Porcine mucin type 3

The previously reported method was used to determine the in vitro mucin adhesion assay [45 (link)]. Flat-bottom 96 well microtiter plates were coated with 300 µL porcine mucin type III (10 mg/mL (Sigma-Aldrich, Inc. St. Louis, MO, USA) suspended in sterile PBS kept overnight at 4 °C. Plates were washed thrice with sterile PBS to remove unbound mucin from the wells. For competitive mucin adhesion, 100 µL of LAB culture with absorbance adjusted to 0.25 ± 0.05 and 100 µL of each of the Salmonella serovars were added to the mucin-coated wells simultaneously and were co-incubated for 90 min. After incubation, the wells were washed five times with PBS. Adhered bacterial cells were then treated with 300 µL Triton X in a sterile phosphate buffer solution to the separate bacterial cells. The viable cell count was measured by enumerating the LAB strains and Salmonella serovars on MRS agar, M17 agar, and SS agar. The % relative adhesion was calculated by (CFU/mL after adhesion/CFU/mL before adhesion) × 100.

A. muciniphila (ATCC-BAA-835) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and cultivated into liquid anaerobe basal medium (OXOID, ThermoFisher), supplemented with 0.05% porcine mucin type III (Sigma) and 4 g/L D-Glucose (Sigma) in an anaerobic workstation (Baker Ruskinn) at 37°C. The concentration of OSH, its precursors, and fibers to test was calculated based on the medium uptake ratio (MUR) of the OSH in these conditions, being that able of occupying half the volume of the culture tube when the hydrogel was fully hydrated in steady state (5 g/L). MUR was determined as described previously¹⁵ (link). All the experiments included a control tube with half the volume of the medium to avoid any concentration bias due to the absorption capacity of the OSH.

To co-culture microbiota and TR146 epithelial cells in non-infectious conditions, we used our recently published model (De Ryck et al. 2014 (link)). Briefly, 75 µL of an agar/mucin solution [5% porcine mucin type III (Sigma-aldrich, Diegem, Belgium), 0.8% agar (BD, Erembodegem, Belgium)] was brought on the porous membrane (0.4 µm) of a 24-well plate Transwell^® system (Corning Inc., NY, USA) and allowed to solidify for at least 30 min after which 20 µL of a microbial suspension was spotted on top of this agar/mucin layer (apical compartment). In the basal compartment, a monolayer of epithelial cells was grown and a wound scratch assay was performed as described below. During co-culture, the inserts with the microbiota were transferred into the wells with the wounded epithelial cells. For experiments with pre-incubation, inserts with microbiota were incubated for 4 h in wells filled with serum-free, antibiotics-free DMEM without epithelial cells, prior to their transfer into the wells with wounded epithelial cells.