Chemical study of the strain Cordyceps spp. from cell fusion between Cordyceps militaris and Cordyceps cicadae.

Chemical investigation on the cultures of the fungus Cordyceps spp., a strain from cell fusion between Cordyceps militaris and Cordyceps cicadae, resulted in the isolation of 13 compounds including 2 new ones named 2-(5-(3-oxobutyl) furan-2-yl) acetic acid (1) and cordycepone (2). Their structures were elucidated from the analysis of 1D/2D NMR and CD data. Among them, compounds 1, 7-9, at a concentration of 50 µg/ml, showed weak inhibitory activity against AChE. Moreover, compounds 6, 9, and 11 showed moderate inhibitory activity against the nematode Panagrellus redivivus with mortality ratio of 79.0, 71.7, and 72.3% at 2.5 mg/ml, respectively.

Isaria takamizusanensis is the anamorph of Cordyceps ryogamimontana, warranting a new combination, Purpureocillium takamizusanense comb. nov.

The entomogenous anamorphic fungus Isaria takamizusanensis has not been resolved clearly in its teleomorphic state. We succeeded in inducing ascostroma formation by incubating conidiomata of I. takamizusanensis on cicada adults in a moist chamber. We observed the ascostroma and conducted a phylogenetic analysis based on ITS rDNA and EF-1α genes. The morphology of the ascostroma was identical to that of Cordyceps ryogamimontana. In the phylogenetic tree inferred from EF-1α, the isolate from the partspores grouped with nine strains derived from conidia of I. takamizusanensis, which was distinct from a clade including Purpureocillium lilacinum. Moreover, a conidial structure identical to that of I. takamizusanensis was rediscovered on the holotype specimen of C. ryogamimontana. As a result, we propose a new name, Purpureocillium takamizusanense, which is a combination of the teleomorph-anamorph connection of C. ryogamimontana-I. takamizusanensis, in accordance with the 'one fungus, one name' concept of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN).

Sub-acute toxicity of cultured mycelia of himalayan entomogenous fungus Cordyceps sinensis (Berk.) Sacc. in rats.

Oral administration of laboratory cultured mycelia powder of C. sinensis did not show any sign of toxicity as no significant change was observed in organ weight and serological parameters in rats. However, there was a significant increase in food intake, body weight gain and hematological parameters like WBC, RBC, Hb and lymphocytes in treated groups. Histopathology of vital organs also supported the non toxic effect of C. sinensis. The results conclude that laboratory cultured mycelia powder of C. sinensis is safe and non toxic up to 2 g/kg body weight dose.

Binding specificity of the recombinant cytoplasmic domain of Cordyceps militaris ß-1,3-glucan synthase catalytic subunit.

The cytoplasmic domain of the medicinal mushroom Cordyceps militaris ß-1,3-glucan synthase catalytic subunit Fks1 was expressed as a fusion protein with an N-terminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and was assayed for binding specificity. The recombinant cytoplasmic domain bound specifically to UDP-agarose and lichenan (ß-glucan), but not to ADP-agarose, GDP-agarose, or other carbohydrates.

Rapid and concise quantification of mycelial growth by microscopic image intensity model and application to mass cultivation of fungi.

The microbial food fermentation industry requires real-time monitoring and accurate quantification of cells. However, filamentous fungi are difficult to quantify as they have complex cell types such as pellet, spores, and dispersed hyphae. In this study, numerous data of microscopic image intensity (MII) were used to develop a simple and accurate quantification method of Cordyceps mycelium. The dry cell weight (DCW) of the sample collected during the fermentation was measured. In addition, the intensity values were obtained through the ImageJ program after converting the microscopic images. The prediction model obtained by analyzing the correlation between MII and DCW was evaluated through a simple linear regression method and found to be statistically significant (R2 = 0.941, p < 0.001). In addition, validation with randomly selected samples showed significant accuracy, thus, this model is expected to be used as a valuable tool for predicting and quantifying fungal growth in various industries.

Optimization of selected cultivation parameters for Cordyceps guangdongensis.

