Bioactivity and Mycochemical Profile of Extracts from Mycelial Cultures of Ganoderma spp.

Fungal mycelium cultures are an alternative to natural sources in order to obtain valuable research materials. They also enable constant control and adaptation of the process, thereby leading to increased biomass growth and accumulation of bioactive metabolites. The present study aims to assess the biosynthetic potential of mycelial cultures of six Ganoderma species: G. adspersum, G. applanatum, G. carnosum, G. lucidum, G. pfeifferi, and G. resinaceum. The presence of phenolic acids, amino acids, indole compounds, sterols, and kojic acid in biomass extracts was determined by HPLC. The antioxidant and cytotoxic activities of the extracts and their effects on the inhibition of selected enzymes (tyrosinase and acetylcholinesterase) were also evaluated. The total content of phenolic acids in the extracts ranged from 5.8 (G. carnosum) to 114.07 mg/100 g dry weight (d.w.) (G. pfeifferi). The total content of indole compounds in the extracts ranged from 3.03 (G. carnosum) to 11.56 mg/100 g d.w. (G. lucidum) and that of ergosterol ranged from 28.15 (G. applanatum) to 74.78 mg/100 g d.w. (G. adspersum). Kojic acid was found in the extracts of G. applanatum and G. lucidum. The tested extracts showed significant antioxidant activity. The results suggest that the analyzed mycelial cultures are promising candidates for the development of new dietary supplements or pharmaceutical preparations.

Effect of Strain, Wood Substrate and Cold Treatment on the Yield and ß-Glucan Content of Ganoderma lucidum Fruiting Bodies.

Wood residues from forestry industries can be potential raw materials for specialty and edible mushroom production. The aim of this study was to evaluate the suitability of wood residues for the cultivation of Ganoderma lucidum originating from boreal forests. The substrates tested included sawdust and wood chips of Betula spp., Populus tremula, Picea abies, Pinus sylvestris and Larix sp. The suitability of the substrates and the ability of the strains to develop fruiting bodies and produce ß-glucan were evaluated. Fruiting body formation was supported by applying two different cold shock treatments to substrate bags. The highest yields were observed with MUS192 strain and Betula spp. and P. tremula wood-based substrates. ß-Glucan content in the fruiting bodies was highest with the MUS75 and P. tremula wood-based substrate. Based on these findings, the combination of P. tremula wood residues and the MUS192 strain is proposed to enhance the yield and ß-glucan content of the fruiting bodies. A cold treatment of 5 °C is suggested to induce primordia formation and to increase fruiting probability. This is the first time that strains of G. lucidum originating from boreal forests have been compared and successfully cultivated simulating commercial indoor cultivation.

Effect of Shading and Forest Type on Morphological Characteristics and Bioactive Compounds of Fruiting Bodies of Songshan Lingzhi Ganoderma tsugae (Agaricomycetes).

Ganoderma tsugae strain MCCCMAS0053 cultivation on short logs results in varied yield and quality under different growth conditions. Thus, growth conditions need optimization to increase yield and quality. An indoor experiment with three shade treatments (A1, two layers of black sun-shade net; A2, one layer of black sun-shade net plus plastic mulch; A3, one layer of black sun-shade net) and a field experiment in two forest types (pine or mixed pine-oak) were conducted. The results showed that shading and forest type significantly affected light intensities and the growth, size, biomass, and bioactive components of G. tsugae fruiting bodies. In the indoor experiment, the mean dry weight of the fruiting body and the diameter of the pileus in A2 increased by 21.51-44.98% and 13.42-22.26%, respectively, compared with those of A1 and A3. Similarly, the accumulation of the bioactive compounds (polysaccharides, total amino acids, and total essential amino acids) in the pileus and stipe were greater in A2 than in A1 and A3. Furthermore, compared with pure pine forest cultivation, fruiting bodies cultivated in mixed pine-oak forest had greater dry weight, pileus diameter, and more bioactive compounds. In addition, no significant difference was found between the A2 and mixed pine-oak treatments, which had a similar light intensity (from 1116 to 2367 lx). Hence, this suggests that the A2 shade treatment or cultivation in mixed pine-oak forest is beneficial for production of G. tsugae fruiting bodies, and light intensity may play a critical role in this process.

Vineyard Pruning Waste Improves Bioconversion and Chemical Composition of Native Ganoderma spp. (Agaricomycetes) Strains from Mexico.

