For the measurement of free sugar contents, 1 ml homogenate of the CG was first boiled in deionized water for 10 min, and then 4 ml ethanol was added to the homogenate. Supernatant was obtained by centrifugation (1,000 × g, 10 min) and dried at 50°C with a stream of filtered air. Dried materials were dissolved in 1 ml distilled water, and then filtered through a membrane (0.45 μm; Gelman). Sample solution (0.5 ml) was introduced into the HPLC system (model 307, Gilson) equipped with a pulsed amperometric detector (Dionex). The column (CarboPac PA-1, Dionex) was eluted with 0.16 M NaOH at a flow rate of 1 ml/min. Fructose was eluted at 4.3 min. The other half sample solution was reduced for 1 h at room temperature by the addition of 0.5 ml NaBH4 (20 mg/ml) in 2 M NH4OH containing 300 μg inositol as an internal standard. Reduced monosaccharides were acetylated for 15 min at room temperature by the addition of 200 μl acetic anhydride and 50 μl 1-methylimidazole as a catalyst42 . Acetylated monosaccharides were analyzed by gas chromatography (GLC; GC-7A, Shimadzu) with a capillary column (SP-2440, Supelco).
Pulsed amperometric detector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pulsed amperometric detector is a specialized analytical instrument used for the detection and quantification of electroactive compounds in various samples. It operates by applying a pulsed voltage to an electrode, which generates an electrical current proportional to the concentration of the target analytes. This technique enables sensitive and selective detection of a wide range of organic and inorganic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Carbopac pa10 column, by Thermo Fisher Scientific (3 mentions)
Ics3000 chromatograph, by Thermo Fisher Scientific (2 mentions)
Carbopac pa1, by Thermo Fisher Scientific (1 mentions)
Model 307, by Gilson (1 mentions)
Gc 7a, by Shimadzu (1 mentions)
Aspartic acid, by Merck Group (1 mentions)
Aminopac pa10, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Carbopac pa20 column, by Thermo Fisher Scientific (1 mentions)
L fuc, by Merck Group (1 mentions)
L rha, by Merck Group (1 mentions)
D gal, by Merck Group (1 mentions)
D xyl, by Merck Group (1 mentions)
D glc, by Merck Group (1 mentions)
D gala, by Merck Group (1 mentions)
Dionex ics 2500, by Thermo Fisher Scientific (1 mentions)
D fru, by Merck Group (1 mentions)
Hpaec, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Chromeleon 7, by Thermo Fisher Scientific (1 mentions)
14 protocols using pulsed amperometric detector
1
Quantitative Analysis of Free Sugars
2
Determination of γ-Carboxyglutamic Acid
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γ-Carboxyglutamic acid (Gla) content was determined by amino acid analysis as described by Price [19 (link)]. One mg of protein was subjected to alkaline hydrolysis in 2 N KOH for 48 h at 110°C. Hydrolyzed amino acids were filtrated through a 0.2 μm filter to remove precipitate. Supernatants were separated by high performance liquid chromatography on an AminoPac PA10 strong anion-exchange column with pulsed amperometric detector (Dionex, Sunnyvale, CA) using an elution buffer of 1 M sodium citrate. Purified γ-carboxyglutamic acid and aspartic acid (Sigma-Aldrich) were used as standards, and samples were compared with a control sample (pdFIX, Enzyme Research Labs, South Bend, IN). Determinations were performed in duplicate, and results are reported as an average.
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γ-Carboxyglutamic acid (Gla) content was determined by amino acid analysis as described by Price [19 (link)]. One mg of protein was subjected to alkaline hydrolysis in 2 N KOH for 48 h at 110°C. Hydrolyzed amino acids were filtrated through a 0.2 μm filter to remove precipitate. Supernatants were separated by high performance liquid chromatography on an AminoPac PA10 strong anion-exchange column with pulsed amperometric detector (Dionex, Sunnyvale, CA) using an elution buffer of 1 M sodium citrate. Purified γ-carboxyglutamic acid and aspartic acid (Sigma-Aldrich) were used as standards, and samples were compared with a control sample (pdFIX, Enzyme Research Labs, South Bend, IN). Determinations were performed in duplicate, and results are reported as an average.
3
HPAEC-PAD Carbohydrate Analysis
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The concentrations of fructose, glucose, maltose, and sucrose were measured by high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) with internal standardization, as described before (De Roos et al., 2018 (link)). Briefly, an ICS3000 chromatograph (Dionex, Sunnyvale, CA, United States) equipped with a CarbopacTM PA10 column (Dionex) and coupled to a pulsed amperometric detector (Dionex) was used. The same mobile phase and eluent gradient were applied. All samples were deproteinized, vortexed, centrifuged (21,912 × g, 15 min, 4°C), and filtered (0.2-μm pore-size Whatman filters; GE Healthcare Life Sciences, Bucks, United Kingdom) before injection (10 μl) into the column.