AIMS: To increase the fruit body production of Cordyceps guangdongensis, selected cultivation conditions, especially nutritional parameters were optimized. METHODS AND RESULTS: Cordyceps guangdongensis was inoculated on potato dextrose agar slants with pH values from 4.5 to 9.0 and cultivated in artificial media with different carbon and nitrogen supplements. Primordium formation in C. guangdongensis was favoured by slightly acidic conditions. Fruit body yields and biological efficiency (BE) recorded were all highest in cultures of C. guangdongensis supplemented with sucrose and KNO3 as carbon and nitrogen supplements, respectively. Highest fruit body yields and BE values were recorded with C : N ratio of 12 : 1. The optimal medium consisted of (g l(-1)) 20.0 sucrose, 4.0 soya bean powder, 5.0 beef extract and 10.0 KNO3. Cultivation experiments using this medium confirmed its reliability; 18.35% of BE was obtained, compared with a calculated maximum BE of 18.65% based on orthogonal test data. CONCLUSIONS: Cordyceps guangdongensis preferred sucrose and potassium nitrate as best carbon and nitrogen supplements. It produced satisfying yield of fruit body with optimized medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimized artificial cultivation conditions could promote the yield of C. guangdongensis and decreased the cost of production.

Enhanced exopolysaccharide production by Cordyceps militaris using repeated batch cultivation.

Cordyceps militaris exo-polysaccharides (EPS) have been reported to possess many benefits, such as anti-tumor, anti-inflammatory and antioxidant activities. In this study, the production of EPS via cultivation in a bioreactor was investigated. Glucose and yeast extract were determined to be the most suitable carbon and nitrogen sources for EPS production. The appropriate levels of glucose and yeast extract were 40 g/L and 10 g/L, respectively, resulting in EPS production of 1.686 g/L in a submerged culture. In the stirred-tank fermentor, an agitation rate of 150 rpm and aeration rate of 1.5 vvm were the most effective for EPS production. Due to the anchoring of mycelial cells on the wall of fermentor, a repeated batch approach was used. EPS production of C. militaris could be enhanced to a maximum of 5.713 g/L, with a productivity of 476 mg/L/day in the second run. The repeated batch approach was expected to generate higher EPS production, increase EPS yield and productivity and further simplify cultivation operations for bio-industrial application.

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice.

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.

Taxonomical Establishment and Compositional Studies of a New Cordyceps (Ascomycetes) Species from the Northwest Himalayas (India).

During a frequent survey in the northwest Indian Himalayan region, a new species-Cordyceps macleodganensis-was encountered. This species is described on the basis of its macromorphological features, microscopic details, and internal transcribed spacer sequencing. This species showed only 90% resemblance to Cordyceps gracilis. The chemical composition of the mycelium showed protein (14.95 ± 0.2%) and carbohydrates (59.21 ± 3.8%) as the major nutrients. This species showed appreciable amounts of P-carotene, lycopene, phenolic compounds, polysaccharides, and flavonoids. Mycelial culture of this species showed higher effectiveness for ferric-reducing antioxidant power, DPPH radical scavenging activity, ferrous ion-chelating activity, and scavenging ability on superoxide anion-derived radicals, calculated by half-maximal effective concentrations.

Morphological Observations and Fatty Acid Composition of Indoor-Cultivated Cordyceps sinensis at a High-Altitude Laboratory on Sejila Mountain, Tibet.

