Preparation,identification And Bioactivities Of Peptides From Mytilus Edulis Protein

Mytilus edulis is a kind of shellfish with high protein content which belongs to mollusca mussel.The annual output of mussel in China is more than 800,000 tons,which is one of the largest producing countries.However,due to the backward processing technology,90% of the mussels in China are used for fresh and frozen products,resulting in a serious low added value of mussels and serious waste of protein resources.In this paper,the protein composition,basic properties,enzymatic hydrolysis characteristics of mussel were systematically analyzed and the extraction of proteins and the preparation of active peptides were studied.The aim is to provide theoretical basis and effective way for the deep development and utilization of mussels,then to realize the efficient increment of mussel resources.Firstly,research on the basis of mussel protein solubility difference,according to the single factor to determine the best extraction technology,three kinds of protein were got,water-soluble protein,salt-soluble protein and acid-soluble protein(namely myogen,myofibril protein,acid soluble matrix proteins),its molecular weight distribution,particle size distribution,and foam ability properties were analyzed.The results showed that the optimal extraction process was as follow.Firstly,the Mytilus edulis meat was suspended(1:6 w/v)in 0.05 M phosphate butter solution(pH 7)and incubated for 60 min at 4 oC to extract the first proteins fraction(P1);The pellet was then suspended(1:9 w/v)in 0.1 M phosphate butter(pH 7)containing 0.6 M NaCL,and incubated for 30 min at 4 oC to extract the second proteins fraction(P2);Finally,the third protein fraction(P3)was extracted from the pellet from the second centrifugation by incubating(1:15 w/v)in 3%(w/v)citric acid solution at 50 oC in a water-bath.Similarly this solution was also centrifuged and the supematant collected.The total protein extraction rate was 83.74% and the results were satisfactory.There were more water-soluble protein,followed by salt-soluble protein,and finally,acid-soluble matrix protein.The molecular weight and particle size distribution of each protein were different,and the P1 protein contains 16.76,26.68,29.51,35.53 and 38.11 kDa protein bands.P2 protein was 99.7,52.13,44.71 and 38.51 kDa protein bands.The P3 molecular weight distribution was 177.33,106.69,88.55 and 75.38 kDa,mainly the collagen strips of 199 kDa.Under the same foaming conditions,the water-soluble protein has the lowest foaming ability,the salt-soluble protein has the best foaming ability,and its foam stability was more stable than the other two proteins.The granularity of P1 and P2 is divided into two parts,and the distribution is relatively dispersed.The particle size distribution of P3 was mainly concentrated in 500-1000 nm.Trypsin,neutral protease and pancreatic were used to digest three kinds of protein in the suitable conditions of enzymatic hydrolysis enzyme solution,respectively,then we got nine kinds of enzymolysis products and measured the degree of hydrolysis under the condition of the enzyme hydrolysis conditions,molecular weight distribution of the enzymatic hydrolysis products were determinate by liquid phase.The enzymatic hydrolysis products were processed by C18 small column in addition to salt and protein,and peptide sequences were identified by mass spectrometry.The number of peptides and the distribution of their amino acids were studied.Venn diagram showed the number of peptides in different enzymolysis products and the intersecting with other products.The results showed that the hydrolysis degree of neutral protease was the highest,peptides with molecular weight less than 3000 Da were more abundant,indicating that the enzymatic hydrolysis was better.Based on the polypeptide sequence in the enzymolysis products obtained in the previous chapter,the angiotensin converting enzyme(ACE)inhibitory activity and anticoagulant activity were carried out by molecular docking to determine the possible biological activity value of each peptide.At the same time,combining with the combination of polypeptide and corresponding protein,such as the site of key amino acids and hydrogen bond,the mechanism of the action of peptide was further understood.The possible bioactive peptides were screened out and their component activity was determined.Apart from the above two kinds of activity,immune activity of product was also studied in this chapter.Cell proliferation of nine groups digestion sample within 24 h,48 h and 72 h were discussed adopting macrophages RAW264.7 mice model,strongest active component was determined by comparison.In the end,the prediction of the toxicity of the confirmed active peptides was made to ensure that the peptide was in a favorable position in the later stage.The results showed that peptide AENELGEVT,ELEDSLESER,RDLDSDVSST and AAQSDYDNL were active peptides with ACE inhibitory activity.At the same time,NP2,PP2 and TP2 were identified as the source of high activity peptides.Peptides DLTKKYNLP,ELEDSLDSER and TDEQVDDIIR were active peptides with anticoagulant activity.PP1 samples had the strongest and stable immune activity after comparison.Among them,small molecular peptides,peptide ISPLYPR and HGADGVPPF were analyzed and identified as active peptides.This study provides a new idea and theoretical basis for the study of active peptides from Mytilus edulis,it laid a foundation for the industrialized application and realize the efficient increment of Mytilus edulis resources.