Isolation and Identification of Antioxidative Peptides from Frog (Hylarana guentheri) Protein Hydrolysate by Consecutive Chromatography and Electrospray Ionization Mass Spectrometry

Frog (Hylarana guentheri) proteins were hydrolyzed by papain and Flavourzyme to obtain antioxidative peptides. The antioxidant activities of the frog protein hydrolysates (FPHs) were measured, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 9.94 +/- 0.13 mg/mL), reducing power (0.39 +/- 0.01 at 5.0 mg/mL), and oxygen radical absorbance capacity (ORAC) value (789.15 +/- 75.10 mu mol Trolox equivalents/g). The hydrolysates were purified by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of ESI-MS/MS, two dipeptides were identified as Leu/Ile-Lys (259.1607 Da) and Phe-Lys (293.1446 Da), respectively.