Abstract
Enzymatic hydrolysis of protein is a principal method to obtain antioxidant peptides. A yak casein hydrolysate (YCH) was prepared by alcalase and trypsin digestion. An ultrafiltration membrane system was used to divide the hydrolysate into four molecular weight fractions; YCH-4 (<3 kDa) had the highest antioxidant activity. Fraction YCH-4 was separated into six subfractions by gel filtration chromatography; reverse-phase high performance liquid chromatography (RP-HPLC) was then used to partition sixteen antioxidant peptide subfractions. Liquid chromatography/electrospray tandem mass spectrometry (LC-ESI-MS/MS) was used to determine the amino acid sequence of a purified antioxidant peptide to be Arg-Glu-Leu-Glu-Glu-Leu (787.41 Da). Finally, a synthetic Arg-Glu-Leu-Glu-Glu-Leu peptide was evaluated for its superoxide anion and hydroxyl radical scavenging activity (IC50 = 0.52 and 0.69 mg mL-1), which confirmed the activity of the native purified peptide. Our results suggested that isolation and purification of antioxidant peptides from yak casein could be an important means to obtain natural antioxidant peptides.