Preparation and evaluation of antioxidant peptide from papain hydrolysate of Sphyrna lewini muscle protein

Inspired by DPPH radical scavenging assay, an efficient method had been developed to acquire the hydrolysate from Sphyrna lewini muscle protein (SPH) by an orthogonal L9(3)4 test. Under optimal proteolysis parameters (proteolysis time 2 h, total enzyme dose 1.2%, proteolysis temperature 60 °C and pH 6), DPPH radical scavenging rate of SPH reached to 91.27 ± 2.69% at the concentration of 25 mg/mL. Proteolysis time showed significant effects on hydrolysate preparation (p < 0.05). One peptide (SAP) was isolated from SPH by using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). The primary structure of SAP was identified as Leu-Asp-Lys with molecular weight of 374.44 Da. SAP exhibited good scavenging activity on hydroxyl radical (EC50 0.17 mg/mL), ABTS radical (EC50 0.19 mg/mL), and superoxide anion radical (EC50 0.12 mg/mL), but moderate DPPH radical (EC50 3.06 mg/mL). SAP was also effective against lipid peroxidation in the model system and peroxyl free radical scavenging in β-carotene linoleic acid assay. These results suggested that SAP could be used as natural antioxidant in enhancing antioxidant properties of functional foods and in preventing oxidation reactions in food processing.

► An efficient method was developed to acquire the hydrolysate from Sphyrna lewini protein (SPH). ► A peptide (SAP) with the highest antioxidant activity was isolated from SPH. ► The sequence of SAP was identified as Leu-Asp-Lys. ► The antioxidant activity of SAP was evaluated by six methods.