Ganoderma neo-japonicum Imazeki revisited: Domestication study and antioxidant properties of its basidiocarps and mycelia

Mushroom cultivation benefits humankind as it deliberately encourages wild mushrooms to be commercially propagated while recycling agricultural wastes. Ganoderma neo-japonicum is a rare polypore mushroom found growing on decaying Schizostachyum brachycladium (a tropical bamboo) clumps in Malaysia. The Malaysian indigenous tribes including the Temuans and Temiars use the basidiocarps of G. neo-japonicum to treat various ailments including diabetes. In this study, the domestication of G. neo-japonicum in artificial logs of different agricultural residues was investigated. Sawdust promoted the mycelia spawn colonisation in the shortest period of 38 ± 0.5 days. However, only sawdust and bamboo dust supported the primodia formation. Complex medium supported mycelium growth in submerged cultures and 27.11 ± 0.43 g/L of mycelia was obtained after 2 weeks of cultivation at 28 °C and 200 rpm. Antioxidant potential in mushroom may be influenced by different cultivation and extraction methods. The different extracts from the wild and cultivated basidiocarps as well as mycelia were then tested for their antioxidant properties. Aqueous and ethanol extracts of mycelia and basidiocarps tested had varying levels of antioxidant activities. To conclude, domestication of wild G. neo-japonicum using agroresidues may ensure a continuous supply of G. neo-japonicum for its medicinal use while ensuring the conservation of this rare species.


Results
Selection of substrate for basidiocarp production based on mycelia growth. All lignocellulosic substrates tested supported mycelia growth (Fig. 1). The support of mycelia growth and colonisation of the different substrates was in descending order: sawdust > oil palm trunk tissue > oil palm leaves ≥ bamboo waste ≥ cotton waste ≥ paddy straw. The growth rate of G. neo-japonicum was the highest (p < 0.05) on sawdust whereby 39.8 ± 1.2 days was taken to colonise a 500 g mushroom bag (Fig. 1).

Mushroom basidiocarp production.
Sawdust was selected as the substrate for preliminary domestication of G. neo-japonicum and basidiocarp production since it supported the highest growth rate of mycelia. The duration of a complete colonisation was about 38.0 ± 0.5 days. The duration recorded for  To test the ability of G. neo-japonicum to produce extracellular enzymes The mushroom showed strong β -glucosidase and avicelase activities 12 2 To determine the optimal media conditions for the detection of cellulase activity in G. neo-japonicum The dye reagent, pH, and temperature for the optimum detection of cellulase activity for G. neo-japonicum were Congo red, pH 7.0, and 25 °C; respectively. 13 3 To investigate the effects of amino acids on the production of total phenolic compounds in the mycelial culture of G. neo-japonicum  The effects of media on mycelia production in SmF. The mycelia production of G. neo-japonicum in the complex media (24.68 ± 0.58 g/L) was 1.6-fold higher than the mycelia produced in malt extract broth (15.07 ± 0.51 g/L) (Fig. 4). The mycelia production was comparable to the 21.53 ± 0.38 g/L of mycelia produced by G. lucidum 25 . However, the dry cell weight (DCW) harvested after freeze drying was only about 10% of the fresh mycelia. Glucose and peptone present in the complex media may have promoted mycelia production.
