Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T21:39:29Z (GMT). No. of bitstreams: 1 Santos_LucielenOliveirados_M.pdf: 943021 bytes, checksum: 7237d5c1a050d0bfae7083ba9026d36f (MD5) Previous issue date: 2004 / Resumo: As bacteriocinas são substâncias protéicas produzidas por bactérias, com atividade bactericida e bacteriostática contra bactérias sensíveis. Estes compostos são produzidos por microrganismos gram-positivos e gram-negativos, incluindo as bactérias láticas. Estas bactérias são extensamente usadas no processamento de alimentos devido a sua contribuição na vida de prateleira, textura e propriedades organolépticas. Algumas cepas de enterococos podem produzir bacteriocinas ativas contra bactérias patogênicas, tal como a Listeria monocytogenes. As bacteriocinas produzidas por Enterococcus faecium e Enterococcus faecalis são peptídeos pequenos, hidrofóbicos e termoestáveis. Dentro deste contexto este trabalho teve como objetivo estudar a produção de enterocina por E. faecium e E. faecalis. Inicialmente, as fermentações foram feitas em frascos Erlenmeyers, incubados a 37°C por 24h, para determinar as melhores condições de fonte de carbono (glicose, sacarose comercial ou açúcar mascavo), pHinicial (4,0; 6,0; 8,0 ou 10,0) e agitação (0 ou 200rpm). O meio basal modificado sem glicose foi usado como meio base sendo complementado com diferentes fontes de carbono. Em um segundo estágio, os experimentos foram conduzidos em fermentador de bancada por 24h. Durante as fermentações foram coletadas amostras em intervalos de 24h ou 1h, para a determinação da atividade de enterocina, massa celular seca, pH e conteúdo de açúcar. Os resultados obtidos a partir dos testes em frascos Erlenmeyers indicam que usando o E. faecium para produção de enterocina as melhores condições de cultivo foram pHinicial 10,0, sacarose e sem agitação. A máxima produção de enterocina foi 2,81UE. Os resultados indicaram que as melhores condições para produção de enterocina usando E. faecalis foram pHinicial 10,0, 200rpm e açúcar mascavo, com atividade de enterocina de 1,68UE. No fermentador, a maior atividade de enterocina foi 2,94UE após 21h de fermentação no experimento com pHinicial 10,0, 200rpm, sacarose e E. faecium. A atividade de enterocina foi de 1,30UE após 12h de incubação no experimento com pHinicial 10,0, 200rpm, açúcar mascavo e E. faecalis. A L. monocytogenes Scott A foi utilizada como microrganismo teste e foi inibida pelas enterocinas produzidas por E. faecium e por E. faecalis / Abstract: Bacteriocins are protein substances produced by bacteria that are bactericidal or bacteriostatic against sensitive bacterial species. Both gram-negative and gram-positive organisms, including the lactic acid bacteria, produce these compounds. These bacteria are extensively used in food processing, for their contribution to shelf life, texture and organoleptic properties. Some strains of entecococci can produce bacteriocins active against pathogenic bacteria such as Listeria monocytogenes. Bacteriocins produced by Enterococcus faecium and Enterococcus faecalis are small, hydrophobic and termostable peptides. The aim of this work was the study of enterocin production by E. faecium and E. faecalis. Initially, fermentations assays were carried out in Erlenmeyers flasks for determining the best conditions of carbon sources (glucose, sucrose and brown sugar), pHinitial (4,0; 6,0; 8,0 or 10,0) and agitation (0 or 200rpm). The modified basal medium without glucose was used as base medium and the different carbon employed. The flasks incubated at 37°C for 24h. In a second stage, the experiments were carried out in laboratory scale fermentor for 24h. During the fermentations samples were collected at intervals of 24 h or 1h, for determination of enterocin activity, biomass (cell dry mass), pH and sugar contents. The first phase in Erlenmeyers flasks indicated that using E. faecium for enterocin production the best culture