Fig. 1: Schematic representation and graphic illustration of the production pipeline for Norway spruce AcGGM.

a Wood chips were milled to increase the surface area exposed for hydrothermal extraction and washing the released manno-oligosaccharides. Spruce wood contains long-chained galactoglucomannan (DP200-400), which is highly acetylated (DA ~ 0.65), predominantly with 2-O and 3-O acetylations⁶⁶. b During hydrothermal pretreatment, the release of acetic acid promoted hydrolysis of glycosidic bonds, thereby relinquishing monosaccharides, oligosaccharides, and other breakdown products. c Ultrafiltration retained the longer, complex oligosaccharides and discarded the monosaccharides, acetic acid, and other steam explosion byproducts. d The purified mannan was incorporated into feed pellets at varying inclusion levels, produced by a conventional feed pelleting process. e The growth performance experiment and a feeding trial were conducted in a randomized block design, with four inclusion levels of AcGGM. f Multi-omic approaches were used to analyze the porcine gut microbiome in response to AcGGM and determine if indigenous mannan PULs matched the glycan structure of the AcGGM fiber. Glycosidic bonds between the β-(1,4)-mannose and glucose units in the backbone of AcGGM require hydrolysis by glycoside hydrolases (GH) from families GH5 and GH26. GH36 and GH27 α-galactosidases are required to remove the α-(1,6)-galactose decorations. Single mannose and glucose units are removed from the non-reducing end of the oligosaccharides by enzymes from families GH1, GH2, GH3, GH113, and GH130, while mannose in the reducing end can be removed by GH113 family mannosidases. 3-O- and 6-O-acetylations on mannan are removed by esterases from family CE2. A unique feature of particular beta-mannans is the axially oriented 2-O-acetylation on mannose, which is the prevalent form of acetylation present on AcGGM used in this study. 2-O-acetylations are removed by esterases homologous to the RiCE17 mannan-specific esterase from Roseburia intestinalis, which was recently characterized by our group⁵.