Functional Properties Of Protease Inhibitor And The Major Yolk Protein From Coelomic Fluid Of Sea Cucumber (Stichopus Japonicus)

Sea cucumber Stichopus japonicus is one of the most important holothurian species in coastal fisheries due to its abundant nutrient elements and high commercial value.Bodyfluid,is a medium for transporting nutrients,metabolic waste products and gases around the body,and is abundant in protease inhibitors.The coelomic fluid is the main by-product in the processing of sea cucumber accouting for 5-10%of the total weight.Based on the fact that the yield of sea cucumber is growing year by year,a mass of coelomic fluid is directly thrown away,resulting in a waste of resources and environmental pollution.Aiming at the protease inhibitors from the coelomic fluid of S.japonicus,the effect of coelomic fluid on the degradation of aquatic proteins,including S.japonicus muscle tissues?sjMT?,turbot and shrimp muscles was investigated.Firstly,the optimal conditions of protein degradation of turbot and shrimp were pH 7.0,50?,4 h and pH 3.0,50?,2 h by SDS-PAGE assay.Then,at the favorite conditions,the effects of S.japonicus coelomic fluid isolation?SC?and freez-dried S.japonicus coelomic fluid isolation?FSC?on the protein degradation of turbot,shrimp muscles and sjMT were determined by combination of SDS-PAGE and TCA-soluble oligopeptide assay.The results showed that SC and FSC had the best inhibitory effect on sjMT with inhibitory rate up to 87.17%by TCA-soluble oligopeptide assay.Moreover,FSC could effectively protect the hydrolysis of myosin heavy chain?MHC?.To further study the effects of SC and FSC on protease activity,the results showed that SChad the inhibitory effect on activities of papain and trypin,as well as exhibited good stabilities on temperature,salt and pH.The properties of protease inhibitors of SC were investigated by SDS-PAGE.The results showed that the inhibitory effects of SC/FSC were not affected by metal ions,heating and enzyme treatment.These results suggest that the inhibitory ingredients in SC are more likely to be a non-protein or small polypeptide with strong thermal stability.The protein from the SC was identified as major yolk protein?through NanoLC-ESI-MS/MS.The suitable conditions for recovery of protein from SC were determined by precipitation with polyethylene glycol?PEG?.The results indicated that the condition was 10%PEG 6000 at pH 8.0 4?for 6 h.By using the methods of in silico,the biopeptides with antioxidant activities were screened with 16 kinds of enzymes.The scavenging activities of polypeptides,including MY,FHH,LWG,and WY,on hydroxyl radical and ABTS free radical were determined by an ESR spectrometer and chemical method,respectively.Meanwhile,the protective effects of the four peptides on oxidative stress in HepG2 cells were detected.The results showed that polypeptides had different degrees of antioxidant activities.LWG had a better scavenging effect on hydroxyl radical(IC₅₀=6.97±0.47 mM).WY could effectively scavenge ABTS free radicals(IC₅₀=0.34±0.15mM),and showed protective effects in HepG2 cells.However,the scavenging abilities of peptides were lower than that of GSH.The results above suggest that the SC has small molecules of protease inhibitors.Moreover,SC also has abundant major yolk protein,which is a good source of antioxidant peptides,suggesting that SC is a promising resources for further development.