The effects of shaking speeds on mycelia production in SmF. The   The growth was measured in days taken to colonise 500 g of substrate using 5% (w/w) of 7-day inoculum at 28 ± 2 °C. Results with the similar alphabets are not significant different at p < 0.01. wild basidiocarp (101.01 ± 0.59 μ gGAE/mg), followed by hot aqueous extract of cultivated basidiocarp (23.76 ± 0.76 μ gGAE/mg) ( Table 3). Ethanolic extract of wild basidiocarp showed the highest DPPH quenching effects with IC 50 value 29.95 μ g/ml ( Table 3). The radical scavenging ability of the different extracts decreased in the following order: ethanolic extract of wild basidiocarp > hot water extract of wild basidiocarp > ethanolic extract of SSF > hot water extract of mycelia > ethanolic extract of mycelia > filtrate > hot water extract of cultivated basidiocarp > ethanolic extract of cultivated basidiocarp. Similarly, the ethanolic extract of wild basidiocarp also showed the highest ferric-reducing power (0.76 ± 0.03 mol Fe 2+ /g), followed by hot water extract of wild basidiocarp (0.36 ± 0.01 mol Fe 2+ /g). In ABTS •+ decolourisation assay, the results were comparable to those obtained in DPPH and ferric-reducing reactions. All the scavenging activities were dose-dependent, whereby the highest ABTS •+ scavenging   was observed for ethanolic extract of wild basidiocarp (720.85 μ g/ml), followed by hot water extract of cultivated basidiocarp (1343.01 μ g/ml) and hot water extract of wild basidiocarp (1801.74 μ g/ml).

Discussion
As shown in Table 4, the higher lignin content in sawdust (14-34%) and oil palm trunk tissue (22.6%) may have enhanced the growth rate of mycelia [26][27][28][29][30][31] . Ganoderma neo-japonicum, a white rot fungus, produces different types of extracellular enzymes to aid in colonising a specific substrate 32 . G. neo-japonicum showed strong β -glucosidase and avicelase activities and moderate ligninase activity 12,13 . Further, G. neo-japonicum has been reported to produce extracellular oxidative enzymes including laccase and lignin peroxidases which may aid in the degradation of lignocellulose-based substrates 17 .
Although the growth cycle of this species is much longer compared to the other well domesticated G. lucidum and G. tsugae, it was still shorter as compared to the reported annual life cycle in the wild 33 . For the production of basidiocarp, the sawdust with an adjusted moisture content of 70% was supplemented with 10% (w/w) rice bran and 1% (w/w) calcium carbonate. Moisture content in substrate is very important for enzymatic hydrolysis processes during mycelia growth, while rice bran provides the organic nitrogen, essential vitamins B and trace elements to boost mycelia growth. Calcium carbonate functions by maintaining a near neutral pH in mushroom bags. It acts as a buffering system to prevent acidic environment due to accumulation of carbon dioxide throughout the incubation period. Shorter   www.nature.com/scientificreports/ lag phase in growth cycle and a near neutral pH will minimise the contamination of mushrooms bags by other fungi, for example Aspergillus sp., Trichoderma sp. and Rhizopus sp. 34 .
Most basidiomycetes prefer complex organic nitrogen sources as selected essential amino acids may not be synthesised from inorganic nitrogen sources in the submerged culture 35 . Mycelia production increased with the shaking speed and this could be due to better aeration which is essential for cell growth. Improvement of efficiency and gradient of gas exchange, as well as reduction of carbon dioxide during agitation may also play important roles in submerged culture. Furthermore, higher shaking speed could inhibit pellet formation of mycelia and hence encourage a homogenous mycelium growth as suggested by Wagner et al. 36 .
Mushrooms are noted for their abundance of polar phenolic compounds especially the phenolic acids, for example gallic acid, tannic acid, protocatechuic acid, and gentisic acids 37 . In general, the wild growing mushrooms have higher concentration of phenolics as suggested by Grangeia et al. 38 . The hot aqueous extract of G. lucidum cultivated in Malaysia was found to contain 63.51 ± 3.11 mg GAE/g extract of TPC 39 . The amount of TPC was higher when compared to the hot aqueous extract of G. neo-japonicum (23.76 ± 0.76 μ g GAE/mg). As reported by Park and Lee 14 , the TPC content of G. neo-japonicum was 5.78 ± 0.68 mg GAE/g dry weight. The TPC was significantly increased by 13.2-fold upon supplementation of an amino acid, tryptophan. As an aromatic amino acid, tryptophan might play a role in the shikimate pathway and phenylpropanoid pathway, hence regulated the TPC in mushrooms. Besides, we observed that the polarity of extraction solvent had a direct influence on the phenolic content of the resulting extracts. In this study, the ethanol extract revealed a higher TPC. On the other hand, Wong et al. 40 showed that the hot aqueous extracts of Hericium erinaceus had a higher TPC than that of methanol extracts.
