Research On Mechanism And Modification Of Protein Degradation In Alfalfa Silage

Alfalfa is an important protein feed resource in livestock production,ensiling is a popular method of preserving nutrients of moist forage based on lactic acid fermentation under anaerobic conditions,especially in moist areas.However,part of true protein is degraded to non-protein nitrogen.Excessive non-protein nitrogen in silage can reduce the efficiency of nitrogen utilization in the rumen,as a result,leading to increasing dietary protein supplementation and potential environmental pollution.Effect of alfalfa material(variety and harvesting stage),additive(inoculants and chemical additives),addition of red clover compound(mixing with red clover and enzyme extract from red clover)and mixing with sorghum-sudangrass were studied on fermentation quality,nitrogen distribution,protein fraction,proteases activities and bacterial community in ensiled alfalfa.The main results are as follows:(1)Compared with Golden Empress,the fermentation quality in Zhongmu No.1 silage was betterwhile the protein degradation was stronger in Zhongmu No.l silage.No differences in protein fractions were found between two varieties in laboratory-scale silos.With the delay of harvesting stage,fermentation quality decreased,bound protein increased,the rate of protein degradation decreased while extent of proteolysis increased.(2)The degradation of protein to non-protein nitrogen took place mainly during the first 3 d,while degradation of peptides and free amino acids occurred throughout the ensiling process.All additives including Lactobacillus plantarum + sucrose(LP+S),Lactobacillus plantarum + cellulase(LP+C),tannin acid 2%DM(TA2%)and tannin acid 5%DM(TA5%)lowered non-protein nitrogen and free amino acids nitrogen proportion during the ensiling.Additive TA 5%was the most effective to inhibit proteolysis among the four additives,followed by LP+S.They inhibited the activities of all three plant proteases and decreased production of non-protein nitrogen,free amino acids and ammonia nitrogen during the ensiling process.The relative abundances of Enterobacter,Enterococcus and Clostridium sensu were decreased and relative abundance of Pantoea was increased by LP+S and TA5%.In addition,TA5%decreased the relative abundance of Weissella and increased the relative abundance of Pseudomonas,LP+S improved the relative abundance of Lactobacillus.Lactobacillus and Weissella were the primairy bacteria produced lactic acid in this study.The production of acetic acid was due to Enterobacter.Enterococcus and Clostridium sensu stricto 12.Enterobacte dominated the production of non-preotein nitrogen.Pantoea was related to hydrolysis of true protein,and Enterobacter,Enterococcus and Clostridium sensu stricto 12 played roles in deamination of amino acids.The true protein of Enterocccus degraded alfalfa is mainly PB2,i.e.true protein degraded intermediumly.Enterobacter may degrade protein by secreting carboxypeptidases.(3)In silage treated with LP+S,with increasing proportion of red clover in silage,total nitrogen content and proportions of non-protein nitrogen,peptide nitrogen,free amino acid nitrogen and ammonia nitrogen decreased linearly,and PC(bound true protein defined by the Cornell Net Carbohydrate and Protein System)proportion increased linearly after ensiling.During ensiling,proteases including carboxypeptidase,aminopeptidase and acid proteinase activities decreased as red clover proportion increased.Moreover,50:50 was the optimal mixing ratio of alfalfa with red clover-that showed good fermentation quality with lower pH and higher lactic acid concentration,reduced proteases activities and proteolysis compared to pure alfalfa silage,and also had more total nitrogen content than pure red clover silage.In silage without additive,fermentation quality was worse in pure red clover silage than in pure alfalfa.Silage pH value increased and lactic acid concentration decreased with increasing red clover proportion in the mixture.Application of LP+S improved fermentation quality,increased peptide nitrogen proportion and decreased free amino acid nitrogen proportion,in addition,PC proportion was reduced.In correlation analyses,Enterococcus,Enterobacter and Sphingobacterium showed positive correlation with lactatic acid concentration.There was a strong positive correlation between Clostridium sensu and butyric acid concentration.Enterobacter,Enterococcus,Pseudomonas,and Sphingobacterium dominated the production of non-protein nitrogen.Pediococcus and Devosia were related to the degradation of amino acids.However,only therelative abundance of Clostridium sensu stricto 12 was positively related to the content of ammonia nitrogen.There were strong positive correlations between Lactobacillus and PB1 and PB2 proportions.The relative abundances of Lactobacillus,Devosia,and Sphingomonas showed positive correlattion with PB1 content.The relative abundances of Pantoea,Pseudomonas and Sphingobacterium were negatively correlated with PB3 content.The relative abundances of Pseudomonas and Sphingobacterium were negatively correlated with PB3 content.The correlation between bacteria abundance and protease activity was weak.Previously-fermented juice(PFJ)and enzyme extract from red clover stood in the air for 0,24 and 48h(E-Oh,E-24h and E-48h,respectively)showed negative effects on fermentation quality in ensiled alfalfa,resulting in higher silage pH value and butyric acid concentration.Compared with the control,all treatments had lower concentration of non-protein nitrogen and free amino acid nitrogen,higher PC concentration.No differences on proteolysis indicators were detected between E-Oh,E-24h and E-48h.(4)Compared with alfalfa ensilied alone,non-protein nitrogen concentration was lower while concentration of free amino acid and ammonia nitrogen was higher in the mixture of alfalfa and sorghum-sudangrass.