Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation

•21 Enzyme recalcitrant oligosaccharides from corn fiber are structurally identified.•Common structural features: gal-xyl-ara side chains, acetyl-, and feruloyl groups.•Branched backbone xylose residues may be acetylated as well.•Ethyl glycosides of hemicellulosic structures identified may originate from SSF.•Recalcitrant structures identified clearly point to lacking enzyme activities. In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MSn. The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an α-l-galactopyranosyl-(1→2)-β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose side chain attached to the O-3 position of the β-1–4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxylan.