Mushroom Fungi Research Articles

EDIBLE MUSHROOMS: A GREEN BIOTECHNOLOGY AND GREAT NUTRITIONAL VALUE FOR IMPROVING HUMAN HEALTH

In recent years, great emergence of environmental issues such as “green biotechnology” or “white biotechnology” (in EUR) is developed. Green biotechnology can be regarded as application of microorganisms or their metabolites in food, medicine, and pharmaceutical fields. Edible mushroom is an ideal food due to the fairly high content of proteins include amino acids which are essential to human health and nutrition. Mushroom fungus is easily digested, free of cholesterol, and provides a source of dietary fiber. Fungal proteins compete successfully with animal protein foods and are healthy alternative to meat. Many studies have found that some species of mushrooms are having therapeutic properties such as antioxidant, antimicrobial, anticancer, cholesterol lowering and immuno-stimulatory effects. Consumption of mushroom-rich foods is associated with a reduced risk of cardiovascular diseases, and certain types of cancer. Mushrooms have nutraceutical properties such as prevention or treatment of Parkinson, Alzheimer, hypertension, and high risk of stroke. However, some of wild mushroom are considered to be poisonous and there has been some concern about the concentration of metals such as arsenic, cadmium, copper and lead in wild fungi. Consumption of wild fungi in normal quantities is, however, did not pose any significant health risk.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Fungal biofermentation of pine bark producing organic acids and their quantification with capillary electrophoresis

Bioprocessing of pine bark was accomplished using oyster mushroom fungi. The organic acids and inorganic anions in the extracts of fermented samples were identified by capillary electrophoresis (CE). Nine organic acids were simultaneously determined in 12min through the use of an electrolyte composed of 20mM pyrazine-2,3-dicarboxylic acid (2,3-PyDC), 1.5mM CaCl2·2H2O and 0.3mM MTAH in methanol:water (10:90, v/v) at pH 9.0. The concurrent determinations of three inorganic anions were performed in less than 10min in an electrolyte consisting of 2.25mM pyromellitic acid, 6.50mM NaOH, 0.75mM hexamethonium hydroxide, and 1.60mM triethanolamine at pH 7.70. The applied analysis methods efficiently separated and identified organic acids and inorganic anions in the extracts. Butyric, maleic and glucuronic acids were the primary organic acids formed via fermentation. Chloride, nitrate and sulphate were detected in all of the samples.

Green synthesis of silver nanoparticles using the mushroom fungus Schizophyllum commune and its biomedical applications

Silver nanoparticles, which are small metallic colloidal particles, ranging 1 ∼ 100 nm in size, have several nano biotechnological applications in medicine, manufacturing and engineering industries. Fungus-mediated synthesis of silver nanoparticles is an ecofriendly, green process. Further, extracellular enzymes and proteins elaborated by fungi are involved in the synthesis of the silver nanoparticle, which makes the downstream processing relatively simpler. In the present investigation, Schizophyllum commune, a mushroom fungus, was tested for its ability to synthesize extracellular as well as intracellular silver nanoparticles. When the fungus was challenged with 1 mM silver nitrate, a change in colour of the broth and the mycelium was observed, indicative of extracellular and intracellular synthesis of silver nanoparticles. The presence of silver nanoparticles was confirmed by studying its Surface Plasmon Resonance absorption band in the visible wavelength. FTIR spectrum analysis of the silver nanoparticles indicated the presence of biomolecules in association with the reduction of silver ions. Scanning Electron Microscopic analysis of the silver nanoparticles revealed the nanorange dimensions of both the extracellular and the intracellular silver nanoparticles. Analysis of biological activities of the silver nanoparticles disclosed their significant antibacterial activity against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas fluorescens. Additionally, the silver nanoparticles inhibited the growth of the dermatophytic fungal pathogens viz. Trichophyton simii, Trichophyton mentagrophytes and Trichophyton rubrum significantly. Anticancer activity of silver nanoparticles, assayed through MTT cytotoxicity assay, uncovered that 27.2 and 64% mortality could be obtained in Human Epidermoid Larynx Carcinoma (HEP -2) cell lines at concentrations between 10 and 100 µg/mL, respectively. The results obtained indicate that Schizophyllum commune is capable of synthesizing silver nanoparticles in shaken broth cultures (120 rpm) at 25 ± 2℃ and pH 7.

