Engineering Aspects in Production of Various Medicinal Mushrooms Biomass in Submerged Bioreactors.

Basidiomycetes of various species and their wide range of pharmaceuticaly interesting products in the past decades represents one of the most attractive groups of natural products in Asia and North America. Production of mushroom fruit bodies using farming technology is hardly covering the market. Development of comprehensive submerged technologies in stirred tank and air lift bioreactors are the most promising technologies for fast and large-amount cultivation of medicinal mushroom biomass and its pharmaceutically active products. Research in physiology, basic and applied studies in mushroom metabolism, process engineering aspects, and clinical studies in the past two decades represent a large cotribution to the development of this potential, which initiates the development of new drugs and some very attractive over-the-counter human and veterinary remedies. The current article is an overview of the most relevant engineering achievements in submerged cultivation of some medicinal mushrooms-Grifola frondosa, Trametes versicolor, Hericium erinaceus, and Cordyceps militaris-and some other species biomass production in bioreactors.

Functions of a Glucan Synthase Gene GFGLS in Mycelial Growth and Polysaccharide Production of Grifola frondosa.

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.

CE-MS-based metabolomics reveals the metabolic profile of maitake mushroom (Grifola frondosa) strains with different cultivation characteristics.

Maitake mushroom (Grifola frondosa [Dicks.] Gray) is generally cultured using the sawdust of broadleaf trees. The maitake strain Gf433 has high production efficiency, with high-quality of fruiting bodies even when 30% of the birch sawdust on the basal substrate is replaced with conifer sawdust. We performed metabolome analysis to investigate the effect of different cultivation components on the metabolism of Gf433 and Mori52 by performing CE-MS on their fruiting bodies in different cultivation conditions to quantify the levels of amino acids, organic acids, and phosphorylated organic acids. We found that amino acid and organic acid content in Gf433 were not affected by the kind of sawdust. However, Gf433 contained more organic acids and less amino acids than Mori52, and Gf433 also contained more chitin compared with Mori52. We believe that these differences in the metabolome contents of the two strains are related to the high production efficiency of Gf433.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Effects of different extraction temperatures on the physicochemical properties of bioactive polysaccharides from Grifola frondosa.

This study examined the effects of different extraction temperatures (70°C, 100°C and 121°C) on the physicochemical properties of water soluble polysaccharides (WSP; GF70, GF100 and GF121, respectively) from Grifola frondosa (GF) fruiting bodies, and evaluating their effects on nitric oxide (NO) production in lipopolysaccharide-stimulated RAW264.7 macrophages. Results showed that GF121 had the highest yield. GF70, GF100 and GF121 contained a similar monosaccharide composition and the predominant monosaccharide was glucose. These polysaccharides contained two major macromolecular populations; the high molecular weight population showed a clear trend of reduced molecular weight with increasing extraction temperature. GF121 contained the highest amount of (1→3, 1→6)-ß-d-glucans, while the degree of branching in all samples was similar. GF WSP possessed NO inhibitory activity, and the strongest was GF121. This study concludes that WSP are good sources of food ingredients, and high temperature extraction could improve the quantity and quality of GF WSP.

In Vitro Studies of Secondary Metabolite-Related Responses in Some Species of Genus Grifola (Agaricomycetes) from Argentina.

Grifola gargal Singer and Grifola sordulenta (Mont.) Singer mushrooms are related to Grifola frondosa (Dicks.) Gray, which is well known for its medicinal properties. In vitro studies were performed to find a useful guide for optimizing the environmental parameters through biotransformation of lignocellulosic materials and basidiome development, also considering secondary metabolism-related responses (SMRRs) associated with these processes and the variability among species and strains; this optimization is necessary to make the mushroom's industrial cultivation profitable. Morphological features of mycelial cultures revealed that intraspecific variability was of taxonomic relevance. A low ligninolytic capacity in studied Grifola species was observed when compared with 2 control species: G. frondosa and Ganoderma lucidum. Experiments with nutrient media containing different carbohydrate sources indicated that G. gargal mycelia grew better in xylulose and G. sordulenta, in xylulose or cellulose; in addition, the latter species presented cellobiose dehydrogenase activity. An additional study of SMRRs under different light conditions (aroma, pigmentation, and morphogenic manifestations) showed that white light was better than blue, green, or red-filtered light at inducing advanced SMRRs. The results of SMRR stimulation are proposed as useful guidance for optimizing the environmental parameters for bioprocesses aimed at metabolite production.

Control of Grifola frondosa Morphology by Agitation and Aeration for Improving Mycelia and Exo-Polymer Production.

