3.6. Trypsin Inhibitor Activity Assay

Trypsin inhibitor activity (TIA) was measured according to ISO 14902 standard method with slight modifications [88]. Trypsin activity was quantitatively determined by using the synthetic substrate Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPA, Merck Life Science, Milan, Italy). Trypsin stock solution was prepared dissolving 27 mg of trypsin (Merck Life Science, Milan, Italy) in 100 mL of 1 mM HCl with 5 mM CaCl₂. This solution was then diluted 1:20 prior to the test execution. BAPA working solution was obtained diluting 1:100 of the stock solution (1.5 mM in DMSO) in 50 mM Tris-HCl, pH 8.2, and 5 mM CaCl₂. The assay was performed by mixing 0.1 mL of protein extract (0,4 mg/mL) with 0.1 mL of BAPA working solution and 0.2 mL of water. After 10 min of incubation at 37 °C, 0.1 mL of trypsin solution was added and the sample was incubated for 10 min at 37 °C. The reaction was stopped by adding 0.1 mL of 30% acetic acid. The absorbance for the sample reading at 410 nm was a measure of the trypsin activity in the presence of the sample inhibitors (As). The reaction was also run in the absence of inhibitors by replacing the sample extract with an equal amount of water (standard). The corresponding absorbance was the reference reading (Ar). In addition, reagent blanks for the sample readings (Abs) and a reagent blank for the reference readings (Abr) were also prepared by adding the acetic acid solution before the trypsin solution.

TIA was calculated as i=[(Ar−Abr)−(As−Abs)Ar−Abr]×100

Each experiment was performed in triplicate.