Growth,fruiting and lignocellulolytic enzyme production by the edible mushroom <Emphasis Type="Italic">Grifola frondosa</Emphasis> (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, β-glucosidase 2.3, endoxylanase 20.3, amylase 0.26, laccase 14.8 and Mn-peroxidase 7.4 U/g dry substrate.     

Modeling Grifola frondosa fungal growth during solid‐state fermentation

Grifola frondosa (maitake) is an edible and medicinal mushroom. Considering its increasing popularity, there are limited references for its cultivation. Previous studies demonstrated that carpophore formation is correlated directly with mycelial biomass. The development of a mathematical model for its growth under solid‐state fermentation (SSF) may help to predict the potential of different substrates for maitake production. G. frondosa growth and basidiome development was studied, using oak sawdust and corn bran as substrates. The fungal biomass content was determined by measuring N‐acetyl‐D ‐glucosamine (NAGA). It increased steadily for the first 80 days, to a maximum in coincidence with the first fruiting (60.5 μg NAGA/mg dry sample). Two mathematical models were selected to evaluate G. frondosa development, measuring reducing sugars consumption and NAGA synthesis, as an indirect assessment of fungal growth. Both models showed a good fit between predicted and experimental data: logistic model (R²=0.8896), two‐stage model (R²=0.8878), but the logistic model required a minor number of adjustment parameters.     

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa based on ligninolytic enzymes

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa using ligninolytic enzymes as markers was evaluated with the detection and analysis of activities and composition of 15 edible fungi. The results showed that the activity and composition of ligninolytic enzyme system varied in response to changes of fungal strains. By analyzing the growth rate of mycelia and their ability to produce ligninolytic enzymes, H. marmoreus and P. geesteranus, G. frondosa and P. sajor-caju were screened for further study. Three colonies of 26 regenerated colonies of H. marmoreus and P. geesteranus protoplast fusion and one colony of 48 regenerated colonies of G. frondosa and P. sajor-caju were selected respectively. At the same time, these four strains were identified using RAPD and ISSR molecular markers. The results showed that the strains HM5G1and PS7F1are new strains and have low similarity to parental strains H. marmoreus and G. frondosa. These results are supported by the results of antagonism tests. These two fusants were significantly higher in their ligninolytic enzyme activity than H. marmoreus and G. frondosa. The growth rates of strains HM5G1and PS7F1 were also noticeably higher than those of H. marmoreus and G. frondosa, by 1.36 and 1.5 times respectively. The biological efficiency of the strain HM5G1 was 11.5 % higher than that of the parental strain H. marmoreus. This work suggests that it is an efficient way of breeding new strains to use the decolorization of ligninolytic enzymes as a preliminary screening marker.     

Hypoglycemic Activity of <Emphasis Type="Italic">Grifola frondosa</Emphasis> Rich in Vanadium

The hypoglycemic activity of fermented mushroom of Grifola frondosa rich in vanadium (GFRV) was studied in this paper. Alloxan- and adrenalin-induced hyperglycemic mice were used in the study. The blood glucose and the HbA1c of the mice were analyzed respectively. After the mice were administered (ig) with GFRV, the blood glucose and the HbA1c of alloxan-induced hyperglycemic mice decreased (p < 0.05, p < 0.01) and ascension of blood glucose induced by adrenalin was inhibited (p < 0.01). Also, the bodyweight of the alloxan-induced hyperglycemic mice was increased gradually. In the fermented mushroom of G. frondosa, vanadium at lower doses in combination with G. frondosa induced significant decreases of the blood glucose and HbA1c levels in hyperglycemic mice.     

Three-dimensional Structure of Victorivirus HvV190S Suggests Coat Proteins in Most Totiviruses Share a Conserved Core

Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300–450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination–reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a “T = 2” icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well.     

