10.22376/ijpbs.2019.10.1.p1-12 Volume 4 Issue 3 2013 (July - September) Mastitis, a severe infectious disease among dairy cattle, is still a challenge for livestock treatment due to the increased infections by antibiotic resistant strains. Antimicrobial peptides could be the alternate for treating such drug resistant strains. In the present study, the whey proteins extracted from buffalo milk was subjected to enzymatic hydrolysis using trypsin, chymotrypsin, pepsin, papain and Proteinase K. Antibacterial assay carried out with these hydrolysates against lessThan i greaterThan Escherichia coli, lessThan /i greaterThan lessThan i greaterThan Staphylococcus aureus, Streptococcus agalactiae lessThan /i greaterThan and lessThan i greaterThan Streptococcus dysgalactiae lessThan /i greaterThan isolated from mastitis milk showed that these hydrolysates have an inhibitory effect on their growth. Papain hydrolysates showed highest antibacterial activity against lessThan i greaterThan E.coli, lessThan /i greaterThan lessThan i greaterThan Staphylococcus aureus, Streptococcus agalactiae lessThan /i greaterThan and lessThan i greaterThan Streptococcus dysgalactiae lessThan /i greaterThan . The fraction 4 (12 min fraction) of RP-HPLC purified papain hydrolysate showed highest antibacterial activity and that fraction has got Minimum inhibitory concentration of 100 µg/ml against lessThan i greaterThan E.coli lessThan /i greaterThan and 150 µg/ml against lessThan i greaterThan Staphylococcus aureus, lessThan /i greaterThan 100 µg/ml against lessThan i greaterThan Streptococcus agalactiae lessThan /i greaterThan and 100 µg/ml against lessThan i greaterThan Streptococcus dysgalactiae lessThan /i greaterThan . MEIGNANALAKSHMI. S AND VINOTH KUMAR. S Whey protein, Buffalo milk, antibacterial activity, Papain hydrolysate 1133-1138