Azocasein solution

Non-specific protease activity of both casein and whey fractions isolated from raw and heated ruminant milk samples were determined as previously described (17 (link)–19 (link)) using azocasein (a protein coupled with diazotized aryl amines) as substrate.
Briefly, 1 ml of 1% azocasein solution (catalog A2765, Sigma-Aldrich, St. Louis, USA) was combined with 100 μl of casein or whey fraction. Samples were incubated at 36°C for 15 min, and the reaction was stopped by the addition of 2 ml of 5% TCA solution (catalog T6399, Sigma-Aldrich, St. Louis, USA). Samples were centrifuged at 2455 g at room temperature for 4 min, and the supernatant was filtered on paper Whatman 1 (Sigma-Aldrich, St. Louis, USA). The absorbance of the filtrate was read at 345 nm in a UV/Visible spectrophotometer (Genesys, Thermo Fisher Scientific, Massachusetts, USA).
Absorbance values were compared to a standard calibration curve, which was generated by mixing 100 μl of protease from Bacillus sp. solution (16 U/g, catalog P3111, Sigma-Aldrich, St. Louis, USA) at different concentrations with 1% azocasein solution. The absorbance was determined as described above at 345 nm, and the coefficient of determination (r²) obtained was 0.9991. The analysis was performed in triplicate.

Proteolytic activity was assayed by using 2.5% (w/v) azocasein solution (Sigma). Briefly, 2 ml of human duodenal secretion from a representative healthy individual or 10 μl of trypsin enzyme solutions (10 mg/ml) (positive control) or without enzyme solutions (negative control) were added in reaction buffer (50 mM ammonium bicarbonate buffer, pH 7.8, 37°C). The mixture was incubated at 37°C for 30 min. The reactions were terminated by adding 800 μl of 5.0% (v/v) trichloracetic acid, followed by centrifugation at 14.000 rpm for 5 min. 100 μl of the supernatant was neutralized by adding 100 μl of 500 mM NaOH and the absorbance at 440 nm was measured. Experiment was repeated using independent experiments. Data are presented as mean +/- SEM of two experiments.

The anti-protease activity was determined as follows [30 (link)]: 10 μL of skin-mucus sample (undiluted) or 5 μL of sample serum (1:2 dilution) were mixed with a standard trypsin solution (20 μL; 5 mg mL⁻¹, Lonza, Basel, Switzerland) and incubated for 10 min (RT). Azocasein solution (60 μL; 1% (w/v), Sigma-Aldrich, St. Louis, MO, USA) was added to the mixture and incubation continued for 1 h (RT). Following this, trichloroacetic acid (TCA) (100 μL, 10% (w/v), Applichem, Darmstadt, Germany) was added and incubated for 30 min. The mixtures were centrifuged (1900× g, 10 min), and 100 μL of each supernatant were transferred to a clean flat-bottom 96-well transparent microplate containing 100 μL of 1 M NaOH per well. The absorbance was measured at 450 nm. Each sample was expressed as the percentage of trypsin inhibition. The percentage inhibition of trypsin activity was calculated by comparing it with a 100% control sample, in which buffer replaced the serum, while for the negative control, buffer replaced both serum and trypsin, based on the equation % Trypsin inhibition = (OD_Trypsin − OD_Sample)/OD_Trypsin × 100.