D xylose xyl

Mulberry leaf was purchased from Tong Ren Tang Co. (Beijing, China, Series: 20180605). Standards of monosaccharide include d‐Mannose (Man), l‐Rhamnose (Rha), d‐Glucuronic acid (GlcA), d‐Galacturonic acid (GalA), d‐Glucose (Glc), d‐Galactose (Gal), d‐Xylose (Xyl), and l‐Fucose (Fuc) were purchased from Sigma‐Aldrich. Phenyl‐3‐methyl‐5‐pyrazolone (PMP), trifluoroacetic acid (TFA), sodium hydroxide (NaOH), and monopotassium phosphate (KH₂PO₄) were purchased from Aladdin. The purity of each standard was no <98%. Chromatographic grade acetonitrile and formic acid were purchased from Fisher Scientific. Potassium phosphate buffer (PBS) was purchased from Solarbio. The other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

S. japonica was harvested on 20 June 2019 in Rongcheng, Shandong province, China. The strain is named Yanza, which is the traditionally hatched and cultured strain in Rongcheng S. japonica cultural area. Diaminofluorophore 4-amino-5-methylamino-2′7′-difluorofluorescein diacetate (DAF-FM DA), 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), and acridine orange were from Sigma company (St. Louis, MO, USA). The monosaccharide standards of D-fucose (Fuc), galactose (Gal), L-arabinose (Ara), D-mannose (Man), L-d-rhamnose (Rha), D-glucose (Glc), and D-xylose (Xyl), as well as Griess reagents, lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were also obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were from company of Gibco (Rockville, MD, USA). ELISA kits were purchased from Dakewei (Shenzhen, China).

For the determination of the monosaccharide composition, the EPS was hydrolyzed as described above. Ten microliters of hydrolysate were auto-injected into a Nexera-i LC2040C Plus UHPLC system (Shimadzu Corporation, Kyoto, Japan), coupled with a Zorbax Carbohydrate column (4.6 × 150 mm, 5 μm-ϕ) and Zorbax Reliance Cartridge guard-column operating at 35 °C. The sample was eluted with a mobile phase composed of a mixture of acetonitrile:H2O (80:20, v/v) at a flow rate of 0.6 mL/min. The eluate was monitored using a refractive index detector (RID)-20A (cell temperature of 35 °C). The concentration of sugars in the sample was deduced using a calibration curve constructed by plotting the peak area (XX-axis) against five different concentrations (YY-axis) for each sugar. The peak corresponding to different sugars in the sample was confirmed through a comparison of the retention time with that of the standards. D(+) Mannose (Man) (99%), L(+) Rhamnose (Rha).H₂O (99%) and D-Glucuronic acid (GlcA) (≥98+%)—purchased from Alfa Aesar, Thermo Fisher GmbH, Kandel, Germany; and D(+) Xylose (Xyl) (≥99%), D(+) Glucose (Glc) (≥99.5%), D(−) Fructose (Fru) (≥99%) and D(+) Glucosamine hydrochloride (GlcN.HCl)—purchased from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany.