Abstract
Yeast cell walls undergo modifications during the brewing process, leading to a remodelling of their architecture. One significant change is the increased insolubility of the cell wall glycogen pool, likely due to the formation of covalent bonds between glycogen and cell wall polysaccharides. To verify this hypothesis, we extracted the brewer's spent yeast with 4 M KOH, obtaining an insoluble glucan fraction (AE.4 M) primarily composed of (α1 → 4)- and (1 → 3)-linked Glc residues. Dynamic nuclear polarization solid-state NMR of AE.4 M revealed distinct glucan resonances that helped to differentiate between α- and β glucosyl (1 → 4)-linked residues, and confirm covalent linkages between (β1 → 3)-glucans and glycogen through a (β1 → 4)-linkage. The hydrolysis with different endo-glucanases (zymolyase, cellulase, and lichenase) was used to obtain solubilized high molecular weight glycogen fractions. NMR analysis showed that covalent links between glycogen and (β1 → 6)-glucans through (α1 → 6) glycosidic linkage, with branching at the C6 position involving (β1 → 3), and (β1 → 6)-glucans. HPAEC-PAD analysis of the enzymatically released oligosaccharides confirmed covalent linkages of (β1 → 3), (β1 → 6)-, and (β1 → 4)-glucan motifs with (α1 → 4)-glucans. This combination of multiple enzymatic approaches and NMR methods shed light into the role of yeast cell wall glycogen as a structural core covalently linked to other cell wall components during the brewing process.