2.10. Protease Activity Assay

Total protease activity was measured in the concentrated filtrates using casein as substrate [25]. In brief, different Cn filtrate volumes (250, 500, and 1000 µL) were adjusted to 1 mL with the enzyme solution (10 mM sodium acetate buffer with 5mM calcium, pH 7.5) to make a final volume of 1 mL. The assay was carried out by using 5 mL casein solution equilibrated at 37 °C in a water bath, and then 1 mL filtrate concentrates was added to casein solution and incubated at 37 °C for 10 min. After incubation, 5 mL TCA solution was added to stop the reaction. The final volume in the blank tubes was adjusted by adding 1 mL of the enzyme solution. Samples were incubated at 37 °C for 30 min in a water bath, then filtered using a 0.45 µm filter. Color was developed by adding 2 mL sample mixture with 5 mL sodium carbonate solution and 1 mL Folin’s reagent. Samples were incubated at 37 °C for 30 min in a water bath, then filtered using 0.45 µm filters. The absorbance of the developed blue color was measured with the Harvard Biochrom Ultrospec 2100 pro UV/Visible Spectrophotometers (ThermoFisher Scientific) at a wavelength of 660 nm. The activity was calculated on the basis of a standard curve generated using l-tyrosine (Sigma-Aldrich, St. Louis, MO, USA). The experiment was carried out with 3 biological replications and the entire experiment was repeated once.