Preparation And Rapid Screening Of ACE Inhibitor Peptides From Channa Striatus

Hypertension is a kind of disease that seriously affects human health.At present,most of the drugs of angiotensin-converting enzyme（ACE）inhibitors for the treatment of hypertension were chemosynthetic.The effect of these drugs was obvious,but the side effects can not be underestimated.Bioactive peptides have significant antihypertensive activities without side effects and are currently the focus of research.The meat of Channa striatus was used as raw materials to prepare hydrolysates with high ACE activity by enzymatic hydrolysis,and best controllable enzymolysis technics was obtained through step-by-step single factor experiments and response surface methodology.Established approach of bio-affinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking to screening and identification of ACEIPs.Molecular docking be used to study peptide and ACE site and interaction.The main research results are as follows:（1）Taking ACE inhibitory activity and degree of hydrolysis as evaluation indexes,the optimal enzyme for preparing ACEIPs from enzymolysis blackfish was screened out from papain,alkaline protease,trypsin and pepsin,and the effects of enzymolysis temperature,enzymolysis time,p H,solid-liquid ratio and enzyme dosage on the optimal enzyme preparation of ACEIPs were investigated.The molecular weight distribution of the enzymatic hydrolysate was determined.Finally,the stability of the enzymatic hydrolysate in the simulated digestive system was explored,which provided a theoretical basis for its application in food development.The results showed that the ACE inhibitory activity of the hydrolysates of alkaline protease was81.29%and the degree of hydrolysis was 23.23%.The alkaline protease was the best enzyme for the preparation of ACEIPs.The enzymatic hydrolysis conditions of alkaline protease were optimized by single factor experiment and response surface optimization experiment.The optimal enzymatic hydrolysis conditions were as follows:enzymatic hydrolysis temperature 55℃,p H 9,enzyme dosage 5%,solid-liquid ratio 1:20,and time 3 h.Under these conditions,the ACE inhibition rate of the enzymatic hydrolysate was 54.35%.The molecular weight distribution of the enzymatic hydrolysate was determined.The molecular weight of the enzymatic hydrolysate was mainly composed of 6 components.The molecular weight of nearly60%of the peptides was less than 900 Da,and the relative contents ofⅢ,mol and acin were 26.3%,49.9%and 10.7%,respectively,accounting for over 85%of the total content.The ACE inhibitory activity of the hydrolysates did not significantly change before and after simulated digestion（P>0.05）,but the molecular weight distribution of the hydrolysates changed,and the content of the components with low molecular weight increased.（2）A rapid screening and identification method of ACEIPs by biological affinity ultrafiltration combined with Nano–LC–ESI–Q–Orbitrap–MS/MS and molecular docking was established,and the inhibition types and mechanism of ACE by LPGPGP and EYFR were discussed.The optimal concentration of the primary screening sample was determined to be 5 mg/m L,and the ACE inhibitory activity of BPAA was the highest,which indicated that bio-affinity ultrafiltration could effectively screen ACEIPs.There are 30 unique peptide fragments（ALC≥99%）in BPAA identified by mass spectrometry,and the binding energy distribution ranges from-11.51 kcal/mol to-3.81 kcal/mol after secondary screening with molecular docking simulation software Auto Dock Tools.Fourteen peptides with the lowest binding energy were synthesized,among which 7 peptides showed the strongest ACE inhibitory activity when the concentration was 1 mg/m L,and the IC₅₀of EYFR and LPGPGP were 179.2 and 186.3μM,respectively.The analysis of amino acid residues binding between peptides and ACE showed that LPGPGP and EYFR mainly bind to amino acid residues of S1 active pocket and S2 active pocket of ACE,but the main forces are different.According to Linweaver-Burk curve,the inhibition types of LPGPGP and EYFR were judged as competitive inhibition.