Heat shock protein 9 mRNA expression increases during fruiting body differentiation in Grifola frondosa and other edible mushrooms

We isolated a homolog of Schizosaccharomyces pombe HSP9 from Grifola frondosa and designated it Gf.HSP9. The Gf.HSP9 gene consisted of four exons and three introns and encoded 84 amino acid residues. We have also identified related HSP9 genes from Pleurotus eryngii (Pe.HSP9), Hypsizygus marmoreus (Hm.HSP9), and Lentinula edodes (Le.HSP9). The predicted tertiary structures of these HSP9s were the same. In addition, the expression pattern of their mRNA increased during fruiting body in a differentiation-dependent manner. These results suggested that HSP9 was associated with the fruiting body differentiation in these four edible mushrooms.     

药用真菌子实体多糖SEC—HPLC图谱的相似度定量评价研究

目的：对灵芝属Ganoderma下4个种共7个菌株及灰树花Grifola ffondosa和云芝Trametes versicolor共9种子实体粗多糖进行了体积排阻HPLC色谱（SEC—HPLC）,并分析了相似度。方法：各样品水提物的HPLC图谱用平方欧氏距离法计算了相似度,用离差平方和法进行聚类分析。结果：灵芝属内各样品聚类距离为8,灰树花与云芝的聚类距离为16,它们与灵芝属的距离为25,说明同一灵芝属内的不同种及同种不同菌株所产生的多糖分子量分布近似度远高于来自树花菌属的灰树花或栓菌属的云芝。结论：此方法可量化描述不同多糖间SEC—HPLC的相似关系,有作为真菌多糖样品质量控制手段的潜在能力。     

A Novel Victorivirus from a Phytopathogenic Fungus,Rosellinia necatrix,Is Infectious as Particles and Targeted by RNA Silencing

A novel victorivirus, termed Rosellinia necatrix victorivirus 1 (RnVV1), was isolated from a plant pathogenic ascomycete, white root rot fungus Rosellinia necatrix, coinfected with a partitivirus. The virus was molecularly and biologically characterized using the natural and experimental hosts (chestnut blight fungus, Cryphonectria parasitica). RnVV1 was shown to have typical molecular victorivirus attributes, including a monopartite double-stranded RNA genome with two open reading frames (ORFs) encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), a UAAUG pentamer presumed to facilitate the coupled termination/reinitiation for translation of the two ORFs, a spherical particle structure ∼40 nm in diameter, and moderate levels of CP and RdRp sequence identity (34 to 58%) to those of members of the genus Victorivirus within the family Totiviridae. A reproducible transfection system with purified RnVV1 virions was developed for the two distinct fungal hosts. Transfection assay with purified RnVV1 virions combined with virus elimination by hyphal tipping showed that the effects of RnVV1 on the phenotype of the natural host were negligible. Interestingly, comparison of the RNA silencing-competent (standard strain EP155) and -defective (Δdcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with hypovirus or transgenic expression of an RNA silencing suppressor, hypovirus p29. Phenotypic changes were observed in the Δdcl-2 strain but not in EP155. This is the first reported study on the host range expansion of a Totiviridae member that is targeted by RNA silencing.     

Grifola frondosa extract and ergosterol reduce allergic reactions in an allergy mouse model by suppressing the degranulation of mast cells

ABSTRACT

The increasing number of patients suffering from allergic diseases is a global health problem. Grifola frondosa is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that G. frondosa extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow–derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells in vivo. Our findings suggest that G. frondosa, including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.     

Ergosterol and its derivatives from Grifola frondosa inhibit antigen-induced degranulation of RBL-2H3 cells by suppressing the aggregation of high affinity IgE receptors

