Preparation And Antioxidant Activity Of Fish Skin Collagen Peptides By Nanospheres-Immobilized Enzyme

Fish skin collagen peptide is a small molecular substance with certain physiological activity prepared by hydrolysis of fish skin collagen.In recent years,it has become popular as an emerging functional food material at home and abroad.Enzymatic hydrolysis is an important method for preparing fish skin collagen peptides.However,in practical industrial production,the acquirement of high enzymatic condition and difficulty of reutilization limit its application in a low-cost way.At the same time,the fish skin collagen peptide is a high-quality antioxidant raw material,but the preparation process is complicated and time consuming,which limits its application in the food industry.In this paper,subtilisin?SU?was immobilized by sulfonated polystyrene nanospheres?SPSNs?,and the influencing factors of immobilized enzyme process were studied.The prepared immobilized enzyme was used to hydrolyze tilapia skin collagen.The tilapia skin collagen is hydrolyzed by the immobilized enzyme to prepared a collagen peptide with good oxidation resistance in vitro.Based on the analysis of the peptide sequence of the product,high-throughput screening was realized by molecular docking and the representative functional peptides were verified by vitro antioxidant activity.The main research contents and results of this thesis are as follows:?1?Study on catalytic activity and molecular structure of subtilisin.The isothermal titration calorimetry?ITC?experiment showed that the catalytic hydrolysis activity of subtilisin on fish skin collagen was better than that of bovine hemoglobin and bovine serum albumin.Subtilisin was demonstrated an alkaline protein with an isoelectric point of 8.66 by analyzing the biological information.It presents positively charged in the solution with pH<7 and is easily electrostatically adsorbed with a negatively charged substance.In addition,its active site is a catalytic triad composed of aspartic acid?137?,histidine?168?and serine?325?,which can catalyze the hydrolysis of internal?-peptide bonds in the polypeptide chain.?2?Study on immobilized subtilisin?SPSNs-Subtilisin,SPSU?by sulfonated polystyrene nanospheres.Polystyrene nanospheres?PSNs?were used as raw materials to modify by concentrated sulfuric acid.It achieved the SPSNs with sulfonation of 2.625±0.05 mmol/g by sulfonation at 33?for 6 h when the ratio of material to liquid was 1:40?m/v?.Subtilisin was immobilized on SPSNs by electrostatic adsorption method.The optimized condition enzymatic step was as following:25?,pH 6.5?Disodium hydrogen phosphate citric acid buffer?for 60min.The immobilized enzyme loading was 397.15 mg/g,and the activity recovery was 77.3%.Under these conditions,the immobilized enzyme loading achieves 397.15 mg/g and the activity recovery accesses 77.3%.In the range of pH 7.0¹0.3 and temperature 25⁶5?,the immobilized enzyme has higher relative enzymatic activity than the free enzyme,and the optimum pH of the immobilized enzyme is increased from 10.3 to 10.6.The immobilized enzyme still had 68.7%relative activity after repeated use for 5 times.?3?Preparation of fish skin collagen peptides and evaluation of their antioxidant activity in vitro.The tilapia fish skin collagen was hydrolyzed by SPSU and the polypeptide conversion rate reached 54.9%.In vitro antioxidant assay showed that the fish skin collagen hydrolysate had good antioxidant activity.The IC₅₀ values of hydrolysate for hydroxyl radical?·OH?,2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid?ABTS?radical and 1,1-diphenyl-2-picrylhydrazyl?DPPH?radical scavenging acticitivs were 1.29 mg/mL,24.94 mg/mL and 12.48mg/mL,respectively.?4?Sequence analysis of fish skin collagen polypeptide and screening of antioxidant active peptides by molecular docking technology.The liquid chromatography-mass spectrometry was used to obtain more than 1500 polypeptide sequences with molecular weight range of 700 Da to 3000 Da,which contains 105 type I collagen peptide sequences.Twelve antioxidant peptides were rapidly screened by molecular docking technique,and three representative high antioxidant activities of PFRMY?PY-5?,MPVPGPM?MM-7?and PGPIGPMGPRG?PG-11?were synthesized by solid phase peptide synthesis,and their activity were verified by in vitro antioxidant activity.The results showed that PY-5,MM-7 and PG-11showed good antioxidant activity compared with the fish skin collagen hydrolysate.The IC₅₀values for·OH scavenging were 0.73 mg/mL,0.81 mg/mL,and 1.42 mg/mL,respectively.The IC₅₀0 values for DPPH free radical scavenging were 10.17 mg/mL,3.85 mg/mL,and 4.14 mg/mL,respectively.The interaction analysis showed that the antioxidant effects of PY-5,MM-7 and PG-11 may be mainly through the regulation of SIRT1-7 and AMPK pathway,especially the effect of SIRT1-7 pathway is obvious.The above results confirmed that PY-5,MM-7 and PG-11 are important components of the antioxidant active ingredient in the fish skin collagen hydrolysate.