Oligosaccharides

HPAEC-PAD was carried out on a Dionex ICS-6000 system equipped with a CarboPac PA210-Fast-4 μm column, a palladium hydrogen (PdH) reference electrode, and a gold working electrode for detection. The flow rate was set to 0.6 mL/min. Elution of mono- and oligosaccharide was achieved by a gradient (from 12 mM to 100 mM) of potassium hydroxide. The auto-sampler was set at 10 °C while the column was at 30 °C. Samples were centrifuged (10,000× g for 5 min), appropriately diluted with dH₂O, and filtered using 0.22 μm syringe filters. Finally, monosaccharides (Sigma-Aldrich, Saint Louis, MI, USA) and oligosaccharides (Megazyme, Bray, Ireland) were identified and quantified through standard curves prepared in the range 25–0.1 mg/L. All experiments were repeated in triplicate and the error was calculated as the standard deviation of the three measurements.

Details about carbohydrates used in this work can be found in Supplementary Table S3. AX polysaccharides (from wheat and rye), oligosaccharides (XA3XX, XA2XX, XUXX, A23XX, A2XX), and chitohexaose (β-1,4-D-(GlcNAc)₆; Chi6) were purchased from Megazyme. Xylan (from birch, beet, and oat) and arabinan (from sugar beet) were purchased from Sigma-Aldrich.