Preparation And Separation Of ACE Inhibitory Peptides From Walnut Meal Protein

Activity peptides, derived from nature, were very important in regulating the human body. In recent years, the ACE(angiotensinⅠ-converting enzyme) inhibitory peptide is attracting more and more attentions during to its special bioactivity.In this study, walnut protein, extracted form skimmed walnut, was used to produce ACE inhibitory peptide. The hydrolysis of walnut protein was investigated by comparing two kinds of methods by using single and binary-enzymes. And the optimal enzymatic methods and conditions of preparing ACE inhibitory peptides were selected using ACE inhibitory rate as an index. On this basis, the simple separation and purification technology of walnut meal ACE inhibitory peptides was studied.(1) Walnut protein was extracted from defatted walnut meal using alkaline extraction and acid isoelectric precipitation technique. And four various protease including alkaline protease, neutral protease,papain and flavor protease were applied for production of antihypertensive peptides. As the indexes, ACE inhibitory rate and the degree of hydrolysis were determined to select the best enzymes with best enzymatic effect and single factor experiments were carried out to study the effects of the factors such as substrate concentration(g/L), enzyme dosage(U/g), hydrolysis time(h), hydrolysis temperature(℃) and p H. On this basis, the response surface methodology was used to optimize the above conditions of the enzymatic hydrolysis. Results showed that substrate concentration of 30 g/L, enzyme dosage of 8000 U/g,temperature of 57℃, p H of 8.6 and enzymatic time of 3 h were optimal when using alkaline protease with a outcome of 64.32% ACE inhibition rate. However, the optimal processing technology of flavor protease was established with 15 g/L substrate concentration, 6000 U/g enzyme concentration, 50 ℃enzymolysis temperature, p H 6.5 and 3 h enzymatic hydrolysis. The final ACE inhibition rate could reach57.92% under the above conditions.(2) In this paper, antihypertensive peptides were prepared by two protease(alkaline protease and flavor protease) with two-step hydrolysis. Single factor experiments were carried out to study the effects of the factors such as substrate concentration(g/L), enzyme dosage(U/g) and adding quantity proportion. The response surface methodology was used to optimize the above conditions of the enzymatic hydrolysis. The results showed that the optimum conditions were 29 g/L substrate concentration, 6000 U/g enzyme dosage,adding quantity proportion of 3:2. And the ACE inhibition rate could reach 67.05% under these conditions.By comparation, the effect of two-step-hydrolysis using alkaline protease and flavor protease was better than that of single alkaline protease hydrolysis.(3) The separation and purification of walnut ACE inhibitory peptides was conducted by ultrafiltration and gel chromatography. At first, enzyme solution was separated into three parts, namely,molecular weight <1 k Da(A), 1-3 k Da(B) and >3 k Da(C) using 1 k Da and 3 k Da ultra filtration membranes. The ACE inhibition rate of different components were determined respectively and component A(75.92%) was much higher than that of component B(39.51%) and component C(16.94%).Then sephadex G-15 was employed on component A and three peaks were obtained eventually. After the determination of ACE inhibition rate on the above peaks, No.2 peak(75.92%) was reserved when comparing the No.1 peak(44.78%) and(58.49%). And walnut ACE inhibitory peptide samples were obtained after freeze drying.