Dextrans

Yingshan Cloud Mist Tea was purchased from Yingshan County, Caotangchun Tea Co. Ltd. (Hubei Province, China). Dextrans of different molecular weight (T-1300, T-6000, T-12000, T-22000, T-50000, T-110000, T-400000, and T-800000) and the standard monosaccharides samples (rhamnose, fucose, arabinose, xylose, mannose, glucose, and galactose) used in this study were purchased from Sigma Chemical Co. (MO, USA). 1,1-diphenyl-2-picrylhydrazyl (DPPH), the ascorbic acid, and trichloroacetic acid solution were purchased from Aladdin Biotechnology Co. Ltd. (Shanghai, China). The commercial assay kits for GSH-PX, SOD, MDA, and T-AOC were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China). All other chemicals and reagents were of analytical grade in this study.

The organic solvents, inorganic salts, and acids used in the study were manufactured by Dia-m (Moscow, Russia). The sorbent for chromatography Macro-Prep DEAE was purchased from Bio Rad Laboratories, Inc. (Hercules, CA, USA). The phosphate-buffered saline (PBS), L-glutamine, penicillin/streptomycin solution (10,000 U/mL, 10 µg/mL), Minimum Essential Medium Eagle (MEM), and reference standards (mannose, rhamnose, glucose, galactose, xylose, and dextrans) were purchased from the Sigma-Aldrich company (St. Louis, MO, USA). The MTS reagent 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide was purchased from Promega (Madison, WI, USA). The trypsin, fetal bovine serum (FBS), 2-deoxy-D-glucose (2-DG), and the protein marker PageRuler^TM Plus Prestained Protein Ladder were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
The cell lysis buffer (10×), glycolysis antibody sampler kit #8337, antibodies against Glut 1, and horseradish peroxidase (HRP) conjugated secondary antibody from rabbit and mouse were purchased from Cell Signaling Technology (Danvers, MA, USA), and β-actin was purchased from the Sigma-Aldrich company (St. Louis, MO, USA).

Vi, freeze dried as the sodium salt, was solubilized in water and H₂O₂ was added to give a final concentration of 2.5 mg/mL Vi and 5% (wt/v) H₂O₂ in water. The mixture was heated at 80±0.5°C for 2h. The mixture was then injected into a Hiscreen Capto Q [GE Healthcare] column (4.7 mL of resin loading up to 100 mg of fragmented Vi mixture) equilibrated with buffer A and populations of different average size were separated using a gradient step method. NaH₂PO₄ 20 mM pH 7.2 and NaH₂PO₄ 20 mM NaCl 1M pH 7.2 were used as buffer A and B respectively. Pools at average size Vi of 8.6 and 43 kDa were eluted at 25 and 37% of buffer B respectively. Each pool was desalted on a Sephadex G-25 column [GE Healthcare] equilibrated with water. The average size of the fragmented Vi pools was determined by HPLC-SEC equipped with a TSK gel 3000 PW_XL column and a TSK gel PW_XL guard column (Tosoh Bioscience). Dextrans (5, 25, 50, 80, 150 kDa) were used as standards (Sigma Aldrich). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). HPAEC-PAD was used to measure Vi content [10 (link), 21 (link)]. ¹H NMR was used to verify Vi identity and confirm O-acetylation levels were >60% [10 (link), 21 (link)].