Our results showed that the extracts of wild basidiocarps showed higher free radicals scavenging effects as compared to the cultivated ones. We also found that solvent extraction by ethanol was the best extraction method to maximise the antioxidative activities of this mushroom. Ganoderma sp. has been shown to be effective in free radical scavenging and chelating. The hot aqueous extract of a locally cultivated G. lucidum exhibited significant DPPH scavenging activity with IC 50 of 5.280 ± 0.263 mg/ml 39 . Our result indicated that the hot aqueous extract of G. neo-japonicum had stronger ability to scavenge free radical, hence a better antioxidant; since its IC 50 value was lower (1343.01 ± 7.33 μ g/ml). Park and Lee 15 reported that the DPPH and ABTS scavenging effects of G. neo-japonicum mycelia extracts were 11.80 ± 0.48 mg/mL and 2.12 ± 0.23 mg/mL, respectively. The discrepancy between their results and ours was maybe due to different cultivation methods. Tseng et al. 2 reported that polysaccharides extracted from the basidiocarp of G. tsugae exhibited a stronger antioxidant properties compared to those extracted from mycelia and filtrate. According to Bhanja et al. 23 , the production of β -glucosidase enzyme during fermentation process was responsible for polyphenol accumulation and radical scavenging properties. There was a significant difference in antioxidant activities across all assays of the extracts of wild basidiocarp when compared to the cultivated basidiocarp extract as well as extracts of mycelium and fermented grains. This observation has not been reported by other studies. The difference in biological activities among ethanolic and hot water extracts of mushrooms were attributed to the differences in their chemical composition, especially polysaccharides, and phenolic content 41 .
On-going studies show that G. neo-japonicum exhibited genoprotective and anti-inflammatory properties (unpublished data). This mushroom which is used in traditional medicine by the indigenous tribes of Malaysia may be developed as a nutraceutical and therapeutic agent. However, caution is to be taken to ensure that the domestication process retains the bioactivities of this mushroom. Besides, we also observed that the antioxidant activities of mycelia and fermented grains were not comparable to the activities detected in wild basidiocarp. Therefore, environmental and nutritional factors in the cultivation process need to be kept as close to the nature as possible. Since G. neo-japonicum is a traditionally used mushroom with promising potentials, further investigations on the domestication process was warranted.
Ganoderma neo-japonicum can be cultivated for basidiocarps as well as cultured in SSF with wheat grains or SmF with complex media tom obtain the mycelia. Among the different agro-residues tested, sawdust was the best substrate for cultivation of this mushroom. Complex medium and shaking speed of 200 rpm were found to support the maximum mycelia production. The ethanolic extract of wild basidiocarps showed the highest phenolic content and antioxidant activities when compared to the cultivated basidiocarps and mycelia in SSF and SmF. The loss of the antioxidant properties of G. neo-japonicum during the domestication processes was noted and the domestication process has to be optimised to retain the nutraceutical potential in the cultivated basidiocarps. Further, the cultivation of basidiocarps, not only will ensure a constant supply of the mushrooms for the tribes but also reduce over harvesting of the wild gene pool in the forests. As this mushroom also contributes to the economic activity of the tribes, cultivation on artificial logs may contribute to their income as well. Mycelia growth and primordia formation by G. neo-japonicum. Bamboo waste, oil palm trunk tissue, oil palm leaves, paddy straw, cotton waste and sawdust were obtained from handicraft centres, oil palm plantation, paddy fields, cotton processing factory and sawmills, respectively. All the biomaterials were chopped into small size (0.1-0.5 cm), mixed with rice bran and calcium carbonate in 89:10:1 ratio and then mixed with water to obtain 70% moisture. Plastic bags were filled with 500 g of well mixed substrates, autoclaved at 121 °C and 15 psi for one hour. The mushroom bags were then left to cool overnight and then inoculated with 5% (w/w) of two-week-old fermented wheat grains prepared as mentioned in SSF below. The bags were incubated at 27 ± 2 °C in the dark 34 . Spawn (mycelia) run was recorded every three days. When the mycelia had been fully colonised the substrates, the bags were transferred to the mushroom house and high humidity (70-90%) and temperature (25 °C) were maintained to induce primordia formation. Yield was calculated as biological efficiency (%). Production of mycelia by solid substrate fermentation (SSF). Wheat grains were soaked overnight in distilled water and excess water was drained. About 250 g of soaked grains were dispensed into each of several 500 mL conical flasks and autoclaved at 121 °C, 15 psi for 15 minutes 42 . The flasks were then cooled to room temperature and inoculated with 3 discs of 10 days old mycelia of G. neo-japonicum grown on MEA plates. The fermented grains were then harvested after two weeks of incubation at room temperature and used to inoculate mushroom bags or freeze dried and kept for antioxidant analysis.

