| Soybean peptides have various physiological activities, showing good development prospect in the fields as foodstuff, pharmaceuticals, healthcare and daily-use chemicals. However, the bitter taste generating from the manufacturing process restricted further development of soybean peptides. Therefore, debitterness of soybean peptides has attracted many concerns at the present time. Many debittering methods have been developed and the enzymatic debittering shows a great effect. In this study, the sprouted soybean was used as raw material to extract a set of proteases, and the debittering function of the proteases was demonstrated. Meanwhile, the antioxidant activites of the debittering products after hydrolysis was investigated.The change of basic nutrients during soybean germination was measured. Based on the change of protease activity and SDS-PAGE trends, the soybean after six-day germination was selected to extract the protease which has the debittering function. Single factor optimum experiments showed that the optimal extracting solution was 0.3 M Na Cl with two times extraction. The optimal enzymatic conditions were carried out at temperature = 50 ℃, p H = 5.5, time = 3 h. Nine components were separated by saturated(NH4)2SO4 segmentation precipitations from the crude enzyme solution of six-day soybean sprouts. High enzyme activity components were obtained from 40 %(0.17±0.01 U/mg), 50 %(0.21±0.02 U/mg), and 60 %(0.45±0.05 U/mg) saturated(NH4)2SO4 precipitation Double-step enzymatic hydrolysis was conducted, alcalase was first used to obtain bitter peptides, then the protease components with the high enzyme activity were used for debittering. The results showed three components of the soybean sprouts proteases had good debitterizing effect and 50 % salting out component was better than that of 40 % and 60 %. The 50 % components could remove bitterness in the 3 h hydrolyzation process. Then the 50 % salting out component protease was isolated and purified, and two purifiedproducts(G1 and G2) were isolated by DEAE-Sepharose FF chromatography and Sephadex G-100 chromatography. The G1 had an excellent debitterizing effect. The enzyme activity of G1 was 2.67±0.1 U/mg and the purifying times increased by 20.54. HPGPC showed the G1 had two peaks and its average molecular weight was 47.74 ku. The FTIR, CD were used to analyze its structure and the results showed that the set of proteases was mainly composed of β-sheet(about 36.44%), random coil(about 30.85%), with a small amount of α-helix(about 16.36%), β-turn(about 16.36%).DH, yield of soluble peptides and protein recovery rate of the debitterizing products obtained from G1(hydrolysate E) was studied, and it was shown that they all increased correspondingly, which could improve the utilization of soybean protein. Additional, the hydrophobicity of hydrolysate E were analyzed by Q value. The result was showed the hydrophobicity of hydrolyzed amino acids reduced to 35.14 k J/mol after hydrolysis, while the hydrophobicity of free amino acids increased to 35.37 k J/mol. The free hydrophobic amino acids(mainly devoted to the bitter taste) reduced largely after G1 hydrolysis, therefore the hydrolysate E was debittering. Moreover, The scavenging ability for free radicals(DPPH·, ·OH, O2-·) and total reducing power of hydrolysate E were prominently enhanced than bitter soybean peptides. The study provides a new protease for the debitterizing technology of soybean peptides. |
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