Vesículas de membrana : um componente inexplorado no rúmem com capacidade de degradação de proteínas, carboximetilcelulose e amido

• Autor(a) principal: Prado, Rodolpho Martin do
• Data de Publicação: 2015
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Estadual de Maringá (RI-UEM)
• Texto Completo: http://repositorio.uem.br:8080/jspui/handle/1/1653
• topic: Amilase; Celulase; Prevotella ruminicola; Protease; Vesículas de membrana; Vesículas de membrana externa; Brasil.; Amylase; Cellulase; Membrane vesicle; Outer membrane vesicle; Prevotella ruminicola; Protease; Brazil.; Ciências Agrárias; Zootecnia

Membrane vesicles production is a ubiquitous process in Gram-negative and rampositive bacteria. Research on membrane vesicles increased during the last 10 years, but it is mainly focused on human pathogens. Also, in most studies only bacterial pure cultures are investigated. With the advance of proteomics, several new functions are now associated with membrane vesicles. Yet, so far no study has isolated membrane vesicles from rumen fluid, or assessed their capacity to hydrolase substrate. Thus, as a first study, outer membrane vesicles were isolated from an axenic culture of Prevotella ruminicola, using a protocol used to isolate outer membrane vesicles from pathogenic bacteria, and hydrolytic activity from the samples was assessed. The protocol was successfully employed as no bacterial contamination was observed on cross-section transmission electron microscopy. The protein profile from the whole culture, washed culture, outer membrane vesicle-free supernatant, and outer membrane vesicles were distinct as observed on SDS-PAGE. Proteolytic activity was assessed on gelatin zymograms and outer membrane vesicle samples had distinct and clearer bands compared to whole culture, washed culture, and outer membrane vesicle-free supernatant. Protease inhibitors to assess protease classes were added to the samples but no clear effect could be observed. Furthermore, no activity could be observed on carboxymethylcellulose and starch zymograms. Although a greater enzymatic activity was expected, results provide evidence that some proteolytic enzymes from P. ruminicola are selectively loaded into outer membrane vesicles from this bacterium. As a second study, membrane vesicles were isolated using rumen liquor and particulate matter from the rumen. Fractions using solid-attached bacteria and liquid-associated bacteria were used to further improve membrane vesicle characterization. Three cows were sampled but only samples from one cow were imaged with negative staining transmission electron microscopy. Protein profiles of samples were similar when assessed on SDS-PAGE, but proteolytic activity was diverse on gelatin and casein zymograms. Furthermore, activity on carboxymethylcellulose and starch zymograms was diverse across the samples. These results provide evidence that membrane vesicles in the rumen have been overlooked as in some cases more activity was observed in vesicle samples compared to within the microorganisms. Understanding fundamentals from microbiome is key to improve feed use in ruminants and research using membranes vesicles isolated from the rumen might add knowledge on rumen microbiome function.