Evaluation of polysaccharides content in fruit bodies and their antimicrobial activity of four Ganoderma lucidum ( W Curt . : Fr . ) P . Karst . strains cultivated on different wood type substrates

Ganoderma lucidum (Curt.: Fr.) Karst. is a species of Basidiomycetes, wich belongs to Ganodermataceae (or Polyporaceae) of Aphylloporales [1]. This mushroom grows on a wide variety of dead or dying deciduous trees, especially Quercus, Acer, Alnus, Betula, Carpinus, Castanea, Fagus, Fraxinus, Juglans, Malus, Populus, Pyrus, and Robinia species. Species of the genus Ganoderma have been reported to occur throughout the world. Over 250 species of this mushroom are known [2]. Investigations of the representatives of the Ganodermataceae in Central European phytocenoses were carried out particularly intensively since the middle of the 20th century. Research in this area has been particularly abundant and there are numerous excellent domestic articles presenting and characterizing proportions of these representatives of cap mushrooms in individual communities of forest and non-forest national parks and nature reserves. The above-mentioned publications describe most frequently Ganoderma applanatum, G. adspersum (frequently in urbicenosis) as well as G. lucidum [3]. Artificial cultivation of G. lucidum began in early 1970s, and since 1980, its production has developed rapidly, particularly in China [4]. This mushroom have been cultivated in solid substrates such as lignocellulosic materials, especially sawdust, supplemented with wheat or rice bran [5]. G. lucidum as a medicinal mushroom has been widely used in China (named Ling Zhi) and Japan (named Reishi, Mannentake) for hundreds of years. It was used as a health tonic to promote longevity for more than two thousand years. Many bioactive components have been identified from its fruit bodies, mycelia, spores, and culture media. It was found that the major bioactive components were polysaccharides and triterpenes. Currently available data suggests that G. lucidum polysaccharides, mostly β-glucanes, activate host's immune responses exerting anti-cancer functions, whereas anti-tumor effect of triterpenes seems to be related to their cytotoxic activity against the tumor cells directly [6-8]. It is also believed that Ganoderma lucidum acts as an immunological agent. It has been used as a natural adjuvant for immunotherapy [9]. The highest attention is paid to polysaccharides from G. lucidum, which activate macrophages, lymphocytes, NK cells, proinflmmatory cytokines such as TNF or interleukins, essential for host survival from infection, and are also required for the repair of tissue injury [10,11]. Polysaccharides extracted from the fruit bodies, mycelium, and spores of G. lucidum can promote the function of macrophages as well as B cells [12]. Abstract


Introduction
Ganoderma lucidum (Curt.: Fr.) Karst. is a species of Basidiomycetes, wich belongs to Ganodermataceae (or Polyporaceae) of Aphylloporales [1].This mushroom grows on a wide variety of dead or dying deciduous trees, especially Quercus, Acer, Alnus, Betula, Carpinus, Castanea, Fagus, Fraxinus, Juglans, Malus, Populus, Pyrus, and Robinia species.Species of the genus Ganoderma have been reported to occur throughout the world.Over 250 species of this mushroom are known [2].Investigations of the representatives of the Ganodermataceae in Central European phytocenoses were carried out particularly intensively since the middle of the 20th century.Research in this area has been particularly abundant and there are numerous excellent domestic articles presenting and characterizing proportions of these representatives of cap mushrooms in individual communities of forest and non-forest national parks and nature reserves.The above-mentioned publications describe most frequently Ganoderma applanatum, G. adspersum (frequently in urbicenosis) as well as G. lucidum [3].Artificial cultivation of G. lucidum began in early 1970s, and since 1980, its production has developed rapidly, particularly in China [4].This mushroom have been cultivated in solid substrates such as lignocellulosic materials, especially sawdust, supplemented with wheat or rice bran [5].
G. lucidum as a medicinal mushroom has been widely used in China (named Ling Zhi) and Japan (named Reishi, Mannentake) for hundreds of years.It was used as a health tonic to promote longevity for more than two thousand years.Many bioactive components have been identified from its fruit bodies, mycelia, spores, and culture media.It was found that the major bioactive components were polysaccharides and triterpenes.Currently available data suggests that G. lucidum polysaccharides, mostly β-glucanes, activate host's immune responses exerting anti-cancer functions, whereas anti-tumor effect of triterpenes seems to be related to their cytotoxic activity against the tumor cells directly [6][7][8].It is also believed that Ganoderma lucidum acts as an immunological agent.It has been used as a natural adjuvant for immunotherapy [9].
The highest attention is paid to polysaccharides from G. lucidum, which activate macrophages, lymphocytes, NK cells, proinflmmatory cytokines such as TNF or interleukins, essential for host survival from infection, and are also required for the repair of tissue injury [10,11].Polysaccharides extracted from the fruit bodies, mycelium, and spores of G. lucidum can promote the function of macrophages as well as B cells [12].
Additionally, it has been shown that the anti-oxidant property of Ganoderma polysaccharide peptide decreased the oxidation of low density lipoprotein and exhibited anti-oxidant effect by scavenging reactive oxygen species in mice [13].Other studies demonstrated that G. lucidum contained antibacterial constituents [14][15][16].The aqueous extract from the carpophores of G. lucidum inhibited 15 types of bacteria [17].
It is known that the content of polysaccharides is different depending on the growth substrate.Nowadays almost all of the important medicinal mushrooms have been subjected to large-scale artificial cultivation by solid substrate or low moisture fermentation.Various substrates for Ganoderma lucidum cultivation have been investigated as a supplement for substrate mixture.Erkel [18], Siwulski and Sobieralski [19] stated that yielding of G. lucidum depended on the type of sawdust, meaning the tree species it originated from.Kim et al. [20] reported that different media in submerged culture of G. lucidum influenced the mycelial growth and exobiopolymer production.The aim of our study was quantitative determination of polysaccharides in G. lucidum fruit bodies from different sawdust cultivation substrates and their antibacterial activity.

