Molecular properties, bioactivities and gut microbial metabolism of exopolysaccharides from Cordyceps Cs-HK1 mycelial fermentation

Abstract

Cordyceps sinensis is a valuable Chinese medicinal fungus with a wide range of physiological activities. Polysaccharides are a major class of active constituents of C. sinensis, which have shown numerous bioactivities such as antioxidant, anti-inflammation, immunomodulation and hypoglycemic activities. Since natural C. sinensis is very rare and expensive, mycelial fermentation has become a more viable process for production of the fungal biomass and polysaccharides. Cs-HK1 is a C. sinensis fungus and capable of producing exopolysaccharide (EPS) in mycelial liquid fermentation. This research aims to investigate the prebiotic activities and potential health benefits on intestinal health through in vitro fecal fermentation of EPS isolated from Cs-HK1 mycelial fermentation liquid. Furthermore, the physicochemical properties and structural characteristics of separated and purified EPS fractions were analyzed to examine the structure-activity relationship. The following are the most significant research results and findings: In the first part of this research, a Cs-HK1 EPS, EPS-LM was assessed together with LBPS isolated from a well-known medicinal plant Lycium barbarum L. and the subsequent influences on human gut microbiota through simulated gastrointestinal systems were investigated. EPS-LM, an EPS isolated from mycelial culture of a medicinal fungus C. sinensis Cs-HK1, was characterized as a heteropolysaccharide consisting of Man(108):Gal(52.7):Glc(29.2) (molar ratio) with an average molecular weight (MW) 5.513×106 Da. LBPS was isolated from a well-known medicinal plant (Lycium barbarum L.) which was also characterized as a heteropolysaccharide (1.236×105 Da). Both polysaccharides were highly resistant to saliva, gastric and small-intestine digestion with negligible MW reduction and release of reducing sugars but were quickly degraded to lower MW during in vitro human fecal fermentation. They were consumed as a carbon source by the gut bacteria to produce short-chain fatty acids (SCFAs). In comparison, the carbohydrate content of EPS-LM was more completely consumed than LBPS and there were also notable differences in consumption of specific monosaccharides and production of specific SCFAs, propionic and butyric acid, and relative abundance of gut bacterial populations between EPS-LM and LBPS group. The results suggested that metabolic outcomes and modulating effects of EPS-LM and LBPS on the gut microbiota were highly dependent on their molecular composition. The second part of the research was to the prebiotic properties and the protective effects of EPS and human fecal fermentation products on gut barrier. Two EPS fractions with different MW and composition, EPS-LM (4.5×106 Da) and EPS-HM (9.4×107 Da) were fractionated through ethanol precipitation. The results revealed that the EPS fractions were highly resistant to digestive enzymes and gastric acid in a simulated human gastrointestinal tract but the EPS fractions were highly fermentable in human fecal culture, serving as a carbon source for the gut microbiota. Over 48 hours of fecal fermentation, EPS fractions were significantly degraded and utilized by the intestinal microbiota, resulting in a notable increase in production of SCFAs including acetic, propionic and butyric acid. The consumption rates of carbohydrates and the production levels of SCFAs varied slightly between the two EPS fractions. The fecal fermentation of EPS fractions increased the abundance of Actinobacteria, Bacteroidetes and Faecalibacterium, which are associated with improved gut health and metabolic function. In lipopolysaccharide (LPS)-stimulated Caco-2/Raw264.7 co-culture model, the fecal fermentation products of the EPS fractions exhibited a potential protective effect against inflammatory damage of gut barrier function, by inhibiting the proinflammatory cytokines, maintaining the trans-epithelial resistance (TEER) and upregulating the tight junction (TJ) proteins. Therefore, these findings suggest that EPS fractions may serve as promising therapeutic agents for enhancing gut barrier function and improving gut health through the modulation of gut microbiota. In the third part of the research, a neutral polysaccharide, EPS-LM-n was isolated from the low-MW Cs-HK1 EPS and further purified through anion-exchange and gel permeation chromatography. EPS-LM-n had a weight-average MW of 4.17×105 Da, a monosaccharide composition of Man:Glc:Gal 1:0.49:0.21 molar ratio, and a backbone structure of →2)-α-Manp-(1→ with branches →4)-α-Glcp-(1→, →3)-α-Galp-(1→. EPS-LM-n showed potential hypoglycemic activities by inhibiting α-amylase and α-glucosidase (IC50 values of 0.64 and 0.74 mg/mL, respectively) and significant antioxidant, and radical scavenging activities by several chemical assays. Furthermore, EPS-LM-n suppressed the ROS level and H₂O₂-induced cell damage and apoptosis in human vascular endothelial cell EA.hy926 culture. The results and findings collectively indicate that EPS-LM-n isolated from the Cs-HK1 EPS may serve as a potential candidate for dietary and therapeutic management of hyperglycemia and oxidative stress-related metabolic and cardiovascular diseases. In summary, the results from this research project are helpful to establish the structure and prebiotic function relationship for EPS from the Cs-HK1 mycelial fermentation. The Cs-HK1 EPS can regulate gut microbiota and alleviate intestinal damage by regulating intestinal flora, thereby maintaining intestinal health. These findings provide a theoretical basis for the future development of the Cs-HK1 EPS as a novel prebiotic food ingredient and for the creation of functional food products specifically designed to enhance human health.