Trypsin activity of crude extract was measured using BAPNA as a substrate according to the method of Khantaphant and Benjakul (2008). A 200 µl of sample was mixed with 200 µl of distilled water and 1000 µl of reaction buffer (50 mM Tris–HCl buffer, pH 8.0, containing 10 mM CaCl2). The reaction was initiated by adding 200 µl of 2 mg/ml BAPNA to the reaction mixture. After incubation for 20 min at 60 °C, 200 µl of 30% acetic acid (v/v) were added to terminate the reaction. Production of p-nitroaniline was measured by monitoring the absorbance of reaction mixture at 410 nm (A410) using a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). A blank was conducted in the same manner except that the sample was added after addition of 30% acetic acid. One unit was defined as the amount of trypsin causing an increase of 1.0 in A410/min.