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In this study, quantitative monitoring of low-frequency (20 kHz) and high-frequency (355 kHz) ultrasound-induced inactivation of Kunitz (KTI) and Bowman-Birk inhibitor (BBI) using RP-HPLC was achieved, and its consistency with a traditional TI activity assay was verified. The effect of TI concentration, ultrasonic frequency, power density and pH on inactivation kinetics of KTI and BBI was explored. Results showed that the pseudo-first-order kinetic rate constants of KTI and BBI were decreased by over 60% when the initial TI concentration was increased from 100 mg/L to 1000 mg/L. Also, the amounts of inactivated KTI and BBI were increased by around 4-fold at the higher TI concentration of 1000 mg/L (20 kHz, 1.71 W/mL and pH 4). The colloidal environment and ultrasonic conditions influenced the secondary and tertiary structure and particle size of TIs in LF-induced inactivation. In comparison, the abovementioned conditions affected the oxidation of methionine and the conformational change of TIs in HF-induced inactivation. |
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