4.2.2. Purification of Crude Polysaccharides

Four hundred mg of each crude polysaccharide (BP, SP and RP) powder was dialyzed (cutoff of 3500), redissolved in deionized water, filtered through a 0.45 μm filter, and applied to a DEAE-Sepharose (Fast Flow, FF) column (50 mm × 40 cm, Beijing Rui Da Heng Hui Science Technology Development Co., Ltd., Beijing, China), as described by Huang et al. [91]. Briefly, neutral polysaccharide fractions were firstly eluted with deionized water, the elutes were collected, concentrated and lyophilized, and BNP, SNP and RNP were obtained. Then, acidic polysaccharide fractions were obtained after being eluted with a linear NaCl gradient solution (0–1.5 mol/L, 2 mL/min). The elution solution was collected using a computer automatic collector (5 min per tube and 10 mL per tube). Take 200 μL of the elution solution, mix with 200 μL phenol and 1 mL sulfuric acid; cool and transfer 200 μL for determination using UV spectrophotometry (Multiskan SkyHigh, Thermo Fisher, Waltham, MA, USA) with the absorbance value at 490 nm. Then, use absorbance as the vertical axis and the number of tubes as the horizontal axis to create an elution curve, and merge the required parts based on the elution peak. After pooling eluates together based on the elution profile, concentrating and dialyzing (cutoff 3500 Da), acidic polysaccharide fractions were obtained and named BAP, SAP and RAP, respectively.