Study On Preparation And Purification Of ACE Inhibitor Peptide By Immobilized Enzyme Hydrolysis Of Bitter Almond Gluten

Bitter almonds are mainly produced in Xinjiang.Although they have extremely high use value,they are not fully developed at present,and their resource advantages are wasted If it can be further studied and utilized,it will play a greater role and create a high income.In this study,the enzymatic hydrolysis of bitter almond was analyzed based on proteases.First,the proteases used were screened,and finally alkaline proteases were selected.Then,the immobilized microspheres were prepared by using relevant technologies,and the optimal process for preparing the corresponding enzymes was selected.Secondly,in order to complete the synthesis of ACE inhibitory peptide,a single factor method was used to determine the most suitable enzymatic hydrolysis conditions and optimize them by response face.Finally,the obtained short peptides were separated and purified by ultrafiltration and chromatography methods,and their amino acid sequences were analyzed by LC-MS/MS.The main conclusions of this study are as follows:(1)Based on the gluten of bitter almond,five common proteases were selected for enzymatic hydrolysis.The optimal conditions were set for the five enzymes respectively to analyze the effects of different enzymes on gluten.The enzymatic hydrolysis ability of various enzymes was compared by the number of short peptides obtained and the degree of gluten hydrolysis.Finally,it was found that under the same conditions,alkaline protease had the best effect,and its ACE inhibitory activity was relatively high,the inhibition rate was up to 68.08±1.96%.(2)The relative activity and properties of alkaline protease immobilized by ferrooxide nanoparticles were studied by analyzing the factors such as pH and time of action.The results showed that the optimal conditions for the preparation of immobilized enzyme were 2 g of enzyme dosage,9.0 pH,11h crosslinking time and 8%glutaraldehyde.The enzymatic properties of the free enzyme were also slightly different from that of the immobilized enzyme.The optimal temperature of the two was 60?,but the optimal pH of the immobilized enzyme was 10.0 and the optimal pH of the free enzyme was 9.0.The optimal pH of the immobilized enzyme was slightly higher.In addition,Mie's constant analysis showed that the immobilized enzyme was milder to the substrate and its activity remained high after repeated use.(3)The bitter almond gluten is hydrolyzed,the single factor method is used for the experiment,and the response surface method is used to optimize it.Analyze the experimental results to get the specific process for preparing the relevant enzymes:the amount of enzyme is 6900 U/g,the enzymatic hydrolysis time,pH and temperature are 4.2 h,9.9 and 58?,respectively.The short peptide generation at this time is84.93%,and the ACE inhibition rate is 65.78%.This condition is suitable for preparing bitter almond gluten ACE inhibitory peptide.(4)The prepared crude peptides were separated and purified,and the content of short peptides reached72.69%after ultrafiltration through a 3 k Da ultrafiltration membrane,which was slightly higher than that before ultrafiltration.After desalting by dextran G-15 gel chromatography,the IC₅₀value of each component after recovery was significantly lower than that before purification,and the IC₅₀value of component II was the lowest(1.576 mg/mL),indicating the ACE inhibitory activity of this component highest.After gel chromatography,the content of short peptides is significantly higher than before desalting,and the desalting rate of each component is above 80%.The amino acid sequence was determined as Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu and Pro-His-Cys-Lys-Arg-Met using ultra-high pressure nano-upgraded liquid chromatography tandem electrospray ionization mass spectrometry system(LC-MS/MS).