Three major free soluble sugars, namely, sucrose, glucose, and fructose, were measured using an HPLC (Dionex UltiMate 3000, Thermo Fisher Scientific, Waltham, MA, United States) equipped with a Shodex RI-101 Detector (Showa Denko, Japan, Tokyo). The glucose reagent was obtained from Junsei Chemical Co., Ltd. (Tokyo, Japan), and the sucrose and fructose reagents were obtained from Sigma-Aldrich (Darmstadt, Germany). Five grams of pulverized frozen sample were mixed with triple distilled water, and the mixed volume was adjusted to 50 mL (5 g/50 ml). Each sample was filtered with a 0.45-μm PVDF membrane filter, and 10 μL of filtrate was injected into the HPLC. The separation was conducted using a Sugar-Pak column (6.5 × 300 mm, Water Corp., Perth, WA, Australia) held at a 70°C oven temperature with 30 min of run time. Ten micromoles of HPLC-grade distilled water was used as the mobile phase at a 0.5 mL min–1 flow rate. The HPLC measurement results were used to obtain the total soluble sugar contents by summing the individual sugar contents.
Shodex ri 101 detector
Manufactured by Resonac
Sourced in United States, Japan
The Shodex RI-101 is a refractive index (RI) detector used in liquid chromatography (LC) applications. It measures the change in the refractive index of a sample as it passes through the detector cell, providing information about the concentration of analytes in the sample. The Shodex RI-101 detector is a versatile and reliable instrument for a wide range of analytical applications.
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Lab products found in correlation
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Jnm ecp 600 nmr spectrometer, by JEOL (1 mentions)
Superaw4000, by Tosoh (1 mentions)
Superaw3000, by Tosoh (1 mentions)
Superaw2500, by Tosoh (1 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
Rid 20a refractive index detector, by Shimadzu (1 mentions)
Spd 40 uv detector, by Shimadzu (1 mentions)
Nexera uhplc system, by Shimadzu (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Varian unity 500, by Agilent Technologies (1 mentions)
Uv 1650pc spectrophotometer, by Shimadzu (1 mentions)
Jms 700 spectrometer, by JEOL (1 mentions)
Ft ir 4100, by Jasco (1 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
8 protocols using shodex ri 101 detector
1
HPLC Analysis of Soluble Sugars
2
Isolation and Identification of Sterols from EKA
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In our previous study, we found that one of the major neurotrophic compounds in EKA was a sterol-related compound (Tirtawijaya et al., 2018 (link)). To identify the sterols present in this study, we fractionated EKA to collect active neutral, moderately polar, and basic fractions in accordance with their polarity (Harborne, 1998 ). We combined the 3 fractions, and then separated them by reverse-phase (RP) high-performance liquid chromatography (HPLC). We then carried out an isocratic program using a mixture of acetonitrile, methanol, and ethyl acetate (1:1:3) as the mobile phase. The mixture was eluted through a reverse-phase C18 column (1 cm i.d.×25 cm) (Ultrasphere; Beckman Coulter, Fullerton, CA, USA) with a flow rate of 2 mL/min and detection was by a refractive index detector (Shodex RI-101 detector; Showa Denko K.K., Tokyo, Japan) and each peak was dried completely under a stream of nitrogen gas. Samples were dissolved in chloroform-d (CDCl3) prior to analysis of 1H (600 MHz) and 13C (151 MHz) nuclear magnetic resonance (NMR) spectra using a JNM-ECP 600 NMR spectrometer (JEOL, Tokyo, Japan). The structures of the isolated compounds were identified and confirmed as being identical to the spectral data reported by Khatun et al. (2017) (link) and Nasir et al. (2011) (link).
3
Molecular Weight Analysis of Polysaccharides
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High performance size-exclusion chromatography with refractive index detection (HPSEC-RI) was used to determine the molecular weight distribution before and after treating wheat arabinoxylan, xylan, CMC, galactomannan, and xyloglucan with secretomes. The system used was an Utimate 3000 system (Dionex Corp., Sunnyvale, CA, USA)) coupled to a Shodex RI-101 detector (Showa Denko K.K. Tokyo, Japan). Three columns, TSK-Gel Super columns (4000AW, 3000AW, and 2500 AW; 6 ID × 150 mm per column, 6 μm), and a TSK Super AW guard column (6 ID × 40 mm) (Tosoh Bioscience Tokyo, Japan) were used at 55°C. Incubated polysaccharide samples were injected (10 μL) and eluted with 0.2 M sodium nitrate at a flow rate of 0.6 mL min−1. Pullulans with molecular weights of 1080, 6100, 9600, 47100, 194000 and 708000 Da were used as standards. The system was controlled with Chromeleon software and data analysis was carried out with Chromeleon software.
