Submitted by Chaylane Marques (chaylane.marques@ufpe.br) on 2015-03-12T18:50:13Z No. of bitstreams: 2 Billy_Manoel_dos_Santos.pdf: 1757421 bytes, checksum: fbd4f87a02c1c73851681e4a75d97970 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T18:50:13Z (GMT). No. of bitstreams: 2 Billy_Manoel_dos_Santos.pdf: 1757421 bytes, checksum: fbd4f87a02c1c73851681e4a75d97970 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / A indústria alcooleira no Brasil perde anualmente vários milhares de reais devido a episódios de contaminação produzidos por bactérias. Além disso, outros milhares são gastos com o uso de antibióticos industriais para a contenção dessas contaminações. Infelizmente, não há ainda um catálogo completo de informações sobre todas as possíveis espécies bacterianas envolvidas na contaminação industrial, seus efeitos no processo e os fatores que levam a essas contaminações. Com a crescente demanda por fontes de energia renováveis, principalmente o etanol, grandes esforços têm sido feitos para aumentar a sua produção. Este trabalho tem como objetivo principal identificar as bactérias lácticas que entram no processo de fermentação alcoólica através do caldo de cana-de-açúcar com a utilização de ferramentas moleculares para uma rápida e eficiente identificação. Para foram realizadas coletas em quatro destilarias do Nordeste do Brasil durante a safra de 2007/2008. A identificação dos isolados foi realizada através da técnica ARDRA e pela análise do sequenciamento de DNA dos genes pheS e 16S. Os resultados obtidos mostram que os lactobacilos são os principais contaminantes do processo, com o Lactobacillus fermentum se destacando entre as demais. Também foi observado que entram no processo bactérias dos gene Weissella e Leuconostoc, com destaque para a W. confusa e o Leuc. mesenteroides que foram identificadas em três das destilarias estudadas. Além da identificação, os isolados de W. confusa e o Leuc. Mesenteroides foram tipados pelo REP-PCR com o primer (GTG)5. Os resultados mostraram que, além da diversidade de espécies bacterianas detectadas, existe uma diversidade de linhagens dessas duas principais espécies no processo. A espécie Leuc. mesenteroides tem sido apontada como contaminante em processo de fermentação alcoólica industrial, sendo algumas linhagens capazes de causar a formação de dextrana, podendo causar danos ao processo fermentativo. Desta forma se faz necessário trabalhos futuros com o fim de correlacionar os perfis destas espécies com possíveis danos à fermentação, para que com este conhecimento possa ser criadas novas estratégias de controle destes microorganismos.

Les bactéries lactiques sont utilisées pour la fermentation d'aliments dans le but d'augmenter leur durée de conservation et d'améliorer leurs propriétés organoleptiques et nutritionnelles. Dans le but de diversifier l'offre d'aliments fonctionnels et de limiter les pertes en produit frais, nous proposons des produits fermentés à base de fruits ou légumes, riches en antioxydants et plaisants pour le consommateur. Pour cela, mes travaux de thèse ont été menés en deux étapes : caractériser la flore bactérienne lactique présente à la surface de fruits et de légumes cultivés à La Réunion, puis sélectionner des bactéries autochtones possédant certaines propriétés fonctionnelles. Ainsi, 77 bactéries lactiques isolées de papayes, de tomates et d'achards de la Réunion, appartenant aux genres Leuconostoc, Lactococcus, Weissella, Lactobacillus et Fructobacillus ont été caractérisées génétiquement et phénotypiquement. Parmi la grande diversité observée, certains isolats ont présenté des caractéristiques technologiques (conditions et vitesse de croissance, résistance aux stress environnementaux) et fonctionnelles (production d'exo-polysaccharides) intéressantes pour l'élaboration d'aliments fermentés, en particulier les isolats Weissellla cibaria 64 et 30, Leuconostoc pseudomesenteroides 60 et Lactobacillus plantarum 75. A partir de ces données, des étapes complémentaires de criblage ont été réalisées sur différents substrats (mangue, papaye, ananas, thé vert, thé noir) fermentés, en examinant leurs propriétés organoleptiques et leurs activités antioxydantes. Deux isolats, Lc. pseudomesenteroides 12b et W. cibaria 64, ont augmenté significativement la teneur en composés phénoliques et l'activité antioxydante de jus d'ananas Victoria au cours d'une fermentation de 48h. Le produit fermenté se conserve 16 jours au froid sans aucune altération et conserve l'ensemble de ses bénéfices nutritionnels. Une odeur et des goûts typiques ont été détectés dans les boissons fermentées obtenues. L'augmentation de l'activité antioxydante observée au cours de la fermentation résulte probablement du métabolisme des composés phénoliques par les bactéries. L'identification des molécules produites et des enzymes impliquées est nécessaire afin de comprendre les mécanismes mis en jeu. / Lactic acid bacteria are used for the production of large variety of fermented foods in order to enhance shelf-life together with improved organoleptic and nutritional properties. In order to create new functional foods and to reduce fresh product waste, the objectives of my PhD were to develop and characterize fruit or vegetables fermented foods, rich in antioxidants and pleasant for consumers. To reach these goals, my PhD work was split in two steps: 1- characterization of