Hydrolysis Character And Immunological Activity Of Bovine Milk By Protease From Pseudomonas Fluorescens

Hydrolytic character and immune biologically active peptide from Pseudomonas Fluorescens Protease hydrolyzed bovine milk were investigated.After enzymatic hydrolytic character on different protein in bovine milk and optimum parameters was chosed,immunological function on hydrolyze defatted milk on mice was studied.The whole study consisted of three experimental parts.1 Study on zymologic character of PFP:Purity,optimum temperature and optimum pH on PFP was investigated.After separation and purification of protease,the simple substance electrophoresis was validated by SDS-PAGE;Purity was detected by methods of Komars-G250;Enzymatic optimum temperature and pH were investigated by methods of azocasein.It drawed the conclusion that:PFP is a single electrophoresis protease;The purity is 62.37μg/mL;Protease activity is 17333 U;Optimum temperature is 40℃and optimum pH is 7.5.2 Study on hydrolytic character of PFP on bovine milk:Hydrolytic character and optimum parameters of PFP hydrolyzed the protein of bovine milk were investigated. The hydrolysate of PFP hydrolyzed the six main protein of bovine milk was detected by SDS-PAGE and RP-HPLC,hydrolytic parameters was optimized by orthogonal experiment.The result showed that:PFP can hydrolyze three kind of casein:α_s-CN,β-CN,κ-CN,but not action on BSA,α-la,β-lg.In this experiment,according to detected absorption area at 210 nm,the optimum condition of formative immune active peptide of reaction time 3 hour;pH value 6.5;reactive temperature 40℃and enzyme levels and substrate concentration is 1:200(v/v).3 Study on immune function of PFP hydrolyzed on bovine defatted milk:The effect of defatted milk PFP hydrolysate on immune function of mice has been studied.The control group was fed physiological brine,GroupⅠwas fed 100 mg/Kg.bw and groupⅡ250 mg/Kg.bw PFP hydrolysate 30 days to the experiment mice.The results showed that the relative weight of spleen in groupⅠwas higher than control group at significant level (P＜0.05),but no difference in relative weight of thymus.Both relative weight of spleen and thymus were no different;Mononuclear macrophage phagocytosis proliferation activity on groupⅡwas higher than control group,at significant level(P＜0.05).Delayed allergy proliferation on groupⅡ24 h and on groupⅠ48 h were higher than control group (P＜0.05),and delayed allergy proliferation on groupⅡ48 h and on groupⅠ24 h and antibody-producting cell proliferation in GroupⅠand groupⅡwere all higher than control group,at significant level(P＜0.01).PFP hydrolyzed defatted milk can enhance the immune function of mice.We can draw the conclusion from three parts of study that PFP is a heat-resistant enzyme.PFP can hydrolyze three kind of casein,but not action on BSA,α-la,β-lg,also the kinds of casein can form immune active peptides,the optimum condition of formative immune active peptide of reaction time 3 hour;PH value 6.5;reactive temperature 40℃and enzyme levels and substrate concentration is 1:200.PFP hydrolyzed defatted milk can enhance the immune organ,stimulate mononuclear macrophage phagocytosis proliferation activity,and accelerate delayed allergy proliferation and antibody-producting cell proliferation,PFP hydrolysate can enhance immune of mice.A method was offered to utilized PFP,and a new way was provided to produce biologically active peptide,also enriched immune active peptides of bovine milk in this study.