Identification and functional analysis of pheromone and receptor genes in the B3 mating locus of Pleurotus eryngii.

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

DNA barcoding for identification of species in mushrooms: A component of product certification

One challenge in the botanical industry is the confirmation of species identification, and this can be compounded by the processing of the materials before reaching the vendor, such as milling or drying. This can be particularly difficult for samples that contain fungal mycelia, where morphological characteristics do not present sufficient variation to differentiate species. However, monitoring the safety and quality of such products is a requirement for the protection of consumer health. DNA barcoding, a genetic-based species identification system, has become popular in the past decade, and it shows great promise for species level identification of fungi. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, we generated ITS barcodes for two representative mushroom fungi, which are being used by consumers for food and dietary supplement purposes. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested the utility of the ITS by performing a BLAST search against both curated and non-curated public databases. Results demonstrated that the ITS region was able to identify both mushrooms used in the present study to species-level. The BLAST search provided 99% similarity of the query sequence to those available in the public database. We anticipate that this presentation will motivate a discussion on DNA barcoding based species identification, particularly as it applies to the verification/certification of mushrooms containing products.

Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi – Assessment of their effect on the final product and spent substrate properties

Nine agro-industrial and forestry by-products were subjected to solid-state fermentation by Agrocybe cylindracea and Pleurotus ostreatus, and the process and end-products were comparatively evaluated. Grape marc waste plus cotton gin trash was the best performing medium for both fungi, while substrate composition had a marked effect on most cultivation parameters. Biological efficiency was positively correlated with nitrogen, lignin and ash, and negatively with hemicelluloses and carbohydrate content of substrates. Spent substrates demonstrated high reductions in hemicelluloses and cellulose in contrast to lignin; fibre fractions were correlated with nitrogen, fat and ash content of initial materials, while residual mycelial biomass was affected by mushroom productivity. Mushroom proximate analysis revealed significant variations of constituents depending on the substrate. Crude protein and fat were correlated with substrates nitrogen for both species. Alternative cultivation substrates of high potential are proposed, while spent material could be exploited as animal feed due to its upgraded properties.

Identification of a Fungi-Specific Lineage of Protein Kinases Closely Related to Tyrosine Kinases

Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

Patterns of major metabolites biosynthesis by different mushroom fungi grown on glucose-based submerged cultures

The biosynthetic potential of four basidiomycetes (Agrocybe aegerita, Flammulina velutipes, Ganoderma applanatum and Pleurotus pulmonarius) and one ascomycete (Morchella esculenta) was examined in regard to biomass, intracellular (endopolysaccharides and lipids) and extracellular (exopolysaccharides) compounds' production in liquid media with glucose as substrate, in static and agitated cultures. Exopolysaccharides' production presented significant negative correlation with biomass, endopolysaccharides and lipids, while biomass was positively related to the production of endopolysaccharides and lipids. Maximum values of biomass, endo- and exo-polysaccharides obtained were quite impressive: P. pulmonarius produced 22.5 g/L of biomass, A. aegerita 60.4 % (w/w) of endopolysaccharides and F. velutipes 1.2 g/L of exopolysaccharides. Polysaccharides and lipids synthesized at the early growth stages were subjected to degradation as the fermentation proceeded. Mycelial lipids of all strains were highly unsaturated, dominated by linoleic acid, whereas glucose was the main building block of endopolysaccharides. The ability of the examined mushroom fungi to synthesize in high quantities biomass and polysaccharides, products with biotechnological and medicinal interest, renders these fungi as potential candidates in sugar-based bio-refineries.