lactic bacteria present on fruits and vegetables grown at Reunion Island and 2- selection of autochthonous bacteria with functional properties. Thus, 77 lactic bacteria from genera Leuconostoc, Lactococcus, Weissella, Lactobacillus and Fructobacillus were isolated from papaya, tomato and sliced cabbage from Reunion Island. They were genetically and phenotypically characterized. A huge diversity in term of genetic and phenotypic characteristics was determined. Furthermore, several isolates exhibiting specific technological and functional properties (growth rate, resistance to environmental stress, production of exopolysaccharides) were identified. These isolates, potentially useful for the production of fermented foods, were Weissella cibaria 64 and 30, Leuconostoc pseudomesenteroides 60 and Lactobacillus plantarum 75. Then, further screening steps were performed on different food substrates (pineapple, mango, papaya, tea infusions) in order to select isolates able to improve antioxidant and organoleptic properties. Two isolates, W. cibaria 64 and Lc. pseudomesenteroides 12b were shown to significantly enhance the phenolic content and the antioxidant activity of Victoria pineapple juice after a 48h-fermentation. The fermented products exhibited a good shelf-life of 16 days without alteration and preserved nutritional benefits. Characteristic odour and tastes were detected in the fermented drinks. The increase in antioxidant activity observed during fermentation was probably the consequence of a depolymerization of phenolic compounds. Further works are required to identify the composition changes over fermentation and to reach a better understanding of the mechanisms involved.

Our large intestine is like a large metabolic organ colonised by microorganisms. Beneficial probiotic bacteria are of interest since they might metabolise certain prebiotic carbohydrates and produce metabolites that are suggested to promote health and prevent diseases.   Strains of Weissella have proven probiotic properties since they, for example, show ability to metabolise prebiotic oligosaccharides, are resistant to a low pH (pH 2-3) and bile salt. In a previous project, six new strains of Weissella were isolated from Indian fermented food and vegetables, and four of them, including strain 92, were able to ferment xylooligosaccharides and form short chain fatty acids (SCFA), especially acetic acid. This strengthened the probiotic potential of these strains.   The aim of this project was to see if previously untested oligosaccharides (arabinooligosaccharides (AOS), laminarioligosaccharides (LOS) and chitooligosaccharides (COS)) could be metabolised by Weissella strain 92.   This study includes the following steps; cell growth in MRS (De Man, Rogosa and Sharpe) medium on different carbohydrates measured with spectrophotometer, pH measurement (analysing the difference of MRS medium (pH 6.42) pre and post cell growth, where reduced pH indicates acid production), and analysis of fermentation products (including SCFA (short chain fatty acids, e.g. acetic acid, butyric acid, propionic acid), lactic acid and ethanol) with an HPLC (high-performance liquid chromatography) instrument.   This research study has shown that Weissella strain 92 produces acetic and butyric acid as a consequence of use of AOS, LOS and COS, this indicates that the oligosaccharides are prebiotic and emphasizes the probiotic potential of Weissella strain 92.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:40:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-BillySantos.pdf: 1715330 bytes, checksum: 928f73e06e0a832aee3a63d319146b43 (MD5) / Made available in DSpace on 2017-04-06T18:40:10Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-BillySantos.pdf: 1715330 bytes, checksum: 928f73e06e0a832aee3a63d319146b43 (MD5) Previous issue date: 2012 / A indústria alcooleira no Brasil perde anualmente vários milhares de reais devido a episódios de contaminação produzidos por bactérias. Além disso, outros milhares são gastos com o uso de antibióticos industriais para a contenção dessas contaminações. Infelizmente, não há ainda um catálogo completo de informações sobre todas as possíveis espécies bacterianas envolvidas na contaminação industrial, seus efeitos no processo e os fatores que levam a essas contaminações. Com a crescente demanda por fontes de energia renováveis, principalmente o etanol, grandes esforços têm sido feitos para aumentar a sua produção. Este trabalho tem como objetivo principal identificar as bactérias lácticas que entram no processo de fermentação alcoólica através do caldo de cana-de-açúcar com a utilização de ferramentas moleculares para uma rápida e eficiente identificação. Para foram realizadas coletas em quatro destilarias do Nordeste do Brasil durante a safra de 2007/2008. A identificação dos isolados foi realizada através da técnica ARDRA e pela análise do sequenciamento de DNA dos genes pheS e 16S. Os resultados obtidos mostram que os lactobacilos são os principais contaminantes do processo, com o Lactobacillus fermentumse destacando entre as demais. Também foi observado que entram no processo bactérias dos gene Weissella eLeuconostoc, com destaque para a W. confusa e o Leuc. mesenteroides que foram identificadas em três das destilarias estudadas. Além da identificação, os isolados de W. confusa e o Leuc. Mesenteroidesforam tipados