Separation And Purification Of Angiotensin I-converting Enzyme(ACE)Inhibitory Peptide From Walnut Dross

The main purpose of this study,walnut dross protein was hydrolyzed by pepsin.Then use a variety of separation methods,extraction and purification of high activity ACE inhibitory peptide.The main method:First,using single factor test,orthogonal experiment to determine the optimal hydrolysis conditions;then using microfiltration,ultrafiltration,Sephadex G-25 chromatography and other methods to extract crude ACE inhibitory peptide,ACE inhibitory activity analysis by HPLC;then RP-HPLC,AKTA protein separation and purification systems for crude ACE inhibitory peptides were isolated and purified,and ultimately high ACE inhibitory activity of pure peptides by N-terminal amino acid sequencing and synthetic,simulated digestion in vitro experiments and validation experiments.The results:the single factor experiment and orthogonal experiment determine the optimal conditions for pepsin hydrolysis process:hydrolysis temperature is 55?,solid-liquid ratio of 1:20,pH value of 2.8,plus 3.5%of enzyme,the enzyme solution time was 4.0h.Verification experiment performed under optimal conditions,the final degree of hydrolysis measured for protein walnut powder 16.4762%.Extracted through microfiltration,ultrafiltration,Sephadex G-25 chromatography and other methods of ACE inhibitory peptides crude extract product,ACE inhibitory activity analysis by HPLC.Get a higher ACE inhibitory activity of the crude peptide with an IC₅₀ of 0.2633 mg/mL.After RP-HPLC,AKTA protein separation and purification system is isolated and purified the highest ACE inhibitory activity of peptides obtained through ACE inhibitory peptide sequence of the protein sequencing test is:Tyr-Val-Pro-His-try-Asp-Leu?YVPHWNL?,its molecular weight is 929.03.Synthetic ACE inhibitory peptides sequenced YVPHWNL,after in vitro gastrointestinal digestion experiments using HPLC measurement of its activity,the IC₅₀ values obtained after digestion of ACE inhibitory peptides are 0.1357?M/mL,0.1732?M/mL.Significance:In this study,we got a higher activity of ACE inhibitory peptides and identified its structure successfully through the extraction and purification from walnut dross protein.This study provides a theoretical basis for the study of lowering blood pressure,but also laid the foundation for the blood pressure lowering health food.