Selective Preparation And Mechanism Study Of Soybean ACE Inhibitory Peptides

Hypertension is a global health problem which takes the responsibility for the death of approximately 94 millions people each year.ACE inhibitory peptides have been attracted extensive attention due to its obvious blood pressure lowering function without toxic side effects.Therefore,it is significative that develop a polypeptide having high ACE inhibitory activity.In the present study paper,the enzymatic hydrolysis process of preparing functional peptides with high ACE inhibitory activity using soy protein isolate was optimized considering the protein recovery rate,peptide yield and hydrolysis degree,and most important the ACE inhibitory activity.The results showed that the optimal enzymatic hydrolysis to prepare peptides with high ACE inhibitory activity was as followed:enzymatic hydrolysis for 8 h at a substrate concentration of 7.5%,Alcalase?2.4 L addition of 0.75%,temperature of 55°C,and pH at 7.5.ISPH prepared by the best process showed an IC₅₀ value of 0.185 mg/mL.In order to build an simple and industrial purification process for ACE inhibitor peptides,the ACE inhibitory peptides in ISPH were selectively enriched by using membrane ultrafiltration,ethanol fractionation and adsorption chromatography.The results showed that the optimal purification process was as follows:the<3 kDa fraction?F4?in SPIH was obtained by membrane ultrafiltration,then be fractionated by ethanol and the fraction dissolved in 75%ethanol solution?F4-2?was collected.The ACE inhibitory peptides in F4-2 with a concentration of 10 mg/mL was selectively enriched using activated carbon added with the half mass of F4-2,and adsorbed for 2 h,at pH3.0 and room temperature,then resolved for 2 h by using75%ethanol solution at room temperature.The IC₅₀ value of ACE inhibitory peptide prepared by the optimal enrichment process was 0.0188 mg/mL,which was 10.4 times higher than that of SPIH,indicating the effectiveness of the selective enrichment of ACE inhibitory peptide.62 peptides were identified and then screened according to hydrophobicity and the predicted activity provided by PeptideRanker.12 peptides were selected for chemical synthesis and activity verification,furthermore the mechanism of action was also discussed.Results shows that 6 peptides including IY,FF,LVF,WMY,YVVF and LVLL all have noteworthy ACE inhibitory activities,and their IC₅₀ values were 0.53±0.02?M,0.27±0.01 mM,0.36±0.01 mM,0.55±0.02 mM,0.72±0.02 mM,0.73±0.02 mM,respectively.Molecular docking results showed that all of the six peptides have better affinity with the C domain of ACE,which indicated that the ACE inhibitory peptide from SPIH have C domain selectivity.Associated with His353,His513,Lys511,Gln281 in the main active conformation S2'of ACE,and Ala354,Tyr523 and Glu384 in the main active conformation S1,and other important active sites such as His383,Glu411,Ala356 and His387 in the form of hydrogen bonds,all together contribute to the ACE inhibitory activity of the 6 peptides selected from SPIH.At the same time,they can also interact with Zn²⁺which located in the active center of ACE.All six peptides were completely hydrolyzed after in vitro gastrointestinal digestion,the ACE inhibitory activity of IY digestion products decreased by only 3.37±0.02%,and the ACE inhibitory activity of WMY digestion products increased by 22.10±1.57%unexpectedly.According to the BIOPEP database,WM,VF,YV and LVL in the digested products are all reported high ACE inhibitory peptides,which means a good ACE inhibitory activity of peptides after digestion.