Cosmosil pbr column

Cell growth was determined by measuring the OD600 nm on a UVmini-1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Glucose concentration was analyzed using a Prominence high-performance liquid chromatography (HPLC) system (Shimadzu) equipped with a Shodex SUGAR KS-801 column (6 μm, 300 × 8.0 mm L × I.D., Shodex). The column was maintained at 50°C, and water was used as the mobile phase at a flow rate of 0.8 ml/min. The HPLC profile was monitored using a refractive index detector. The concentration of shikimic acid was determined using HPLC equipped with COSMOSIL PBr column (5 μm, 4.6 mm × 250 mm, I.D. × L, Nacalai Tesque). A 98:2 mixture of 0.2% phosphoric acid and methanol was used as the mobile phase at a flow rate of 1.0 ml/min, and the column was maintained at 40°C. The HPLC profile was monitored using a UV-VIS detector at 240 nm.

The anthocyanin compositions of petal extracts were analyzed by HPLC. Fresh petals were collected from greenhouse-grown plants and stored at −80 °C until used. The petals were soaked in 80% methanol containing 0.1% (v/v) trifluoroacetic acid to extract the anthocyanins, which were then separated with an HPLC–photodiode array detector system [PU-4180 PUMP, MD-4015 photodiode array detector, and ChromNAV (version 2.02) software; JASCO, Tokyo, Japan] equipped with a COSMOSIL PBr column (4.6 mm internal diameter × 250 mm; Nacalai Tesque, Kyoto, Japan). We used a linear gradient elution (2 ml min⁻¹) of 10%–80% acetonitrile in 1% aqueous phosphoric acid over 50 min.
Total anthocyanin concentrations were estimated based on the molar absorptivity of delphinidin (ε mol = 27,940 at 520 nm, evaluated in 80% MeOH containing 0.1% TFA). Delphinidin chloride, delphinidin 3-O-glucoside and delphinidin 3,5-O-diglucoside were purchased from Funakoshi Co. (Tokyo, Japan). Additionally, delphinidin 3-O-β-d-glucoside 5-O-(6-O-caffeoyl-β-d-glucoside) 3′-O-β-d-glucoside, delphinidin 3-O-β-d-glucoside 5-O-(6-O-caffeoyl-β-d-glucoside), delphinidin 3,5,3′-O-triglucoside (kindly provided by Drs. Tanaka and Nakamura, Suntory Ltd.), and gentiodelphin [delphinidin 3-O-β-d-glucoside 5-O-(6-O-caffeoyl-β-d-glucoside) 3′-O-(6-O-caffeoyl-β-d-glucoside)] were used as authentic standards.

The oligosaccharide S4 was obtained from gum arabic AGP (final concentration, 5.0%) following incubation with the bacterial cell fraction of B. longum subsp. longum JCM7052 grown in larch AGP in 400 mL of 50 mM sodium acetate buffer (pH 5.0) for 53 h. The released oligosaccharides were obtained by ethanol precipitation, and the supernatant was evaporated to dryness. S4 was separated via gel-filtration chromatography on a Bio-Gel P-2 column (φ, 25 by 830 mm; Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with water and high-pressure liquid chromatography using a Cosmosil PBr column (φ, 4.6 by 250 mm; Nacalai Tesque Inc.) and was eluted using 20 mM sodium phosphate (pH 2.5). Finally, the sample was desalted by gel filtration chromatography on a Bio-Gel P-2 column equilibrated with water. The freeze-dried sample was dissolved in deuterium oxide (D₂O). ¹H and ¹³C NMR spectra and two-dimensional spectra (¹H-¹H COSY, HMQC with/without ¹³C-¹H decoupling, HMBC) were measured in D₂O at room temperature on a Jeol ECX 400 spectrometer (400 MHz). For MS, MALDI-TOF mass spectra were recorded on Shimadzu Kompact MALDI Axima-CFR spectrometer with 2,5-dihydroxybenzoic acid as the matrix. ESI-TOF mass spectra were recorded on Jeol AccuTOF JMS-T700LCK with CF₃CO₂Na as the internal standard.