Application of Mushroom Fungi in Solid Waste Management

Application of Mushroom Fungi in Solid Waste Management

On the asymmetry of mating in natural populations of the mushroom fungus Schizophyllum commune

Before a mycelium of a mushroom-forming basidiomycete develops mushrooms, the monokaryotic mycelium needs to become fertilized. Although the mechanistic details of mating in mushrooms have been studied thoroughly in laboratory research, very little is known on mating patterns in nature. In this study, we performed fine-scale analyses of three populations of Schizophyllum commune from their natural substrate (i.e. dead beech branches). From the three branches, 24, 12, and 24 fruiting bodies were isolated and for each mushroom, the origins of its nuclei and cytoplasm were reconstructed using DNA markers. Nuclear genotypes were determined using sequencing data and mating types, and mitochondrial haplotypes using SNP markers. From these combined data we reconstructed colonization and mating patterns of the mycelia. On each branch, we found multiple dikaryons (3, 3, and 8, respectively); in two instances one nuclear haplotype was shared between two dikaryons and in two other cases a nuclear haplotype was shared between three dikaryons. Each dikaryon always had a single mitochondrial haplotype. These findings indicate that mating usually is not symmetrical and that a monokaryon is most likely fertilized by a small monokaryon, a spore or a dikaryon. Sharing of nuclear haplotype between different dikaryons resulted either from multiple fertilizations of a single monokaryon, if the dikaryons had identical mitochondrial haplotypes, or, if the dikaryons had different mitochondrial haplotypes, most likely from secondary matings between a monokaryon and a dikaryon (Buller phenomenon). We conclude that mating in S. commune between same-sized monokaryons with reciprocal migration, as generally described in textbooks, is rare in nature. We discuss the implications of non-symmetric mating for basidiomycete evolution.

AnIn VitroEvaluation of Antioxidant and Colonic Microbial Profile Levels following Mushroom Consumption

The biological activity of mushroom consumption is achieved by the antioxidant effect of constituent biomolecules released during digestion. In the following study, the consumption of mushroom fungi was determined to increase the number of Lactobacillus and Bifidobacterium strains within the colon. The main phenolic antioxidant compounds identified were both gentisic and homogentisic acids. Moreover, the flavonoid catechin as well as a significant amount of δ- and γ-tocopherols was determined. The amount of Lactobacillus and Bifidobacterium strains from different sections of the human colon was significantly correlated with levels of antioxidative biomolecules. The experimental data clearly demonstrate a significant impact of mushroom consumption on the fermentative function of microorganisms in the human colon, resulting in the homeostasis of normal physiological colonic functions.

Lanostane triterpenes from cultures of the Basidiomycete Ganoderma orbiforme BCC 22324

Seven lanostane triterpenoids, ganorbiformins A-G, together with twelve known compounds, were isolated from cultures of the mushroom fungus Ganoderma orbiforme BCC 22324. Ganorbiformin A is an unusual rearranged analog, whereas the other compounds share the same lanostane skeleton with known ganoderic acids. The C-3 epimer of ganoderic acid T also exhibited significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MIC 1.3 μM).

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement.

Lignolytic Enzymes of a Mushroom Stereum ostrea Isolated from Wood Logs.

Production of lignolytic enzymes by the mushroom fungus Stereum ostrea in liquid medium under conditions of vegetative growth was examined for 10 days in comparison to the reference culture Phanerochaete chrysosporium. Though growth and secretion of extracellular protein by S. ostrea were comparable to those of P. chrysosporium, yields of laccase enzyme by S. ostrea were higher than laccase titres of P. chrysosporium by more than 2 folds on the peak production time interval (IVth day of incubation). S. ostrea yielded titres of 25 units of laccase/ml as against 8.9 units of laccase/ml on the IVth day of incubation. Stereum ostrea also exhibited activities of other lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), higher than the reference culture. Growth of S. ostrea on the medium in the presence of Remazol orange 16 resulted in the decolourisation of dye, confirming the presence of lignolytic enzymes. S. ostrea appears to be a promising culture with complete lignolytic system.

