Chromeleon version 6

Colon content (25 mg) from piglets of two breeds (n = 7) was extracted with 1 mL of 0.3 M HClO₄, and sonicated once for 5 min after vortex mixing, followed by centrifugation for 5 min (16,090 g, 4°C). The supernatant (160 μL) was then transferred to a new tube containing 2 M KOH solution and equilibrated at 4°C for 3 h. After centrifugation (5 min, 16,090 g, 4°C), the supernatant was passed through a membrane filter (0.22 μm), and 150 μL was transferred into a 2 mL glass vial for HPLC analysis. HPLC analysis was performed using methanol: 50 mM KH₂PO₄ buffer solution (9:91, v:v) (pH 6.5) as the mobile phase with a flow rate of 0.5 mL/min, and detected using a UV detector at a wavelength of 259 nm. Finally, data processing was performed using Chromeleon version 6.8 (Thermo Fisher).

Samples were run on a Dionex ICS-3000 analytical system controlled by Chromeleon version 6.8 software (Thermo Scientific, Sunnyvale, CA). The autosampler injected 25μL of sample onto a 4 x 250 mm IonPac CS12A cation exchange column preceded by an IonPac CG12A guard column (4 x 50 mm) (Thermo Scientific, Sunnyvale, CA). Isocratic separation of the ammonium ion was achieved using a 20mM methanesulphonic acid (MSA) eluent prepared by the EG40 eluent generator (Thermo Scientific, Sunnyvale, CA) that required only a deionized water source. The column temperature was maintained at 25°C. The pumps and eluent generators were turned on at least 60 min before the first injection to allow baseline stabilization. Ammonium ions were detected using a conductivity detector. Prior to detection by the conductivity detector, the MSA eluent conductance was suppressed by the cation self-regenerating suppressor (CSRS). All ammonium values generated in Chromeleon were transferred to Microsoft Excel 2010 for further analysis. Statistical evaluations were done using JMP Software (SAS Institute Inc., Cary NC).

Labelled OS were analysed using a Dionex Ultimate 3000RS Ultra High Performance Liquid Chromatography (UHPLC) system coupled to a Dionex RF2000 fluorescence detector (both Thermo, Sunnyville, USA) and a Q-Trap 4000 Mass spectrometer (AB Sciex, Framingham, MA). Separation was achieved on a BEH Glycan analytical column (2.1×150 mm, 1.7 µm, Waters, Milford, MA) using one of three different methods described below. For two of the methods (LC methods 1 and 2) a Van Guard BEH Amide guard column (1.7 µm; 2.1×5 mm, Waters) was used as a trapping column to capture the 2AB labelled OS and remove excess labelling reagents [23] (link). For the third method (LC method 3) excess reagents were not removed and a guard column was not employed. For methods 1 and 2 the system was controlled by Analyst version 1.5 (AB Sciex) with DCMS-link (Thermo) to control the chromatography system. Method 3 was used without the mass spectrometer and in this case the system was controlled by Chromeleon version 6.8 (Thermo).

Fecal SCFAs extraction was performed following De Baere et al.,^{66 (link)} protocol. SCFAs concentrations were measured using high-performance liquid chromatography UltiMate 3000 UHPLC system (HPLC-UV) (Thermo Scientific, Breda, The Netherlands) with external calibration standards curve method as previously described in details.^{67 (link)}Briefly, calibration standards were prepared at concentrations ranging from 0.5 mM to 50 mM for acetic acid (AA), butyric acid (BA), propionic acid (PA), and succinic acid (SA) as internal standard (all purchased from Sigma-Aldrich, St. Louis, MO, USA). After chromatographic separation testing on a Hypersil Gold aQ column (150 × 4.6 mm i.d.) with a 3 μm particle (Thermo Scientific, Breda, The Netherlands), HPLC-UV was performed on thermostated and guard column protected HPLC columns using UV detection at 210 nm. The mobile phase consisted of 20 mM of sodium dihydrogen phosphate (Merck, Darmstadt, Germany) in HPLC water (pH 2.2) (Merck) (A) and HPLC grade acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) (B).
HPLC-UV data were processed using Chromeleon version 6.8 software (Thermo Fisher Scientific, MA, USA). SCFAs concentration were calculated using mathematical equation: SCFA (AA, BA, PA) = (organic acid in fecal sample × 6 × 10⁻³)/(succinic acid in fecal sample × fecal sample mass) × 1000 [mmol/kg]. All measurements were done in triplicate.

The monosaccharides released by enzymatic hydrolysis were analyzed using a high-performance anion-exchange chromatography system coupled with a pulsed amperometric detector (HPAEC-PAD) (Dionex ISC-3000, Sunnyvale, CA, USA). A CarboPac PA1 analytical column (250 mm × 4 mm) and a guard column (50 mm × 4 mm) were used. Prior to analysis, the samples were boiled for 10 min to denature proteins, filtered through a 0.2 µm nylon filter, and the samples were diluted with Milli-Q water. Water was used as eluent at 1 ml min⁻¹, and 300 mM NaOH was added at 0.5 ml min⁻¹ before the detector. The column was cleaned with 200 mM NaOH dissolved in 170 mM sodium acetate. Sample injection was 25 µl, and the column was maintained at 30 °C during analysis. Calculations were performed using Chromeleon version 6.8 (Dionex).

The glucose and organic acids were measured by High Performance Liquid Chromatography—HPLC, Ultimate 3000 (Dionex, Sunnyvale, CA, USA). Samples were diluted five or ten times in sulfuric acid (50 mM) and filtrated in the membrane at 0.45 μm before being loaded automatically in the aminex column HPX—87H (300 mm × 7.8 mm; Bio Rad, Hercules, CA, USA) at 60 °C, with an UV (210 nm) and RI detector; the mobile phase was sulfuric acid at 5 mM, and a flow rate of 0.6 mL/min was used. The integration of chromatographic peaks to determine the concentration determination was calculated through the software Chromeleon, version 6.8 (Dionex, Sunnyvale, CA, USA).

Separation of the carotenoids and vitamins from all the extracts was accomplished using a high-performance liquid chromatography (HPLC) system (Dionex^TM, Thermo Fisher Scientific, CA, USA) equipped with a Diode Array Detector UltiMate 3000 (Dionex, Thermo Fischer Scientific, CA, USA) monitored at 450 nm for carotenoids and 290 and 220 nm for vitamins. Separation of the carotenoids was achieved using a YMC Carotenoid C30 column (250 × 4.6 mm, 3 μm, YMC America, Devens, MA, USA) at ambient temperature. Two mobile phases of A (MeOH/MTBE/H₂O = 81/15/4) and B (MeOH/MTBE/H₂O = 6/90/4) were used at elution gradient of 1–100% B for 90 min and at a flow rate of 1 mL/min. Separation of the vitamins was achieved using a XTerra MS C18 column (250 × 4.6 mm, 5 μm, Waters Limited, Wilmslow, Cheshire, England) at 40 °C. Phosphate buffer (10 mM TBAOH and 10 mM KH₂PO₄) with a pH of 5.2 and acetonitrile with the ratio of 90:10 v/v were used as mobile phases for the water-soluble vitamins at a flow rate of 0.8 mL/min. For the fat-soluble vitamins, acetonitrile and MeOH with a ratio of 60:40 v/v were used at a flow rate of 1 mL/min. Dionex Chromeleon version 6.8 software was used for data interpretation.