Isolation and identification of antioxidative peptides from crocodile meat hydrolysates using silica gel chromatography

Crocodiles are cultured in large numbers in Asia and other places in order to protect wild resources and meet the needs of human life. In this study, crocodile ( Crocodylus siamensis ) meat proteins were extracted and hydrolyzed into peptides, their antioxidant peptides were isolated and purified by silica gel chromatography and identified by LC/MS. Crocodile meat proteins were optimally extracted with water and hydrolyzed by papain based on the degree of hydrolysis and antioxidant activity. The hydrolysates were fractionated by ultrafiltration into 3 kDa, 3–30 kDa, and ≥ 30 kDa fractions. The 3 kDa fraction showed most antioxidant activity of the hydrolysates. Its active peptides were separated by silica gel column chromatography and purified by silica gel TLC, based on TLC bio-autographic assays of the activity. Four highly active peptides were identified by LC/MS as SSLTIQFVEGQFVDSYDPTIENTFTK, VPPHIY, VAPEEHPVLLTEAPLNPK, and RNGLPGPIGPAG. The identified peptides were synthesized and showed 50% free radical scavenging activities at 1.0 mg/mL, equal or higher to ascorbic acid at 0.5 mg/mL, in both DPPH and ABTS assays. The results indicated that the 3 kDa hydrolyzed peptides of crocodile meat had high antioxidant activity and the active peptides can be effectively separated and purified by silica gel column chromatography and TLC.