Purification and Characterization of a Radical Scavenging Peptide from Rapeseed Protein Hydrolysates

We previously reported that crude rapeseed peptides (CRPs) and peptide fractions (RP25 and RP55) prepared from aqueous enzymatic extraction of rapeseed exhibited marked antioxidant activities, among which RP55 showed the most potent effects. In the present study, RP55 was further purified using consecutive chromatographic methods for identification of antioxidant peptides. The alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging effects of peptides were measured at each purification step to evaluate the antioxidant activities. RP55 was first fractionated by anion-exchange chromatography, and three fractions (E1, E2, and E3) were obtained. All of them showed significantly lower scavenging activities compared to RP55, which was very probably due to the remarkable loss of tannin during the separation. Next, the active fraction E2 with higher protein content was sequentially purified by gel filtration chromatography (GFC) and reversed-phase high performance liquid chromatography (RP-HPLC). The purified peptide, of which the median effective dose (ED50) value for DPPH radical scavenging was 0.063 mg/mL, was identified to be Pro-Ala-Gly-Pro-Phe (487 Da) using electrospray ionization (ESI) mass spectrometry. The unique amino acid composition and sequence in the peptide might play an important role in expression of its antioxidant activity.