Novel structures and properties of polysaccharides derived from exopolysaccharides produced by a cordyceps sinensis fungus in liquid fermentation

Abstract

Polysaccharides (PS) from edible and medicinal fungi are valuable nutraceuticals because of their functional properties and bioactivities. Cordyceps (Ophiocordyceps) sinensis, generally known as the Chinese caterpillar fungus, is a precious medicinal fungus and widely used as a favorable tonic in China. Because natural caterpillar fungi are very rare and expensive, mycelial fermentation of a C. sinensis fungus Cs-HK1 has been established in our lab, and applied to liquid fermentation for production of mycelial biomass and exopolysaccharides (EPS). This research project aims to discover and characterize novel PS structures from the EPS produced by Cs-HK1 mycelial fermentation and their functional properties and bioactivities. The crude EPS from Cs-HK1 mycelial fermentation was a mixture of PS and PS-protein (PSP) complexes in a wide molecular weight (MW) range. Two-step ethanol precipitation was applied to separate the EPS into a high-MW and a low-MW EPS fractions. The high-MW EPS, EPS1, was obtained in the first step with 40% ethanol and the low-MW EPS, EPS2, was obtained in the next step with 70% ethanol. The high MW EPS1 had poor solubility and high viscosity in water. To improve its solution properties, EPS1 was partially degraded by high-intensity ultrasound (US), yielding a water-soluble polysaccharide EPS1U. EPS1U exhibited a single, symmetrical peak on HPGPC with an average MW about 730 kDa by light scattering analysis. EPS1U had a much lower intrinsic viscosity (1.7 versus 15.6 dL/g) but a much higher solubility in water (77.5 versus 5.1 g/L) than EPS1. Methylation analysis and NMR spectrometry were performed to investigate the structural feature of EPS1U. The structure of EPS1U was elucidated as a (1→3)-β-D-glucan with glucose side chains attached to O-6 position at the branching points. This is the first report on a high-MW (1→3)-β-D-glucan isolated from the EPS produced by Cs-HK1. EPS1U showed a high moisture absorption capability comparable to chitosan and urea, suggesting its potential use as moisturizing agent for food and cosmetic applications. In RAW264.7 murine macrophage cell cultures, EPS1U showed significant immunomodulatory activities by stimulating the production of nitric oxide, and release of tumor necrosis factor α and interleukin 6. A protein-containing PSP complex, EPS2BW, was isolated and purified from the low-MW EPS fraction EPS2. EPS2BW was mainly composed of galactomannan with about 16% (w/w) protein and 50 kDa average MW. The galactomannan part consisted of mannose and galactose at a molar ratio of 1.7:1.0, and the protein segments were composed of sixteen amino acids with 12.5% proline and 16.6% threonine (mol%) being the most abundant. Based on the analytical results from NMR, methylation analysis, partial acid hydrolysis and GC-MS, the galactomannan structure was elucidated as a (1→2)-α-D-mannopyranosyl (Manp) backbone with O-6-linked galactopyranosyl (Galp) branches. EPS2BW exhibited high antioxidant capacity in both chemical and cell culture assays, with a Trolox equivalent radical scavenging activity of 44.7 μmol Trolox/mg, a Fe³⁺ reducing power of 38.9 μmol Fe²⁺/mg, and significant cytoprotective effect against H₂O₂-induced PC12 cell death at 50-250 μg/mL. Silver nanoparticles (AgNPs) have attracted much attention because of their unique functional properties and potent antimicrobial activity. Natural PS have been widely exploited as biocompatible carriers for green synthesis of AgNPs. As a novel application of EPS produced by Cs-HK1, EPS1 was used for the synthesis of AgNPs in water. The formation and properties of AgNPs were evaluated at various temperatures, time periods, and silver nitrate/EPS1 concentrations in water. At suitable conditions (100 °C, 60 min and 10 mM AgNO₃ with 1.0 mg/mL EPS1), AgNPs were formed with an average diameter of 50 nm and a narrow size distribution, and they had stable dispersion that lasted for at least 2 months. EPS1 might have acted as a reducing and stabilizing agent in the formation of AgNPs, which were attached to the hydroxyl groups of EPS1. The AgNPs formed in EPS1 solution exhibited concentration-dependent inhibition of both Gram-negative and -positive bacteria but very low cytotoxicity on the RAW264.7 murine macrophage cells. The results demonstrated the potential of EPS produced by Cs-HK1 for green synthesis of AgNPs. The production of PS in microbial fermentation can be affected by the major nutrients. Therefore we further evaluated the effects of alternative carbon sources on the productivity and properties of EPS in the Cs-HK1 mycelial culture. Galactose or mannose was used (at 5 g/L) as a secondary carbon source together with glucose (35 g/L) at the mass ratio of 1:7. The yield and composition of EPS produced by Cs-HK1 varied as the component of carbon source changed. The addition of galactose improved the total yield of EPS in culture while mannose increased the glucan content of EPS. In summary, the results from this project showed that the mycelial fermentation of Cs-HK1 fungus produced a variety of EPS structures with interesting and useful properties, and bioactivities for nutraceutical applications and green synthesis of AgNPs. The major findings from this project has laid the foundation for further development and application of EPS from the Cs-HK1 in the pharmaceutical, food and cosmetic industries.