Isolamento e caracterização de bactérias ruminais com alta atividade específica de produção de amônia

• Autor(a) principal: Lima, Janaina Rosa 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/123456789/1543
• topic: Amônia; Rúmen; Desanimação; DGGE; Bacteriocinas; Ammonia; Rumen; Deamination; DGGE; Bacteriocin; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS

The objectives of this study was to evaluate the production of ammonia in cattle fed diets containing increasing levels of crude protein and investigate the impact of diet on the genetic diversity of the rumen ecosystem, isolate and characterize physiological and genetically rumen bacteria with high specific activity, production ammonia and determine the effect of monensin and bovicina HC5 on deamination of the bacterial isolates hiperprodutores ammonia.The total production of ammonia and specific activity of ammonia production (SAAP) of pure and mixed cultures obtained from the rumen of animals fed diets containing increasing levels of protein were analyzed. Cultures were incubated in anaerobic mineral medium containing Trypticase (15 g/L) and deamination was determined. The genetic diversity of the rumen microflora of the animal fed a diet containing increasing concentrations of protein and the enrichment cultures transferred in vitro in the presence of Trypticase was analyzed by denaturing gradient gel electrophoresis (DGGE). It was found that the ability of deaminated amino acids varied between the cultures of ruminal bacteria harvested from cattle fed with 8, 18 and 24% crude protein (CP). Aliquots of rumen fluid were diluted successively (increments of 10 times) reduced deamination of Trypticase. The SAAP decreased linearly with the dilution of the rumen microbiota, was less than 10 nmoles ammonia / mg protein / min after dilution 10-4 (8 and 18% CP) or 10-5 (24% CP). Analysis of genetic diversity of the rumen microbiota by DGGE technique did not indicate significant changes in microbial community composition between the first and last days of feeding with each diet. For transfers from the diet with 8, 18 and 24% CP, there were changes in the profiles of bands corresponding to different microbial species present in the samples. The culture originated from the diet containing 24% CP showed the highest production of ammonia and adapt more quickly to Trypticase. Bacteria that showed high specific activity of ammonia production were isolated and metabolic parameters were evaluated. The strategies used in isolation has been obtained from fourteen isolates (cultures 27B, 35, 53P, 57A, 86A, 86C, 92B, 99A, 102, 102B, 103, 15A, 15B and CT2) with high specific activity of ammonia production. These organisms were characterized as Gram-positive, capable of producing heat-resistant spores and grow under anaerobic conditions. The results indicated that the isolates differ in the rate of deamination and growth rate in medium containing Trypticase. However, it was observed that these isolates shared cultural characteristics and fermentation products in common. The biochemical analysis of microorganisms involved in this work showed that several isolates were able to ferment carbohydrates. When the isolates were incubated with amino acids, there was high specific activity of ammonia production from phenylalanine, glutamine, isoleucine, leucine, serine, tyrosine and threonine. Analysis of genetic diversity among fourteen isolates with high specific activity of ammonia production by the technique of BOX-PCR resulted in different band patterns among the isolates and cultures hiperprodutoras ammonia previously described in the literature. Based on data from the sequencing of 16S rDNA was determined that the isolates 57A, 86A, 99A, 102 and 15A showed identity of 96%, 99%, 93%, 94% and 99% respectively with Clostridium botulinum and isolated RF2B, coming feces of pigs, but not yet characterized. Isolates 15B and CT2 had 94% identity and 88% respectively with Clostridium hastiforme. The sensitivity of rumen microbes and isolated hiperprodutores ammonia rumen bacteriocin bovicin HC5 and the ionophore monensin was evaluated and the results showed that bovicin HC5 inhibited the production of ammonia by ruminal microbes and the isolated hiperprodutores ammonia characterized in this work. Considering the results obtained in this study, it appears that ruminal microbes of cattle tropical bacteria present with high specific activity of ammonia production related to the genus Clostridium that may contribute to the loss of dietary nitrogen in the rumen.