Inactivation of Soybean Bowman–Birk Inhibitor by Stevioside: Interaction Studies and Application to Soymilk

Abstract

In this work, the interaction of the soybean Bowman-Birk inhibitor (BBI) with stevioside (STE) was studied by stopped-flow-fluorescence and molecular docking. STE's inactivation of protease-inhibitor activities in soymilk and the influence of STE addition on the sensory character of soymilk were also evaluated. The results indicate that STE binds BBI with a binding constant ( K_a) of 3.38 × 10⁵ L mol^-1 to form a 1:1 complex. The docking study reveals that two hydrogen bonds are formed between the side-chain of Lys16 (reactive site 1) of BBI and the glucose-ring hydroxyl groups of STE, which may block BBI from contacting trypsin and thus deactivate the trypsin-inhibitor activity (TIA) of BBI. Moreover, the residual TIA in soymilk could also be inactivated by STE. A mixture of 159 mg/L STE and 60 g/L sucrose could be used for sweetening soymilk without affecting the sensory characteristics when compared to a reference product sweetened with 9% sucrose.