| Low-value marine fish decapterus maruadsi, due to the lack of effective processing methods, mostly be processed into feed,snack foods,etc., and resulted in low value-added, and protein resources are not fully utilization. In this paper, preparation of angiotensin I-converting enzyme inhibitory peptides from decapterus maruadsi protein with six kinds of protease in their respective appropriate conditions, and ultrafiltration, gel filtration chromatography, ion exchange chromatography were used to isolated and purified the active component of the hydrolysates. This provided a theoretical basis for the development of new bioactive peptides, improve the utilization and deep processing of decapterus maruadsi protein.The component protein of decapterus maruadsi was analyzed with protein content and amino acid, and the result show that decapterus maruadsi protein content of79.98%(dry basis), and18kinds amino acid in proteins of decapterus maruadsi was about850.39mg·g-1, in which the total quality of8essential amino acid was about46.28%, aromatic amino acid contained11.35%, polar amino acid contained35.07%. this conformed to the structure of food source of protein antihypertensive peptides, so decapterus maruadsi protein canbe used to prepared antihypertensive peptides. The ACE inhibitory activity of the six kinds hydrolysates were compared with the evaluation of IC50, and the papain hydrolysates provided with the highest IC50. The relationship between DH and IP of papain hydrolysates was investigated, the DH between13.0%-16.0%with the highest ACE inhibitory activity of the hydrolysates, and the highest ACE inhibitory activity was64.7%while the DH was13.1%. The optimized condition for hydrolyzed was:initial enzyme dosage at7000U·g-1Pro, initial substrate concentration at2.5%, the hydrolysis temperature at45℃, pH7.0, and hydrolysis time240min with the degree of hydrolysis was16.0%, in this condition the hydrolysates with a good ACE inhibitory activity. The hydrolysates with a molecular weight less than14.4KDa, which was proved by SDS-polyacrylamide gel electrophoresis.Accoroding to hydrolyzate process, enzymatic kinetic model formula was derived, and deduced papain enzymatic kinetic model of decapterus maruadsi protein,R=(202.785e0+0.896S0) exp [-0.6513(DH)] and which based on the result of hydrolysis, and kinetic parameters were obtained:enzymatic of papain inactivation constant kd was112.33min-1, hydrolysis activation energy ΔEA was33.85KJ·mol-1, the enzyme deactivation activation energy ΔED was38.49KJ·mol-1. Finally, validated the hydrolysis kinetic model, the theoretical results in accordance with the actual results proved there was practical application value with the model.The10KDa and5KDa ultrafiltration membranes were used to separate the ACEI initially. And the optimum process parameters were determined:ultrafiltration time for10KDa memebrane was80min,ultrafiltration temperature at30℃, pH7.0, feed concentration was22.4mg·L-1; ultrafiltration time for5KDa memebrane was100min,ultrafiltration temperature at30℃, pH7.0, feed concentration was22.4mg·L-1. Compared the ACE inhibitory rate of different components before and after ultafiltration, the hydrolysates inhibition rate was51.26%, ultrafiltrate under10KDa with ACE inhibition rate was62.71%, ultrafiltrate under5KDa with ACE inhibition rate was88.47%.the result showed that ultrafiltration can be used to separated the active component of which can decreased blood pressure efficiently.Gel filtration chromatography and ion exchange chromatographic were used to separate the molecular weight less than5KDa ultrafiltration. And the optimum separatin conditions of gel Sephadex G-15were:ultra pured water was used as eluted solution, sample volume was1mL, eluted rate was0.8mL·min-1, and the sample protein concentration was10.2mg·mL-1. Four components were got, ACE inhibitory activity of G1#and G2#were35.32%and37.79%separately. ACE inhibitory activity of G4#was41.38%, and ACE inhibitory activity of G3#was56.38%, which was the highest ACE inhibitory activity, all those achieved the purpose of separation. The Mono Q4.6/100preloaded column was used to separated decapterus maruadsi, and the components that with different inhibitory activity would be separated in different elution conditions. The optimum separation conditions were that buffer pH was7.0, and ionic strength was10mM. In the congditions, three components were got, and with a higher inhibition rate to ACE separately, the transmitted peak with a inhibitory rate92.95%to ACE, the eluting peak T#inhibitory rate to ACE was95.71%, and the eluting peak2#inhibitory rate to ACE was96.23%, a higher total recovery of protein and the activity of lowing blood pressure were got, they were64.85%and86.85%separately. |
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