Study On The Analysis And Functional Property Of The Enzymatic Polypeptides From Small Miscellaneous Fish

The small miscellaneous fish as a low economic value marine fish has become one kind of main marine capture fisheries of Zhejiang province. The small miscellaneous fish is rich in protein.At present,the proteins haven't been used widely and industrially.Preparation ofpolypeptides by hydrolyzing small miscellaneous fish protein can fully use the protein resource and enhance its product value.In this dissertation,the physical and chemical properties,amino acid composition and nutritional value of the polypeptides prepared from small miscellaneous fish protein were determined.The molecular weight distribution and iso-electric point of the polypeptides were determined by electrophoresis.Taking the ACE inhibitory activity as an index,the polypeptides were purified through ultra-filtration,gel chromatography and ion exchange chromatography.In addition,the antioxidant activity of the polypeptides was determined.The results from the physical and chemical property analysis showed that polypeptides were rich in crude protein and the content of fat was low(0.32%).Polypeptides had a good solubility and low viscosity property.The amino acid composition analysis of the polypeptides showed that the total content of essential amino acid was 47.14%.Among essential amino acids,Contents of Lys,Iie,Leu,Thr,Val,Phe+Tyr and Met+Cys were much higher than that of the FAO/WHO recommended pattern.The desalting of the polypeptides was performed using Sephadex G-10.The molecular weight distribution and iso-electric point were determined by SDS-PAGE,Tricine-SDS-PAGE and Isoelectrofocusing. The results showed that compared with SDS-PAGE,Tricine-SDS-PAGE obtained better separation effect of the polypeptides.The polypeptides had the peptides which molecular weight was less than 14.4kDa by Tricine-SDS-PAGE with 16%polyacrylamide gel and 5%cross-link.The main composition of the polypeptides was acid peptides.Purification of the polypeptides through ultrafiltration,gel filtration and ion-exchange was carried out and the ACE inhibitory activity was measured at each purification step.The concentration of half inhibitory rate(IC₅₀) value of polypeptides was 0.203mg/ml.The polypeptides was passed through three different ultra-filtration(UF) membrane system having molecular weight cut-offs of 1000,5000 and 10000 Da in turn. Four fractions were obtained,among which,Fraction I had the highest ACE inhibitory activity and the IC₅₀ value was 0.176mg/ml.Fraction I was then loaded onto a Sephadex G-25 column and four fractions were obtained which were a,b,c and d.A had no ACE inhibitory activity and d had the highest ACE inhibitory activity that the IC₅₀ value was 0.088mg/ml.Fraction d was loaded on a DEAE ion exchange column and Fraction Id(1) and Id(2) were obtained.The IC₅₀ value of the two fractions was 0.084mg/ml and 0.080mg/ml.The study on the in vitro antioxidant activity of the enzymatic peptides from small miscellaneous fish protein indicated that polypeptides could significantly inhibit DPPH,and showed good dose-effect relationship.The IC₅₀ of polypeptides on MDA was 9.8mg/ml. The polypeptides had inhibitory effects on lipid peroxidation of the linoleic acid while negative inhibitory effects on·OH and O₂^._</sup>.