Cordyceps sinensis, a caterpillar entomopathogenic fungus-host larva complex, is a rare medicinal herb found in the Qinghai-Tibetan Plateau and its surrounding high-altitude areas. The alternation of generations in the life cycle, whatever the fungus or its host insect, requires special growth conditions. However, it is difficult to simulate the growth conditions of C. sinensis, which hinders its artificial cultivation. In this work, the life cycle from the host larva to C. sinensis was observed in an indoor-cultivation laboratory at 4,200 m a.s.l. on Sejila Mountain, Tibet. Comparative examinations between indoor-cultivated and wild C. sinensis demonstrated that the indoor-cultivated C. sinensis preferred to germinate multiple long, slim stromata at diverse positions on dead larvae, including but not limited to their heads. Their fatty acid composition shows a significant difference in the levels of polyunsaturated fatty acids (PUFAs). In indoor-cultivated C. sinensis, PUFAs constituted 24.59% and 49.43%, respectively, of neutral and polar lipids; meanwhile, in wild C. sinensis, PUFAs represented 34.34% and 61.25% of neutral and polar lipids, respectively. These observations and fatty acid data suggest that environmental factors, particularly temperature, soil pressure and light intensity, strongly affect the growth of C. sinensis. Our new findings may provide important information for improving techniques for the large-scale artificial cultivation of C. sinensis.

Ultrasonic disruption of fungal mycelia for efficient recovery of polysaccharide-protein complexes from viscous fermentation broth of a medicinal fungus.

High-intensity ultrasound (US) was applied to facilitate the extraction of intracellular and extracellular polysaccharide-protein complexes (PSPs) from the viscous mycelial fermentation broth of a medicinal fungus Cordyceps sinensis Cs-HK1. The US treatment caused the disruption of fungal mycelia, a dramatic reduction of the apparent broth viscosity, and the release of intracellular products into the liquid medium. The degree of mycelium disruption and the rate of intracellular product release were dependent on US power intensity, treatment period and biomass concentration of broth. The extraction or release kinetics of total water-soluble products and PSPs (yield Y versus time t) under the effect of US was fitted closely to the Elovich model Y=Yo+Y1 lnt and parabolic model Y=Yo+Y1t(½), respectively. Another interesting effect of the US treatment was a notable increase in the antioxidant cytoprotective activity of PSP against H2O2 induced cell death.

Production and characterization of exopolysaccharides from an enthomopathogenic fungus Cordyceps militaris NG3.

Optimization of the submerged culture conditions for the mycelial growth and production of exopolysaccharide (EPS) from a newly isolated Cordyceps species (C. militaris NG3) was studied in flask cultures. The optimal temperature and initial pH for EPS production were 30 degrees C and 8.0, respectively. Sucrose (30 g/L) and corn steep powder (10 g/L) were the most suitable carbon and nitrogen source for both mycelial growth and EPS production. There was a distinguishable morphological changes in mycelium grown between organic and inorganic nitrogen sources. A smooth pellet growth with heavy hyphal thickness was observed in the medium containing organic nitrogen sources, whereas irregular pellets with less hairy region were formed in the medium containing inorganic nitrogen sources. More highly branched cells appearing in the medium of organic nitrogen sources seemed a favorable morphological form for both EPS production and mycelial growth. Under optimal culture conditions, the maximum concentrations of mycelial growth and EPS were 17.6 and 3.4 g/L in a 5-L stirred-tank fermenter. Four groups of EPSs (designated as Fr-I, Fr-II, Fr-III, and Fr-IV) were obtained from the culture filtrates by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The weight-average molar masses of the Fr-I, Fr-II, Fr-III, and Fr-IV of EPS were determined to be 2.262 x 10(6), 3.348 x 10(5), 1.049 x 10(5), and 5.059 x 10(4) g/mol, respectively. All four EPSs showed very low polydispersity indices ranging from 1.00 to 1.18. The SEC/MALLS analysis revealed that the molecular shape of the Fr-I was a rigid sphere suspected to be an aggregate of complex polysaccharides, the Fr-II and Fr-III were nearly globular in shape, and the Fr-IV was an almost rodlike structure.

Cordyceps fungi as natural killers, new hopes for medicine and biological control factors.

