Hpx 87h

In the last week of experiment, the animals were fasted for 12 h and then received, by gavage, 2 mL of the solution (200 mg of lactulose and 100 mg of mannitol); samples of their urine were collected for 24 h. At the end, the collected urine volume was measured, recorded and stored at −80 °C [26 (link)]. Then, for the analysis, the urine was diluted 1:2 with distillated water, filtered (0.45 µm) and analyzed by the HPLC method.
Furthermore, the colonic feces of animals were quantified for their levels of SCFA, acetate, propionate, and butyrate. The extraction of the SCFA was performed by mixing 100 mg of luminal contents with 2 mL of perchloric acid (10%) and centrifuged (9000× g, 10 min, 25 °C). The supernatant was filtered (0.45 µm) and analyzed by HPLC [27 (link)].
The HPLC conditions for the analyses of lactulose, mannitol and SCFA were a Shimadzu HPLC system (Kyoto, Japan) with a degasses (Model DGU-14A), pump (Model LC-10AT), auto-sampler (Model SIL-20A), column oven (Model CTO-10AS), UV–vis detector (Model SPD-10AV), and a refractive index detector (Model RID-10A). The column used was Aminex HPX-87H (300 cm × 8.7 mm; BIO-RAD) in 55 °C with a pressure of 1920 psi, using H2SO4 0.005 mM as a mobile phase under isocratic conditions [28 (link)]. Lactulose, mannitol and SCFA levels (mg/g of feces) were determined by standard curves using commercial standards (Sigma-Aldrich).

The supernatatant was used to determine glucose, xylose and arabinose by high-performance liquid chromatography (HPLC) (Detector: Shimazu RID-10 A; Column: BioRad (Hercules, CA, USA) Aminex HPX-87H (300 × 7.8)) at 65 °C. The eluent was 5 mmol/L sulphuric acid, and the flow rate was 0.5 mL/min.
The recoverable sugars measurement was carried out in triplicate. The glucan, xylan and arabinan contents of the fibre samples were determined, taking into account the depolymerisation factor of the monosaccharides [8 ]. The glucan includes all polysaccharides consisting of glucose monomers.

Cell density: The cell density was followed by measuring the optical density at 620 nm (OD_620nm) using a spectrophotometer (Ultrospec 1000, Pharmacia Biotech, Uppsala, Sweden) after proper dilution of the sample.
Cell dry weight: For measuring the cell dry weight (CDW - g/L), 10 mL of the fermentation broth was collected in a 15-mL Falcon tube, centrifuged at 5000×g for 10 min at room temperature. The supernatant was discarded, and the cell pellet was dried overnight at 105 °C.
$$Celldryweight(mg/mL)=(weightofthedrytubewithcellpellet-weightofthedrytube)/10$$
Finally, the optical density was correlated with the cell dry weight (g/L) where 1 OD_620nm unit was equivalent to 0.366 g_CDW/L.
Analytes: The concentration of the substrates (glucose and glycerol) and products (PA, AA, and SA) was determined using JASCO HPLC (Tokyo, Japan) as described elsewhere [61 (link)]. Briefly, 50 µL of properly diluted and acidified samples were injected into the mobile phase (0.5 mM sulfuric acid), flowing at a rate of 0.4 mL/min. Separation of the different components was done at 55 °C using the Biorad column, Aminex HPX-87H (Richmond, CA, USA). The detection was done using an ERC refractive index (RI) detector (Kawaguchi, Japan).

Five hundred-microliter samples were taken at each sampling point, centrifuged in Eppendorf tubes at 5,000 × g at 4°C for 5 min, and supernatants were stored at −20°C until analysis. Supernatants were analyzed for the presence of ethanol, glucose, glycerol, and acetate using HPLC UltiMate 3000 (Thermo Fisher) with Aminex HPX87H ion exclusion column. Samples were run for 30 min at 0.600 ml/min at 60°C using 5 mM H2SO4 as eluent. Compounds were detected using a Dionex RI101 and DAD-3000 detectors (Dionex) for RI and UV detection, respectively.

Cell growth experiments were conducted in triplicate for each of the three species investigated. 1 mL samples were extracted from experimental cultures hourly for OD₆₀₀ measurements (Ultrospec 10/Amersham Biosciences). An additional 1 mL sample extracted, sterile filtered through a 0.2 µm syringe filter (Corning), then stored at + 4 °C for metabolite analysis via HPLC. HPLC analysis for the metabolic output was completed using an Agilent 1200 equipped with a refractive index detector and an Aminex HPX-87H cation exchange column (300 × 7.8 mm i.d. × 9 µm; Bio-Rad)^{41 (link)}. 100 µL of each sample was injected into the system for isocratic elution, with a mobile phase of 3.25 mM H₂SO₄ at 0.6 mL/min and 35 °C.