2.4. Qualitative Analysis of Monosaccharide Composition

Monosaccharide composition of the polysaccharide was analyzed by reverse-phase HPLC after PMP derivatization [2,18,20,21]. To hydrolyze polysaccharides, 300 μL polysaccharide solution (BCG-1 or BCG-2, 1 mg/mL in deionized water) was mixed with 300 μL TFA (4 M) at 110 °C for 4 h in the sealed COD tube. Then, the reaction solution was evaporated to dryness at 70 °C to remove the residual TFA. Then, 100 μL of the hydrolyzed sample solution was incubated with 100 μL sodium hydroxide (0.6 M) and 200 μL PMP (0.5 M in methanol) at 70 °C for 60 min. After reaction, the solution pH was adjusted to 7.0 by adding HCI (0.3 M). Then, the solution was added and mixed with 2 mL of chloroform, and the chloroform layer was discarded; this step was repeated for at least six times and the top aqueous layer was collected for HPLC analysis. The standard monosaccharides (Man, Rib, Ara, Glc, and Gal) were processed by the same procedures.

The analysis of the PMP-labelled monosaccharides was carried out using an Agilent technologies 1260 series (Agilent, CA, USA) which was equipped with DAD detectors and a ZORBAX SB C18 column (4.6 mm × 150 mm, 5 μm). Mobile phase A and B (v/v, 83:17) were ammonium acetate (0.1 M, pH 5.5) and acetonitrile, respectively, at a flow rate of 1 mL/min, and UV absorbance of the effluent was monitored at 250 nm.