Preparation And Purification Of ACE Inhibitory Peptides From Soybean Protein By Enzymatic Hydrolysis

The soybean peptides derived from the enzymatic hydrolysate under the moderate condition depress the blood pressure by inhibiting the activity of Angiotensin-Ⅰconverting enzyme (ACE), and are really safe to diet and patient suffering in hypertension.In this paper, firstly, the five enzymes concluding alcalase pepsin trypsinase papain and neutrase were individual and multiple applied to hydrolyze the soybean protein. The pre-treat temperature reaction time and quantity of enzyme addition effected the hydrolysis degree of soybean. The single and multiple enzymatic hydrolysate were both presented the ACE inhibitory activity with different scales. Among enzymes, mensurating the ACE inhibitory activity on up-hudrolysate, the highest ACE inhibitory activity of hydrolysate from alcalase was 45% and the pepsine hydrolysate took the second place with 40% ACE inhibitory activity.The membranes with MWCO of 10K Da 3K Da and 1K Da were used to classify the hydrolysate derived from the five enzymes.Different enzymatic hydrolysate collocated same thickness. The content of <1K Da fraction were 71.25% and 69.35% of the total hydrolysate from alcalase and pepsin respectively. All hydrolysate with different molecular grades were both appeared the ACE inhibitory activity and which was increased with the lowering of molecular mass. The <1K Da exhibited the higher ACE inhibitory activity of 64.57% (alcalase) and 78.49% (pepsin) respectively. The ACE inhibitory activity of fraction <1K Da from pepsin was higher than all that of others and that of trypsinase papain and neutrase were a bit of equal. The Tricine-SDS-PAGE showed that the classification by ultrafitration was apparently and the molecular mass of the three grades was obviously less than that of the original soybean protein, what's more, the molecular mass distribution of them gave a descending tendency.After the cross experiments to hydrolyze the soybean protein by pepsin, the optimal condition to produce the hydrolysate with the highest hydrolysis degree of 17.1% was under conditions of 3% substrate concentration, 2% enzyme quantity addition and 2 h reaction time. The solubility of P1000 from pepsin under the acid environment was better than that of alkaline and which was improved with the increase of temperature.The ACE inhibitory peptides P_D and P_J isolated by continuous chromatographic technique of gel filtration chromatography on a Superdex Peptide 10/300 GL column and twice reverse chromatography on Sephasil peptide 5μm C18 ST 46/250 column from the <1K Da fraction of pepsin hydrolysate exhibited the IC50 of 0.714 and 0.626 mg/ml, respectively. However, the molecular mass of P_J could not be confirmed by SDS-PAGE. The ACE inhibitory activity of P_J was susceptible to pH. The ACE inhibitory activity under acid environment was prone to loss than that of alkaline. After the treatment of different temperatures, more than 90% of ACE inhibitory activity was retained and there was almost no loss of ACE inhibitory activity, which suggested the P_J was thermal stable.