The Screening And Activities Research Of Sea Cucumber-derived Peptides Based On Peptidomics And Bioinformatics

The preparation of aquatic proteins-derived bioactive peptides(APBPs)is an important direction of high-value intensive processing.APBPs have many sources and various functions,but they often exist in the form of mixtures.The traditional research strategy "enzymatic preparation,separation and purification,sequence identify,activity verification" is time-consuming and laborious,moreover,the low-abundance BPs are easy to be missed.At present,the online combination of high separation efficiency liquid chromatography(HPLC)and high resolution mass(MS)spectrometry has become one of the main methods for the separation and identification of peptides.In addition,peptide databases and molecular docking can be used to simulate protein digestion under given conditions,which can predict and screen BPs.This research strategy can improve the discovery efficiency of BPs.Sea cucumber is a general designation of Echinodermata and Holothuroidea.The BPs with novel sequence structures can be obtained from sea cucumber protein by enzymolysis.Therefore,three kinds of sea cucumbers,Apostichopus japonicus(A.japonicus),Cucumaria frondosa(C.frondosa)and Acaudina molpadioides(A.molpadioides),were used as raw materials in this study.Besides,the difference of peptide profiles of three sea cucumbers were compared by Nano liquid chromatograph(Nano-LC)and Orbitrap Fusion Lumos Tribrid mass spectrometer.At the same time,the potential BPs were screened by bioinformatics analysis and enzymatic prediction in silico.Then molecular docking was used to analyze the interactions between peptides with ACE and DPP-Ⅳ.The main contents of this study are as follows:(1)Alcalase and papain were selected to hydrolyze three sea cucumbers under the optimal conditions,then the degree of hydrolysis(DH)of enzymatic products were compared.It was found that the DH of three sea cucumber alcalase groups were slightly higher than papain after 4 h.On this basis,Nano-LC-Orbitrap Fusion Lumos Tribrid mass spectrometer was used to compare the peptide profiles of different proteases.The results showed that for the same sea cucumber,the number of peptides released by the hydrolysate of papain was more than that in alcalase.For different sea cucumbers,the number of peptides released by hydrolyzed products of alcalase was C.frondosa >A.japonicus > A.molpadioides.The number of peptides released from the enzymatic hydrolysis products of papain was A.japonicus > C.frondosa > A.molpadioides.After simulated gastrointestinal digestion in vitro,the number of peptides released by three sea cucumbers was C.frondosa > A.japonicus > A.molpadioides.There were 868 common peptides in the three hydrolysates of A.japonicus,and 1471 peptides were identified in three hydrolysates of C.frondosa in common.There were 663 common peptides in the three hydrolysates of A.molpadioides.The difference of peptide profiles from three sea cucumbers by the same exogenous protease was analyzed.The results showed that 717 common peptides were identified after hydrolysis by alcalase.1510 common peptides were identified by papain hydrolysis.After simulated gastrointestinal digestion in vitro,744 peptides were identified from three sea cucumbers in common.(2)Taking the peptide library established by peptiomics,oligopeptides with molecular weight less than 1000 Da were selected for bioinformatics analysis.Based on biotoxicity,sensitization,solubility,and(absorption,distribution,metabolism,excretion,toxicity,ADMET)prediction.The binding activity of the peptides to DPP-Ⅳ and ACE was analyzed by molecular docking.The results showed that the hexapeptide K6M(KFPPPM)derived from the yolk protein of A.japonicus could bind to the active pocket of ACE through hydrogen bonding and hydrophobic interaction with the lowest binding energy of-1.85 k J/mol,which is expected to be a potential ACE inhibitory peptide.(3)In order to obtain ocean-derived DPP-Ⅳ and ACE inhibitory peptides with unique sequence characteristics,the potentially bioactive peptides were screened by bioinformatics and enzymatic hydrolysis in silico,and the interaction between the peptide with DPP-Ⅳ and ACE was studied by molecular docking.The results showed that papain had a higher potential to release bioactive peptides than alcalase,this was consistent with the results of actual enzymatic hydrolysis.Six oligopeptides with nontoxicity,non-allergenicity and good solubility were screened.Among them,dipeptides CS and SM can bind to amino acid residues of the S1 and S2 active pockets of DPP-Ⅳthrough hydrogen bonding and hydrophobic interactions,and experiments in vitro showed that at a final concentration of 0.5 mg/m L,the DPP-Ⅳ inhibition rate was 22.5%and 33.2%,respectively.Four oligopeptides could successfully dock with ACE,at a final concentration of 0.2 mg/m L,the ACE inhibition rate of CD was the highest,reached to 43.3%.In summary,in this study,we explained the differences from two low-value sea cucumbers peptides profiles,C.frondosa,A.molpadioides,and the most valuable one,A.japonicus,then the difference of peptide profiles of three sea cucumbers were compared by Nano-LC and Orbitrap Fusion Lumos Tribrid mass spectrometer.This study provided a scientific basis for the selection of raw materials and proteases in the preparation of sea cucumber-derived bioactive peptides(SCBPs).Besides,a method for virtual enzymatic hydrolysis and activity prediction of SCBPs was established.In addition,the interaction between peptides and target proteins was analyzed using molecular docking.This study provided a viable strategy for high-throughput screening of bioactive peptides from aquatic animals.