(177a) Preparation of Functionalized Tilapia Fish Skin Collagen Peptide By Spsn-Immobilized Protease and Their Anti-Oxidation Component Analysis

Collagen polypeptide has been proved to possess the wide applications in functional foods, cosmetics, medical materials and pharmaceuticals. The production of collagen polypeptide is deeply affected by enzymes and raw material proteins. In this study, a protease extracted from Bacillus subtilis was used to enzymatic hydrolysis tilapia fish skin collagen in a immobilized method. Take advabtage of the database and analysis software, the elaborate molecular structure of the protease has been determined, which benifits its further immobilization by sulfonated polystyrene nanospheres (SPSN). Its active site is a catalytic triad composed of aspartic acid (137), histidine (168) and serine (325), which can catalyze the hydrolysis of internal ɑ-peptide bonds in the polypeptide chain. The protease was immobilized on SPSM by electrostatic adsorption with 397.15 mg/g enzyme loading and 77.3% activity recovery. The tilapia skin collagen was hydrolyzed by immobilized protease. Results showed that the polypeptide conversion percent achieved 54.9 % and the IC50 values of collagen polypeptide for •OH, DPPH and ABTS radical scavenging were 1.29 mg/mL, 12.48 mg/mL and 24.94 mg/mL, respectively. The tandem mass spectrometry was used to obtain more than 1500 polypeptide sequences with molecular weight range of 700 Da to 3000 Da. Combined with the important signal pathways of antioxidant-related NAD+ and Keap1-Nrf2 pathways, three representative anti-oxidation peptides PFRMY (PY-5), MPVPGPM (MM-7) and PGPIGPMGPRG (PG-11) were selectively synthesized and their antioxidant capacity was verified by in vitro antioxidant activity assay. Compared with collagen hydrolysate, PY-5, MM-7 and PG-11 showed better antioxidant activity, resulting from their regulation of the SIRT1-7 and AMPK pathways. Such a result suggested the potential application of the tilapia fish skin collagen peptide on anti-oxidation and immune-mediation fields.