The present study describes the improved mycelia and exo-polymer production under control of Grifola frondosa morphology by changing the aeration rate and agitation intensity in a 25-L stirred fermentor. The aeration rate of 1.0 vvm yielded a highest mycelia biomass of 24.754 g/L with the lowest pellet percentage of 20.5 %. The maximum exo-polymer (2.324 g/L) was achieved at 0.75 vvm with mycelia polysaccharide production (0.321 g/g), whereas clumps and filaments dominated the ratios of 45.6 and 33.9 %, respectively. The change of aeration rate and agitation intensity had slight influence on the monosaccharide compositions in exo-polymers and significantly affected glucose and mannose contents in the mycelia polysaccharides. These findings will provide a clue for exploring the relationship between fermentation parameters, morphologies, and polysaccharide synthesis pathway of G. frondosa.

[Study on suitable distribution areas of Grifola umbellate in Sichuan province based on remote sensing and GIS].

Grifola umbellate is the important medicinal materials in China which has a very high medicinal value. This study analyzedthe suitable distribution areasof G. umbellate and provided scientific basis for determining G. umbellate planting regions and planning production distribution reasonably. The suitable distribution areas of G. umbellate in Sichuan province was researched based on TM, ETM+, and DEM data,the key ecological factors that affect the growth of G. umbellate were extracted, including elevation, slope, aspect, average annual temperature,average annual precipitation,forest information,soil information, following remote sensing and GIS techniques, combining field researchdata. The results showed that the G. umbellate resources in Sichuan province were mainly distributed in Pingwu, Beichuan, Licountry, Yanyuan, Xichang, Dechang, Yanbian, Miyi, Huidong, Panzhihua and so on, the suitability distribution areas is 276.214 4 km² approximately and accounting for more than 0.143 3% of the total area.According to the related document information and the field investigation, showed that the suitability distribution based on RS and GIS were corresponded with the actual distribution areas of G. umbellate.

Submerged-Culture Mycelia and Broth of the Maitake Medicinal Mushroom Grifola frondosa (Higher Basidiomycetes) Alleviate Type 2 Diabetes-Induced Alterations in Immunocytic Function.

Type 2 Diabetes mellitus (T2DM), a disease with impaired glucose, protein and lipid metabolism, low-grade chronic inflammation, and immune dysfunction, is a global public health crisis. We previously demonstrated that Grifola frondosa has bioactivities in improving glycemic responses in diabetic rats. Herein, we investigated the immunomodulatory effects of the submerged-culture mycelia and broth of G. frondosa on the peripheral blood cells (PBL) and splenocytes. Male Wistar rats were administered with saline (normal rats) or streptozotocin plus nicotinamide (T2DM rats) and were intragastrically administered with placebo, fermented mycelia, broth, or mycelia plus broth (1 g kg-1 day-1) for two weeks. In normal rats, ingestion of mycelia significantly decreased monocytes and ingestion of mycelia and broth significantly decreased the productions of interferon (IFN)-γ and interleukin (IL)-4 from the PBL and splenocytes. In T2DM rats, ingestion of mycelia, broth, and mycelia plus broth significantly alleviated the increases in 2 h postprandial blood glucose and the productions of IFN-γ from the T-leukocytes, IL-4, and IL-6 from the monocytes and IL-4 from the T-splenocytes, as well as significantly improved the productions of tumor-necrosis factor-α from the macrophages. In conclusion, submerged-culture mycelia and broth of G. frondosa may decrease cell-medicated immunity in normal rats and improve hyperglycemia and diabetes-induced alterations in cell-medicated and innate immunities in T2DM rats.

Submerged Culture of Grifola gargal and G. sordulenta (Higher Basidiomycetes) from Argentina as a Source of Mycelia with Antioxidant Activity.

Submerged culture is an alternative mycelium source for Grifola gargal and G. sordulenta, two rare edible mushrooms related to Grifola frondosa. This work studies their mycelia as a source of antioxidants. The efficient concentrations of methanolic extracts in both radical scavenging (RS) and reducing power (RP) abilities in G. gargal and in G. sordulenta showed a high antioxidant activity. In the experimental design used, the antioxidant activity mainly depended on the culture conditions rather than on the media composition. Irrespective of the basal culture medium, mycelium methanolic extracts of G. sordulenta obtained from culture in Erlenmeyer flasks showed equivalents to ascorbic acid (EQ(AA)) RS-EQ(AA) and RP-EQ(AA) contents higher than the corresponding values obtained with jar cultures. Under stationary cultivation, G. sordulenta produced approximately 50% higher content in both RS-EQ(AA) and RP-EQ(AA) than the medicinal mushroom G. frondosa. Phenolics correlated with RS-EQ(AA) and RP-EQ(AA) in G. gargal and with RP-EQ(AA) in G. sordulenta; besides, thin-layer chromatography showed these compounds to be at least in part related to the RS capacity. It is concluded that G. gargal and G. sordulenta mycelia are excellent sources of antioxidant metabolites.

