Structural Characterization of a Neutral Β-D-Glucan-Containing Fraction Isolated from the Basidiomycete Fungus Fomes fomentarius By NMR Spectroscopy

Abstract

Background: Fungal β-glucans are structurally diverse polysaccharides whose linkage patterns and branching characteristics influence their physicochemical properties. Fomes fomentarius contains chemically heterogeneous polysaccharide fractions; however, fraction-specific structural information on neutral polysaccharides isolated from naturally occurring fruiting bodies remains limited. Detailed NMR-based characterisation is therefore important for clarifying the glycosidic linkage and substitution patterns of purified β-glucan-containing fractions. Aims: This study aimed to isolate a water-soluble melanin–glucan complex from the fruiting bodies of the basidiomycete fungus Fomes fomentarius collected in Uzbekistan, purify its polysaccharide components, and elucidate the structural features of the neutral polysaccharide fraction using nuclear magnetic resonance (NMR) spectroscopy. Methodology: Dried and powdered fruiting bodies were defatted by Soxhlet extraction for 24 h and subsequently extracted with hot water at solid-to-liquid ratios of 1:20, 1:15, and 1:10. The combined aqueous extracts were concentrated and lyophilised to obtain the crude water-soluble melanin-glucan complex (BTW). The crude fraction was further purified by ion-exchange chromatography, yielding neutral (BTW-1) and anionic (BTW-2) polysaccharide fractions. The purified BTW-1 fraction was structurally investigated by 1H NMR, 13C NMR, HSQC, and HMBC spectroscopy. Results: Soxhlet pretreatment yielded 2.45% of extractable material, whereas subsequent hot-water extraction afforded 4.85% of the crude water-soluble fraction. BTW was obtained as a cream-coloured, water-soluble powder that exhibited opalescence at low concentrations and increased viscosity at higher concentrations. UV spectroscopy showed an absorption band around 230 nm, and the total carbohydrate content was 30.46%. Ion-exchange chromatography yielded 20.00% of the neutral BTW-1 fraction and 53.20% of the anionic BTW-2 fraction. The 13C NMR and HSQC spectra of BTW-1 revealed characteristic signals of carbohydrate residues, including anomeric correlations at δH/δC 4.373/103.058 and 5.250/99.855 ppm. The signal at δH/δC 3.604/84.342 ppm was consistent with a downfield-shifted C-3 environment indicative of O-3 substitution, whereas C-6 resonances at δC 68.269-69.587 ppm suggested possible O-6 substitution. Conclusion: The chromatographic and NMR data indicate that the neutral BTW-1 fraction contains β-D-glucopyranosyl residues with structural features compatible with a β-(1→3)-linked glucan backbone and possible C-6 substitution or branching. The proposed linkage pattern is considered a provisional structural motif.