Fatores que interferem na produção de bovicina HC5 e obtenção de mutantes não produtores de bacteriocina

• Autor(a) principal: Carvalho, Ana Andréa Teixeira de
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1546
• topic: Streptococcus bovis HC5; Bovicina HC5; Produção de bacteriocina; Mutagênese insercional; S. bovis HC5; Bovicin HC5; Bacteriocin production; Insertional mutagenesis; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS

This work was performed to investigate the factors that influence bovicin HC5 production by S. bovis HC5 and to obtain mutants unable to produce bovicin. S. bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l-1 of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when 0.5 g l-1 yeast extract and 1.0 g l-1Trypticase® were added to the basal medium. S. bovis HC5 cultures were able to produced bovicin HC5 in media containing cheese whey and sugar cane juice as carbon sources. S. bovis HC5 grew in basal media, at aerobic and anaerobic conditions, but bacteriocin production was higher under anaerobic conditions. S. bovis HC5 grew faster and produced more biomass when cultivated at pH 7.0 and 45ºC, but bovicin HC5 production was higher when the initial pH was 6.5 and the temperatures ranged from 30°C to 39ºC. When the pH was maintained at constant values, bacteriocin production was inversely proportional to lactate production, glucose consumption and the increase in pH. Continuous culture experiments with dilution rates varying from 0.07 to 1.20 h-1 under uncontrolled pH resulted in homolactic fermentation and greater bacteriocin production at lower dilution rates. In this latter case, residual glucose was never completely consumed, but the media pH was approximately 5.0. When the pH was maintained at 7.5, mixed-acid fermentation occurred at dilution rates below 1.20 h-1. Bacteriocin production increased at higher dilution rates but was at least 10 times lower when compared with uncontrolled pH conditions. Residual glucose was only detected in the fermentation vessel at dilution rates above 0.60 h-1. These results indicate that bovicin HC5 production is not directly related to S. bovis HC5 growth, and can vary depending on extracellular pH and growth rate. S. bovis HC5 cells were transferred 40 times in basal media and bovicin HC5 production was a stable phenotype in S. bovis HC5. No plasmids were detected in S. bovis HC5 genome, indicating that the genes encoding for bovicin HC5 biosynthesis are located in the chromosome. Thirty mutants that showed reduced ability to produce bovicin HC5 were isolated in this study. The presence of the plasmid vector used for insertional mutagenesis in the genome of some mutants was confirmed by molecular techniques and these mutants can be used for characterization of the bovicin HC5 genetic determinants.