C. psychrerythraea strain 34H was grown at 4°C in Marine Broth medium; the bacteria were removed by centrifugation (11.4 g, 30 min, 4°C), and the supernatant (2 L) was dialyzed against water (Spectra-Por, cut-off 10-12000 Da) obtaining 1.9 g.
An amount of 200 mg of growth broth was hydrolyzed with 5% aqueous CH3COOH (20 mL, 100 °C, 3 h). The resulting suspension was then centrifuged (11.4 g, 4 °C, 30 min). The pellet was washed twice with water, and the supernatant layers were combined and lyophilized (170 mg). The supernatant portion (160 mg) was then fractionated on a Sephacryl S-400 HR column (GE Healthcare, 1.5 × 95 cm, flow 16.8 mL/h, fraction volume 2.5 mL), eluted with 0.05 M ammonium hydrogen carbonate obtaining two fractions. The first, eluted with the void volume, contained a mixture of saccharidic material (11 mg), while the second was constituted by medium components.
The saccharidic portion was further purified on a Q-Sepharose fast flow ion exchange chromatography (GE Healthcare, 1 × 35 cm, flow rate 23 mL/h, fraction volume 2 mL) eluted with 0.1 to 1 M NaCl, obtaining a pure fraction containing the EPS (0.8 mg, yield 0.04%).
An amount of 200 mg of growth broth was hydrolyzed with 5% aqueous CH3COOH (20 mL, 100 °C, 3 h). The resulting suspension was then centrifuged (11.4 g, 4 °C, 30 min). The pellet was washed twice with water, and the supernatant layers were combined and lyophilized (170 mg). The supernatant portion (160 mg) was then fractionated on a Sephacryl S-400 HR column (GE Healthcare, 1.5 × 95 cm, flow 16.8 mL/h, fraction volume 2.5 mL), eluted with 0.05 M ammonium hydrogen carbonate obtaining two fractions. The first, eluted with the void volume, contained a mixture of saccharidic material (11 mg), while the second was constituted by medium components.
The saccharidic portion was further purified on a Q-Sepharose fast flow ion exchange chromatography (GE Healthcare, 1 × 35 cm, flow rate 23 mL/h, fraction volume 2 mL) eluted with 0.1 to 1 M NaCl, obtaining a pure fraction containing the EPS (0.8 mg, yield 0.04%).