Plant material
Four strains of Ganoderma lucidum (Fr.)Karst.fructifications were used in experiment, i.e.GL01, GL02, GL03 and GL04.All strains originated from the Culture Collection of the Department of Vegetable Crops of Poznań University of Life Sciences.As a growth substrate birch (Bo), maple (Kl) or alder (Ol) sawdust were used and they were amended with 10, 20 and 30% (w/w) of wheat bran, correspondingly assigned as (1), ( 2) and (3).After mixing the components, the substrates were watered up to the moisture content of 65%.In experiment the substrates were packed into PP foil bags with filter (0.02 μm).Each bag contained 1.5 kg of substrate.Bags with the substrates were sterilized at 121°C for 2 h.After cooling to the room temperature (ca.21°C) the substrates were inoculated with the mycelium of tested Ganoderma lucidum strains in the amount equal to 5% of the substrate dry weight.Next, substrates were incubated at 25°C and 80-85% RH until all the substrate volume was overgrown by the fungus.After incubation bags were opened by cutting in the upper part and substrates were transferred into cultivation room with temperature of 17-18°C and RH 85-90%.Cultures were illuminated with fluorescent light (Day-Light) of 500 lx intensity for 12 h a day.Fruiting bodies were set on the upper surface of the substrate.They were harvested once, after the end of maturation, by cutting them back at the substrate surface.The experiment was set in a randomised design with 3 replicates during the spring of 2009.

Reagents and instruments
Ethanol used for preparation of the extract from fungal material was of analytical grade and purchased together with phenol and sulphuric acid from the Polish Reagents (POCH, Gliwice, Poland).Water was purified using a Millipore laboratory ultra-pure water system (SimplicityTM system, Millipore, Molsheim, France).A Specol-11 spectrophotometer (Carl Zeiss, Germany) was employed for reading the absorbance.

Extraction
Polysaccharides were isolated by the method proposed by Pillai [21] with some modifications.Ten grams of fungal material was extracted three times with 200 mL of hot water (85°C) for 5 h.Water extracts were filtered, combined and concentrated to 100 mL.Then 300 mL of chilled ethanol was added and it was left in a cold place (4°C) for 24 h.The precipitate was collected after centrifugation (5000 rpm, 10 min), washed with ethanol and dried.Crude polysaccharides were obtained.

Determination of total sugar in crude polysaccharide
The total sugar in a sample of crude polysaccharide of G. lucidum was determined using a phenol-sulphuric acid method [22].One milligram of each sample was dissolved in 10 mL Tab. 1 The content of determined polysaccharides in examined samples of Ganoderma lucidum.
Skalicka-Woźniak et al. / Polysaccharides of Ganoderma lucidum.Antibacterial activity of distilled water.One milliliter of this solution was mixed with 1 mL of 5% phenol solution and 5 mL of concentrated sulphuric acid.The mixture was shaken for 30 min and then the absorbance was measured at 490 nm.The total sugar was calculated based on the standard curve of glucose.All experiments were repeated three times and the SD (standard deviation) and RSD value (the percentage of relative standard deviation) were calculated.

Calibration procedure
The calibration curve was analyzed three times with five different concentrations as follow: 0.055, 0.114, 0.155, 0.239, 0.475 mg/mL.The quantification was performed by measuring the absorbance.The calibration curves were characterized by their regression coefficient, slope of the line (b) and intercept of the straight line with y axis (a).

