Ultrahydrogel linear

The ¹H NMR analysis was carried out on a 400 MHz Bruker NMR spectrometer (Figures S1 and S2) [12 (link)]. The aldehyde group content of A-HA was determined with 3,5-dinitrosalicylic acid assay method [13 (link)]. The substitute degree was determined as 23.4%. The FT-IR spectra were performed on an iS5 FT-IR instrument (Thermo Fisher, Waltham, MA, USA) and scanned from 400 to 4000 cm⁻¹.
The relative molecular weight and distributions of the polysaccharides were determined on a Waters 1525 high performance liquid chromatograph equipped with the column of Ultrahydrogel™ Linear (300 mm × 7.8 mm) (Table S1). The column temperature was kept at 40 °C. Sodium nitrate solution (0.1 M) was used as the mobile phase with the flow rate of 0.8 mL/min. The standards (dextran T-2000, Dextran T-300, Dextran T-150, Dextran T-10 and Dextran T-5) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, with the Mw of 2,000,000, 30,060, 135,030, 9750 and 2700, respectively.

The clear juice (prepared as described in Section 2.2) was precipitated with 4 volumes of 80% ethanol, and the precipitate was freeze-dried to prepare water-soluble polysaccharide samples. The water-soluble polysaccharide samples were redissolved in deionized water, and filtered with 0.45 µm membrane. The molecular size distribution of the water-soluble polysaccharides was analyzed using a GPC system (Shimadzu LC-20AT) equipped with a refractive index detector (Kyoto, Japan). Samples were separated using Ultrahydrogel linear (Waters, Milford, MA, USA) and G3000PWXL (Tosoh Corporation, Tokyo, Japan) columns. The elution buffer was 0.2 mol/L sodium nitrate at a flow rate of 0.4 mL/min.

The molecular weight of HWE-VVP or UAE-VVP was determined by high performance size exclusion chromatography (HPSEC) on a Waters 1525 HPLC system (Waters Corporation, Milford, MA, USA) equipped with an Ultrahydrogel Linear (7.8 mm × 300 mm, Waters Corporation) and a 2414 refractive index detector. HWE-VVP or UAE-VVP (2.0 mg) were dissolved by 1.0 mL NaNO₃ (0.1 M) and loaded into the chromatography system (15 μL). The columns were maintained at 45 °C and eluted with 0.1 M NaNO₃ at a flow rate of 0.9 mL/min. Preliminary calibration of the column was conducted by using standards dextrans (Sigma-Aldrich, St. Louis, MO, USA) with different molecular weights. The molecular weight (Mw) of HWE-VVP or UAE-VVP was calculated by comparison with the calibration curve [49 ].

The molecular weight of H. erinaceus polysaccharide was determined by High-performance Gel Permeation Chromatography (HPGPC, 6000 high performance liquid chromatograph, Waters, MA, USA). A 300 mm × 7.8 mmid × 2 Ultrahydrogel™ Linear (Waters, Milford, MA, USA) was eluted with 0.1 M NaNO₃ (0.9 mL/min, 45 °C) and determined by a Waters 2410 RI detector. Calibrating the average molecular weight was performed using a pullulan polysaccharide calibration kit (Agilent Technologies, Santa Clara, CA, USA).