Methods
Preparation of aqueous and ethanol extracts. Freeze dried mycelia, fermented wheat grains, and air dried basidiocarps (both wild and cultivated) were blended, soaked in 95% ethanol at 1: 100 (w/v) on 150 rpm shaker and incubated in room temperature (25 ± 2 °C) for seven days 43 . The aliquots were then filtered through filter paper Whatman No.1 and evaporated to dryness using Eyela vacuum rotary evaporator at 40 °C and 173 Hpa. Hot aqueous extracts were prepared by boiling freeze dried samples (100 g/L) at 100 °C for two hours 43 . The aliquots were filtered through filter paper Whatman No.1 and the filtrate was freeze dried.
Quantification of total phenolic content (TPC). Total phenolic content was quantified by using the method of Bhanja et al. 23 . The extracts ranging from 10-1000 μ g/mL were mixed with 50 μ L of 10% Folin-Ciocalteu reagent in 96-well microplate and incubated at room temperature for three minutes. Then, 100 μ L of 10% sodium carbonate was added and the absorbance was measured at 750 nm after one hour incubation at room temperature. Gallic acid was used as a standard and all data are expressed in mg of gallic acid (GAE)/ g of extracts.
Antioxidant activity of different extracts of Ganoderma neo-japonicum. 2.9.1 2,2-diphenyl-1picrylhydrazyl (DPPH)radical scavenging properties. The various extracts were dissolved in DMSO and diluted to yield concentrations ranging from 1-1000 μ g/mL. Diluted extracts (5 μ L) was mixed with DPPH reagent (195 μ L) in a 96-well microplate and the absorbance was measured at 515 nm every 20 minutes for two hours using a microplate reader. Ascorbic acid was used as a standard and the assays were conducted in quadruplicates. Radical scavenging activities of the various extracts were expressed as percentage (%) of DPPH quenched 44 . IC50 was determined using GraphPad Prism software version 5.0.
Scientific RepoRts | 5:12515 | DOi: 10.1038/srep12515 (%) = ( ) / × -DPPH quenched absorbance blank absorbance of sample absorbance blank 100 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging properties. ABTS radical scavenging assay was carried out according to the method of Bhanja et al. 23 . The ABTS reagent was prepared by mixing 5 mL of 7 mM ABTS with 89 μ L of 140 mM potassium persulfate. The solution was incubated in the dark, at room temperature for 12 to 16 hours in order to generate ABTS radicals. The radical-containing reagent was then diluted with absolute ethanol to yield an absorbance in the range of 0.68 to 0.72 at 734 nm wavelength. The assay was continued by mixing 10 mL of extract with 100 μ L of the appropriately diluted ABTS reagent. The absorbance was measured after one minute at 734 nm using a microplate reader. Trolox was used as a standard and radical scavenging activities of the various extracts were expressed as percentage (%) of ABTS quenched. IC50 was determined using GraphPad Prism software version 5.0.
Statistical analysis. Results were from three independent experiments performed in triplicates. All data were subjected to analysis of variance (ANOVA) using Prism Graphpad Statistical Software version 5. The differences between each sample were evaluated by Tukey multiple range test where p < 0.05 was considered significantly different.