Comprehensive insights into how glycoside hydrolases combinations tailor arabinoxylo-oligosaccharides (AXOS)

Arabinoxylo-oligosaccharides (AXOS) are structure-dependent prebiotics. However, mainly because structurally different (purified) AXOS are lacking, this structure-dependence has not yet been thoroughly examined. Therefore, this study evaluated the enzymatic tailoring and production of AXOS in sufficiently large amounts for fermentation studies, and highlights encountered production challenges. AXOS produced from wheat arabinoxylan (AX) by xylanases from glycoside hydrolase (GH) families 5_21, 5_34, 8, 10, and 11 were first examined by HPAEC, of which AXOS produced by GH5 and GH8 xylanases show distinct structures compared to those produced by the previously well-characterized GH10 and GH11 xylanases. Afterwards, using the GH5_21, GH8, GH10, and GH11 xylanases, in combination with a β-xylosidase and specific arabinofuranosidases, we produced and purified (removed monosaccharides and XOS) ten AXOS fractions, that are purely mono- and/or disubstituted, and are within the “low” DP range of 3 to 15. Four more AXOS fractions, that have DP ranges of 3 to 9, 8 to 14, 12 to 23, and 18 to 34, were enzymatically produced and were separated by preparative gel permeation chromatography. Up to half a gram of each AXOS fraction was produced, and are now ready for the future assessment of their prebiotic potential.