4
Molecular Weight Analysis of Xyloglucan
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Xyloglucan and corresponding digests were analyzed by high performance size exclusion chromatography (HPSEC) for their molecular weight distribution. The analysis was performed on an Ultimate 3000 system (Thermo Scientific, Waltham, MA, USA) coupled to a Shodex RI-101 detector (Showa Denko K.K., Tokyo, Japan). Three TSK-Gel columns (SuperAW4000, SuperAW3000, SuperAW2500; 6 mm × 150 mm per column) in series combined with a TSK Gel super AW guard column (6 mm ID × 40 mm) (Tosoh Bioscience, Tokyo, Japan) were used with the column temperature of 55 °C. Supernatant (10 μL) of LPMO-xyloglucan digests was injected and eluted with 0.2 M NaNO3 at a flow rate of 0.6 mL min−1. Pullulans (Associated Polymer Labs Inc., New York, NY, USA) in the range of 0.4–708 kDa were used as calibrants.
5
HPLC Analysis of Carbohydrates and Organic Acids
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Two independent HPLC systems were used to avoid cross‐contamination. A Dionex UltiMate 3000 HPLC System (Thermo Scientific, Germany) was applied for the mobile phases containing H2SO4 (5 mM) and TFA (0.1%). Signals were detected with a Dionex UltiMate 3000 Variable Wavelength Detector and a SHODEX RI‐101 detector (Showa Denko Europe GmbH, Germany). When using formic acid (FA) as an additive, experiments were performed with a Shimadzu Nexera UHPLC system coupled with a RID‐20A Refractive Index detector and an SPD‐40 UV detector (Shimadzu, Japan). The cell temperature of all UV and RI detectors was set to 40°C. The UV detectors were set at 210 nm. Two identical columns Isera Metab‐AAC 300 × 7.8 mm column (Isera, Germany) were installed in both HPLC. The injection volume was 5 μL. The flow rate was 0.4 mL/min, and the column oven was set at 40°C unless otherwise noted. The whole system was purged and equilibrated when a parameter was changed before analysis. An authentic standard mixture was used in all optimization steps. This mixture included 20 g/L cellobiose, 20 g/L glucose, 20 g/L quinic acid, 20 g/L xylitol, 20 g/L glycolaldehyde, 20 g/L itaconic acid, 10 g/L adipic acid, 20% MeOH, and 20 g/L BDO. All chemicals were obtained from Carl Roth GmbH & Co. KG (Germany) or Sigma Aldrich Chemie GmbH (Germany) with a purity higher than 99%.
6
Quantification of Fermentation Metabolites
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The concentrations of glucose, fructose, sucrose, ethanol, glycerol and organic acids (acetic, lactic, succinic and formic) during fermentation were quantified by high performance liquid chromatography (ultimate 3000; Dionex, Sunnyvale, CA, USA) using a Rezex ROA H+ column (300 mm×7.8 mm; Phenomenex, Torrance, CA, USA) and Shodex RI-101 detector (Showa Denko, Tokyo, Japan) for sugars and alcohol, using 0.005 M H2SO4 as eluent, at a flow rate of 0.6 mL/min and column temperature of 45 °C.
7
Analytical Techniques for Compound Characterization
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HPLC was performed using the YMC-Pack Silica or YMC-Pack Pro C18 columns and a Shodex RI-101 detector (Showa Denko K. K., Tokyo, Japan). 1H-NMR spectra were recorded using a Varian Unity 500 (500-MHz) spectrometer (Varian Inc., Palo Alto, CA, USA). Chemical shifts are reported in ppm from tetramethylsilane, used as the internal references (CDCl3: δH 7.26 ppm), with solvent resonance resulting from incomplete deuteration. 13C-NMR spectra were recorded using a Varian Unity 500 (125-MHz) spectrometer with complete proton decoupling (Varian Inc., Palo Alto, CA, USA). Chemical shifts are reported in ppm from tetramethylsilane with the solvent as the internal reference (CDCl3: δC 77.26 ppm). High-resolution mass spectra (HRMS) were obtained with a JEOL JMS-700 spectrometer at the Korea Basic Science Institute (JEOL Ltd., Tokyo, Japan). The optical rotations were measured using a JASCO digital polarimeter (JASCO International Co. Ltd., Tokyo, Japan), using a 5-cm cell. IR spectra were recorded using a JASCO FT/IR-4100 (JASCO International Co. Ltd., Tokyo, Japan). UV spectra were obtained using a Shimadzu UV-1650PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
8
Acetate Quantification by HPLC
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The concentration of acetate in the supernatant of cultures from both early exponential (3-hour cultivation) and stationary phase (24-hour cultivation) was determined by Ultimate 3000 HPLC system (Dionex, Sunnyvale, USA) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, USA) and a Shodex RI-101 detector (Showa Denko KK, Tokyo, Japan). The column oven temperature was set as 60 °C and the mobile phase consisted of sulfuric acid (5 mM) with a flow rate of 0.5 ml/minute.
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