Study Four Staphylococcal Enzyme Production Capacity And Protein Degradation Mechanisms

Seven functional microbial strains has been isolated from Rugao ham, and then the microbial phylogenetic relationship among them was analysed by the establishment of phylogenetic tree. According to the results, Staphylococcus auricularis RG-1, Staphylococcus auricularis RG-3and Staphylococcus auricularis RG-4all had the closest relationship with Staphylococcus auricularis strain DSM20345. Phylogenetic relationship of Staphylococcus saprophyticus RG-2, Staphylococcus saprophyticus RG-6and Staphylococcus saprophyticus strain F-2was the nearest. And Staphylococcus carnosus RG-10had the closest relationship with Staphylococcus carnosus subsp. carnosus strain13. Finally, Staphylococcus epidermidis RG-5had the closest relationship with Staphylococcus epidermidis strain DSM20345but fruther relationship with Staphylococcus lentus.The results of the enzyme activity showed that the protease enzyme activity of Staphylococcus auricularis RG-1was about85U/mg, and it’s the highest among the three strains. And the aminopeptidase activity of Staphylococcus saprophyticus RG-2was the highest when using LNA as substrate, the activity could reach18U/mg. So it was a high-yielding strain of aminopeptidase. By comparison, the aminopeptidase activity of the other three strains was less than5U/mg. The esterase-producing capacity of Staphylococcus saprophyticus reached more than200U/mg while the capacity of the other three Staphylococcus strains was lower relatively. The glutaminase enzyme activity of Staphylococcus saprophyticus reached14U/mg while the activity of the remaining two strains was about10U/mg, and the difference between Staphylococcus auricularis RG-3and Staphylococcus carnosus RG-10was not significant. The capacity of Staphylococcus carnosus RG-10producing glutamine transaminase was6.4U/mg while the activity of Staphylococcus saprophyticus RG-2was twenty percent lower. The catalase enzyme activity of Staphylococcus carnosus RG-10was about80U/mg, while the activity of Staphylococcus saprophyticus RG-2was followed by68U/mg.The activity of Staphylococcus saprophyticus RG-2was about320U/mL that was cultured18hours. Secondly, the activity of staphylococcus auricularis RG-1was about300U/mL. The nitrate-reductase producing capacity of Staphylococcus strains decreased rapidly when the concentration of sodium chloride exceeded5.5%. With the increasing concentration of sodium nitrite, the nitrate-reductase activity of Staphylococcus strains decreased at first and then tended to be stable, of which the nitrate-reductase producing capacity of Staphylococcus carnosus RG-10was the highest.Utilizing Staphylococcus saprophyticus RG-2which was resistanted to nitrite for optimization of fermentation conditions of aminopeptidase, the results showed that the best fermentation conditions was pH6.8,41℃and1%concentrations of sodium chloride. The aminopeptidase enzyme activity could be reached23.85U/mg under the condition.The effects of Staphylococcus strains on sarcoplasmic protein were investigated. The results showed that the ability of degradation of sarcoplasmic proteins of Staphylococcus auricularis RG-1was higher than the other three strains. In terms of the degradation of myofibrillar protein, the degradation ability of Staphylococcus saprophyticus RG-2was the highest. And free amino acids (FAA) content in myofibrillar protein degradation products by Staphylococcus saprophyticus RG-2was generally higher than the other three strains. The totalvolatile basic nitrogen(TVB-N) content in myofibrillar protein degradation products was analysised. The TVB-N value in sarcoplasmic proteins and myofibrillar protein degradated by Staphylococcus auricularis RG-1was the highest while the TVB-N value in sarcoplasmic proteins by Staphylococcus saprophyticus RG-2was the lowest (4μg/mL), and the value in myofibrillar protein by Staphylococcus epidermidis RG-5was the lowest (9μg/mL)after cultured for8days,By LC-MS method, twelve kinds of polypeptide components in sarcoplasmic protein without treatment (CK) were detected. The retention time of maximum peptide peak area was3.24min and the peak area took up54.6%. The peak area of polypeptide took up19.73%when retention time was7.25min. After degradated by Staphylococcus strains, peptides content increased significantly and the maximum changes occured when the retention time was7.25min. Under the same conditions, the maximum changes of polypeptide of myofibrillar protein degradation by four strains occured when retention time of7.93min.In the myofibrillar protein solution, the total FAA in CK and the production degradated by Staphylococcus saprophyticus RG-2, staphylococcus carnosus RG-10, Staphylococcus auricularis RG-1and Staphylococcus epidermidis RG-5was8mg/100g,13mg/100g,15mg/100g,11mg/100g and12mg/100g respectively.