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Gastrointestinal conditions influence the solution behaviour of cereal β-glucans in vitro.The solution behaviour of β-glucans in a gastrointestinal model was investigated in order to explore the mechanisms explaining the physiological effects of the soluble fibre. Asymmetrical flow field-flow fractionation was used to determine the molar mass distribution, and in-line calcofluor labelling allowed specific detection of β-glucans in complex samples. When dispersed in water, weight-average molar mass (Mw) was determined to 1 × 106 g/mol for pure oat and barley β-glucans, and 200 × 106 g/mol for β-glucans in oat bran, indicating that the β-glucans were aggregating. Samples from the gastric digestion displayed disrupted aggregates, while samples from the small intestinal digestion contained re-formed aggregates. Additionally, the aggregates from pure β-glucans were considerably denser after intestinal digestion. This may be construed as gel-formation in the small intestine, which should be tested for its relevance to health effects. Our results signal the difficulties in predicting β-glucan activity in the gastrointestinal tract purely from analysis of the fibre-rich product.