Antibacterial Activity Of Casein Hydrolysates By Trypsin

In this study, we detect the bioactive effect of antibacterial peptides in milk. We established the method to product the antibacterial peptides hydrolisated from casein and separated the hydrolysates by SDS-PAGE and Sephadex G-15. Study the quality and antibacterial activity of antibacterial peptides and detect the effect of it on rats experimental mastitis.1 Preparation of casein hydrolysates by trypsinCasein from milk was separated with isoelectric precipitation.Casein was hydrolyzed by bovine trypsin at 37℃ ,with the ratio of trypsin and casein 1:30(W/W), the initial pH is8.0 and hydrolysis 5min,15min,30min,45min,lh,2h,3h,4h,5h. If hydrolysising time over 15min, regulated pH to 8.0 when enzymolysis had proceeded 15 min. Hydrolysates were analyzed by SDS-PAGE .The results indicated that hydrolysates for 2 hours was completly digested.The degree of hydrolysis(DH)was measured with the method of triketohydrindene hydrate.The results indicated that the nitrogen concentration ,DH,the average length of peptides of hydrolysates for 2 hours was 4.608 ± 0.005mg/mL ( n=3 ) ,10.5%,9.52 respectively.2 Antibacterial activity of casein enzymatic hydrolysates2.1 Primary study on antibacterial activity of hydrolysatesTo determine antibacterial activity of hydrolysates against Staphylococcus aureus(S.aureus) (ATCC25923) and Escherichia coli(E.coli) (ATCC25922) utilizing a microdilution test. The results demonstrated that hydrolysates for 2 and 3 hours had antibacterial activity,but the others did not show the same effect. Casein can produce antibacterial peptides through hydrolyzing.2.2 To determine MIC of hydrolysates for 2 hours against different bacteriaMIC of hydrolysates for 2 hours against S.aureus ( ATCC25923 ) ,S.aureus ( CMCC46801 ) ,E.coli ( ATCC25922 ) ,E.coli ( 0138 ) ,E.coli ( CMCC46103 ) ,Streptococcus,Bacilli subtilis,Pasteuralla,Salmonella was 0.045mg/mL,0.045mg/mL,0.09mg/mL,0.09mg/mL,0.09mg/mL,0.36 mg/mL,0.18mg/mL, 0.36mg/mL,0.36mg/mL respectively. The results depicted that hydrolysates for 2 hours had antibacterial peptides. They had antibacterial activity against Gram postive and negative bacteria.2.3 Separation of antibacterial peptide fraction and determination of its antibacterial activityHydrolysates for 2 hours were separated by gel filtration chromatography. Six fractions were achieved through Sephadex G15 in column. MIC of fraction 5# against S.aureus (ATCC25923 ) ,S.aureus (CMCC46801) ,E-coli (ATCC25922) ,E.coli ( O₁38 ) ,E coli ( CMCC46103 ) ,Streptococcus,Bacilli subtilis,Pasteuralla,Salmonella was 0.017mg/mL,0.017mg/mL,0.034mg/mL,0.034mg/mL,0.034mg/mL,0.034mg/mL,0.009-0.017mg/mL,0.034mg/mL,0.017-0.034mg/mL. The other fractions did not have antibacterial activity.2.4 Analysis of part characterization of antibacterial peptidesHydrolysates for 2 hours repeated freezing-melting. Boiling water bath with 5min,10min,15min,20min,30min,40min. MIC of them against S.aureus (ATCC25923) and E.coli (ATCC25922) were 0.045-0.09mg/mL and 0.09mg/mL. Their antibacterial activity did not remarkably change. The results showed that antibacterial peptides were freezing-melting- resistant and heat-resistant.3 Effect of antibacterial peptides from casein on rats experimental mastitisThirty lactating SD rats were randomly divided into Con (n=6), positive control group (Ps.aureus P_E.coli, n=12) and treatment group (T_S.aureus,T_E.coli, n=12) . The 100 μ L of physiological saline (Con,Ps.aureus,PE.coli) or hydrolysates for 2 hr (Ts.aureus,T_E.coli) were injected intramuscularly into the left biceps femoris of the rat after parturition respectively.The 100 μ L of physiological saline (Con), bacterial suspension of S.aureus (Ps.aureus Ts.aureus) or E.coli (P_E.coli T_E.coli) were inoculated into the fourth (abdominal) mammary gland via the teat duct 72 hours after parturition respectively. After 24 hours of inoculation ,all rats euthanatized. Some enzymes activity in the mammary glands and serum were determined respectively.In comparison with those of normal control group,N-acetyl- β -D-glucosaminidase(NAGase) a...