Peptide Fractionation and Free Radical Scavenging Activity of Zein Hydrolysate

Alcalase-treated zein hydrolysate (ZH) was separated by gel filtration, ultrafiltration, and reversed-phase HPLC, and the scavenging activities for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(center dot+)), 1,1-diphenyl-2-picrylhydrazyl (DPPH center dot), and superoxide anion (O-2(center dot-)) radicals of different peptide fractions were measured to establish the antioxidant efficacy. Results showed that the ability to stabilize water-soluble free radicals (ABTS(center dot+)) by ZH components was insensitive to the peptide size, whereas that against ethanol-soluble free radicals (DPPH center dot) and O-2(center dot-) was molecular weight dependent. Antioxidative peptides of <1 kDa were further separated by HPLC into 30 components, of which those with great hydrophobicity exhibited strong DPPH center dot and O-2(center dot-) scavenging ability and those with intermediate hydrophobicity displayed the maximum ABTS(center dot+) scavenging activity. Two dominant components (fractions 8 and 17) were further purified and identified by LC-PDA-ESI-MS to be Tyr-Ala and Leu-Met-Cys-His, respectively. The results demonstrated that the free radical scavenging activity of ZH depended on the radical species and was strongly related to the molecular weight and hydrophobicity of the constituting peptides.