Analysis and glycosyl composition of the exopolysaccharide isolated from submerged fermentation of Ganoderma lucidum CG 144

Ganoderma lucidum (Curtis) P. Karst is a famous fungus (mushroom) in Chinese herbal medicine. Recently, polysaccharides from G. lucidum have been identified as active ingredients responsible for its biological activities. For instance, polysaccharides from G. lucidum fruiting bodies have been shown to have antimicrobial property [1]. In the same way, G. lucidum mycelium have been demonstrated to possess (1,3;1,6)-beta-D-glucans which are recognized for their many health claims [2]. Finally, after the biological evaluation of intracellular polysaccharides (polysaccharides from fruiting bodies and mycelium), studies also have reported biological activities such as antioxidant properties of the exopolysaccharides (extracellular polysaccharides) from G. lucidum cultured in submerged fermentation [3]. In addition, there are only a few reports available on biological activities of the exopolysaccharides from G. lucidum and new biological activities cannot be carried out randomly. Similarly, the glycosyl composition is a key factor to understand the underlying biological activities and possible metabolic mechanisms of fungal polysaccharides. For example, studies have been reported the immunostimulatory and antitumor activities of glucans and mannans [4]. Galactose may prevent cognitive deficits associated with glucose hypometabolism in Alzheimer’s disease [5]. Recent studies have also focused on the effect of glucans on redox balance [6]. Acetylglucosamine may modulate function of the skin fibroblasts and it also has anti-inflammatory and anti-senescent activity in mesothelial cells [7]. Thus, the glycosyl composition is an essential step to guide future research designs applied in bioactivity. Moreover, a great lack of knowledge exists concerning the glycosyl composition of the exopolysaccharides from G. lucidum. Considering the glycosyl composition as an essential step to guide future researches applied in bioactivity and the unexplored bioactivity of exopolysaccharide from Ganoderma lucidum, here we investigated the glycosyl composition of the exopolysaccharide isolated from submerged fermentation of Ganoderma lucidum to serve as guide for future studies on bioactivity.


Introduction
Ganoderma lucidum (Curtis) P. Karst is a famous fungus (mushroom) in Chinese herbal medicine.Recently, polysaccharides from G. lucidum have been identified as active ingredients responsible for its biological activities.For instance, polysaccharides from G. lucidum fruiting bodies have been shown to have antimicrobial property [1].In the same way, G. lucidum mycelium have been demonstrated to possess (1,3;1,6)-beta-D-glucans which are recognized for their many health claims [2].Finally, after the biological evaluation of intracellular polysaccharides (polysaccharides from fruiting bodies and mycelium), studies also have reported biological activities such as antioxidant properties of the exopolysaccharides (extracellular polysaccharides) from G. lucidum cultured in submerged fermentation [3].In addition, there are only a few reports available on biological activities of the exopolysaccharides from G. lucidum and new biological activities cannot be carried out randomly.
Similarly, the glycosyl composition is a key factor to understand the underlying biological activities and possible metabolic mechanisms of fungal polysaccharides.For example, studies have been reported the immunostimulatory and antitumor activities of glucans and mannans [4].Galactose may prevent cognitive deficits associated with glucose hypometabolism in Alzheimer's disease [5].Recent studies have also focused on the effect of glucans on redox balance [6].Acetylglucosamine may modulate function of the skin fibroblasts and it also has anti-inflammatory and anti-senescent activity in mesothelial cells [7].Thus, the glycosyl composition is an essential step to guide future research designs applied in bioactivity.Moreover, a great lack of knowledge exists concerning the glycosyl composition of the exopolysaccharides from G. lucidum.
Considering the glycosyl composition as an essential step to guide future researches applied in bioactivity and the unexplored bioactivity of exopolysaccharide from Ganoderma lucidum, here we investigated the glycosyl composition of the exopolysaccharide isolated from submerged fermentation of Ganoderma lucidum to serve as guide for future studies on bioactivity.
Bioprocess Engineering and Biotechnology Division at Federal University of Paraná (UFPR), Curitiba -PR, Brazil.It was maintained on potato-dextrose agar (PDA) medium, incubated at 30°C for 10 days and subsequently stored at 4°C.Sub-cultures were made every 2 months to maintain the strain active.

Inoculums preparation
For preparation of inoculums, the G. lucidum CG 144 was transferred from agar PDA slants into 125 ml Erlenmeyer flasks containing 20 ml of potate dextrose broth and incubated in a shaker at 30°C, agitated at 120 rpm for 7 days.

Flask culture
The culture medium was composed (in grams per liter) of: glucose 35, peptone 5, yeast extract 2.5, KH 2 PO 4 0.88 and MgSO 4 •7H 2 O 0.5; the initial pH of the medium was adjusted to 5.5.Erlenmeyer flasks (250 ml) containing 120 ml medium were inoculated at 30°C in a rotatory shaker, at 120 rpm, for 10 days.A 4% (v/v) of inoculum was added to the flask culture.Cells were removed from the culture medium by centrifugation at 12 000 g for 15 min at 4°C [8].

Isolation and purification of exopolysaccharide
Exopolysaccharide was obtained by diluting the fermented culture medium with 3 volumes of cold 95% ethanol and leaving for 2 h at 4°C for precipitation to be complete, followed by centrifuging (7500 g for 10 min at 5°C) and dialysis through a membrane with 10 kDa cutoff.The exopolysaccharide was isolated as a lyophilized material [9].

Glycosyl content and composition
Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/ MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.An aliquot (10 μg) was taken from the sample and added to separate screw-cap tubes with 40 µg and 60 µg of inositol as the internal standard.The samples were lyophilized.Methyl glycosides were then prepared from the dry sample (0.3 mg) following the mild acid treatment by methanolysis in 1 M HCl in methanol (1 M) at 80°C (16 h), followed by re-N-acetylation with pyridine and acetic anhydride in methanol.The sample was then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80°C (0.5 h).GC/MS analysis of the TMS methyl glycosides was performed on an AT 6890N GC interfaced to a 5975B MSD (mass selective detector), using a Supelco EC-1 fused silica capillary column (30 m × 0.25 mm ID) [10][11][12].

Results and discussion
The main objective of this study was to evaluate the glycosyl composition of the exopolysaccharide from G. lucidum CG144.Certainly, the glycosyl composition is an essential step to guide future researches applied in bioactivity and must be studied in promising bioactive molecules such as exopolysaccharides, especially from G. lucidum.
Glycosyl content and composition were evaluated by combined GC/MS of the TMS derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.This technique has been used extensively to evaluate the glycosyl composition of polysaccharides.Detection limits of a few nanograms can be achieved with this methodology [10][11][12].
Interestingly, the carbohydrate composition of the exopolysaccharide resembles the carbohydrate compositions in other fungal systems (other heteroglycans), which are known about their immunostimulatory activity and antioxidant properties [13,14].Thus, research focused on exopolysaccharide from G. lucidum and all aspects of the immune system is certainly promising and should guide future studies.Exopolysaccharide lyophilization yielded 0.45 g l −1 of submerged fermentation.Cell dry weight reached a maximum value of 7.22 g l −1 .

Conclusion
In summary, these experiments have shown that the exopolysaccharide from G. lucidum CG 144 was composed of glucose (58.1%), mannose (26.6%) and galactose (12.5%), which can be referred to as heteroglycan.Further studies should be made in order to evaluate their immunostimulatory activity.
Glycosyl content and composition of exopolysaccharide isolated from submerged fermentation of Ganoderma lucidum CG 144.