Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates

•Casein hydrolysate <1 kDa by alcalase had higher antioxidant activity than trypsin.•Casein hydrolysate with high ABTS ability consisted mainly of Arg, Leu, Ala, Ser.•Buffalo casein hydrolysate contained antioxidant peptides RELEE, MEDNKQ, TVA, EQL.•Bovine casein hydrolysate contained antioxidant peptides PYPQ, EMPFPK, YFYPE, PQSV.•Mechanism and reaction pathways of RELEE, TVA and EQL with ABTS were proposed.

Buffalo and bovine caseins were hydrolysed by alcalase and trypsin to produce novel antioxidant peptides. The casein hydrolysates were purified using ultrafiltration (UF) and further characterized by RP-HPLC. The fractions produced higher antioxidant activities were identified for their peptides using LC MS/MS. All UF-VI (MW < 1 kDa) fractions showed higher antioxidant activity. Hydrolysate produced by alcalase for buffalo casein (UF-VI with 54.84-fold purification) showed higher antioxidant activity than that obtained by trypsin. Trypsin hydrolysate contained high amount of hydrophobic amino acids while alcalase hydrolysate consisted mainly of Ser, Arg, Ala and Leu. The antioxidant peptides identified by LC MS/MS were RELEE, MEDNKQ and TVA, EQL in buffalo casein hydrolysates produced by trypsin and alcalase, respectively. Mechanism and reaction pathways of selected antioxidant peptides with ABTS were proposed. Conclusively, buffalo casein provided antioxidant peptides similar to bovine, suggesting that buffalo casein is a novel source of antioxidant.