4
Monosaccharide Composition Analysis of Polysaccharides
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Samples (2 mg) of six polysaccharide components were hydrolyzed with 3 mL 2 M trifluoroacetic acid (TFA) at 110 °C for 4 h. After hydrolysis, hydrolysates were dried with a Termovap sample concentrator, and then 3 mL methanol was added and dried repeatedly three times until the TFA was completely removed. The monosaccharide compositions were determined by a high-performance anion exchange chromatography (HPAEC) system (Dionex ICS-2500, Dionex, Sunnyvale, CA, USA) equipped with a CarboPac™ PA20 column (3 mm × 150 mm, Dionex, USA) and a pulsed amperometric detector (Dionex, USA). The column was eluted with 2 mM NaOH (0.45 mL/min) followed by 0.05 to 0.2 M NaAc at 30 °C. The monosaccharide compositions and content of polysaccharide components were determined using d -Gal, d -Glc, d -Ara, l -Fuc, l -Rha, d -Man, d -Xyl, d -Fru, d -Rib, d -GluA, and d -GalA (Sigma-Aldrich, St. Louis, MO, USA) as the standards.
5
Enzymatic Modification of Amylose by GBEs
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To determine the action pattern of CbGBE and PhGBE on AM, 0.2% of AM (w/v) in 50 mM sodium acetate buffer (pH 6.0) was incubated with 5 U of PhGBE or 2 U of CbGBE at 60 °C for 20 h. The products were analyzed using a high-performance anion-exchange chromatography (HPAEC) column with a pulsed amperometric detector (Dionex, Sunnyvale, CA, USA). The detailed method was described previously [18 (link)]. The side chain length distribution of GBE-modified starches is described in the Supplementary Materials and Methods .
6
Quantification of Soluble Sugars and Starch
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Soluble sugars and starch were extracted as described in Lira et al.74 (link). The supernatant residue was measured in a HPLC system equipped with a pulsed amperometric detector (Dionex, Sunnyale, USA) and a CarboPac PA1 (4 × 250 mm) column as described in Purgatto et al.76 (link). The endogenous metabolite concentration was obtained by comparing the peak areas of the chromatograms with commercial standards. Starch content was determined from dried pellets as described in Suguiyama et al.77 (link).
7
Monosaccharide Composition Analysis of ECC and ECC_CPSs
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The monosaccharide composition of ECC and ECC_CPSs was analyzed using high-performance anion-exchange chromatography (HPAEC) [20 (link)]. The used detector and column were pulsed amperometric detector (Dionex, Sunnyvale, CA, USA) and CarboPac™ PA10 column (I.D.: 2 mm, length: 250 mm, particle size: 10 μm, Dionex) for separation. All ECC and ECC_CPSs were hydrolyzed using trifluoroacetic acid (Sigma-Aldrich). The injection volume was 20 μL, flow rate was 1 mL/min, and eluent A/B was 18 mM NaOH/200 mM NaOH. The eluent program was as follows: 0–20 min, 0% B; 20–35 min, 100% B; 35–45 min, 0% B.
8
HPAEC-PAD Analysis of Maltooligosaccharides
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The concentrations of maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were measured by HPAEC-PAD with internal standardization, as described before for maltotriose (De Roos et al., 2018 (link)). Briefly, an ICS3000 chromatograph (Dionex) equipped with a CarbopacTM PA100 column (Dionex) and coupled to a pulsed amperometric detector (Dionex) was used. The same mobile phase and eluent gradient were applied. All samples and standards were deproteinized, vortexed, centrifuged, and filtered, as described above, before injection (10 μl) into the column.
9
HPAEC-PAD Analysis of Carbohydrates
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Samples were diluted to 20 μg mL−1 (DFβCD and βCD) or 10 μg mL−1 (2′-FL and 3-FL) and centrifuged at 19 000g for 10 min. The supernatant (10 μL injection volume) was analyzed using an ICS 3500 HPAEC system from Dionex (Sunnyvale, USA), in combination with a CarboPac PA-1 (2 × 250 mm) column, with a Carbopac PA-1 guard column (Dionex). Carbohydrate peaks were detected by an electrochemical Pulsed Amperometric detector (Dionex) after elution with 0.3 mL min−1 at 25 °C. The eluents consisted of A (0.1 M NaOH solution) and B (1 M NaOAc in 0.1 M NaOH). Two different gradients were used. For DFβCD and βCD the gradient used was: 2.5–25% B (0–30 min), 25–100% B (30–40 min), 100% B (40–45 min), 2.5% B (45–60 min). For 2′-FL and 3-FL the gradient used was 0–15% B (0–15 min), 15–100% B (15–20 min), 100% B (20–25 min), 0% B (25–45 min). Substrates were quantified using 2.5–10 μg mL−1 2′-FL and 3-FL or 5–20 μg mL−1 DFβCD and βCD. In addition, mono-Fuc-β-CD (MFβCD) was injected as a pure compound to detect it within the DFβCD mix. Data analysis was performed with Chromeleon™ 7.2.6 software from Thermo Fisher Scientific (Waltham, Massachusetts, USA).
10
Monosaccharide Composition Analysis by IC
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Twenty mg of SLNP was mixed with 4 mL of trifluoroacetic acid in a sealed test tube and was put into an oven to hydrolyze at 120°C for 6 h. Then the hydrolysate was added methanol to evaporate trifluoroacetic acid completely and dissolved in 1 mL of distilled water. Afterward, the obtained solution was diluted 10 times to measure the monosaccharide composition by ion chromatography (IC) with Dionex Carbopac PA10 column (250 mm × 4 mm) and Dionex pulsed amperometric detector (California, United States) with Au electrode. The detection conditions were as follows: the column temperature was 30°C, the injection volume was 25 μL, the flow rate was 0.45 mL/min, and elution mode was 10% 200 mmol/L NaOH and 90% ultrapure water. Glucose, fructose, mannose, xylose, galactose, and arabinose were selected as standard monosaccharide.
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