*2.6. Determination of Xylo-Oligosaccharides Pattern Profiles*

In order to determine the hydrolysis product profiles from synergy studies, XOS were analyzed by thin-layer chromatography (TLC). Five µL of hydrolysate sample and a mixture of XOS standards were applied on a silica gel 60 F<sup>254</sup> plate (Merck, Darmstadt, Germany). The migration was repeated twice using a mobile phase consisting of 1-butanol, acetic acid and water in a 2:1:1 ratio, respectively. The plate was then submerged in Molisch's Reagent (0.3% (*w*/*v*) α-naphthol dissolved in methanol and sulphuric acid in a 95:5 ratio (*v*/*v*), respectively). The spots corresponding to the different XOS were visualized by heating the plate in the oven at 110 ◦C for 10 min.

The XOS were quantified by a Shimadzu HPLC system (Shimadzu Corp, Japan) equipped with a refractive index detector (RID) using a CarboSep CHO 411 column (Anatech, South Africa) with water as the mobile phase in isocratic mode. The column oven was set at 80 ◦C and separation was performed within 35 min at a flow rate of 0.3 mL per min. An injection volume of 20 µL was employed for all samples and XOS standards.
