Preparation and identification of galacto-mannooligosaccharides generated from guar gum by a recombinant β-mannanase from Bacteroides fragilis (BF0736)

Guar gum (GG) containing mainly galactomannan (GM) is one of the most used hydrocolloids in food industry. β-Mannooligosaccharides (β-MOS) as building blocks generated from GG are potential prebiotics, yet their structures are still unclear. Preparation of GG-derived β-MOS and decoding the galactose substitution information of β-MOS are an ongoing challenge. Here, a novel β-mannanase BF0736 that belongs to the glycoside hydrolase family 26 from Bacteroides fragilis NCTC 9343 strain was cloned and applied to degrade GG for preparation of β-MOS mixture with a yield of 81.3 ± 2.2% (w/w; relative to GG). Eleven major galacto-mannooligosaccharides (G-MOS) with degree of polymerization (DP) 2–11 were purified from the mixture by SEC and HPAEC techniques. A new strategy for decoding the galactose substitution information of G-MOS was developed by using two-step labeling (derivatizations of anomeric site in mannose and primary hydroxyl group site in galactose) combined with tandem mass spectrometry analysis methods. Precise structures of these G-MOS were characterized, and eight oligosaccharides including 6³-α-D-galactosyl-β-D-mannotriose, 6¹,6³-di-α-D-galactosyl-β-D-mannotriose, 6³,6⁴-di-α-D-galactosyl-β-D-mannotetraose, 6¹,6³,6⁴-tri-α-D-galactosyl-β-D-mannotetraose, 6³,6⁴,6⁵-tri-α-D-galactosyl-β-D-mannopentaose, 6¹,6³,6⁴,6⁵-tetra-α-D-galactosyl-β-D-mannopentaose, 6³,6⁴,6⁵,6⁶-tetra-α-D-galactosyl-β-D-mannohexaose and 6¹,6³,6⁴,6⁵,6⁶-penta-α-D-galactosyl-β-D-mannohexaose were prepared and identified from GG for the first time, which provided critical insights into the distribution pattern of galactose in GG. Our results contribute to the utilization of GG and its derived β-MOS in functional foods.