Isolation,Purification And Identification Of ACE Inhibitory Peptide From Walnut Protein

In this research,defatted walnut kernel cake was used as the raw material.After extracting the walnut protein isolate,alkaline protease was used for enzymolysis.The highest ACE inhibitory active components after isolation and purification were sequenced to predict the activity in order to obtain high activity ACE inhibitory peptide.The main research contents and results are as follows:(1)The detection conditions for rapid detection of uric acid by reversed-phase high-performance liquid chromatography were established.The specific conditions are: acetonitrile: water = 25: 75(v/v),adding 0.1% trifluoroacetic acid.Keep the flow rate to 1m L / min and set column temperature to 30 ?.The detection wavelength is 228 nm and the injection volume is 10 ?L.Under these conditions,the detection of uric acid has good stability,high repeatability,fast and easy operation.(2)The alkaline protease was used to hydrolyze the walnut protein isolate.The ACE inhibition rate was used as the factor to investigate.The optimal proteolysis process was obtained through single factor and response surface optimization experiments.The substrate concentration was 5%,and the amount of enzyme was 6400 U/g.The p H of the enzymatic hydrolysis was 9.45.Hydrolysis temperature was 53 ?.And hydrolysis time was 2.6 h.The ACE inhibitory activity was 32.17% under those process conditions.(3)The antioxidant properties and physicochemical stability of walnut protein ACE inhibitory peptides were measured.The results showed that high concentration of ACE inhibitory peptides had strong antioxidant activity.The inhibitory peptide had good stability at 4 � C and remained stable for 3 hours at 25 � C.It is more sensitive to metal ions.The 100 g/m L metal ions could greatly reduce the activity of the inhibitory peptide,so high concentration of salt should be avoided.The activity decreased rapidly in strongly acidic environments and remained stable in neutral and weakly alkaline environments.The simulated gastrointestinal digestion in vitro showed that the inhibitory peptide has good antidigestibility,and it could maintain a 53.9% inhibition rate after the degradation of gastrointestinal enzymes.(4)After ultrafiltration and Sephadex G-25 gel chromatography of the enzymatic hydrolysate of walnut protein isolate,the semi-inhibitory concentration of the most active component was 0.16 mg /m L.After mass spectrometry sequencing and AHTpin prediction,the predicted inhibitory activities of the six peptide sequences were significantly different from other peptides,namely YLPP,PPPL,PKPP,PPKP,YPQY and PPPT.Molecular docking results showed that YLPP and YPQY peptide hold the best performance in ACE inhibition.