Purification And Properties Assessment Of Amaranth ACE Inhibitory Peptides

As a kind of drought-resistant, salt-resistant, alkali-resistant and barren-enduring plant, Amaranth is the common name of the types in the Amaranthaceae, whose grain can be used as food. Being rich in Amaranth, China is one of the main producer countries in the world. Constituting about17%protein,6.2%fat,60%starch, and being abundant in essential amino-acid, unsaturated fatty acid, Amaranth is very high nutrient. However, the researches and production of Amaranth in our country fall further behind the other countries. Besides the studies of extracting protein, starch, pigment and developing of health food with Amaranth powder, the development and utilization of Amaranth in foreign countries are also under study, such as researching on the property and modification of protein and starch, the antioxidant, hypotensive, hypolipidemic or hypoglycemic effect of Amaranth seeds. Nowadays, producing activity peptides by enzymatic hydrolysis is on focus in functional food. According to this theory, Amaranth protein can be used to produce activity peptides by enzymatic hydrolysis. Hypertension is one of the serious chronic disease but most of the medicine is synthetic, which will lead to side effect. Along with their health consciousness improving, people do not feel satisfied with such medicine. As a result, the safe ACE inhibitory peptides coming from food protein becomes more and more important. Seeking for the cheap and high quality protein is the research trends for the enzymatic preparation of ACE inhibitory peptides. Developing ACE inhibitory peptides from Amaranth will offer a new kind medicine for hypertension, reduce the cost of antihypertensive peptides, and improve the economic value of Amaranth, which will promote the extension of Amaranth in the wasteland.This article used Amaranth protein as raw stuff, optimized the processes of protein extraction and enzymatic preparation of ACE inhibitory peptides, and purified high activity ACE inhibitory peptide by macroporous resin absorption, gel filtration chromatographic and semi-preparative RP-HPLC, which may provide basis theory for the development and utilization of Amaranth. The effect of NaOH concentration, solid-liquid ratio, temperature and time to the extraction rate of protein extraction by alkali-solution and acid-isolation method were studied. The optimal conditions were NaOH concentration0.018%, solid-liquid ratio1:35,35℃and30min by orthogonal experimental design. In that way, the extraction rate was90.21%.The hydrolysis effect of Alcalase, Neutrase, Protamex, soybean peptide hydrolysis enzyme, flavor enzyme and papain was evaluated, and Alcalase was chose to hydrolyze Amaranth protein. The affect factors such as substrate concentration, protease dosage, pH value and temperature were studied by single factor experiment, resulting that the optimized process was:oncentration4g/100mL, protease dosage4.0%, pH value9.0, temperature55℃. The relevant regression equation Y=45.66371-5.353013X1-2.827188X2-10.53672X12-11.77593X22-5.268773X32+2.41875X1X2-1.67125X1X3was established by orthogonal rotation design. On the basis of the equation, the factors were optimized:substrate concentration4g/100mL, protease dosage3.50%, pH value8.78, temperature54.4℃and3h, while the ACE inhibitory IC50of the enzymatic hydrolysate was0.76mg/mL.The macroporous resin adsorption technology was applied to purify the enzymatic hydrolysate of Amaranth protein. Six kinds of resin were sifted by adsorption-desorption experiment, and the DA201-C resin was chose as the best one. After studying the effect of concentration of samples, pH value, and the volume fraction of ethanol by the static adsorption-desorption experiment, the optimal conditions were got as:concentration of samples10mg/mL, pH value5, and the volume fraction of ethanol75%. Basing on it, the effect of loading quantity, loading velocity, elution velocity and elution volume were studied by dynamic adsorption-desorption experiment, and the optimal conditions were:loading quantity1BV, loading quantity6mL/min, elution velocity5mL/min, elution volume5BV, by which the adsorption rate and desorption rate of resin was83.69%and98.69%respectively. After purified, the protein purity and salt rejection of ACE inhibitory peptide was89.47%and88.92%, together with the improvement of ACE inhibition by27.63%.ACE inhibitory peptides with strong activity were isolated from Amaranth protein by gel filtration chromatographic and semi-preparative RP-HPLC. The higher activity peptide AP-II was got after gel G25filtration chromatographic, which ACE inhibition was43.53%, when the quality concentrate was0.17mg/mL. The highest activity peptide AP-Ⅱ-2, which ACE inhibition was66.98%(quality concentrate=0.10mg/mL), was got after semi-preparative RP-HPLC. By RP-HPLC analysing, AP-Ⅱ-2may be made up with four ingrediens.The solubility, pH stability, temperature stability and in vitro anti-digestion stability of AP-Ⅱ were assessed, which all showed good stability, meaning that Amaranth ACE inhibitory peptide can be utilized in food industry widely.