Preparation Of ACE Inhibitory Peptides From Stichopus Japonicus Viscus And Study Of Their Activity

Hypertension and its associated cardiovascular diseases are seriously affecting people’s health.Currently,the chemically synthesized drugs used to treat hypertension have more or less side effects,while small molecule peptides with ACE-inhibiting activity extracted from food are usually mild,stable and safe,providing new ideas and ways for the prevention and adjunctive treatment of hypertensive diseases.In this paper,the preparation of ACE-inhibiting peptides from the Stichopus japonicus viscera and the study of their activity provide an idea to explore the medical effects of ginseng and help promote the comprehensive and high-value utilization of marine biological resources.Objective:Establish and optimize the enzymatic preparation process of active peptides from Stichopus japonicus viscera.To purify peptide fragments with ACE inhibitory activity;To analyze the physicochemical properties and stability of small molecule peptides and to study the physiological functions of purified peptides using cellular models;To screen the active fragments and identify their molecular structures in combination with bioinformatics analysis and to validate the activity of chemically synthesized ACE peptides.Methods:（1）The ACE inhibitory peptide was prepared by enzymatic digestion using stichopus japonicus viscera as the raw material;the effects of material-liquid ratio,enzyme addition and enzymatic digestion time on the activity of the peptide prepared by enzymatic digestion were optimized by single-factor and response surface analysis;the active peptide was purified by ultrafiltration and chromatographic chromatography,and the ACE inhibitory activity of each component was analyzed.（2）The highly active peptide was characterized by protein electrophoresis and infrared spectroscopy;The stability of the peptide was also investigated.The peptides were characterized by protein electrophoresis and infrared spectroscopy;The stability of acid,alkali,heat,and anti-digestion were also investigated.（3）A model of oxidative stress injury cells was established to verify the physiological functions of the purified peptides,and the changes of related regulatory factors such as Nitric Oxide（NO）,Superoxide Dismutase（SOD）and Malondialdehyde（MDA）were detected to explore the protective effects of ACE inhibitory peptides on injured cells in ginseng.（4）Mass spectrometry（LC-MS/MS）was used to The peptides were analyzed by mass spectrometry（LC-MS/MS）,combined with molecular docking technique and compared with relevant databases to obtain peptide fragments with good ACE inhibitory activity;And the peptides were validated by solid-phase chemical synthesis.Results:（1）The preparation process of ACE-inhibiting peptide by enzymatic digestion of the stichopus japonicus viscera was established and optimized:the reaction temperature was 37℃,the reaction p H 8.5,the feed-to-liquid ratio was 1:1.98,the amount of trypsin added was 2.56%,and the enzymatic digestion time was 1.0 h.The highest ACE inhibition rate of 56.22%was obtained from the enzymatic digestion solution.（2）The peptide was purified by ultrafiltration and Sephadex G-25 gel chromatography,and the IC₅₀value of the purified peptide was 535.33±30.24μg/m L.It was verified that the peptide had good stability at low and medium temperatures（20～50℃）and neutral or weak acidic conditions（p H 4～7）,but it has strong hygroscopicity and ACE inhibitory activity in the presence of metal ions such as Zn²⁺and Fe³⁺.The peptide was also shown to have a certain degree of antagonism to the digestion of artificial gastrointestinal fluid,as shown by simulated gastrointestinal digestion.（4）Mass spectrometric analysis combined with molecular docking screening resulted in a peptide fragment with strong ACE inhibitory activity,whose amino acid sequence was PPPVFDAK and molecular weight was 435.742Da;Molecular docking showed that it could successfully pair to the active site of ACE receptor and the IC₅₀of the peptide on ACE was 54.70μM.Conclusion:We extracted ACEIP from stichopus japonicus viscera,which has good antihypertensive effect,stable performance,and the ability to protect HUVEC from oxidative damage.We screened and identified ACE inhibitory sequences with high activity.The results of this study provide reference significance for the high-value application of stichopus japonicus visceral waste and the exploration of foodborne antihypertensive peptides.