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*+), 1,1-diphenyl-2-picrylhydrazyl (DPPH*), and superoxide anion (O2*-) radicals of different peptide fractions were measured to establish the antioxidant efficacy. Results showed that the ability to stabilize water-soluble free radicals (ABTS*+) by ZH components was insensitive to the peptide size, whereas that against ethanol-soluble free radicals (DPPH*) and O2*- 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* and O2*- scavenging ability and those with intermediate hydrophobicity displayed the maximum ABTS*+ 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.