Study On The Extraction And Bioactivity Of Muscles Polypeptides From Takifugu Flavidus

Polypeptides are obtained from the decomposition of proteins and have abundant biological functions.China is rich in marine living resources,so it has a good application prospect to study marine biological resources with polypeptides as a starting point.The muscles of puffer fish is rich in proteins.In this paper,Takifugu flavidus muscles was used as raw materials,and the hydrolysis level of enzymolysis solution was used as the index.The experiments of proteases screening,single factors experiment and response surface experiment were conducted to determine the best enzymolysis process:Flavor protease was used at 55?,pH 7,enzyme amount 4521.96 U/g and 1:10(g/mL)of materials to liquid ratio,the muscles was hydrolyzed for 4.5 h.The hydrolysate was treated by ceramic membrane and ultrafiltration membrane step by step,and the 0-1 KD component was collected for vacuum freeze-drying,and the sample was TFMP-F.It showed that TFMP-F had antioxidant activity,the IC50 of DPPH radical was 1200 ?g/mL,the IC50 of ·OH radical was 260?g/mL,and it also had reducing power to Fe3+.TFMP-F cound significantly improve the time of loaded-swimming and the contents of glycogen in liver of fatigue mice.and reduced the contents of urea nitrogen and lactic acid in serum of fatigue mice.The results showed that TFMP-F could improve the athletic performance of mice and alleviate sports fatigue.The acute toxicity test in mice confirmed that TFMP-F was nontoxic.We used the muscles of Takifugu flavidus as the raw materials for enzymolysis experiment,and the inhibition rate of mushroom tyrosinase(mTYR)activity in vitro as the index.Through the screening of different proteases,single factors experiment and response surface analysis,we determined the best enzymolysis technology:Trypsin was used at 36.9?,pH 7.0,enzyme concentration 3011 U/g and 1:10(g/mL)of materials to liquid ratio for 8 hours.The hydrolysate was treated by ceramic membrane and ultrafiltration membrane step by step.0-1 KD components were collected and freeze-dried to obtain TFMP-T.The inhibition mechanism of TFMP-T on mTYR activity was irreversible.0-2000 ?g/mL TFMP-T did not affect the proliferation of B16F10 melanoma cells,but inhibited the melanin synthesis and tyrosinase(TYR)activity in B16F10 cells with concentration dependence.Western blot was used to further explore the mechanism of TFMP-T inhibiting melanin synthesis.The results showed that TFMP-Tcound decrease the expression of MITF by regulating MC1R pathway,and then acted on TYR family proteins,which decreased its expression and inhibited the synthesis of melanin.Then,using zebrafish model.we found that 0-200 ?g/mL TFMP-T is not toxic to zebrafish embryos,but could inhibit the melanin contents,TYR activity and the expression of TYR family proteins in zebrafish embryos,and ultimately inhibited the formation of melanin in zebrafish embryos.The acute toxicity test in mice confirmed that TFMP-T was nontoxic.In this study,two kinds of muscles polypeptides of Takifugu flavidus were obtained.TFMP-F had antioxidant activity and anti-fatigue effects and was expected to be developed into anti-fatigue health care products.TFMP-T had the effects of inhibiting melanin formation and cound provide theoretical basis for the development of whitening cosmetics.