Growth, fruiting and lignocellulolytic enzyme production by the edible mushroom Grifola frondosa (maitake).

Cultivation of specialty mushrooms on lignocellulosic wastes represents one of the most economical organic recycling processes. Compared with other cultivated mushrooms, very little is known about the nature of the lignocellulolytic enzymes produced by the edible and medicinal fungus Grifola frondosa, the parameters affecting their production, and enzyme activity profiles during different stages of the developmental cycle. In this work we investigated the enzymes that enable G. frondosa, to colonize and deconstruct two formulations based on industrial lignocellulosic by-products. G. frondosa degraded both substrates (oak-sawdust plus corn bran, and oak/corn bran supplemented with coffee spent-ground) decreasing 67 and 50% of their lignin content, along with 44 and 37% of the polysaccharides (hemicellulose and cellulose) respectively. 35.3% biological efficiency was obtained when using oak sawdust plus corn bran as substrate. Coffee spent-ground addition inhibited mushroom production, decreased growth, xylanase and cellulase activities. However, taking into account that G. frondosa successfully colonized this residue; this substrate formula might be considered for its growth and medicinal polysaccharide production. Although G. frondosa tested positive for Azure B plate degradation, a qualitative assay for lignin-peroxidase, attempts to detect this activity during solid state fermentation were unsuccessful. Enzyme activities peaked during colonization but declined drastically during fruiting body formation. Highest activities achieved were: endoglucanase 12.3, exoglucanase 16.2, ß-glucosidase 2.3, endoxylanase 20.3, amylase 0.26, laccase 14.8 and Mn-peroxidase 7.4 U/g dry substrate.

Preparation of culinary-medicinal maitake mushroom, Grifola frondosa (Dicks.: Fr.) S.F. Gray (Aphyllophoromycetideae)-fermented wheat and its antioxidant properties.

The optimization of solid-state fermentation conditions for mycelial growth in wheat by culinary-medicinal maitake mushroom, Grifola frondosa (Dicks.: Fr.) S.F. Gray was studied. The results from the center of the design analysis showed that the recommended optimal conditions for carbon, nitrogen sources, and temperature were 19 mg lactose/g, 2.0 mg malt extract/g, and 30 degrees C, respectively. Under these conditions, the mycelial biomass of 533.1 mg/g was obtained in fermented wheat, comparable to that (545.7 mg/g) obtained using the one-factor-at-a-time method. Based on the results obtained, EC50 values of ethanolic and hot water extracts from fermented wheat were 1.13-15.27 mg/mL, indicating that fermented wheat was effective in antioxidant activity, reducing power, scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals, and chelating ability on ferrous ions. Total phenol contents of both extracts were 13.35 and 23.01 mg/g, respectively. Accordingly, the information obtained would be applicable for the cultivation and consumption of G. frondosa-fermented wheat to provide its beneficial health effects.

Cultivation and bioactivity assessment of Grifola frondosa fruiting bodies on olive oil press cakes substrates.

Grifola frondosa, a medicinal and edible mushroom, was cultivated on substrates composed of olive oil press cakes with different supplements. Crude extracts were prepared from mature fruiting bodies and tested for their capacity to stimulate splenocyte proliferation. Two wild-growing fruiting bodies were extracted for comparison. Olive oil press cakes reduced the mushroom yield, and the best biological efficiency was obtained on substrates supplemented with wheat bran and without olive oil press cakes. All extracts were capable of inducing splenocyte proliferation and were half as effective as the positive control (6.0 microg/mL phytohaemagglutinin). No correlation between substrate composition and bioactivity could be established. Extracts from wild-growing G. frondosa were superior to cultivated ones in respect to biological activity.

Chemical characteristics and immuno-modulating activities of exo-biopolymers produced by Grifola frondosa during submerged fermentation process.

The immuno-modulating activities and chemical characteristics of exo-biopolymer (EX-GF) produced by a submerged mycelial culture of Grifola frondosa were studied. The EX-GF was fractionated into EX-GF-Fr.I, II, and III by Sephadex G-100 gel chromatography. Anti-complementary activity of EX-GF-Fr.III was highest (71.1%) among them, and its activation system occurred through both classical and alternative pathways, where the classical pathway found to be major one. Lysosomal enzyme activity and nitric oxide production ability of macrophage were also found to be mediated by EX-GF-Fr.III. The molecular weight of the EX-GF-Fr.I, II, and III was estimated to be about 163, 40, and 2.8 kDa, respectively. Total sugar and protein contents of the three fractions were 80.3, 61.9 and 89.3%, and 17.3, 35.2, and 10.7%, respectively. The sugar and amino acid compositions of the EX-GF-Fr.I, II, and III were also analyzed in detail.