Grifola frondosa is an edible mushroom consumed as a health food and/or traditional medicine in Asia. However, the anti-allergic effects of G. frondosa are not yet understood. In this study, we demonstrated the effects of G. frondosa extract (GFE) on IgE-mediated allergic responses, using antigen-stimulated RBL-2H3 cells. Three active compounds: ergosterol, 6β-methoxyergosta-7,22-dien-3β,5α-diol (MEDD), and 6-oxoergosta-7,22-dien-3β-ol (6-OXO) were isolated from GFE and shown to inhibit the antigen-induced release of β-hexosaminidase and histamine. Among the three active components, we focused on ergosterol because of its high content in GFE. Ergosterol inhibited the aggregation of high-affinity IgE receptor (FcεRI), which is the first step in the activation of mast cells and antigen-induced tyrosine phosphorylation. Furthermore, ergosterol suppressed antigen-increased IL-4 and TNF-α mRNA. Taken together, our findings suggest that G. frondosa, including ergosterol and its derivatives as active components, has the potential to be a novel functional food that prevents type I allergies.     

Grifola frondosa (Maitake) extract activates PPARδ and improves glucose intolerance in high-fat diet-induced obese mice

Grifola frondosa is a mushroom that has anti-obesity effects, but the detailed mechanism is poorly understood. In this study, we found that lipid soluble extracts derived from G. frondosa (GFE) had peroxisome proliferator-activated receptor δ (PPARδ) agonist activity, inducing the expression of PPARδ-target genes in vitro. Furthermore, administration of GFE to high-fat diet-induced obese mice lowered the total blood cholesterol levels, upregulated the expression of PPARδ-target genes in skeletal muscles and improved glucose intolerance. Additionally, analyses of C₂C₁₂ myotubes revealed that GFE restored glucose uptake, which was inhibited by sodium palmitate, to normal levels. Unexpectedly, such acceleration was not abolished by a PPARδ antagonist. These results suggest that G. frondosa is a novel functional food that may prevent life-style related diseases like obesity and diabetes, and that these beneficial effects are likely to be mediated through the activation of PPARδ and a PPARδ-independent insulin signaling pathway.     

Purification and molecular cloning of a serine protease from the mushroom Hypsizigus marmoreus

A protease with a molecular mass of 28 kDa, designated as hmsp, was isolated from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on CM-cellulose. hmsp was thermolabile, and exhibited a temperature optimum at 50 °C and a pH optimum at pH 7.5. The activity of the protease was adversely affected by PMSF, EGTA and aprotinin, indicating that it is a serine protease. Based on the N-terminal sequence, the cDNA of hmsp was cloned by using RACE combined with the TAIL-PCR method. The deduced protease sequence contained a signal peptide with 19 amino acids, a pro-region with 82 amino acids, and a mature protease with 285 amino acids and a molecular mass of 28.07 kDa. It possessed the three active sites characteristic of the subtilisin family (S8A). hmsp demonstrated 63%, 57% and 44% identity in amino acid sequence respectively to Absp1, Absp2, and Gf-spr1, which are serine proteases from Agaricus bisporus and Grifola frondosa.     

灰树花提取物清除氧自由基的研究

采用化学发光法比较了灰树花各提取物对超氧阴离子自由基、羟基自由基的清除效果。研究结果表明,灰树花子实体和菌丝体的提取物对羟基自由基均有很好的清除作用,灰树花菌丝体水提物(GFME)、灰树花子实体水提物(GFE)、灰树花子实体水提多糖(GFP)、灰树花子实体碱提多糖(GFAP)清除羟基自由基的IC50均小于1.0 mg/mL,其他提取物清除羟基自由基的IC50也均小于3.0 mg/mL。而对超氧阴离子自由基仅GFME、GFMP、GFE有清除作用,其他的提取物清除作用很弱。     

灰树花生长发育过程中的几种胞外酶活性变化

胞外CMC酶、FP酶、HC酶活性高峰出现于子实体的成熟期;胞外淀粉酶活性在菌丝生长的前期阶段较高,以后逐渐下降;胞外漆酶活性在菌丝生长阶段一直较高,原基形成后逐渐下降;胞外过氧化物酶活性在菌丝生长阶段较高,子实体生长发育阶段明显下降,子实体成熟后消失.     

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0 kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130 kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.     

Suppression of D-galactosamine-induced liver injury by mushrooms in rats

Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5%) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.