|Title:||Anti-inflammatory activities of exopolysaccharides from a medicinal fungus cordyceps sinensis Cs-HK1 and enhanced by bifidobacterial fermentation|
|Advisors:||Wong, Wing-tak (ABCT)|
Wu, Jian-yong (ABCT)
|Subject:||Cordyceps -- Therapeutic use|
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xx, 170 pages : color illustrations|
|Abstract:||This research project aims to investigate the anti-inflammatory potential of exopolysaccharides (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1, to fractionate the bioactive EPS fraction, and study the interaction of EPS fermentation by probiotic bifidobacterium, as well as the modified EPS molecular properties and enhanced bioactivity after bifidobacterial metabolization.|
In the first part of this project, the whole and crude EPS (CEPS) was recovered from the Cs-HK1 mycelial fermentation liquid by ethanol precipitation in a single step. After deproteinization and dialysis of the crude EPS, the partially purified EPS had a total sugar content of 74.8% with a maximum average molecular weight (MW) over 107 Da, consisting mainly of glucose, mannose, and a trace amount of galactose and ribose. In THP-1 and RAW264.7 cell models in vitro, the EPS fraction significantly inhibited lipopolysaccharide (LPS)-induced inflammatory responses including the release of NF-kB and several pro-inflammatory factors such as NO, TNF-α and IL-1β. In the murine model of LPS-induced acute intestinal injury, the oral gavage of EPS alleviated the intestinal injury and also suppressed the expression of major inflammatory cytokines TNF-α, IL-1β, IL-10 as well as iNOS in the treated animals. These results suggest that the EPS from Cs-HK1 has notable anti-inflammatory activity and can be a potential candidate for further development of new anti-inflammatory therapeutics.
The second part of the project was then to assess the anti-inflammatory activities of different EPS fractions, to isolate and purify the most active EPS fraction and to characterize its physicochemical properties. The EPS was first isolated from the Cs-HK1 fermentation liquid by two-step ethanol precipitation, using 40% ethanol in the first step to attain the higher MW EPS fraction (EPS-HM) and 80% in the second step to attain the lower MW EPS (EPS-LM). As the EPS-LM fraction showed higher anti-inflammatory activity than both the higher MW fraction EPS-HM from the two-step precipitation and the whole EPS from single step precipitation at 80% v/v ethanol, it was chosen for further characterization and anti-inflammatory assessment. The low-MW EPS-LM was further purified by columns, giving a purified EPS designated EPS-LM-1 with a homogenous average MW of 360 kDa. EPS-LM-1 was composed of three monosaccharide residues including mannose, glucose, and galactose at 3.9:6.9:1 mole ratio. EPS-LM-1 had a main chain of [6)-β-D-Glcp-(1→4)-α-D-Manp-(1→4)-α-D-Glcp-(1]2→3,6)-α-D-Glcp-(1→[3)-α-D-Manp-(1→4)-α-D-Glcp-(1]2, which was branched at the O-3 position of 3,6)-α-D-Glcp-(1→ with β-D-Galf-(1→ side chains (~8% degree of branching). In addition, the anti-inflammatory effects of EPS-LM-1 in in vitro were evaluated. In human monocytic THP-1 cell model, EPS-LM-1 significantly inhibited LPS-induced release of major pro-inflammatory factors with IC50 values below 5 µg/mL. Excessive protein expression levels of IKBα (nuclear factor-kappa-B inhibitor alpha) and NF-κB complex were both dose-dependently inhibited by EPS-LM-1 as well. In summary, EPS-LM-1 showed a notable anti-inflammatory activity probably through suppressing NF-kB signalling pathway. EPS-LM-1 may play a crucial role for its be a promising candidate for further development of anti-inflammatory therapeutics or nutraceuticals.
The third part of the project was to assess the anti-inflammatory activity of fermented EPS and metabolic products by selected Bifidobacterium species. The results indicated that the digesta (supernatant) from the bifidobacterial fermentation of EPS-LM showed much higher activity than the EPS-LM alone on LPS-stimulated proinflammatory responses in THP-1 cell model. This suggests that some metabolic products derived from the bifidobacterial fermentation of EPS-LM may produce stronger anti-inflammatory activity. The EPS-LM could enhance the growth of two bifidobacteria strains (B. breve and B. longum) at 1 g/L (p < 0.05) and also increased the acetic acid production significantly. Analytical results from the fermentation digesta suggested that EPS-LM was partially degraded to lower molecular weight (MW) products with modified structures during the bifidobacterial fermentation. More interestingly, the higher MW digesta fraction containing the partially degraded EPS-LM showed even stronger inhibiting activity than the original EPS-LM on the LPS-induced inflammatory THP-1 cell model. These results indicated that the fermentation by selected bifidobacterial strains is effective to modify natural polysaccharides with enhanced bioactivities. Another interesting effect from the bifidobacterial fermentation of EPS-LM was the notable antibacterial activity on the growth and biofilm formation disruption of Escherichia coli compared to the conventional digesta (RCM) as well as EPS-LM without fermentation, indicating the EPS-LM metabolism by bifidobacteria is responsible for potent bioactive components production.
This thesis presents the interesting and useful effects of Cs-HK1 EPS and its metabolite products from bifidobacterial fermentation and the new findings of highly active EPS molecular characteristics and the possible mechanisms for their anti-inflammatory effects. These results and findings will help pave the way for further development and application of Cs-HK1 EPS as new and effective ingredients for anti-inflammation therapy as well as maintaining human gut health. The findings will also make new and valuable contributions for discovery of novel nutraceutical and therapeutic products from probiotic fermentation of natural polysaccharides.
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