Formation of branched oligosaccharides during sake brewing via transglycosylation by Aspergillus oryzae α-glucosidases

Sake, a traditional Japanese alcoholic beverage made from rice, contains a variety of oligosaccharides; however, the mechanisms underlying their formation remain poorly understood. In this study, we brewed sake using Aspergillus oryzae strains with disrupted α-glucosidase gene(s) and analyzed oligosaccharides with degrees of polymerization (DP) up to eight. We identified AgdA and AgdB as the primary α-glucosidases in sake brewing, and found that disruption of these genes significantly altered the oligosaccharide composition. Structural elucidation of six oligosaccharides was achieved through purification followed by NMR spectroscopy. We revealed that AgdA predominantly forms consecutive α-1,6-glucosyl linkages at the non-reducing end, while AgdB mainly forms α-1,2 and α-1,3 linkages. Based on these findings, sake oligosaccharides could be classified into four distinct types based on their structural features. Furthermore, we propose a model for the oligosaccharide formation process in sake brewing, in which oligosaccharides are diversified via transglycosylation.