Sterostreins A–E, New Terpenoids from Cultures of the Basidiomycete Stereum ostrea BCC 22955

Sterostreins A-E (1, 2, 3a/3b, 4, and 5), five novel terpenoids, were isolated from cultures of the mushroom fungus Stereum ostrea BCC 22955. Sterostrein A (1) exhibited antimalarial activity (IC(50) 2.3 μg/mL) and cytotoxicity (IC(50) 5.3-38 μg/mL).

Antimicrobial properties of protein extracts from wild mushroom fungi and native plant species against hospital pathogens

Protein extracts of either native or exotic rare mushroom fungi and plants that are normally known for novel therapeutics including immune modulation were investigated for their potential antimicrobial effects. Data obtained using the Kirby-Bauer’s disc-diffusion assay methods showed that a number of locally sourced wild mushroom fungi (e.g. Ganoderma resinaceum, Russula fragilis and Inocybe grammata) had proteins with inherent antimicrobial properties against a number of typical hospital pathogens. The wild type fungus Mycena pura exhibited strong antagonism against Escherichia coli, an organism often commonly associated with nosocomial infections both locally and worldwide. Polyacrylamide gel electrophoresis (PAGE) of protein extracts revealed unique protein banding patterns for the exotic fungal species and possessed significant inhibitory effects against a range of nosocomial pathogens including MRSA, Salmonella, Candida and Aspergillusspecies. This small-scale study revealed the occurrence of wild fungal peptides of potential therapeutic significance and antimicrobial potential for exploitation in complementary therapies in clinical and veterinary medicine. Key words: Exotic fungi and medicinal plants, antibacterial activity.

A tetrahydrobenzofuran derivative from the fermentation broth of Lentinus squarrosulus BCC 22366

A new tetrahydrobenzofuran derivative, (6 S,7 S)-6,7-dihydroxy-3,6-dimethyl-2-isovaleroyl-4,5,6,7-tetrahydrobenzofuran ( 1), together with the known 1,2-dihydroxymintlactone ( 2), was isolated from the fermentation broth of the edible mushroom fungus Lentinus squarrosulus BCC 22366. The structure was elucidated by interpretation of NMR spectroscopic and mass spectrometry data. The absolute configuration of 1 was addressed by application of the modified Mosher's method.

Phallus indusiatus Vent. & Pers. (Basidiomycetes) - a new generic record for Eastern Ghats of India

A mushroom fungus Phallus indusiatus Vent. & Pers. (commonly called Long Net Stinkhorn) belonging to the order Phallales in the fungal phylum Basidiomycotina was collected from a bamboo thicket near the foothills of the Kutrumali Hill range in Koraput District of Orissa, representing a new distributional record for the Eastern Ghats of India.

The Comparative Immunomodulatory Effects of β-Glucans from Yeast, Bacteria, and Mushroom on the Function of Macrophages

The comparative immunomodulatory effects of β-glucans isolated from mushroom fungi (Coriolus versicol), yeast (Saccharomyces cerevisiae) and bacteria (Agrobacterium) on the major functions of macrophages were evaluated. As parameters of macrophage functions, we examined tumoricidal activity, phagocytosis, nitric oxide (NO) production, and the induction of inducible NO synthetase (iNOS) in RAW264.7 cells, following treatments with β-glucans from the three different sources. The results indicated that all β-glucan treatments significantly induced tumoricidal activity in the RAW264.7 cells, with a remarkable effect shown by the beta-glucan from Agrobacterium at a concentration of 10 ㎍/mL. There was also a significant increase in iNOS-NO system activity in macrophages treated with β-glucans extracted from yeast; however, iNOS-NO system activity was not markedly changed by the treatment of β-glucans from C. versicolor mushroom fungi or Agrobacterium. Furthermore, the β-glucans from C. versicolor had a significant phagocytotic effect at concentrations of 1, 10, and 100 ㎍/mL. Taken together, the present data suggest that these β-glucans, isolated from three different sources, have different effects on macrophage function, and therefore, may have different clinical uses in different for various types of diseases.