Rid10

The extraction of polysaccharides was performed according to a previously described method by a previously described procedure [17 (link)]. Briefly, 200 mg of powdered seaweed was incubated in 20 ml of 85% ethanol at 80 °C for 4 h to remove lipids. This extraction operation was repeated three times. Polysaccharides in the residue were extracted with 30 ml of cold water, dialyzed against distilled water, and then freeze-dried (32.0 mg). The total sugar content was determined by phenol–sulfuric acid method using galactose as a standard [18 (link)]. The sulfate content was determined by the BaCl₂-gelatin method [19 (link)] using κ-carrageenan (TCI, Tokyo, Japan) as a positive control. The relative molecular weight of the polysaccharides was determined by high-performance size exclusion chromatography with Shodex OHpak SB-807G (Guard), SB-807 HQ, and SB-806 M HQ (8.0 mm ID × 300 mm length; Showa Denko KK, Tokyo, Japan) at 40 °C and estimated using dextran standards (150, 270, and 670 kDa from Sigma Aldrich Corp., 3,755 kDa from American Polymer Standards Corp., Mentor, OH, USA). Sample (injected volume: 20 μl) was eluted using 0.3 M NaNO₃ at a flow rate of 1 ml/min and was detected using a refractive index (RI) detector RID10 (Shimadzu Corp., Kyoto, Japan).

The concentration of released sugars (glucose, cellobiose, xylose and arabinose) was determined by HPLC (Shimadzu, Kyoto, Japan) using a Supelcogel C610H column (300 mm × 7.8 mm; Supelco Analytical, Bellefonte, PA, USA) with matching guard column at 55 °C and 0.1% phosphoric acid as eluent at a flow rate of 0.5 mL min⁻¹. The sugars were detected by a refractive index detector (Shimadzu RID-10).

Extraction and purification of polysaccharide from G. furcata was carried out via the previously reported procedure (Hu et al., 2012 (link)). In brief, the powdered 100 mg of G. furcata was extracted with 10 ml of 85% ethanol at 80°C for 4 h (three times) to remove lipids. Polysaccharide in the residue was extracted with 10 ml of cold water, dialyzed against distilled water, and then freeze‐dried (13.0 mg). The total sugar content was determined by the phenol‐sulfuric acid method using galactose as a standard (DuBois et al., 1956 ). Sulfate content was determined by the BaCl₂‐gelatin method (Ji et al., 2013 (link)) using κ‐carrageenan (TCI, Tokyo, Japan) as a positive control. The protein content was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). The relative molecular weight of polysaccharide was determined by high‐performance size exclusion chromatography with Shodex OHpak SB‐807G (Guard), SB‐807 HQ, and SB‐806M HQ (8.0 mm ID × 300 mm length; Showa Denko KK, Tokyo, Japan) at 40°C and estimated using dextran standards (150, 270, and 670 kDa from Sigma Aldrich Corp., 3755 kDa from American Polymer Standards Corp., Mentor, OH, USA). Samples (injected volume: 20 μl) were eluted using 0.3 M NaNO₃ at a flow rate of 1 ml/min and were detected using a refractive index (RI) detector RID10 (Shimadzu Corp., Kyoto, Japan).