黑豆胰蛋白酶抑制剂性质分析及高温对其消化特性的影响

胰蛋白酶抑制剂(trypsin inhibitor, TI)在食品科学和生物技术中具有重要的研究价值。为探讨黑豆胰蛋白酶抑制剂(black soybean trypsin inhibitor, BSTI)的性质及胃肠液消化特性,本研究通过脱脂、热处理、硫酸铵盐析以及阴离子柱层析等方法,从黑豆中纯化得到BSTI。SDS-PAGE显示BSTI呈单一条带,相对分子质量约为21 kDa。在60℃以下与pH2~11的范围内,BSTI呈现较高稳定性。当BSTI与胰蛋白酶的摩尔比达到1时,胰蛋白酶的活力被抑制在15%以下。抑制动力学结果表明,BSTI对胰蛋白酶的抑制属非竞争性抑制,其抑制常数Ki=0.24 nmol·L^-1。圆二色谱分析发现,在常温下BSTI的二级结构为:β-折叠43.4%,无规则卷曲29.0%,β-转角21.2%,α-螺旋8.1%。BSTI的变性温度为61±0.9℃。BSTI在模拟胃肠液消化过程中表现出很强的抗酶解作用,但经121℃热处理30 min后,其抗酶解作用显著降低,易被胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶等主要消化酶消化。

Purification,Characterization of a Trypsin Inhibitor from Black Soybean [Glycine max (L.) merr.] and Effect of High Temperature on Its Digestion

Trypsin inhibitor(TI) has significant research value in food science and biotechnology. In order to investigate the properties of black soybean trypsin inhibitor(BSTI) and the digestive properties of gastrointestinal fluids, BSTI was purified from black soybeans by degreasing, heat treatment, ammonium sulfate salting out and anion column chromatography. SDS-PAGE showed a single band of BSTI with a relative molecular mass of approximately 21 kDa. BSTI was stable below 60℃ and in the range of pH2-11. When the molar ratio of BSTI to trypsin reached 1, the activity of trypsin was suppressed to 15% or less. Inhibition kinetics indicated that the inhibition of trypsin by BSTI was non-competitive type, and its inhibition constant Ki=0.24 nmol·L-1. The results of circular dichroism analysis showed that the secondary structure of BSTI at room temperature was β-sheet 43.4%, random coil 29.0%,β-turn 21.2%,α-helix 8.1% and its denaturation temperature was 61±0.9℃. BSTI was resistant to simulated gastrointestinal fluid digestion. However, after heat treatment at 121℃ for 30 min, the anti-enzymatic action of BSTI was significantly reduced, and was easily hydrolyzed by major digestive enzymes such as pepsin, trypsin and chymotrypsin.