Antibacterial assay in vitro
Minimal inhibitory concentration (MIC) of the polysaccharide fractions obtained from G. lucidum fruit bodies from different sawdust cultivation substrates for eight reference strains, including four Gram-positive bacteria (Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 10240) and four Gram-negative bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027, Proteus mirabilis ATCC 12453), was performed by the micro-dilution broth method according to Skalicka-Woźniak et al. [23].Briefly, the stock solutions of polysaccharides fractions were prepared in dimethyl sulphoxide (DMSO).The series of two-fold dilution of this stock solutions, ranging from 0.075 to 5 mg/mL, were prepared in Mueller-Hinton broth (Biocorp, Poland) in 96-well microtiter plates.Bacterial inoculum was added to each well to obtain final optical density corresponding to 5 × 105 CFU/mL.After incubation at 35°C for 24 h, the MIC was assessed visually as the lowest concentration of polysaccharides showing total inhibition of bacterial growth.Appropriate DMSO, growth and sterile controls were carried out.Minimal bactericidal concentration (MBC) of polysaccharide fractions towards all bacterial strains tested was defined by subculturing 0.1 mL from each well that showed bacterial growth inhibition onto Mueller-Hinton agar (Biocorp, Poland) plates.The plates were incubated at 35°C for 24 h and the MBC was defined as the lowest concentration of essential oil at which there was no bacterial growth.Each experiment was repeated in triplicate.Representative data are presented.

Results and discussion
The calibration curve was linear (R 2 = 0.9993) in a concentration range 0.055-0.475mg/mL and was y = 1.7465x + 0.0106.The RSD value as a measure of repeatability was lower than 0.003.The amounts of polysaccharides in the analyzed samples are shown in Tab. 1.
Thirty six samples were analyzed.Four strains of Ganoderma lucidum (GL01, GL02, GL03 and GL04) were cultivated on the growth substrates of three different sawdust types: birch, maple or alder amended with wheat bran in three different concentrations: 10, 20 and 30% (w/w).Even though the highest production of polysaccharides was noticed in GL01 strain, the highest amount of polysaccharides were calculated in GL04Kl3 sample.The lowest level of production of polysaccharides was in the GL03 strain, low level was noticed also for GL04 strain.However, it was the GL04Kl3 sample that appeared to be the richest in interesting compounds.Alder sawdust was found as the best growth substrate for GL01 and GL03 strains, while for strains GL02 and GL04 it was a maple sawdust.Sugars, as the carbon sources, can influence mycelium growth and polysaccharide production [24].It is known that lactose exerts more favourable impact on cell growth and production of intercellular polysaccharide than glucose.The effect of different carbon sources on the biomass production in G. lucidum was reported by Erkel [25], Avtonomova et al. [26] and Yang et al. [27].Baabitskaia et al. [28] maintain that polysaccharide production by G. lucidum depends on such conditions as the initial pH of the substrate, C:N ratio and incubation temperature.The influence of substrate pH on polysaccharide production by G. lucidum was also reported by Kim et al. [29] and Lee et al. [30].According to Prosiński [31], wood of various tree species differs with regard to the content of cellulose, lignin, hemicellulose, sugars, protein as well as other substances and shows differences in pH.It is possible that these properties led to differences in the polysaccharide content of the fruiting bodies of G. lucidum obtained from the substrates used in our own experiments.
According to our results presented in Tab. 2 the analyzed samples of polysaccharides exhibited inhibitory effect against all bacterial strains tested with MIC values ranging from 0.62 to 5.0 mg/mL, however, M. luteus was the most sensitive strain (MIC = 0.62 − 1.25 mg/mL).The MBC values for all analyzed samples were comparable (2.5 or 5.0 mg/mL).Only slight differences of MICs and MBCs for polysaccharide samples obtained from the particular G. lucidum strains and the cultivate conditions were observed.The low values of MBC/MIC ratio (1-8) suggest that polysaccharides acted as bactericidal agents.
The screening of antibacterial activity indicates that there were no significant differences in the spectrum or the power of activity between the polysaccharides obtained from four strains of G. lucidum fruit bodies from different sawdust cultivation substrates.All the polysaccharide samples tested in our study show the broad spectrum of antibacterial activity, covering both Gram-positive and Gram-negative strains, however, the power of this activity is moderate.Our results are in agreement with other studies reporting the moderate activity and the broad antimicrobial spectrum of the different extracts from G. lucidum [17,32,33].Its polysaccharides are confirmed to be the bioactive components, which show the significant antibacterial activity.Similarly to data obtained by Yoon et al. [17], the polysaccharides tested in our study exerted the strongest inhibitory effect towards M. luteus (MIC 0.62 or 1.25 mg/mL).

Tab. 2
Minimal inhibitory concentrations and minimal bactericidal concentrations of tested polysaccharides isolated from Ganoderma lucidum against bacterial reference strains.MIC -minimal inhibitory concentration; MBC -minimal bactericidal concentration.Skalicka-Woźniak et al. / Polysaccharides of Ganoderma lucidum.Antibacterial activity