisin Z production. Continuous prefermentation of milk with immobilized cells carried out over 3 weeks leads to high inoculation of milk with Lc. diacetylactis and Lb. rhamnosus . In fermented milk, obtained after an additional batch incubation, populations of these two strains remained at high concentrations leading to high nisin production and higher exopolysaccharide concentration compared to traditional batch fermentation of milk. The real-time PCR method, developed in this project, was successfully used to quantify very low cell concentrations of Lc. cremoris in prefermented and fermented milks. Immobilization and continuous culture also lead to important cell physiological adaptations for Lb. rhamnosus and Lc. diacetylactis. Lb. rhamnosus became much more tolerant to nisin Z, and both strains exhibited a large increase in milk acidification capacity.

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EfeitoUsoDiferentes.pdf: 239802 bytes, checksum: 5b39de29c260f7db7fb4293678fffc0e (MD5) Previous issue date: 2008 / Na conservação de alimentos para animais, os inoculantes microbianos são empregados na busca da melhoria do padrão de fermentação de silagens, por meio do estímulo ao desenvolvimento populacional dos microrganismos benéficos deste processo conservativo, como acontece para as bactérias produtoras de ácido lático, em detrimento a inibição dos microrganismos indesejáveis, tais como leveduras e clostrídios. O estudo proposto avaliou o efeito do uso de diferentes inoculantes microbianos à fresco e liofilizados utilizando a cultura do sorgo [Sorghum bicolor (L.) Moench], como matéria-prima para ensilagem, a fim de indicar a possibilidade do emprego de inoculantes microbianos desenvolvidos no nosso país. Foram realizados dois experimentos, em um mesmo silo, Experimento 1 (tratamentos com inoculantes liofilizados, na região superior do silo) e Experimento 2 (tratamentos com inoculantes à fresco, na região inferior do silo), com cinco tratamentos e três repetições por silo, sendo os tratamentos caracterizados como controle (sem inoculante), inoculante microbiano comercial (IC) e distintos inoculantes confeccionados à partir de bactérias láticas isoladas da planta de sorgo: Lactobacillus plantarum, Lactobacillus paracasei e Lactobacillus rhamnosus. Quando da ensilagem, foram utilizados três silos experimentais de madeira, que foram abertos em distintos períodos, ou seja, 1, 3 e 28 dias após a ensilagem. Foi utilizado o delineamento experimental em parcelas subdivididas no tempo, no qual os três períodos de abertura foram às parcelas e os cinco tratamentos as subparcelas, em delineamento inteiramente casualizado. No experimento 1, os teores de fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) sofreram efeito dos inoculantes microbianos no 3º e 28º dia de abertura dos silos, obtendo menores valores nos tratamentos IC e LPP (L. plantarum + L. paracasei. No experimento 2, os teores de FDN, apresentaram efeito no 28º dia de abertura do silo, demonstrando que os tratamentos IC e LPP diferiram entre si, sendo estatisticamente iguais aos demais. A combinação dos isolados microbianos liofilizados de L. plantarum e L. paracasei mostrou potencial para uso prático, pois foi tão efetivo quanto o tratamento IC. / In the conservation of animal feed, the microbial inoculants are used in pursuit of improving the standard fermentation of silages, by encouraging the population development of the beneficial microorganisms in this process conservative, as the bacteria that produce lactic acid, rather than using inhibition of undesirable microorganisms such as yeasts and clostridia. The proposed study evaluated the effect of using different microbial inoculants to using fresh and freeze-dried sorghum [Sorghum bicolor (L.) Moench], as raw material for silage, to indicate the possibility of using microbial inoculants developed in the Brazil country. Were conducted two experiments in the same silo, Experiment 1 (treatment with freeze-dried inoculants in the upper region of the silo) and Experiment 2 (treatment with inoculants to cool in the lower silo), with five treatments and three replicates per silo the treatments being characterized as a control (without inoculum), commercial inoculant (IC) and the different inoculants made from lactic acid bacteria isolated from the sorghum plant: Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus rhamnosus. When silage were used three experimental silos of wood, which were opened at different periods, 1, 3 and 28 days after ensiling. We used the experimental design in split plot in time, in which the three periods were open on plots and subplots the five treatments in a completely randomized design. In experiment 1, the levels of neutral detergent fiber (NDF) and acid detergent fiber (ADF) had the effect of microbial inoculants on the 3rd and 28th day of opening the silos, getting lower values in treatments IC and LPP (L. plantarum + L. paracasei. In experiment 2, NDF, had an effect on the 28th day of opening of the silo, showing that the IC and LPP treatments differed significantly, being statistically equal to others. The combination of freeze-dried microbial isolates of L. plantarum and L. paracasei showed potential for practical use because it was so effective as treatment failure.

The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.

Increasing evidence implicates the presence of bacteria in intrauterine tissues as an important risk factor for spontaneous preterm labor. Epigenetic alterations of innate immunity genes may increase the mother’s sensitivity to subclinical levels of bacteria. This study examined the presence of TET2, TLR-2, and TLR-9 in intrauterine tissue, and evaluated whether epigenetic alterations of these genes, as well as IL-8, changed their expression in human decidual tissue and a macrophage cell culture. Immunohistochemicalstaining was used to detect the presence of these proteins in intrauterine tissue. Gene expression changes were evaluated in stimulated monocytes and macrophages. Fluorescence immunohistochemistry was used to track translocation of TET2 in stimulated monocytes and macrophages. Secreted IL-8 concentration was detected with ELISA. Decidual expression of TET2, TLR-2, and TLR-9 increased in the order TNL < TL < sPTL < iPTL. This study found that TET2, TLR-2, TLR-9, and IL-8 are regulated by epigenetic mechanisms. This study was the first to report activation of TET2 involves its translocation from the cytosol to the nucleus in macrophages.