Glucoronic acid

Cholesterol, cholic acid, human thrombin (EC: 3.4.21.5), human fibrinogen, O-Phthaldialdehyde reagent (OPA), pepsin (EC: 3.4.23.1), pancreatin (EC: 232-468-9), bile salts, α-amylase (EC: 3.2.1.1), lysozyme (EC: 3.2.1.17), sodium taurocholate, bile assay kit, heparin sodium salt, mucin, bovine serum albumin (BSA), glucoronic acid, trichloroacetic acid, L-leucine, cholestyramine, linoleic acid, trichloroacetic acid, galactose and glucosamine were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). M17 broth, lactose and dextrose were purchased from DIFCO (Sparks, MD, USA). Thrombin time and pro-thrombin time kits were purchased from Wiener Lab (Rosario, Argentina). A Cholesterol assay kit was purchased from RANDOX Laboratories (Crumlin, UK). The DC Lowry protein assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Extracellular polymeric substances were extracted according to Barranguet et al. (2004) (link). Briefly, 2 mL biofilm samples were resuspended in bi-distilled water, vortexed and centrifuged (5 min, 3500 rpm). The supernatant was collected and represents the soluble EPS fraction. The bound EPS fraction was extracted from the residual pellet after incubation at 95°C for 30 min in 2 mL 0.1 M H₂SO₄. Both fractions were analyzed for four groups of substances by comparing the spectrophotometric measurements to the ones of the standard substances. The carbohydrate content of the EPS was quantified according to the phenol-sulphuric acid assay by DuBois et al. (1956) (link) with D-Glucose (Sigma-Aldrich, CAS 50-99-7) as a standard. For protein and humic acid analysis the Folin phenol assay by Lowry et al. (1951) (link) was used. Results had to be corrected due to interferences of humic acids after Frolund et al. (1995) (link). Bovine Serum Albumin (Biorad, CAS n.a.) was used as standard substance for the protein assay, and humic acid (Roth, CAS 1415-93-6) for the humic acid assay. A hydroxydiphenyl assay by Blumenkrantz and Asboe-Hansen (1973) (link) and its modification by Kintner and Van Buren (1982) (link) was used to assess the uronic acid content. Glucoronic acid (Sigma, CAS 6556-12-3) was used as the standard substance.