The aim of this work was to evaluate wood chips from vineyard prunings (VPs) as a potential substrate in cultures of Ganoderma spp. Biological efficiency (BE), production rate (PR), yield, and protein and fat contents of basidiomes increased when the wild strains G. oerstedii and G. subincrustatum, which were isolated from the Sonoran Desert, were cultivated on VPs. The mineral content of the basidiomes varied depending on the strain and substrate. The carbohydrate and phenol contents of the different substrate combinations and of the basidiomes were similar among strains. However, the carbohydrate and phenol contents of the substrates did not correlate with an increase in BE. Conversely, the high availability of protein, fat, and hemicellulose in VPs positively correlated with increases in BE, in yield, and in the protein and fat contents of the basidiomes of the wild strains. Our results clearly demonstrate that, in comparison with the traditional substrate of oak wood, VPs improve the production and biochemical composition of basidiomes. Therefore, cultivation of Ganoderma spp. on VPs is an environmentally friendly strategy for increasing their nutritional value and for cultivating these mushrooms for other biotechnological applications.

Extracts of Polypore Mushroom Mycelia Reduce Viruses in Honey Bees.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

Antioxidant Properties of the Artist's Conk Medicinal Mushroom, Ganoderma applanatum (Agaricomycetes), upon Cultivation with para-Substituted Phenolic Compounds and Tea Leaf Extracts.

In this study we elucidate antioxidative properties of the mushroom Ganoderma applanatum (Pers.) Pat. enhanced by submerged culture with para-hydroxyphenolic compounds and tea leaf extracts. The tea extract has been shown to increase to different extents the antioxidative efficiency of para-substituted phenolics, with the most profound effect for 2-(4-hydroxyphenyl)ethane-1-ol (tyrosol). Within the range of physiological concentrations, the symbatic correlation of the antioxidative action of the fungal samples with the volume of tea extract in the submerged culture medium was observed. We propose an approach to obtain, through the use of black tea extracts as the nutrient medium component, large amounts of G. applanatum seeding mycelia; the extract exerts a profound positive effect on the level of phenolic-type antioxidants.

Effects of Culture Conditions on Antimicrobial Activity of Ganoderma resinaceum (Agaricomycetes) Extracts.

Among many sources of natural bioactive substances, mushrooms constitute a huge and mostly unexplored group. Biologically active secondary metabolites of Ganoderma, a group of wood-degrading mushrooms, have recently been reviewed. Our previous study revealed the antimicrobial activity of extracts from G. resinaceum grown in submerged culture against phytopathogens. Different factors can influence the production of secondary metabolites, including nutritional factors. In this study we evaluated the influence of different culture conditions on the antimicrobial activity of extracts from liquid cultures of G. resinaceum, through use of a factorial design. Minimum inhibitory concentrations for extracts produced under different culture conditions were determined against Staphylococcus aureus and Xanthomonas vesicatoria. Based on the results of these assays, larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation time should be performed in order to isolate from G. resinaceum antibiotic compound(s) that are potentially useful against S. aureus. In addition, pH 5 should be considered for the production of antimicrobial metabolites against X. vesicatoria from supernatant broths or extracts from G. resinaceum.

Improved ganoderic acids production in Ganoderma lucidum by wood decaying components.

Ganoderma lucidum is a legendary Traditional Chinese Medicine (TCM) over a few thousands of years and one kind of its major active components are Ganoderic acids (GAs). GAs are largely produced in the mushroom primordium and fruiting body but much less in mycelium stage. However, little is known on the underlying regulatory mechanism. As a saprophytic fungus, G. lucidum solely obtains nutrients by wood decaying. Wood in general contains sophisticated chemical components with diverse structural units. To explore a strategy that extensively leads to GAs induction in the submerged liquid fermentation, all chemical components that might be possibly from the wood decaying were tested individually as GAs inducers. It was found that GAs production increased 85.96% by 1.5% microcrystalline cellulose (MCC) and 63.90% by 0.5% D-galactose. The transcription level of a few rate-limiting or chemically diverting enzymes responsible for GAs biosynthesis was greatly induced by MCC and D-galactose. The concentration and time-course titration study indicated that these two chemicals might not be utilized as carbon sources but they played a comprehensive role in the secondary metabolites synthesis. Our data indicated that MCC and D-galactose might be further industrialized for higher GAs production in G. lucidum in submerged fermentation.

[Effect of Selenium-Containing Biocomposites Based on Ganoderma Mushroom Isolates Grown in the Presence of Oxopropyl-4-Hydroxycoumarins, on Bacterial Phytopathogens].

