Purification, Identification And Characterization Of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived From Walnut Protein Hydrolysate

Cardiovascular disease has now become the first killer of people and hypertension is a major risk factor for cardiovascular disease such as stroke, kidney failure, myocardial infarction. So a better control of the incidence of hypertension can help to reduce the incidence of cardiovascular disease. The angiotensin I-converting enzyme(ACE) inhibitors are the main drugs for the clinical treatment of hypertension. These ACE-inhibitory peptides derived from food protein show a broad application value because of their extensive sources, efficient antihypertensive effects, high safety factor, and no side effects. In this research walnut protein is hydrolyzed by Alcalase 2.4 L to release ACEinhibitory peptides. Walnut protein hydrolysates are purified by consecutive purification methods and two ACE-inhibitory peptides are isolated which might provide a experimental evidence for the related research and development of ACE inhibitory peptides.The main experimental results are as follows:Optimization of purification of ACE-inhibitory peptide derived from walnut protein by size exclusion chromatography. The results showed that the distilled water was the best eluent, and when the flow rate was 0.4m L/min, the sampling volume was 1.5m L, the sampling concentration was 150mg/m L. The purification effect was appropriate under these conditions. Meanwhile, walnut protein hydrolysates could be divided into four fractions by Sephedax G-15. The ACE inhibitory activity of the highest active fraction(fraction G4) was 90.35±0.25%, whose IC50 value was 0.158 mg/m L. The hydrolysates were effectively purified by RP-HPLC and they were mainly concentrated on the 10, 13, 16-18, 21 min location, respectively. The hydrophobic amino acids were significantly enriched by the size exclusion chromatography, from which the concentration of the aromatic acids Tyr, Phe were 10.46% and 20.15%, respectively.Two-step RP-HPLC(semi-preparative and analytical RP-HPLC) was used for isolation of ACE-inhibitory peptides. Fraction G4 was further puri?ed by semi-preparative RP-HPLC and it was separated into six fractions, from which fraction C has the highest ACE-inhibitory activity. Then, the fraction C was further puri?ed by analytical RP-HPLC and it was separated into five fractions, from which fraction C3 has the highest ACE-inhibitory activity.Structure identification and chemical synthesis of ACE-inhibitory peptides from walnut protein. Two ACE-inhibitory peptides from fraction C3 were identified by UPLC-MALDI-TOF-MS. Their amino acid sequences were Leu-Tyr and Tyr-Leu-Ala, with a calculated molecular weight of 295.19 Da and 366.24 Da, respectively. Peptide Leu-Tyr and Tyr-Leu-Ala were synthesized by solid-phase synthesis method. Both of them have efficient ACE inhibiting effects, whose IC50 value were 42μmol/L and 396μmol/L, respectively.Characterization of peptide Leu-Tyr and Tyr-Leu-Ala. Peptide Leu-Tyr and Tyr-Leu-Ala demonstrated good thermal stabilities and their saving rates were above 80%, when they were heated 3h at 37-80℃ condition. Meanwhile, both of them had good resistant of digestion. Under artificial gastric juice condition, they had good resistance to pepsin digestion. After 5h reaction, their saving rates were above 90%. Under artificial intestinal conditions, both of them also had a good resistance to trypsin digestion. After 5h reaction, their saving rates were above 85%. Finally, the antioxidant of peptide Leu-Tyr and Tyr-Leu-Ala were studied. The result showed that peptide Tyr-Leu-Ala had a weak antioxidant but peptide Leu-Tyr revealed a good ABTS?+ radical scavenging capacity. Peptide Leu-Tyr’s scavenging activities of ABTS? +radical was 76.4% when its concentration was 1.00mg/m L and it was 75.2% the capability of reduced glutathione’s scavenging activity under the same condition.ACE-Inhibitory mechanism of peptide Tyr-Leu-Ala. The inhibition mode of peptide Tyr-Leu-Ala was determined to be non-competitive. It represented that the peptide Tyr-Leu-Ala could bind to the ACE enzyme, regardless of whether a substrate molecule was present or not. Peptide Tyr-Leu-Ala might inhibit the enzyme by causing a conformational change, which prevented the enzyme from converting substrate to product, so the inhibitor worked equally well at low and high concentrations of the substrate.