Methylation analysis was conducted according to the method of Needs and Selvendran (35). Uronic acid was firstly reduced into neutral sugars using NaBD4 prior to methylation analysis. Subsequently, the reduced polysaccharide (5 mg) was dissolved in DMSO (0.5 ml) and methylated with a suspension of NaOH/DMSO (0.5 ml) and iodomethane (1.0 ml). The reaction mixture was extracted with CH2Cl2, and then the solvent was removed via vacuum evaporation. Complete methylation was confirmed by the disappearance of the -OH band (3,200-3,400 cm−1) in the FT-IR spectrum. The per-O-methylated polysaccharide was subsequently hydrolyzed using HCOOH (85%; 1 ml) for 4 h at 100°C and then CF3COOH (2 M; 1 ml) for 6 h at 100°C. The partially methylated sugars in the hydrolysate were reduced using NaBH4 and were then acetylated. The resulting alditol acetates were analyzed using gas chromatography-mass spectrometry (GC-MS; 7890B-5977B; Agilent Technologies, Inc.) with a DB-35 ms capillary column (30 m × 0.32 mm × 0.25 mm), as previously described (27). The oven temperature was programed from 120°C (hold for 1 min) to 210°C (hold for 2 min) at 3°C/min, then up to 260°C (hold for 4 min) at 10°C/min. The temperature of both the inlet and detector was 300°C. Helium was used as the carrier gas. The mass scan range was 50.0-500.0 m/z.

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