conditions were pHinitial 10,0, sucrose and without agitation. The maximum enterocin production was 2,81UE. The results indicated that the best conditions for enterocin production using E. faecalis were pHinitial 10,0, 200rpm and brown sugar, with enterocin activity of 1,68UE. In fermentor the highest enterocin activity was 2,94UE after 21h of fermentation in experiment using pHinitial 10,0, 200rpm, sucrose and E. faecium. The enterocin activity was 1,30UE after 12h of incubation in experiment with pHinitial 10,0, 200rpm, brown sugar and E. faecalis. When it was used L. monocytogenes Scott A as microorganism test occurred its inhibition by enterocin of E. faecium and E. faecalis / Mestrado / Mestre em Engenharia de Alimentos

Bacillus amyloliquefaciens FZB42 ist ein grampositives Bodenbakterium. Es kann in der Rhizosphäre das Wachstum von Pflanzen fördern und durch die Produktion von Sekundärmetaboliten phytopathogene Organismen hemmen. Aus der Genomanalyse und den dazugehörigen Arbeiten war bekannt, dass Bacillus amyloliquefaciens FZB42 nicht-ribosomal je drei antimikrobielle Polyketide und Lipopeptide herstellt, sowie zwei Siderophore und das Dipeptid Bacilysin. Für Bacillus typische Lantibiotika oder große Bacteriocine wurden nicht gefunden. In dieser Arbeit wird erstmalig gezeigt, dass Bacillus amyloliquefaciens FZB42 auf ribosomale Weise antibakterielle Peptide herstellt. Zwei bisher unbekannte Bacteriocine, Amylocyclicin A und Plantazolicin, und deren dazugehörigen Gencluster konnten identifiziert und charakterisiert werden. Amylocyclicin A ist ein unmodifiziertes Peptid, dessen N- und C-Terminus kovalent verbunden sind. Es wurde der Gruppe I der zirkulären Bacteriocine zugeordnet, dessen Mitglieder sich durch schwache Homologie untereinander, aber durch wahrscheinlich ähnliche 3D-Strukturen auszeichnen. Die Masse beträgt 6381 Da und die Substanz ist stark aktiv gegen grampositive Bakterien. Das Biosynthesecluster umfasst sechs Gene für die Synthese, den Export, die Zyklisierung und die Immunität. Plantazolicin ist ein hydrophobes, stark modifiziertes Peptid aus der TOMM-Gruppe, einer Gruppe aus Microcin B17-ähnlichen Peptiden, die nach neueren Erkenntnissen verbreiteter ist, als bisher bekannt. Plantazolicin ist schwach aktiv gegen grampositive Bakterien und besitzt die Masse 1335 Da. Das Biosynthesecluster umfasst zwölf Gene, mit allen nötigen Genen für Synthese, Modifikation, Regulation, Immunität und Export. / Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Five gene clusters direct the non-ribosomal synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide and the iron-siderophore bacillibactin. Three gene clusters direct the non-ribosomal synthesis of the antibacterial acting polyketides macrolactin, bacillaene and difficidin; in addition to the non-ribosomal synthesis of the antibacterial dipeptide bacilysin. Genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Only two incomplete gene clusters directing immunity against mersacidin and subtilin were found. In this work two ribosomally synthesized antibacterial peptides, amylocyclicin A and plantazolicin, and their corresponding gene clusters were identified. Amylocyclicin A is a circular peptide with a mass of 6381 Da and strong activity against Gram-positive bacteria. Six genes are responsible for the synthesis, maturation, export and immunity of this peptide belonging to group I of circular bacteriocins. Plantazolicin is a strongly modified hydrophobic peptide bearing a molecular mass of 1,335 Da and displaying antibacterial activity toward closely related Gram-positive bacteria. Essential modification contains the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide and addition of two methyl groups. Twelve genes are responsible for synthesis, modification, export and immunity of this peptide belonging to the TOMM group of thiazol/oxazol modified microcins.

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