Production Process Optimization And Angiotensin I-Converting Enzyme Activity Confirmation Of Soybean-derived Bioactive Peptides

This research relies on the Jilin Province Science Development Plan Project?20180520045JH?.Soybean meal as the raw material,using neutral protease,complex flavor protease,papain,trypsin,pepsin to prepare soybean meal angiotensin I-converting enzyme?ACE?inhibitory active peptides,via single factor tests and response surface tests.Fixing the optimal pH and the optimal temperature of neutral protease,compound flavor protease,papain,trypsin,pepsin,and optimize the type of enzyme,the time of hydrolysis,and the amount of enzyme.Taking the yield of soybean peptides as the index,the best process for preparing soybean meal peptides by traditional enzymolysis method is:the type of enzyme is compound flavor protease,the enzymolysis temperature is 50?,the enzymolysis pH is 6.5,the enzymolysis time is 7h,and the enzyme amount is 1%.The substrate concentration is 4g/100mL,and the yield of the enzymatically hydrolyzed soybean meal peptide is 12.122±0.06%.Then conducting a single-factor test of ultrasonic-assisted compound flavor protease enzymolysis and fixing enzymolysis time,enzymolysis temperature,type of enzyme,amount of enzyme,substrate concentration parameters,optimize ultrasonic time and ultrasonic power,and using soybean meal peptides yield as the evaluation index,the test conditions for the ultrasonic assisted complex flavor protease enzymatic hydrolysis of soybean meal with the highest yield of soybean meal peptides were selected:ultrasonic power,ultrasonic time factor:ultrasonic power is 150 W,ultrasonic time is 12 min,the type of enzyme is complex flavor protease,the temperature of enzymolysis is 50?,the pH of enzymolysis is 6.5,the time of enzymolysis is 7h,the amount of enzyme is 1%,and the concentration of substrate is4g/100mL.The yield of soybean meal peptides obtained by enzymatic hydrolysis method was significantly improved,and the yield of soybean meal peptides was increased by 10.748%.Soybean meal enzymolysis solution obtained by ultrasonic assisted enzymolysis was combined with ultrafiltration and AKTA chromatography to separate and purify the enzymolysis solution,and the purified components were subjected to in vitro ACE inhibitory activity test,with IC₅₀ as the evaluation Indicator,screening out components with ACE inhibitory activity in vitro for subsequent LC-MS/MS amino acid sequence identification.Among them,the existing laboratory 200Da,1kDa,3kDa,10kDa,30kDa filter membranes were used to separate the peptide enzymolysis solution obtained by ultrafiltration auxiliary enzymolysis.In the ultrafiltration of<200Da with the best ACE inhibitory activity in vitro The components(the IC₅₀0 is:217.33±25.32?mol/L)were subjected to AKTA chromatography,and 5 peaks A,B,C,D and E were obtained,which were collected and measured for in vitro ACE inhibitory activity,and finally found the peak B in vitro ACE inhibitory activity is the best:136.33±25.15?mol/L,so the components of peak B were identified by LC-MS/MS and compared with the protein database?UniProt?to obtain 843 amino acids sequence.According to the relationship between ACE inhibitory activity and amino acid structure,the screening scope is narrowed,and then the obtained amino acid sequence is used as a small molecule ligand and ACE macromolecular receptor for computer simulation docking based on molecular docking technology.Peptide sequences with the lowest binding energy,6 peptide sequences IIVTP,GVRP,GLVP,LFEDP,LSEEY,VTPSP,which may have ACE inhibitory activity,were screened.After FMOC solid phase synthesis,the synthesized tetrapeptide and pentapeptide were subjected to ACE inhibitory activity according to the measurement,the IC₅₀0 of GVRP and IIVTP were the smallest,and the IC₅₀0 values were 84±0.06?mol/L and77±0.08?mol/L,respectively.Through molecular docking,GVRP-1O86 can be simulated.It can form traditional hydrogen bonds,carbon-hydrogen bonds,salt bridges and other intermolecular interactions with the ACE crystal structure 1O86 amino acid residues Glu403,Glu384,Tyr523.IIVTP can form traditional hydrogen bonds,carbon-hydrogen bonds,salt bridges and other intermolecular interactions with the amino acid residues Lys511,Gln281,His353,His513,Glu162 and Asp377 of 1O86 crystal structure of ACE.This experiment provides a certain experimental basis for the production of enzymatic hydrolysis soybean peptide yield process and soybean meal ACE inhibitory peptide sequence,and provides a new idea for the development of soybean meal peptides functional food.