2.3. Oligosaccharide analysis

XOS and gluco-oligosaccharides (GOS) were analysed simultaneously by High-Performance Anion Exchange Chromatography (Thermofischer ICS-5000) coupled with Pulse Amperometric Detector (HPAEC-PAD), using the Dionex CarboPac PA200 guard (3 × 50 mm) and analytical (3 × 250 mm) columns. An injection volume of 25 μL of each sample was analysed by HPAEC-PAD with the column temperature set at 30 °C, and an eluent flow rate of 0.3 mL/min. Analytical reagent grade sodium hydroxide (50 % w/w) and sodium acetate were obtained from Sigma-Aldrich. The HPAEC-PAD elution program for XOS and GOS quantification was as follows: from 0 to 9 min 100 % A (0.1 M NaOH) and 0 % B (0.5 M NaOAc/0.1 M NaOH), from 9.1 to 32 min 92 % A (0.1 M NaOH) and 8 % B (0.5 M NaOAc/0.1 M NaOH), from 32.1 to 46 min 50 % A (0.1 M NaOH) and 50 % B (0.5 M NaOAc/0.1 M NaOH) and from 46.1 to 56 min 100 % A (0.1 M NaOH) and 0 % B (0.5 M NaOAc/0.1 M NaOH). XOS standards (X₂) xylobiose, (X₃) xylotriose, (X₄) xylotetraose and (X₅) xylopentaose as well as GOS standards (G₂) cellobiose, (G₃) cellotriose and (G₄) cellotetraose (Megazyme) were run as calibration standards using serial dilution concentration ranges of 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL and 1.25 μg/mL.