Serratia and Pseudomonas as important heat-resistant protease producers in cold raw milk

• Ano de defesa: 2015
• Autor(a) principal: Machado, Solimar Gonçalves
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa
• Palavras-chave em Português: Bactérias psicrotróficas; Proteólise; Leite cru; Leite cru - Deterioração; Ciências Agrárias
• Link de acesso: http://www.locus.ufv.br/handle/123456789/6784

The storage of raw milk at cold temperatures does not prevent growth of psychrotrophic bacteria, which can produce heat-resistant proteases that compromise the quality and reduce the shelf life of dairy products. To minimize the technological problems resulting from proteolytic activity, these enzymes should be detected and quantified in raw milk before processing by a rapid method. However, there is no efficient method for this purpose. This work aimed to identify the predominant psychrotrophic species with spoilage potential in Brazilian raw milk, to identify and characterize the heat-resistant proteases produced by this microbiota and to developed a rapid method to detect these proteases in raw milk samples. The cold storage conditions were simulated and the psychrotrophic proteolytic bacteria isolated. The heat-resistant protease-producing bacteria were typing by Rep-PCR and clustered. Biochemical tests, 16S rDNA and rpoB gene sequencing were used for identifying one representative isolate from each cluster. The heat-resistant protease produced by Serratia liquefaciens was characterized. The encoding gene was identified after mass spectrometry analysis of tryptic peptides from the heat-resistant protease. The biotinylated casein was coated on microtiter plates and used as substrate to quantify proteolytic activity in solution and milk samples. Highly proteolytic strains were identified and characterized. The isolates were separated into eight different clusters and four solitary fingerprints. The most proteolytic isolates belonged to Serratia liquefaciens and Pseudomonas species. The S. liquefaciens isolates may produce Ser2, which is a a metalloprotease resistant to the heat treatment of 95 °C for 8.45 min. This metalloprotease showed a molecular weight of approximately, 52 kDa and a heat- resistance similar to AprX from Pseudomonas spp. Although nucleotide sequences of ser2 gene were conserved among S. liquefaciens isolates, the spoilage potential among them was heterogeneous indicating differences in enzyme expression levels or post-transcriptional modifications. The developed immunoassay was efficient for quantification of trypsin, papain, pepsin, thermolysin and protease from bovine x pancreas activity. However, further research should be performed to minimize the influence of milk components on the developed assay for detecting proteases in milk samples. This work highlighted the poor conditions of hygiene in milk farms and the high spoilage potential of the microbiota found in raw milk samples besides the development of a promising assay for detection and quantification of proteolytic activity useful for dairy industry.