Study On The Extraction Of Antihypertensive Peptides From Egg Yolk Protein

Angiotensin-I-converting enzyme (ACE) is by definition associated with therenin-angiotensin system, which regulates peripheral blood pressure. The enzyme canincrease blood pressure by converting angiotensinâ… to the potent vasoconstrictor, angiotensinâ…¡, and catalyse the degradation of bradykinin and enkephalins. Inhibition of ACE maytherefore exert an antihypertensive effect and potent synthetic inhibitors of ACE are usedextensively in the treatment of hypertension in humans. A number of ACE-inhibitory peptideshave been isolated and characterised from foods and protein hydrolysates, facilitatingimproved understanding oftheir structure-activity relationship.As a raw materals, chicken egg yolk was degreased and purified by supercriticalextraction of carbon dioxide and ethanol. The egg yolk protein was obtained in purity of 74.68wt% which was 24 wt% higher than that by traditional method. It was manifested that themain components in the protain were composed of over 35kDa, 40kDa, 67kDa and 100Kdamolecules. The product protein was hydrolyzed by 12 types of protease for preparingantihypertensive peptides following alkaline dephosphorylation, respectively. IC₅₀ of thehydrolysates for ACE was determined by high performance liquid chromatographic (HPLC).The stability against ACE for six kinds of hydrolyte which were of lower IC₅₀ values amongtwelve were investigated following preincubation. The results showed that 12 types ofhydrolyte had the inhibition effect to ACE. Further more, the preincubation experimentalresults showed that the IC₅₀ values of 5 kinds of the hydrolyte had no effect or increaseddespite of preincubation with ACE, and they can be regarded as inhibitor and substrate type.The hydrolyte obtained only from protease L showed that its IC₅₀ value (IC₅₀=0.59mg.mL^-1)against ACE was smaller than that before preincubation (IC₅₀=1.19mg.mL^-1) and could be regarded as a prodrug-type ACE inhibitor.The relation between degree of hydrolysis (DH) of hydrolysate and its inhibition for ACEwas also investigated. The result showed that the optimal technologic conditions forhydrolyzing egg yolks were as following: hydrolysis time was 300 min, hydrolysistemperature 45℃, pH 9.5, the concentrate of substrate (S) 60g/L, and the ratio of enzyme tosubstrate (E/S) 0.42g/kg and then the DH, Nitrogen Solution Index (NSI) and the peptidechain length(PCL) were 17.2wt%, 89.09% and 6.5, respectively. Furthermore, the kineticmodel for the dehydration of egg yolk protein was concluded as:r=(270.2E₀-0.4125S₀)exp[-0.23(DH)], which the experimental results.The hydrolyte of egg yolk protein was decolored with the active carbon and purified withultrafiitrate membrane which can block the molecules of 10 K and 3.5 K, respectively. Theinhibiting ability of the obtained product to ACE showed an evident increase. The hydrolyteof egg yolk protein with less than 3.5 kDa molecule was adsorbted by a D61 ion exchangeresin. The suitable separation conditions of ion exchange resin adsorption were as following:pH 4.1, the concentration of sodium acetate buffer in 0.05 mol/L plus sodium chloride in1.0mol/L, gradient washing with sampling in 25mg/mL (wet volume), and washing velocityat 0.5mL/min. As results, the yield and activity of the aimed peptide were 63.7% and 73.59%,respectively.The penetration and desorption sections from ion exchange separation were furtherpurified by Sephadex G-15. The highest active component in the product was enriched andidentfied by RP-HPLC and HPCE. The ACEI sequence was finally testified as Leu-Tyr-Proby thin layer Chromatography and electron spray mass spectrum.The functional test of egg yolk protein hydrate showed that it was of better heat and acidresistant, and also holds better antioxidant ability in a certain range of molecular weight. Theinteraction result with pepsin would cause its IC₅₀ change from 1.19mg/mL to 0.94mg/mL,but that with steapsin would change its IC₅₀ from 1.19mg/mL to 3.19mg/mL.