Trypsin Inhibition Activity Assay

Modified azocasein protease assay, as described in our previous works, was used to test the trypsin inhibition activity of the extracted ATIs [31 (link)]. Briefly, 75 µL of extracted ATIs was mixed with 30 µL of 0.5% sodium bicarbonate buffer pH 8.3 and 20 µL of 10 mg/mL trypsin solution (Sigma Aldrich,). For the control, 20 µL of trypsin was mixed with 105 µL of buffer. 125 µL of 2.5% azocasein solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was added and the mixture was incubated at 37 °C for 30 min. Then, 100 µL of the mixture was mixed with 400 µL of 5% trichloroacetic acid solution, incubated at room temperature for 5 min, and centrifuged for 5 min at 10,000× g. A total of 400 µL of the above solution was mixed with 1200 µL of 500 mM NaOH solution and the absorbance was taken at 440 nm. The trypsin activity was expressed as the amount of activity that gave a change of one unit of absorbance at 440 nm and the inhibition of trypsin activity was calculated as reported in [3 (link)]. The results were expressed as percentage of inhibition.