Mechanism Of Protein Degradation By Microbial Proteinases During Fermentation Of Total Mixed Ration

In this experiment, the characterization of protein degradation, succession of predominant proteolytic microorganisms and their enzymology were examined to exclude the mechanism of protein degradation in fermented total mixed ration (TMR).In experiment1, moisture levels of TMR were adjusted to375g/kg (low moisture level, LML),432g/kg (medium moisture level, MML), and483g/kg (high moisture level, HML), and the dynamic changes of proteolysis in fermented TMR with different moisture levels were deterimined. The extent of proteolysis can be significantly inhibited by the reducing moisture level in fermented TMR. NPN concentrations were significantly increased when fermentation prolonged to56d and most of the NPN was presented in the form of peptide nitrogen (peptide-N). Free amino acid nitrogen (FAA-N) was degraded in the later stage of fermentation, while ammonia nitrogen (NH3-N) concentrations increased throughout the fermentation period.In experiment2, TMR was mainly formulated with soybean residue and alfalfa hay or L. chinensis hay to make two treatments of this experiment (A-TMR and L-TMR). Characterization of protein degradation was examined during the TMR fermentation. Protein degradation occurred in both treatments and the contents of NPN, FAA-N and NH3-N increased significantly during anaerobic fementation, Peptide-N content increased during the initial phase of fermentation, and then maintained relatively stable in the prolonged fermentation stage.In experiment3, dynamic changes of microbial proteinase were determined during anaerobic fermentation of TMR. In A-TMR and L-TMR, the microbial protease activity increased significantly and then decreased gradually, and the serine proteinase and metalloproteinase played the most important roles in the protein degradation during the process of fermetnation. For A-TMR, the proteinase activity reached its maximum at1d, with the relative activity of metalloproteinase approximately53%, and then, serine proteinase dominated the protease in this fermented TMR. For L-TMR, in the first3d of ensiling, the protease activity significantly increased and the metalloproteinase played the major role in protein degradation. With the progressing of fermentation, the ratio of serine proteinase gradually increasedIn experiment4, dominate proteolytic microorganisms were isolated and determined during TMR fermentation. In the early stage of anaerobic fermentation, the increase of the microbial protease activity can be the result of the multiplicaton of the aerobic bacteria, and in the later fermentation stage, lactic acid bacteria was predominate proteolytic bacteria. In A-TMR, the population of Curtobacterium flaccumfaciens significantly increased along with the increase of protease acitivity. C. flaccumfaciens was the dominate strain in A-TMR at Id, and it showed activity in pH6.0and pH5.0, so, C. flaccumfaciens was the main resource of the microbial protease in the early fermentation process of A-TMR. In L-TMR, Paenibacillus amylolyticus、 Pae. borealis、 Pae. xylanexedens and Pae. turicensis were the dominant proteolytic strains in the early stage of fermentation. With the prolonged fermentation, Enterococcus faecium became the mian resource of proteinase in A-TMR, and in L-TMR, E. faecium and E. faecalis dominated in the later fermentation stage and exhibited relatively high proteolytic activity.In conclusion, protein degradation occurred during the process of fermentation, and the activity of microbial proteinse increased at first and then decreased gradually. The increase of the proteinase activity can be the result of the multiplicaton of the aerobic bacteria, and in the later fermentation stage, lactic acid bacteria was the predominate proteolytic bacteria. In A-TMR, C. flaccumfaciens was the main resource of microbial protease in the early fermentation process, and E. faecium became the mian resource of proteinase in the later stage fermentation. In L-TMR, Pae. amylolyticus、 Pae. borealis、 Pae. xylanexedens and Pae. turicensis were the dominant proteolytic strains during the early fermentation, and E. faecium and E. faecalis played the most important role in the protein degradation in the later stage of fermentation.