Study On The Preparation And Activity Of ACE Inhibitory Peptides From Tartary Buckwheat Protein

Hypertension is a chronic disease that endangers human health.The changes in blood pressure are closely related to angiotensin-converting enzyme(ACE),which inactivates bradykinin and promotes the conversion of angiotensin I to angiotensin II,thus causing an increase in human blood pressure.ACE inhibitory peptides obtained from natural products are safer and more efficient than chemical drugs,and are widely used in drug development,which has a broad research potential.Tartary buckwheat protein peptides have been shown to inhibit ACE activity,but the ACE inhibitory activity and structural relationships of Tartary buckwheat graded proteins have not been adequately studied.In this study,the crude protein was extracted by alkali solubilization and acid precipitation using Tartary buckwheat as the raw material.The ACE inhibition rate was used as the main index,combined with the degree of hydrolysis and peptide yield,and the Tartary buckwheat protein pepsin-trypsin continuous hydrolysis process was optimized using single-factor and orthogonal experiments.Subsequently,Tartary buckwheat albumin,globulin,prolamin and glutelin were extracted by Osborne method,and the four graded proteins were hydrolyzed by the optimal hydrolysis process,with ACE inhibition rate as the main index,combined with structural analysis of the difference in inhibition effect and the reasons.Then,the bioactive peptides with the highest ACE inhibitory activity were separated and purified by gel filtration chromatography and reversed-phase high performance liquid chromatography,and the sequences identification of the ACE inhibitory peptides were carried out by liquid chromatography-tandem mass spectrometry.The specific experimental results are as follows:(1)Optimization of Tartary buckwheat protein hydrolysis processPepsin and trypsin were selected for continuous hydrolysis of Tartary buckwheat proteins and the best hydrolysis process is as follows: Tartary buckwheat protein dosage of 0.5 g,the amount of trypsin added to the Tartary buckwheat protein 5.0%.Hydrolysis time for pepsin 2h,trypsin 1 h.Experimentally verified that the optimal level of combination of Tartary buckwheat protein hydrolysate ACE inhibition rate of 76.36 ± 0.41%,9.37 ± 0.36% hydrolysis degree,peptide yield of 71.36 ± 0.10%.(2)Tartary buckwheat graded protein hydrolysate of ACE inhibition activity and structural analysisStructures of albumin,globulin,prolamin and glutelin from Tartary buckwheat before and after hydrolysis were analyzed to explore the reasons for the differences in ACE inhibitory activity of the four graded protein hydrolysates.The results showed that Tartary buckwheat albumin hydrolysate had the highest ACE inhibition rate of 79.89 ± 4.32% at a concentration of 0.2 mg/m L,followed by globulin(71.84 ± 6.32%),prolamin(49.43 ± 1.15%),and glutelin(40.80 ± 3.23%).Among the four graded proteins,Tartary buckwheat albumin had the highest hydrolysis degree of 5.92 ± 0.28% after hydrolysis,and the highest peptide yield of 82.28 ±0.13%.And albumin hydrolysate in the smallest peptide molecular weight,of which 60% is less than 1 k Da peptide,more likely to play an inhibitory effect on ACE.Through structural analysis,Tartary buckwheat albumin in the hydrolysis of β-folded content decreased,β-turn angle and the relative content of the random curl are increased to varying degrees,part of the albumin β-folded by the ordered structure into a disordered structure,the expansion of the protein molecular structure,which is conducive to its ACE inhibitory activity.Fluorescence spectroscopy results showed that the Tartary buckwheat protein advanced structure was destroyed,the surface hydrophobicity of the four graded proteins after hydrolysis were significantly reduced,the surface hydrophobicity of the albumin was significantly higher than the hydrolysis products of the other three proteins,the peptide with higher surface hydrophobicity has better ACE inhibitory activity.Comprehensive analysis of the above,the Tartary buckwheat protein hydrolysate was selected for further isolation and purification.(3)Tartary buckwheat protein ACE inhibition peptide isolation and purification and identificationThree fractions(F1,F2 and F3)were isolated from Tartary buckwheat albumin hydrolysate by gel filtration chromatography,with the F3 fraction showing the highest ACE inhibition rate of 71.83 ± 4.97%% at a concentration of 0.1 mg/m L.Subsequently,reversed-phase HPLC separation of the F3 fraction was performed to obtain eight new subfractions(F31-F38),and the results showed that the F34 fraction exhibited the highest ACE inhibition rate of 58.11 ±3.18%%.the decrease in the inhibition rate of the single subfraction of F3 was probably due to the synergistic effect between the different subfractions.Finally,the F34 fraction was analyzed by high-performance liquid chromatography-mass spectrometry and found to contain 42 peptides with ACE inhibitory potential,14 of which were the latest ACE inhibitory peptides identified in this study.The results of the study provide a valuable reference for the further development and application of Tartary buckwheat protein as a food-derived hypotensive peptide.