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Short chain fatty acids (SCFA) are the major products of carbohydrate fermentation by gut bacteria. Different carbohydrates are associated with characteristic SCFA profiles although the mechanisms are unclear. The individual SCFA profile may determine any resultant health benefits. Understanding determinants of individual SCFA production would enable substrate choice to be tailored for colonic SCFA manipulation. To test the hypothesis that the orientation and position of the glycosidic bond is a determinant of SCFA production profile, a miniaturized in vitro human colonic batch fermentation model was used to study a range of isomeric glucose disaccharides. Diglucose α(1-1) fermentation led to significantly higher butyrate production (p