# 2D-NMR characterization of higher substituted oligosaccharides isolated from enzymatic wheat flour arabinoxylan hydrolysates

## TL;DR

This paper describes the isolation and characterization of complex, highly substituted arabinoxylooligosaccharides from wheat flour using NMR techniques.

## Contribution

The study reports the first complete NMR data sets for four complex AXOS and provides a comprehensive NMR library for 17 AXOS.

## Key findings

- Higher-substituted arabinoxylooligosaccharides with consecutive substitutions were identified in wheat hydrolysates.
- GH10 and GH11 endo-xylanases can cleave densely substituted regions of wheat arabinoxylans.
- Complete NMR data sets were obtained for four complex AXOS and a library for 17 AXOS was compiled.

## Abstract

Arabinoxylans (AXs) play a substantial role in the cell walls of cereals, contributing to their structural integrity and stability. The physicochemical and physiological properties of AX structures vary depending on the degree and pattern of substitution. AX structures are based on a linear β-(1→4)-linked d-xylopyranose backbone, being partially substituted with α-L-arabinofuranose in O2 and/or O3 positions of the xylopyranose units. Alkaline-extracted wheat flour AX were hydrolyzed with endo-β-1,4-xylanases of glycoside hydrolase (GH) families 10 or 11. The resulting arabinoxylooligosaccharides (AXOS) were isolated and purified using various chromatographic techniques, including gel permeation chromatography, semi-preparative hydrophilic interaction chromatography, and high performance anion exchange chromatography. The isolated, purified compounds were characterized by their monosaccharide composition, molecular weight, and monomer binding positions via one- and two-dimensional NMR experiments. In addition to smaller AXOS, higher-substituted oligosaccharides with consecutive mono- and disubstituted xylose residues were identified in the hydrolysates, proving that GH10 and GH11 endo-xylanases can cleave more densely substituted regions of wheat AX, in which disubstitution is preferred. These oligosaccharides were obtained in quantities and purities sufficient for their use as standard compounds. We report complete NMR data sets for the four most complex AXOS (A2+3XA2+3XX, A2+3A2+3XX, XA3A2+3XX, A3A2+3XX) for the first time and also provide a complete NMR library for 17 (A)XOS that were either isolated here or commercially available.