2.8. Analysis of total carbohydrates, RPS and CPS

Total carbohydrates and RPS contents were determined as described previously (Mota et al., 2013). For the quantification of CPS, 5 ml of dialyzed cultures was centrifuged at 3,857 g for 15 min at room temperature, resuspended in water, and boiled for 15 min at 10°C to detach the CPS from the cells’ surface. After new centrifugation as described above, CPS were quantified from the supernatants using the phenol‐sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Total carbohydrate, RPS, and CPS were expressed as mg per L of culture or normalized by optical density. Data were statistically analyzed as described below.

To determine the RPS’ monosaccharidic composition, dialyzed cultures were centrifuged at 3,857 g for 10 min at room temperature to remove the cells and the supernatant was further centrifuged at 75,000 g for 1 hr 30 min at 15°C to remove LPS prior to lyophilization. 2–5 mg of isolated RPS was hydrolyzed with 1 ml of 2 M trifluoroacetic acid (TFA) at 120ºC for 1 hr. Samples were analyzed by ion exchange chromatography using a Dionex ICS‐2500 ion chromatograph with an ED50 pulsed amperometric detector using a gold working electrode (Dionex) and a Carbopac PA1 column (Dionex). The eluents used were (A) MilliQ‐grade water, (B) 0.185 M sodium hydroxide solution, and (C) 0.488 M sodium acetate solution. The gradient consisted of a first stage with 84% solution A, 15% solution B, and 1% solution C (for 7 min); a second stage with 50% solution B and 50% solution C (for 9 min); and a final stage with 84% solution A, 15% solution B, and 1% solution C (for 14 min). The flow rate was 1 ml/min.