Hydration of Wheat Flour Water-Unextractable Cell Wall Material Enables Structural Analysis of Its Arabinoxylan by High-Resolution Solid-State 13C MAS NMR Spectroscopy.

To enable its structural characterization by nuclear magnetic resonance (NMR) spectroscopy, the native structure of cereal water-unextractable arabinoxylan (WU-AX) is typically disrupted by alkali or enzymatic treatments. Here, WU-AX in the wheat flour unextractable cell wall material (UCWM) containing 40.9% ± 1.5 arabinoxylan with an arabinose-to-xylose ratio of 0.62 ± 0.04 was characterized by high-resolution solid-state NMR without disrupting its native structure. Hydration of the UCWM (1.7 mg H2O/mg UCWM) in combination with specific optimizations in the NMR methodology enabled analysis by solid-state 13C NMR with magic angle spinning and 1H high-power decoupling (13C HPDEC MAS NMR) which provided sufficiently high resolution to allow for carbon atom assignments. Spectral resonances of C-1 from arabinose and xylose residues of WU-AX were here assigned to the solid state. The proportions of un-, mono-, and di-substituted xyloses were 59.2, 19.5, and 21.2%, respectively. 13C HPDEC MAS NMR showed the presence of solid-state fractions with different mobilities in the UCWM. This study presents the first solid-state NMR spectrum of wheat WU-AX with sufficient resolution to enable assignment without prior WU-AX solubilization.

1H Diffusion-Ordered Nuclear Magnetic Resonance Spectroscopic Analysis of Water-Extractable Arabinoxylan in Wheat (Triticum aestivum L.) Flour.

The structural heterogeneity of water-extractable arabinoxylan (WE-AX) impacts wheat flour functionality. 1H diffusion-ordered (DOSY) nuclear magnetic resonance (NMR) spectroscopy revealed structural heterogeneity within WE-AX fractions obtained via graded ethanol precipitation. Combination with high-resolution 1H-1H correlation NMR spectroscopy (COSY) allowed identifying the relationship between the xylose substitution patterns and diffusion properties of the subpopulations. WE-AX fractions contained distinct subpopulations with different diffusion rates. WE-AX subpopulations with a high self-diffusivity contained high levels of monosubstituted xylose. In contrast, those with a low self-diffusivity were rich in disubstituted xylose, suggesting that disubstitution mainly occurs in WE-AX molecules with large hydrodynamic volumes. In general, WE-AX fractions precipitating at higher and lower ethanol concentrations had higher and lower self-diffusivity and more and less complex substitution patterns. Although 1H DOSY NMR, as performed in this study, was valuable for elucidating WE-AX structural heterogeneity, physical limitations arose when studying WE-AX populations with high molecular weight dispersions.

13C-DOSY-TOSY NMR Correlation for In Situ Analysis of Structure, Size Distribution, and Dynamics of Prebiotic Oligosaccharides.

Arabinoxylan oligosaccharides (AXOS) are a complex mixture of cereal derived, water-soluble prebiotics, obtained by enzymatic hydrolysis of arabinoxylan, a group of dietary fibers exerting numerous nutritional and health-beneficial effects. Such complex biomolecular mixtures are notoriously difficult to characterize without initial physical fractionation. Here we present the in situ analysis of AXOS using a variety of state-of-the-art sensitivity-enhanced 13C-DOSY methods, enabling virtual separation and identification of the components. Three dimensional correlation plots displaying 13C diffusivity (DOSY: Diffusion Ordered SpectroscopY), relaxation parameters (TOSY: raTe of relaxation Ordered SpectrscopY), and chemical shift offer a unique way to elucidate the composition of mixtures. We have demonstrated this multifaceted 13C probed correlation strategy in standard mixtures of aliphatic and aromatic compounds, before implementing it on AXOS. These 3D-DOSY-TOSY plots in combination with 2D-NMR correlation experiments offer unprecedented clarity for assigning chemical functions, molecular size distribution, and dynamics of oligosaccharide mixtures.

The Impact of Hydro-Priming and Osmo-Priming on Seedling Characteristics, Plant Hormone Concentrations, Activity of Selected Hydrolytic Enzymes, and Cell Wall and Phytate Hydrolysis in Sprouted Wheat (Triticum aestivum L.).

Priming improves the seed germination rate and grain yield. Before this work was executed, little, if any, research has been reported on priming wheat for improving its nutritional properties. The impact of hydro-priming and osmo-priming using solutions with different water potentials on selected hydrolytic enzyme activities and their breakdown of starch, cell wall materials, and phytates during subsequent sprouting was studied here. A higher germination rate in the early growth stage of seedlings was found for hydro-primed or osmo-primed (-0.3, -0.6 MPa) grains. Hydro-primed sprouted grains had the longest radicles and coleoptiles and the highest hydrolytic enzyme activities. The latter lead to a 90% increase in reducing sugar, a 20% increase in water-extractable arabinoxylan, and an 8% decrease in phytate contents after 5 days of sprouting. This study thus offers opportunities for optimizing agricultural practice. The presence of different plant hormones and their concentrations are generally not affected by priming. However, the plant hormone concentrations in grains primed at -1.2 MPa and subsequently sprouted were lower than those in all other samples under study. The induction of too high osmotic stresses in these grains leads to disruption of the sprouting processes. Finally, it was for the first time found, based on the known biosynthesis pathways of wheat, that gibberellic acid (GA)20-oxidase in (primed) sprouted wheat is more active than GA3-oxidase and much more active than GA13-oxidase.