Identification And Activity Of ACE Inhibitory Peptides In Distilled Spent Grain

Distilled spent grain,the residue of sorghum and other crops after solid fermentation and distillation,which is a valuable resource.Most of the early researches focused on the analysis of conventional nutritional components in DSG,but the research on peptides,especially angiotensin converting enzyme?ACE?inhibitory peptides,has not been reported.Hypertension is recognized by the World Health Organization?WHO?as an important source of cardiovascular disease,and ACE inhibitory peptides can lower blood pressure due to their ability to inhibit ACE.In recent years,there have been reports of ACE inhibitory peptides found in jiupei and baijiu,so there are likely to be more in DSG.This research took Chinese strong type flavor DSG as object,followed by using ACE inhibitory activity as the guide.Ultrafiltration and reversed phase performance liquid chromatography?RP-HPLC?,combined with ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry?UPLC-Q-TOF-MS?were used to identify the free and bound peptides in DSG.Ultra-performance liquid chromatography-triple quadrupole mass spectrometry?UPLC-QQQ-MS?was used to quantitatively analyze the ACE inhibitory peptides in DSG.And this research also determined the activities of ACE inhibitory peptides,which indirectly characterizes the contribution of to the whole ACE inhibition;ProteinPilot was further used combined with the protein search database Uniprot in this study for the traceability analysis of free ACE inhibitory peptides.The research of ACE inhibitory peptides in DSG provided important theoretical references for the comprehensive utilization of DSG and increase of high added value.The main research contents and results are as follows:?1?UPLC-Q-TOF-MS combined Biolynx was used for the first time to identify free ACE inhibitory peptides in DSG.4 peptides of the eluted fractions with higher ACE inhibitory activity were identified?including newly found peptide NGGPPT?.UPLC-Q-TOF-MS combined with ProteinPilot software were firstly used to identify the structures of peptides and traceability analysis of eluted components with higher ACE inhibitory activity in DSG.It was planned to use common functional microorganisms such as those in the process of making strong flavor type liquor:Lactobacillus,Acetate bacteria,yeast,etc.A total of 22 peptides were identified in each of the eluted fractions,of which 17 and 5 peptides were identified in the 20%and 50%ethanol-eluted fraction,respectively.The largest number?12?of peptides were identified in Saccharomyces cerevisiae,while the smallest number?2?of peptides were identified in Acetobacter.?2?Based on the ACE inhibitory activity-oriented principle,ultrafiltration combined with semi-preparative RP-HPLC was established in this study for the hydrolysate of alcalase,followed determined IC₅₀ of ultrafiltration and RP-HPLC fractions in different molecular weight ranges.The active fractions were screened for MS identification.The results showed that the ultrafiltration fraction<1 kDa had the highest ACE inhibitory activity(IC₅₀=6.75?g?m L^-1),and the recovery rate was as high as 84.18%.After RP-HPLC optimization,the ACE inhibitory activity of II??were above 70%,the activity of fraction VI was the highest,reaching 88.99%.???were analyzed by UPLC-Q-TOF-MS/MS.?3?Firstly using UPLC-Q-TOF-MS and Biolynx software,the???fractions purified from hydrolysate of alcalase were used to identify the ACE inhibitory peptides.Six new ACE inhibitory peptides were identified,which were NQL,AVQ,YPQ,AYLQ,VLPVLS,and VLPSLN;AVQ showed the highest ACE inhibitory activity with IC₅₀ value of 181.00?mol?L^-1.?4?Firstly using UPLC-QQQ-MS was used in DSG to optimize the mass spectrometry parameters of the target peptide and quantify the bound ACE inhibitory peptide,and the quantitative mass spectrometry data was collected in multiple reaction monitoring?MRM?mode.The results showed that the content of ACE inhibitory peptides in DSG was between0.17 and 16.96 mgg^-1DW,with the highest content of VLPVLS(16.96 mgg^-1DW).?5?The results of enzyme kinetic studies indicated that peptides AVQ and YPQ are non-competitive inhibitors of ACE.Based on the computer molecular docking technology,AutoDock software was used to dock AVQ and YPQ,to the receptor protein,and to analyze their interaction sites with the ACE protein.The results of molecular docking indicated that the peptides AVQ and YPQ could stably bind to the active center of the ACE protein,and Gln281,His353,Ala354,Ser355,Glu384,Lys511,His513,Tyr523 residues could all have an important effect on the effective binding of the polypeptide to the ACE.