Toyopearl deae 650m

All chemicals used in this study were of the highest grade available and were obtained from Sinopharm Chemical Reagent (Nanjing, China). Toyopearl DEAE-650 M and butyl-Toyopearl 650 M were obtained from Tosoh (Osaka, Japan). Chitopearl BCW-2605 and Chitopearl BCW-2503 (chitosan beads) were purchased from Fujibo (Tokyo, Japan).

Fresh roots of P. heterophylla were obtained from a P. heterophylla planting base in Zherong, Fujian, China. The fungal strains, F. oxysporum and C. gloeosporioides, were kindly provided by the Institute of Agricultural Bio-Resources Research, Fujian Academy of Agricultural Sciences, Fuzhou, China. Toyopearl DEAE-650M and Sephadex G50 Fine were purchased from TOSOH Co., Ltd. (Tokyo, Japan) and GE Healthcare (Gothenburg, Sweden), respectively. Trypsin and BAPNA were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Prior to the molecular mass and constitution sugar analyses, the GI-containing fraction was further purified based on the EPS purification procedure described previously [25 (link)]: the concentrated GI fraction prepared from the culture supernatant of BM53-1 was mixed with the same volume of acetone and incubated overnight at 4 °C. The precipitate was collected and dissolved into a 50 mM Tris-HCl buffer (pH 8.0) and treated with nuclease and proteinase K. The GI collected by ethanol precipitation was dissolved into distilled water and applied to the anion exchange resin (TOYOPEARL DEAE-650M, Tosoh Bioscience, Tokyo, Japan). The column through fraction was pooled and then dialyzed against distilled water using an Amicon Ultracel-10K ultrafiltration device.
The molecular mass of the GI was estimated with gel-filtration column (Sephacryl S-500 HR, GE Healthcare, Chicago, IL, USA) chromatography using a high-performance liquid chromatography (HPLC) system as follows: a 50 mM Tris-HCl buffer (pH 8.0) was used as a mobile phase at a flow rate of 0.5 mL/min. The eluent was divided into 95 fractions every 3 min from 0 to 285 min, and the GI content was monitored using a phenol-sulfuric acid method [37 (link)]. The molecular mass was calculated from the calibration curve made by dextran standards of known molecular weights: 25, 50, 270, and 670 kDa.

The stock culture of the IJH-SONE68 strain stored at −80 °C was inoculated into a fresh MRS medium. After the two-day standing cultivation at 28 °C, the cells collected by centrifugation were washed with a sterilized phosphate-buffered saline (PBS: pH 7.4) and resuspended into the same volume of PBS. The resulting cell suspension was inoculated into a modified SDM with a 0.2% volume and grown at 28 °C for 2 days. After the cultivation, at a final concentration, a 4% (v/v) volume of trichloroacetic acid (TCA) solution was added to the culture broth. The culture supernatant obtained by centrifugation was added with the same volume of acetone. The resulting precipitate containing the EPS was dissolved in a 50 mM Tris–HCl buffer (pH 8.0). After treatment with nuclease and protease, the neutral and acidic EPSs were separated by column chromatography using the anion exchange resin (TOYOPEARL DEAE-650M, Tosoh Bioscience, Tokyo, Japan) as described previously [44 (link)]. Each of the purified EPSs was stored at −20 °C until use. Prior to the oral administration, the EPS samples were dialyzed against sterile distilled water by using Amicon Ultra (MWCO = 10 kDa, Merck Millipore Ltd., Carrigtwohill, Co., Cork, Ireland), and the concentrations (1.0 and 0.1 mg/mL) were adjusted with the sterile distilled water.