3.1. LPS and O-Specific Polysaccharide Extraction

Bacteria were grown to late log phase in 8 L of LB media under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described [23] and the LPS was isolated from dried cells by the phenol-water method [24]. The bacterial cells were extracted by stirring under 120 rpm with 50% aqueous phenol for 30 min at 65 °C. The water phase collected by low-speed centrifugation (4000 rpm, 30 min, 4 °C) was dialyzed in distilled water until free from phenol. The dialyzate was lyophilized and dissolved in distilled water. The aqueous solution was treated sequentially with deoxyribonuclease, ribonuclease, and protease K, followed by ultracentrifugation at 8000 rpm for 30 min at 4 °C. The supernatant was then extracted with 50% aqueous phenol, followed by dialysis and lyophilisation to give the LPS. The extracted LPS was bathed in 2% (v/v) acetic acid in the quantity of 2 mg/mL at 100 °C for 3 h. The precipitated lipid A was removed by freezing ultracentrifugation (13,000× g, 30 min, 4 °C). Then, the O-specific polysaccharide was obtained after purification on Sephadex G-50 column with 0.05 M pyridine acetate buffer (pH 4.5).

The molecular weight of the O-specific polysaccharide was determined by high-performance size-exclusion chromatography (HPSEC) [24]. Waters 1525 HPLC equipped with a Ultrahydrogel Linear (7.8 mm × 30.0 cm) column was used to analyze the polysaccharide. A solution of 0.1 mol/L NaNO₃ was used as the mobile phase and the flow rate was kept at 0.5 mL/min. The eluent was monitored by a Waters 2410 refractive index detector. The column temperature was kept at 40 °C. The concentration of polysaccharide test solution was 5 mg/mL in the mobile phase and the injection volume was 50 μL. Five dextran standards (Mw 2.70, 9.75, 135.03, 300.60, and 2000 kDa) and glucose (Mw 180) were used to plot the calibration standards.

An O-specific polysaccharide sample was treated with 12% aq ammonia at 50 °C for 6 h, ammonia was flushed out, and the following lyophilization [25] afforded the O-deacetylated polysaccharide.