Effect of selenium-containing biocomposites obtained from submerged cultures of macrobasidiomycetes Ganoderma applantum, G. cattienensis, G. colossus G. lucidum, G. neojaponicum, and G. valesiacum, on plant pathogenic bacteria Clavibacter michiganensis ssp. sepedonicus (Cms), Micrococcus luteus, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas fluorescens, Pseudomonas viridiflava, and Xanthomonas campestris was studied. Oxopropyl-4-hydroxychromenones were used as components of the fungal nutrient media. The bacteriostatic and bactericidal activity of the Se-containing and Sefree substances of fungal origin against plant pathogenic bacteria was determined using colony-forming units count, the agar well diffusion method, and by turbidity measurements of bacterial suspensions. The composites produced from the extracellular metabolites of G. cattienensis SIE1302 with 4-hydroxy-3-(3-oxo-1,3- diphenyl propyl)-chromen-2-one (S(45)), and of G. lucidum SIE1303 with 4-hydroxy-3-(3-oxo-1-(3-nitrophenyl)- 3-phenylpropyl)-chromen-2-one (S(NO2)) possessed the most pronounced antibacterial action against Cms. The composites produced from the isolates of G. valesiacum 120702 with S(NO2) showed the maximal antibacterial activity against Xanthomonas campestris B-610. High antimicrobial effect of G. lucidum 1315 with S(NO2) against Xanthomonas campestris B-610 and of G. colossus SIE1301 against Pseudomonas fluorescens EL-2.1 was revealed. The pioneering information on the biological activity of coumarin series compounds in their application for producing the substances of fungal origin was obtained.

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.

Effect of cost-effective substrates on growth cycle and yield of lingzhi or reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) from Northwestern Himalaya (India).

To find a cost-effective alternative substrate, the medicinal mushroom Ganoderma lucidum was grown on sawdusts of sheesham, mango, and poplar. Optimum spawn level was determined by spawning in substrates at various levels (1, 2, 3, and 4%). To determine the effect of supplementation, substrates were supplemented with wheat bran, rice bran and corn flour at different concentrations (10, 20, and 30%). Duration of growth cycle, mushroom yield, and biological efficiency data were recorded. Among substrates, mango sawdust was superior, with 1.5-fold higher yields than poplar sawdust, which was the least suitable. However with respect to fructification, mango sawdust produced the first primordia earlier (21±1 days) compared with the other investigated substrates. 3% spawn level was found to be optimal irrespective of the substrate. Yield and biological efficiency (BE) were maximally enhanced by supplementation with wheat bran, whereas rice bran was the least suitable supplement among those tested. Growth cycle shortened and mushroom yield increased to a maximum at the 20% level of supplements. Mango sawdust in combination with 20% wheat bran, if spawned at the 3% level, resulted in a high yield (BE = 58.57%).

Mycelial growth rate and macro- and micromorphological characteristics of medicinal species of genus Ganoderma (higher Basidiomycetes) from Iran.

Mycelial growth rate is a distinguishing quality that demonstrates continuous variation in different isolates collected from various hosts and locations. The objectives of this research were (1) to reinvestigate the previous identification of Iranian species, and (2) to recognize the best native isolate(s) for cultivation of different Ganoderma species. Of 78 samples collected from different hosts and sites, only 43 mycelia could be purified and examined for further study. Growth rate (GR; Δd/Δt) and growth coefficient (GC; dgh/t) were analyzed by growing isolate culture on 2% malt-extract agar medium (pH 5.5) incubated at 25°C. Macro- and micromorphological studies on mycelia and fruiting bodies such as basidiospore and cutis microcharacters as well as fruiting body quality were used for precise identification. Results revealed that samples belonged to 4 species: G. lucidum, G. applanatum, G. resinaceum, and G. australe. Among all samples, the isolate morphologically identified as G. applanatum showed the best GR (12 mm/day) and good GC (128 mm/day), followed by the 2 other isolates identified as G. resinaceum (GRs and GCs of 11 and 55 mm/day and 10.9 and 43.6 mm/day, respectively).

Solid-substrate fermentation of wheat grains by mycelia of indigenous species of the genus Ganoderma (higher Basidiomycetes) to enhance the antioxidant activities.