The Cordyceps genus includes many species of fungi, most of which are endoparasitoids on arthropods.The distribution of these fungi is cosmopolitan, but many occur in regions such as Asia with a hot, humid climate. These pathogens of insect pests are promising candidates for use as biological control factors. Entomopathogenic fungi including the famous Cordyceps sinensis produce bioactive compounds. Lately Cordyceps sinensis was renamed Ophiocordyceps sinensis. This fungus has a long history as a medicinal fungus. It germinates in a living host, kills and mummifies the larva, and then grows from the body of the host. Is known in Tibet as the "winter worm, summer grass",or "Caterpillar fungus" (Yartsa gunbu). Collecting Ophiocordyceps has become an important source of money for local households in Nepal. Ophiocordyceps sinensis is cultivated as an anamorph for its medicinal and pharmaceutical properties in an artificial medium on an industrial scale. Ophiocordyceps compounds have immunostimulating properties and antitumor activity.

Alteration of media composition and light conditions change morphology, metabolic profile, and beauvericin biosynthesis in Cordyceps bassiana mycelium.

Metabolic alterations of Cordyceps bassiana mycelium were investigated under the following culture medium and light conditions: dextrose agar supplemented with 0.5% yeast extract (SDAY) medium with light (SL), SDAY medium without light (SD), nut medium without light (ND), and iron-supplemented SDAY medium without light (FD). The levels of asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols, saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for the enhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C. bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceutical and medicinal resource.

Genome-wide transcriptome and proteome analysis on different developmental stages of Cordyceps militaris.

BACKGROUND: Cordyceps militaris, an ascomycete caterpillar fungus, has been used as a traditional Chinese medicine for many years owing to its anticancer and immunomodulatory activities. Currently, artificial culturing of this beneficial fungus has been widely used and can meet the market, but systematic molecular studies on the developmental stages of cultured C. militaris at transcriptional and translational levels have not been determined. METHODOLOGY/PRINCIPAL FINDINGS: We utilized high-throughput Illumina sequencing to obtain the transcriptomes of C. militaris mycelium and fruiting body. All clean reads were mapped to C. militaris genome and most of the reads showed perfect coverage. Alternative splicing and novel transcripts were predicted to enrich the database. Gene expression analysis revealed that 2,113 genes were up-regulated in mycelium and 599 in fruiting body. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to analyze the genes with expression differences. Moreover, the putative cordycepin metabolism difference between different developmental stages was studied. In addition, the proteome data of mycelium and fruiting body were obtained by one-dimensional gel electrophoresis (1-DGE) coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). 359 and 214 proteins were detected from mycelium and fruiting body respectively. GO, KEGG and Cluster of Orthologous Groups (COG) analysis were further conducted to better understand their difference. We analyzed the amounts of some noteworthy proteins in these two samples including lectin, superoxide dismutase, glycoside hydrolase and proteins involved in cordycepin metabolism, providing important information for further protein studies. CONCLUSIONS/SIGNIFICANCE: The results reveal the difference in gene expression between the mycelium and fruiting body of artificially cultivated C. militaris by transcriptome and proteome analysis. Our study provides an effective resource for the further developmental and medicinal research of this promising fungus.

Anti-asthmatic activities in mycelial extract and culture filtrate of Cordyceps sphecocephala J201.

This study investigated the effects of mycelial extract and culture filtrate of Cordyceps sphecocephala J201 on airway hyper-responsiveness, pulmonary immune cell infiltration, and Th2 cytokine expression in animal models of asthma. After Concanavalin A (Con A) activation of mouse primary spleen cells, the IL-4 and IL-13 cytokine expression were significantly decreased in the presence of the mycelial extract and culture filtrate of Cordyceps sphecocephala J201. The asthma model was induced by sensitization to ovalbumin by intraperitoneal (i.p.) injection treatment in mice. The Cordyceps sphecocephala J201 mycelial extract was injected in order to assess the effects of anti-asthmatic activity by comparing lung cell infiltration in ovalbumin-induced asthmatic mice. The results revealed that the increased IL-4, IL-13 and IL-25 expression were controlled by the mycelial extract and culture filtrate of Cordyceps sphecocephala J201, indicating that the extracts reduced the undesirable immune responses and/or cytokine expression exhibited in asthma.

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein.

In the accompanying paper [Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006). Microbiology 152, 2843-2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2,500 5' end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1,069 singlets, and 306 contigs with 1,064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45-50 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. Approximately 20-25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4,360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109.

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.