Mucin from porcine stomach type 3

Elastolytic, glutenasic, and mucolytic activities were measured in small intestinal contents and in bacterial culture supernatants. Glutenasic activity was also determined in human duodenal biopsies. Glutenasic and mucolytic activity were measured by bioassay (1.6% agar plates) using gluten 1% (Sigma-Aldrich) or mucin from porcine stomach type III 0.5% (Sigma-Aldrich), respectively^{62 (link)}. Samples were incubated in the gluten or mucin-agar plated for 14 h. Amido black was used to stain protein in the mucin bioassay. Positive proteolytic activity was determined by the presence of a halo surrounding the inoculation site on substrate-containing media. Elastase activity was analyzed using Suc-Ala3-pNa (Sigma-Aldrich) or FITC-elastin (AnaSpec) substrates. Briefly, small intestinal washes or bacterial supernatants were incubated in 50 mM Tris-HCl buffer pH 8.2 supplemented with 1 mM CaCl₂, 50 mM NaCl, and Triton 0.25% at 37 °C with the different substrates. Absorbance and fluorescence were measured at various time points. Units of enzyme were determined using standard curves of elastase from porcine pancreas (Sigma-Aldrich). Tryptic activity was determined using Trypsin Activity Assay Kit from Abcam following the manufacturer's recommendations (Abcam, Cambridge, UK).

E80 egg phospholipids (80% phosphatidylcholine) and S100 soybean lecithin lipids (>94% phosphatidylcholine) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Acetic acid, bovine serum albumin, calcium hydroxide, chitosan (low molecular weight, 75–85% deacetylated), chloroform, D(+)-glucose, glycerol solution, ibuprofen, lactic acid, mucin from porcine stomach type III, potassium phosphate monobasic, propylene glycol, sodium chloride, sodium hydroxide, and sodium phosphate dibasic dodecahydrate were products of Sigma Aldrich Chemie GmbH (Steinheim, Germany). Ethanol, mEthanol, and Acetic acid were obtained from VWR Chemicals (Fontenaysous-Bois, France). Potassium hydroxide was obtained from Norsk Medisinaldepot (Oslo, Norway).

Meloxicam (MEL, 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-benzothiazine-3-carboxamide-1,1-dioxide) was donated by EGIS Pharmaceuticals Plc. (Budapest, Hungary) for research work. Human serum albumin (HSA, lyophilized powder, purity > 97%), fluorescein isothiocyanate-labelled HSA (FITC-HSA), Tween 80 (Tween), P407, disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), polar brain lipid extract, cholesterol, mucin from porcine stomach (Type III), and all reagents for cell line studies were purchased from Sigma Aldrich Co. Ltd. (Budapest, Hungary) if not indicated otherwise. Analytical grade solvents such as methanol, dimethyl sulfoxide (DMSO) and dodecane were purchased from Molar Chemicals (Budapest, Hungary). Sodium hyaluronate (NaHA, Mw = 1400 kDa) was obtained from Gedeon Richter Plc. (Budapest, Hungary). In all experiments, water was purified by the Millipore Milli-Q^® 140 Gradient Water Purification System.