Porcine stomach type 3

Total proteolytic, elastolytic and mucolytic activities were measured using fecal supernatants from mice. Non-specific proteolytic activity was determined using the substrate azocasein (Sigma-Aldrich) as previously described.^{13 (link)} Fecal supernatants were incubated in 50 mmol/L Tris-HCl buffer pH 8.2 supplemented with 1 mmol/L CaCl2, 50 mmol/L NaCl, and Triton 0.25% (weight/volume (w/v)), and 0.5% (w/v) azocasein at 37ºC. The reaction was stopped by adding 10% trichloroacetic acid, and absorbance was measured at 366 nm. Elastolytic activity was analyzed using the substrate Suc-Ala3-pNa (Sigma-Aldrich). Fecal supernatants were incubated in the same buffer at 37ºC with the substrate. Absorbance at 410 nm was measured at several timepoints. Enzyme activity was determined by using a standard curve of pancreatic porcine elastase (Sigma-Aldrich). Mucolytic activity was determined by a bioassay using Brain Heart Infusion (BHI) media enriched with 0.05% (w/v) of mucin from porcine stomach type III (Sigma-Aldrich). Fecal supernatant was spotted into agar wells and incubated for 24 h. Zones of clearance were measured after staining agar plates with 10% amido black for 24 h. A standard curve was used to determine mucin-degrading activity of the samples using collagenase type I (0.25–1.0 FALGPA units/mg) from Clostridium histolyticum (Sigma-Aldrich).