Species of the genus Ganoderma are a cosmopolitan wood decaying white rot fungi, which has been used by the Asians for therapeutic purposes for centuries. In the present study, solid-substrate fermentation (SSF) of wheat grains (Triticum aestivum L.) was carried out with indigenous Ganoderma australe (KUM60813) and G. neo-japonicum (KUM61076) selected based on ethnomycological knowledge. G. lucidum (VITA GL) (a commercial strain) was also included in the study. Antioxidant activities of the crude ethanol and aqueous extracts of the fermented and unfermented wheat grains were investigated by ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging ability, and lipid peroxidation assay. Among the six mycelia extracts tested, the ethanol extract from wheat fermented with KUM61076 mycelia showed the most potent antioxidant activities, whereas the ethanol extract of wheat grains fermented with KUM60813 mycelia has a good potential in protecting frying oils against oxidation. Total phenolic content (TPC) in the ethanol extracts were higher than that in the aqueous extract. The wheat grains fermented with G. australe (KUM60813) and G. neo-japonicum KUM61076 have greater antioxidant potential compared to the commercially available G. lucidum (VITA GL). The antioxidant activities of the mycelia extracts had a positive correlation with their phenolic contents. Thus phenolic compounds may play a vital role in the antioxidant activities of the selected Ganoderma spp.

Comparative growth characteristics and yield attributes of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (Higher Basidiomycetes) on different substrates in India.

The present study investigated the effects of four forestry byproducts (sawdust of oak, mango, khair, and tuni) and three agricultural residues (paddy straw, wheat straw, and soybean waste) along with four supplements (wheat bran, rice bran, corn flour, and gram powder) on growth characteristics (spawn run and primordial formation) and yield of Ganoderma lucidum. There were significant differences (P=0.05) in yield regardless of substrates and supplements used in experimentation. Among substrates, agriculture residues supported better yield and biological efficiency of G. lucidum compared to forestry byproducts irrespective of the supplements. The highest yield (82.5 g) and biological efficiency (27.5%) were recorded from paddy straw supplemented with wheat bran, which invariably resulted in significantly higher yield compared to the unsupplemented check(s) or other supplements used in this study.

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

Applied modern biotechnology for cultivation of Ganoderma and development of their products.

A white-rot basidiomycete Ganoderma spp. has long been used as a medicinal mushroom in Asia, and it has an array of pharmacological properties for immunomodulatory activity. There have been many reports about the bioactive components and their pharmacological properties. In order to analyze the current status of Ganoderma products, the detailed process of cultivation of Ganoderma spp. and development of their products are restated in this review article. These include the breeding, cultivating, extracting bioactive component, and processing Ganoderma products, etc. This article will expand people's common knowledge on Ganoderma, and provide a beneficial reference for research and industrial production.

[Cultural characteristics of mycelia of Ganoderma gibbosum].

To study the cultural characteristics of mycelia of Ganoderma gibbosum, a medicinal fungus used in China. The growth rate and biomass of G. gibbosum mycelia were measured under different temperature, lightning carbon and nitrogen sources conditions. It showed that the optimal growth temperature for mycelia was 25 degrees C. Darkness was beneficial for mycelium growth. The initial pH 5.5 was suitable. The sucrose was the best carbon source and yeast extract the best nitrogen source, the optimal carbon-nitrogen ratio 60:2. These conclusions will offer references for further artificial cultivation and liquid fermentation.

[Study on collector for spores of Ganoderma spp].

OBJECTIVE: To develop a convenient, practical low-cost and efficient Ganoderma spore collector. METHOD: The spore collector was made from common materials such as white cardboard and oil-lustrous paper, temperature and humidity were used as indexes to study the effect of the collector on the growth environment of Ganoderma and spore collection. RESULT: The spore collector developed could effectively separate Ganoderma fruit bodies from the outside to form an independent closed space and stop free flow of spores. The use of the collector had few effects on temperature and humidity that influenced the growth of G. spp. and development of the fruit bodies. In addition, the fluctuation of the relative humidity inside the collector tended to be small. CONCLUSION: This collector could efficiently collect quality spores and the yield of spores accounted for 38.3% of the total yield of spores and fruit bodies when this collector was applied on a large scale.

Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences.

The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSU) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than 99.48% homologous, and the consensus sequences of 3 different medicinal mushrooms were more than 97.80% homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms.

[Correlations between the characters of the mycelium vegetative growth and the formation of the fruiting body of Ganoderma luciderm].

OBJECTIVE: To select a proper Ganoderma luciderm strain for the fruiting body production. METHOD: The strains were cultivated on the agar media and in the liquid media, respectively. Then the strains were inoculated onto the solid medium made from agricultural products (such as wheat bran, corn powder, wood meal, etc.) and cultured for a certain period. RESULT: Strains, which were easier to produce polyporic tissues at the vegetative growth stage, would be more quickly to form fruiting body with high quality and yield of the spores. CONCLUSION: Appearance of the polyporic tissues at the mycelium vegetative growth stage could be used as a marker for the strain selection for the G. luciderm substituted cultivation.