Inter-laboratory analysis of cereal beta-glucan extracts of nutritional importance: An evaluation of different methods for determining weight-average molecular weight and molecular weight distribution

Simon Ballance,Yudong Lu,Hanne Zobel,Anne Rieder,Svein Halvor Knutsen,Vlad T Dinu,Bjørn E Christensen,Ann-Sissel Ulset,Marius Schmid,Ndegwa Maina,Antje Potthast,Sonja Schiehser,Peter R Ellis,Stephen E Harding

doi:10.1016/j.foodhyd.2022.107510

Abstract

In an interlaboratory study we compare different methods to determine the weight-average molecular weight (Mw) and molecular weight distribution of six cereal beta-glucan isolates of nutritional importance. Size-exclusion chromatography (SEC) with multi-angle light scattering (MALS), capillary viscometry, sedimentation velocity analytical ultracentrifugation and one asymmetric flow field-flow fractionation (AF4)-MALS method all yielded similar Mw values for mostly individual chains of dissolved beta-glucan molecules. SEC with post-column calcofluor detection underestimated the Mw of beta-glucan >500 × 103 g/mol. The beta-glucan molecules analysed by these methods were primarily in a random coil conformation as evidenced from individual Mark-Houwink-Kuhn-Sakurada (MHKS) scaling coefficients between 0.5 and 0.6 and Wales-Van Holde ratios between 1.4 and 1.7. In contrast, a second AF4-MALS method yielded much larger Mw values for these same samples indicating the presence and detection of beta-glucan aggregates. Storage of the six beta-glucan solutions in the dark at 4 °C for 4 years revealed them to be stable. This suggests an absence of storage-induced irreversible aggregation phenomena or chain-scission. Shear forces in SEC and the viscometer capillary and hydrostatic pressure in analytical ultracentrifugation probably led to the reversable dissociation of beta-glucan aggregates into molecularly dissolved species. Thus, all these methods yield true weight-average molecular weight values not biased by the presence of aggregates as was the case in one of the AF4 based methods employed.

Highlights

Oat and barley 1,3, 1,4-β-D-glucans (BG) are dietary fibres with positive health benefits endorsed by the European Food Safety Authority (EFSA) and U.S Food and Drug Administration (FDA)
The polysaccharide extracted from a BG enriched barley fraction (RT-9) was confirmed to have a high betaglucan content (88% BG, 1.5% protein) while the two samples extracted from oat (RT-7 and RT-10) contained slightly less BG, 79 and 77% respectively, and between 5.0 and 9.5% protein (Table 1)
It would appear that molecular dissolved BG in typical dilute solu tion conditions dominate during the Sizeexclusion chromatography (SEC)-based analysis methods (DMAc/lithium chloride (LiCl) and aqueous mobile phases), capillary viscometry, Sedimentation velocity analytical ultracentrifugation (SV-AUC), and to a large extent, one of the AF4 based methods (Lab 5)

Summary

Introduction

Oat and barley 1,3, 1,4-β-D-glucans (BG) are dietary fibres with positive health benefits endorsed by the European Food Safety Authority (EFSA) and U.S Food and Drug Administration (FDA) These benefits include a capacity to lower fasting blood cholesterol and attenuate postprandial glycaemic responses (EFSA, 2010; 2011a; 2011b). Mechanistic studies have indicated that the retention of high M in BG is important in reducing postprandial gly caemia and insulinaemia This metabolic effect seems to be linked to a decrease in the rate of starch digestion and glucose absorption caused by an increase in digesta viscosity in the proximal gut (Wang & Ellis, 2014). Another area of interest in the M of BG is associated with brewing where barley BG may cause filtration diffi culties during malting and mashing (Jin, Speers, Paulson, & Stewart, 2004)

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Conclusion