Immunomodulatory properties of Grifola frondosa in submerged culture.

Maitake (Grifola frondosa) is a popular mushroom in Asia for its tasty flavor and immune-stimulating property. The aim of the study is to investigate the innate immunity augmentation effects of different extracts of mycelia and culture filtrate from G. frondosa in submerged cultures. The hot water extract of mycelia showed the strongest cytokine induction effect as a function of its concentration in human whole blood culture. The most potent fractions of hot water extract, Fr. I and II, were mainly composed of polysaccharides with molecular masses of 43-140 and 13-38 kDa, respectively. These fractions (0.025 mg/mL) showed marked activity in enhancing phagocytosis of human polymorphonuclear neutrophils (PMN). In parallel, the expression of CD11b, an early marker of PMN activation, was also up-regulated dose dependently. This result suggested that complement receptor 3 was primed by these fractions. In addition to activation of phagocytes, these bioactive fractions also increased human peripheral blood natural killer cell cytotoxicity. These results imply that the relatively low molecular mass polysaccharides isolated from mycelia of G. frondosa can enhance innate immunity in vitro and therefore may serve as biological response modifiers.

Optimization of the medium composition for production of mycelial biomass and exo-polymer by Grifola frondosa GF9801 using response surface methodology.

In this work, a three-level Box-Behnken factorial design was employed combining with response surface methodology (RSM) to optimize the medium composition for the production of the mycelial biomass and exo-polymer in submerged cultures by Grifola frondosa GF9801. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of mycelial biomass and exo-polymer. The model estimated that, a maximal yield of mycelial biomass (17.61 g/l) could be obtained when the concentrations of glucose, KH2PO4, peptone were set at 45.2 g/l, 2.97 g/l, 6.58 g/l, respectively; while a maximal exo-polymer yield (1.326 g/l) could be achieved when setting concentrations of glucose, KH2PO4, peptone at 58.6 g/l, 4.06 g/l and 3.79 g/l, respectively. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yields of mycelial biomass and exo-polymer. Maximum mycelial biomass yield of 22.50 g/l was achieved in a 15-l fermenter using the optimized medium.

[Effects of companion fungus on several enzymatic activities of Grofola umbellata].

OBJECTIVE: To study the effect of companion fungus on several enzymatic activities of Grifola umbellata. METHOD: Chitinase, beta-1,3-glucanase, proteinase and extracellular enzymes of G. umbellata were measured during dual culturing with companion fungus. RESULT: Companion fungus could induce the increase of chitinase and beta-1, 3-glucanase activities of G. umbellata. noevident changeswere found in proteinase activity. When in liquid culture, the activities of extracellular enzymes in dual cultured filtrate were between of these of G. umbellata and companion fungus in monocultures. CONCLUSION: Sclerotia differentiation related materials supplied by mutual nutritional supplement between G. umbellata and companion fungus conduce to sclerotial formation of G. umbellata.

Characterization of phytase activity from cultivated edible mushrooms and their production substrates.

Phytase is used commercially to maximize phytic acid degradation and to decrease phosphorus levels in poultry and swine manure. To determine phytase content in edible mushrooms, basidiomata of Agaricus bisporus and three specialty mushrooms (Grifola frondosa, Lentinula edodes, and Pleurotus cornucopiae) and spent mushroom substrate (SMS) were surveyed. Enzyme activity ranged from 0.046 to 0.074 unit/g of tissue for four A. bisporus types (closed and open whites and closed and open browns) grown at The Pennsylvania State University's Mushroom Test Demonstration Facility (MTDF). The addition of various nutrient supplements to phase II mushroom production substrate did not alter phytase activity in A. bisporus. Portabella mushrooms (open brown) obtained from a commercial farm had significantly higher levels of phytase activity (0.211 unit/g of tissue) compared to A. bisporus grown at the MTDF. Of the specialty mushrooms surveyed, maitake (G. frondosa) had 20% higher phytase activity (0.287 unit/g of tissue) than commercial portabella mushrooms. The yellow oyster mushroom (P. cornucopiae) ranked second in level of phytase activity (0.213 unit/g of tissue). Shiitake (L. edodes) contained the least amount of phytase in basidiomata (0.107 unit/g of tissue). Post-crop steam treatment (60 degrees C, 24 h) of SMS reduced phytase activity from 0.074 to 0.018 unit/g. Phytase was partially purified from commercially grown portabella basidiomata 314-fold with an estimated molecular mass of 531 kDa by gel filtration chromatography. The optimum pH for activity was 5.5, but appreciable phytase activity was observed over the range of pH 5.0-8.0. Partially purified A. bisporus phytase was inactivated following a 10-min incubation at > or =60 degrees C.