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Advances in nuclear magnetic resonance spectroscopy for the structure elucidation of water-extractable and water-unextractable arabinoxylan from wheat (Triticum aestivum L.) flour

The consumption of dietary fiber is associated with several health benefits. As a staple crop, wheat (Triticum aestivum L.) is a major source of dietary fiber in the diet of a large portion of the world population.

The predominant dietary fiber in wheat is arabinoxylan (AX). It consists of a linear backbone of β-(1,4)-linked ß-D-xylopyranosyl (xylose) residues that can be unsubstituted (uXyl), mono-substituted (mXyl) or di-substituted (dXyl) with α-L-arabinofuranosyl (arabinose) residues, some of which in turn carry a phenolic acid (in essence ferulic acid) residue. AX molecules differ in molecular weight and degree of arabinose and ferulic acid substitution. Part of the wheat flour AX (20-30%) is water-extractable (WE-AX), while the major part (70‑80%) is water-unextractable (WU-AX). Notable AX-derived products are AX oligosaccharides (AXOS). These are obtained via enzymatic hydrolysis of AX and have been classified as prebiotics. Since AX structural features determine its overall functionality during product making and its health effects, it is essential to gain insight in AX structure and in the structural heterogeneity within the AX population of a sample.

High-resolution nuclear magnetic resonance (NMR) spectroscopy has over the past decades been instrumental in gaining much of the current knowledge on wheat AX structural features. Still, part of the structural heterogeneity of wheat AX remains unresolved. Prior to this work, AX have not been studied by applying recent advances in NMR spectroscopy.


Against this background, this doctoral dissertation aimed to explore advanced NMR technologies for characterizing the structural heterogeneity of the AX population of the white flour fraction of wheat. Two sub-objectives were as follows: (i) to explore the use of 2D diffusion-ordered NMR spectroscopy (DOSY) for mixture analysis of wheat bran AXOS and wheat flour WE‑AX; (ii) to structurally characterize wheat flour WU-AX with solid-state NMR spectroscopy, while conserving its unextractable nature.

In-depth elucidation of WE-AX structural heterogeneity benefits from fractionation of WE-AX before further analysis by NMR spectroscopy. Although such approach is useful, the obtained WE‑AX fractions still contain compounds which from a structural point of view are heterogeneous.

In the first part of this work, it was reasoned that mixture analysis of AXOS by NMR spectroscopy might allow detailed AXOS structure elucidation and could be a first step towards mixture analysis of larger and more complex WE-AX compounds. Different 13C INEPT DOSY NMR approaches were explored for mixture analysis of aliphatic alcohols and aromatic molecules before applying a similar approach to more complex AXOS samples. H-INEPT-C-DOSY-STE NMR was shown to be an effective technique for structure elucidation of a mixture of AXOS components. Three main AXOS fractions were observed with different diffusion properties. The component in the fraction with the highest diffusion rate was xylobiose, whereas the two components in slower diffusing fractions were identified as unsubstituted xylotriose and xylotriose mono- and di-substituted with arabinose residues. Using 13C DOSY NMR, it was thus possible to distinguish between signals of xylobiose and xylotriose based on diffusion properties, which is very difficult, if not impossible, in standard 1D and 2D NMR analyses due to chemical shift overlap. In addition, AXOS relaxation properties were exploited to yield a 3D correlation NMR spectrum. In-depth identification of the mixture of AXOS compounds based on chemical structure, size and motion dynamics could be performed by combining spectral, diffusion, and relaxation properties.

The above work on AXOS suggested that DOSY NMR might also be valuable for characterizing the structural heterogeneity of wheat flour WE‑AX. In the second part of this work, the value of DOSY NMR spectroscopy was therefore explored for further elucidating WE-AX structural heterogeneity. To this end, wheat flour WE-AX was isolated and fractionated by graded ethanol precipitation from wheat flours (Evina, Claire) following existing protocols. The obtained fractions (F0-30%, F30-50%, F50-65%) were characterized thoroughly in terms of WE-AX yields and purities, arabinose-to-xylose ratios, apparent molecular weight distributions, and substitution patterns (uXyl, mXyl, dXyl). In addition, 1H DOSY NMR revealed the presence of distinct subpopulations differing in self-diffusivities within WE-AX fractions F30-50% and F50-65%. Generally speaking, WE‑AX structures with a high proportion of dXyl had slightly lower diffusivities than structures with a high proportion of mXyl. Furthermore, fractions precipitating at higher ethanol concentrations had a higher degree of structural heterogeneity with more neighboring dXyl residues in the AX structure. 1H DOSY NMR analysis of unfractionated WE-AX isolates and fraction F0-30% was impaired due to high sample viscosity, which were at the physical limits of the NMR equipment.


Earlier detailed structure elucidation of WU-AX required prior solubilization followed by liquid-state NMR spectroscopy, disrupting the original WU-AX structure. Attempts to analyze WU-AX structure by NMR in solid-state thus far have resulted in low resolution spectra, which limits detailed structure elucidation.

In the third part of this work, advanced high resolution solid-state NMR spectroscopy was employed for in-depth structural analysis of WU-AX without prior solubilization. Unextractable cell wall material (UCWM) was isolated from wheat flour (Evina), containing about 40% WU-AX. Limited hydration allowed to obtain 13C HPDEC MAS NMR spectra with sufficient resolution for detailed analysis of wheat flour WU-AX substitution patterns. The proportions of uXyl, mXyl, and dXyl in WU-AX were thus determined with this approach without WU-AX solubilization.


This doctoral study is the first in which 1H DOSY NMR was employed to study WE-AX structural heterogeneity within a fraction without prior physically separating the WE-AX molecules therein. Combined with fractionation by graded ethanol precipitation, 1H DOSY NMR has here been shown to be an effective tool for discriminating WE‑AX structures varying in size, but also in substitution degrees and patterns. Nevertheless, efforts should be undertaken for analyzing WE‑AX isolates exerting high sample viscosity. Also, it was the first time that solid-state NMR spectra could be obtained with sufficient spectral resolution to allow detailed structure elucidation of wheat flour WU-AX present in isolated UCWM.

The research performed in this dissertation has led to an increased insight in the value of several NMR approaches that are novel for AX structural characterization. This has led to detailed insights in the structural heterogeneity and diffusion properties of AXOS and WE-AX, and showed that solid-state NMR can be employed for structure elucidation of WU-AX. The NMR technologies used in this dissertation are presented in the form of an NMR toolbox for studying wheat flour AX structural characteristics and AX structure-function relationships.

Date:1 Oct 2017 →  18 Nov 2022
Keywords:arabinoxylan, wheat flour, dietary fiber, solid state NMR, DOSY NMR
Disciplines:Microbiology, Systems biology, Laboratory medicine, Other chemical sciences, Nutrition and dietetics, Agricultural animal production, Food sciences and (bio)technology, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering
Project type:PhD project