Preparation And Activity Analysis Of Angiotensin-converting Enzyme Inhibitory Peptide From Tilapia Skin Gelatin

Hypertension is a risk factor for a number of other cardiovascular disease such as stroke,coronary heart disease,myocardial infarction and diabetes,which has become one of the most common risk diseases.At present,some blood pressure lowering drugs commonly used in clinical practice have a remarkably effective at regulating blood pressure,but also have certain adverse side effects such as allergic reactions,skin rashes,coughing,taste disturbances,leukopenia and drug dependence.Nevertheless,food-borne Angiotensin I-converting enzyme inhibitory?ACEI?peptide is a promising peptide substance.It not only has antihypertensive activity,but also has high safety and without other obvious side effects.It has become one of the research hotspots in the field of bioactive peptides.The tilapia skin gelatin can be hydrolyzed by protease to obtain a small molecule polypeptide with high security,low toxicity side effects and reducing blood pressure.In this paper,tilapia skin,a by-product of tilapia,was used as the research object.The ACEI peptide was prepared by protease hydrolysis of fish skin gelatin.The gelatin hydrolysate was isolated and purified,and the structure was identified,the finally obtained high ACEI activity of small peptides.The major findings were presented as follows:1.The tilapia skin was used as a raw material,and the two proteases were used for hydrolysis,and the gelatin hydrolysates obtained by hydrolysis was subjected to successive simulated gastrointestinal digestion,and the degree of hydrolysis and ACEI activity of the hydrolysates in each digestion stage were determined.At the same time,considering the effect of intestinal absorption in vitro,the ACEI peptide was prepared using a Caco-2 cell monolayer model.It was separated by Sephadex G-25 gel filtration column and reversed-phase high performance liquid chromatography.The high-resolution mass spectrometry identified two new ACEI peptides,VGLPNSR and QAGLSPVR,with molecular weights of 741.4133 Da and826.4661 Da,respectively.2.The two novel ACEI peptides identified were chemically synthesized for their in vitro and in vivo antihypertensive activity studies.The in vitro ACEI activity of VGLPNSR and QAGLSPVR was determined by reacting different concentrations of ACEI peptide solution with a fixed concentration of substrate HHL.The regression equations of VGLPNSR and QAGLSPVR were determined as follows:y=26.30ln?x?-65.56?R²=0.991?and y=26.40ln?x?-61.53?R²=0.977?,and the IC₅₀0 values were calculated according to the regression equation.They were 80.90 and 68.35?M,respectively.The in vivo antihypertensive activity of VGLPNSR and QAGLSPVR was studied in a model of spontaneously hypertensive rats?SHR?.Captopril was used as a positive control,the systolic?SBP?and the diastolic?DBP?blood pressure of the rats were measured before administration of the treatment?time 0?and after the treatments.The results showed that both of them significantly reduced SBP and DBP in rats,and reached the maximum value of antihypertensive after 3 hours of administration.3.The enzyme kinetics Lineweaver-Burk plot model was used to determine the inhibitory effect types of the novel ACEI peptides VGLPNSR and QAGLSPVR,and the molecular docking software SYBYL-X 2.1.1 was used to study the structural activity relationship between ACEI peptide and ACE.The experimental results showed that the two ACEI peptides identified in this study were noncompetitive inhibitors.Docking simulation results showed that VGLPNSR formed bonds with Ser526,Asp415,Gly386,Ile388,Val380,and Gln369,and QAGLSPVR can form hydrogen bonds with Asp465,Asp415,Val380,and His410.However,VGLPNSR and QAGLSPVR could not interact with the key amino acid residues in the ACE active site.These results suggested that the interaction between two key peptides and ACE is noncompetitive.This finding was in accordance with the result about their ACE inhibition kinetic pattern determined.