Preparation And Functional Properties Of Fish Scale Collagen Peptides

China is a big fishery country.In the process of fish processing,a large number of by-products such as fish scale,fish skin and fish bone are produced.The ash content of fish scale is abundant,which mainly contains hydroxyapatite,and the protein content accounts for50～70%of the total scale weight.Therefore,fish scale can be used as a high-quality raw material for extracting collagen.It can not only solve the soil,air and water pollution caused by landfill,but also realize the goal of high value utilization of fish by-products,and promote the green,low-carbon and sustainable development of fishery.Collagen polypeptide was extracted from silver carp scales by alkaline protease.Response surface methodology（RSM）was used to optimize the extraction process based on the yield of polypeptide,and dialysis membrane separation was used to separate and purify the polypeptide.The antioxidant activity,whitening properties and other physicochemical properties of fish scale peptide enzymatic hydrolysate and its nanofiltrate were systematically studied by in vitro antioxidant experiment and cell experiment.The main research results are as follows:（1）Adding 4%citric acid and using ultrasonic to optimize the decalcification of fish scale:the decalcification rate of fish scale is 96.10±2.97%after ultrasonic was acted for120min.Five kinds of proteases（alkaline protease I,alkaline protease II,alkaline protease III,neutral protease and flavor protease）were used to hydrolyze fish scales with the yield of polypeptide as the index.The results showed that alkaline protease I had the highest extraction rate of polypeptide from fish scales.（2）Taking the polypeptide yield as the index,the preparation conditions of fish scale polypeptide were optimized by response surface methodology.The theoretical optimum conditions were as follows:the ratio of material to liquid was 1:50.61 g/m L,the enzyme dosage was 48.18μL,and the p H value was 8.14.Under these conditions,the polypeptide yield was 88.57%.Combined with the actual conditions:the ratio of material to liquid was1:50.6 g/m L,the dosage of enzyme was 48.2μL,and the p H value was 8.14.The experiment was repeated three times,and the yield of polypeptide was 88.77±0.32%.（3）Nanofiltration membranes were used to separate and purify the enzymatic hydrolysate of fish scales,and three types of nanofiltrates were obtained:nanofiltrate（>5000 Da）,nanofiltrate（3000～5000 Da）,and nanofiltrate（<3000 Da）.Ninhydrin post column derivatization ion exchange chromatography was used to determine amino acids in fish scale hydrolysate and its nanofiltrate.The results showed that the contents of alanine,glycine,cysteine and aspartic acid in the nanofiltrates were significantly higher than those in the enzymatic hydrolysate after nanofiltration separation.（4）MALDI-TOF-MS was used to determine the molecular weight of peptides from fish scale hydrolysate and nanofiltrates.The molecular weight distribution of enzymatic hydrolysate ranged from 399.83 to 1404.62 Da;nanofiltrate（>5000 Da）the molecular weight ranged from 399.83 to 957.47 Da;nanofiltrate（3000～5000 Da）the molecular weight ranged from 399.99 to 699.39 Da;nanofiltrate（<3000 Da）the molecular weight ranged from 399.99to 695.32 Da.（5）UPLC-MS/MS was used to determine the amino acid sequence of the fish scale enzymatic hydrolysate and its nanofiltrate.A total of 13 peptides were obtained,and their amino acid sequences were inferred to have strong antioxidant activity.GMRGPRGA、GARGDKGETGEA、RGDVGPA、GRVGPA、GERGEQGPA may have strong tyrosinase inhibitory effect,which can inhibite melanin production and have a strong whitening effect.（6）The physicochemical properties,antioxidant activity and whitening effect of fish scale enzymatic hydrolysate and its nanofiltrate were determined.The results showed that the solubility of the nanofiltrates was higher than 90%;the emulsifying and emulsifying stability of the hydrolysate were 66.89±0.23%and 91.74±1.36%respectively.When the peptide concentration was 10 mg/m L:nanofiltrate（>5000 Da）had a 1,1-diphenyl-2-trinitrophyde hydrazine（DPPH·）clearance rate of 66.75±2.58%,nanofiltrate（3000～5000 Da）had a hydroxy free radical（·OH）clearance rate of 95.15±3.57%,nanofiltrate（<3000 Da）reducing power absorbance up to 0.99±0.01,nanofiltrate（<3000 Da）scavenging rate of superoxide anion radical（O₂^-·）was 82.49±1.28%.Four concentrations（0.01,0.1,0.5 and 1 mg/m L）of the hydrolysate and its nanofiltrate were selected to test the cell proliferation activity of mouse B16 cells,andα-arbutin was used as positive control,The results showed thatα-arbutin significantly inhibited cell proliferation at all concentrations,and the fish scale collagen polypeptide was safer thanα-arbutin at all concentrations.At low concentration of0.01 mg/m L,nanofiltrate（<3000 Da）had a low TYR activity of 65.37±2.81%,and had a strong whitening effect.