pelo REP-PCR com o primer(GTG)5. Os resultados mostraram que, além da diversidade de espécies bacterianas detectadas, existe uma diversidade de linhagens dessas duas principais espécies no processo. A espécie Leuc. mesenteroides tem sido apontada como contaminante em processo de fermentação alcoólica industrial, sendo algumas linhagens capazes de causar a formação de dextrana, podendo causar danos ao processo fermentativo. Desta forma se faz necessário trabalhos futuros com o fim de correlacionar os perfis destas espécies com possíveis danos à fermentação, para que com este conhecimento possa ser criadas novas estratégias de controle destes microorganismos. / The alcohol industry in Brazil loses several thousand dollars each year due to episodes of contamination produced by bacteria. In addition, thousands more are spent on the industrial use of antibiotics to contain these contaminants. Unfortunately, there is still no complete catalog of all possible information about bacterial species involved in industrial pollution, its effects on the process and the factors that lead to these contaminants. With the growing demand for renewable energy sources, especially ethanol, great efforts have been made to increase production. This work has as main objective to identify lactic acid bacteria that enter the process of fermentation through thejuicesugarcane with the use of molecular tools for rapid and efficient identification. For this, samples were taken at four distilleries in northeastern Brazil during the harvest of 2007/2008. The identification of isolates was performed using the ARDRA technique and analysis of DNA sequencing of the pheSand 16S genes. The results show that lactobacilli are the main contaminants of the process with Lactobacillus fermentumstood out among the others. We also observed that bacteria enter the process of genus Weissellaand Leuconostoc, with emphasis on the W. confusedand Leuc. mesenteroidesthat were identified in three distilleries studied. Besides identification, the isolates of W. confusaand Leuc. mesenteroideswere typed by rep-PCR with the primer (GTG)5. The results showed that, in addition to the variety of bacterial species found, there are several lines of these two major species in the process. The species Leuc. mesenteroideshas been identified as a contaminant in industrial fermentation process, some strains can cause the formation of dextran, causing damage to the process. Thus further work is required in order to correlate the profiles of these species with possible damage to the fermentation, so that this knowledge can be created new control strategies such microorganisms.

Deoxynivalenol (DON) contamination of grain destined for animal feeds is a major toxicological risk to monogastrics and is suspected of restricting productivity in ruminants. Whereas bacterial additives have been developed that can detoxify DON in the rumen and lower intestine, there are currently no commercial inoculants able to perform this task in crimped grain (CG) silage, a regionally important method of moist grain preservation based on homo- and heterofermentative lactic acid bacteria or chemical additives. Determining whether this ensiling process alongside the action of detoxifying bacteria has the potential to remove DON in CG prior to ingestion, was explored in mini-silo ensiling experiments. CG was heat treated (100 °C, 60 min) or ensiled fresh in triplicate 50 g silos, spiked with 5 mg/kg DON and inoculated with lactic acid bacteria derived from wild birds, natural epiphytic inoculants and commercially sourced silage additives (21 d). DON recovery was only significantly reduced (31.2 ± 14.4% recovery, p < 0.001, n= 30) by heat treatment, as determined by IAC-RP-HPLC-UV. Bacterial assemblage analysis by 16S rRNA PCR-DGGE-SEQ identified Weissella cibaria, Pantoea agglomerans, Bacillus subtilis, B. licheniformis and Hafnia alvei as candidate detoxification agents, of which W. cibaria and H. alvei decreased DON recovery in vitro (11.3 and 6.2% recovery respectively, p < 0.05, n = 18), which translated to inoculated W. cibaria yielding a decrease in DON recovery (67.2± 14.4%, 28 d) in naturally contaminated crimped wheat (13.5 ± 1.0 mg/kg, 35-40% moisture, p < 0.05, n =15). As W. cibaria is a lactic acid bacteria already associated with fermented CG by default it has promise as a novel DON detoxification agent in CG silage. DON is however just one of many hepatotoxic co-contaminants. Retrorsine, a DNA-crosslinking pyrrolizidine alkaloid derived from Ragwort (Senecio sp.) was investigated for interactive toxicity with DON in an in vitro co-exposure experiment. HepG2 cells were exposed to Log10 multifactorial binary exposures for 48 h followed by a suite of assays to elucidate mechanisms of interactive cytotoxicity, genotoxicity and modulation of the proteome. Retrorsine was tentatively confirmed to form DNA/protein crosslinks in the comet, micronucleus and crosslinking assays, whilst DON was found to potently induce cytotoxicity and apoptosis. Co-exposure yielded a complex toxicity response, with low doses yielding antagonistic effects and high doses trending towards additive effects, although DON dose was generally the principle component. The difficulties associated with undertaking an interactive toxicity study where both toxins have multiple metabolic and cellular targets are highlighted.