2.10. Evaluation of Mucin Degradation Activities

The ability of the selected potential probiotic LAB to degrade mucin was measured using two different methods, agar plate assay and test tube assay, according to a previous report with modifications [63]. Human fecal bacteria (HFB), used as a positive control in this study, were obtained from one of the coworkers under the regulations of the ethics committee on human-related studies at OUAVM. Autoclaved HFB (AHFB) was used as the negative control. Hog gastric mucin (HGM Type III; Merck) was purified and used in both experiments. Briefly, small circles of the bacterial cells were drawn on agar plates containing the following: 0.5% (w/v) HGM Type III, 7.5 g tryptone, 7.5 g casitone, 5.0 g yeast extract, 5.0 g beef extract, 5.0 g NaCl, 3.0 g K₂HPO₄·3H₂O, 0.5 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 0.5 g L-cysteine HCl, 0.002 g resazurin, 15 g agarose, and 0 or 30 g glucose per liter of deionized water (pH 7.2). The agar plates were incubated under anaerobic conditions at 37 °C for 72 h. After incubation, agar plates were stained with 0.1% (w/v) amido black dissolved in 3.5 M acetic acid for 30 min at 25 °C, and then washed with 1.2 M acetic acid solution until clear zones were observed around colonies. Next, for the test tube assay, bacterial cells were incubated at 37 °C for 18 h in MRS, harvested, washed twice with PBS, inoculated in a medium with a similar composition to that used in the above assay, but without agar, and incubated at 37 °C for 48 h. After incubation, the remaining mucin in the cultivated medium was collected and subjected to 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB) and periodic acid–Schiff (PAS) reagents.