Purification and characterization of Se-enriched Grifola frondosa glycoprotein, and evaluating its amelioration effect on As3+ -induced immune toxicity.

BACKGROUND: Selenium (Se)-enriched glycoproteins have been a research highlight for the role of both Se and glycoproteins in immunoregulation. Arsenic (As) is a toxicant that is potentially toxic to the immune function and consequently to human health. Several reports suggested that Se could reduce the toxicity of heavy metals. Moreover, more and more nutrients in food had been applied to relieve As-induced toxicity. Hence glycoproteins were isolated and purified from Se-enriched Grifola frondosa, and their preliminary characteristics as well as amelioration effect and mechanism on As3+ -induced immune toxicity were evaluated. RESULTS: Four factions, namely Se-GPr11 (electrophoresis analysis exhibited one band: 14.32 kDa), Se-GPr22 (two bands: 20.57 and 31.12 kDa), Se-GPr33 (three bands: 15.08, 20.57 and 32.78 kDa) and Se-GPr44 (three bands: 16.73, 32.78 and 42.46 kDa), were obtained from Se-enriched G. frondosa via DEAE-52 and Sephacryl S-400 column. In addition, Se-GPr11 and Se-GPr44 are ideal proteins that contain high amounts of almost all essential amino acids. Thereafter, the RAW264.7 macrophage model was adopted to estimate the effect of Se-GPr11 and Se-GPr44 on As3+ -induced immune toxicity. The results showed that the pre-intervention method was the best consequent and the potential mechanisms were, first, by improving the oxidative stress state (enhancing the activity of superoxide dismutase and glutathione peroxidase, decreasing the levels of reactive oxygen species and malondialdehyde); secondly, through nuclear factor-κB and mitogen-activated protein kinase-mediated upregulation cytokines (interleukin-2 and interferon-γ) secretion induced by As3+ . CONCLUSION: The results suggested Se-enriched G. frondosa may be a feasible supplement to improve health level of the As3+ pollution population. © 2021 Society of Chemical Industry.

Grifola frondosa may play an anti-obesity role by affecting intestinal microbiota to increase the production of short-chain fatty acids.

Background: Grifola frondosa (G. frondosa) is a fungus with good economic exploitation prospects of food and medicine homologation. This study aims to investigate the effects of G. frondosa powder suspension (GFPS) on the intestinal contents microbiota and the indexes related to oxidative stress and energy metabolism in mice, to provide new ideas for developing G. frondosa weight loss products. Methods: Twenty Kunming mice were randomly divided into control (CC), low-dose GFPS (CL), medium-dose GFPS (CM), and high-dose GFPS (CH) groups. The mice in CL, CM, and CH groups were intragastrically administered with 1.425 g/(kg·d), 2.85 g/(kg·d), and 5.735 g/(kg·d) GFPS, respectively. The mice in CC group were given the same dose of sterile water. After 8 weeks, liver and muscle related oxidative stress and energy metabolism indicators were detected, and the intestinal content microbiota of the mice was detected by 16S rRNA high-throughput sequencing. Results: After eight weeks of GFPS intervention, all mice lost weight. Compared with the CC group, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents in CL, CM, and CH groups were increased, while Succinate dehydrogenase (SDH) and Superoxide Dismutase (SOD) contents in the liver were decreased. The change trends of LDH and SDH in muscle were consistent with those in the liver. Among the above indexes, the change in CH is the most significant. The Chao1, ACE, Shannon, and Simpson index in CL, CM, and CH groups were increased. In the taxonomic composition, after the intervention with GFPS, the short-chain fatty acid (SCFA)-producing bacteria such as unclassified Muribaculaceae, Alloprevotella, and unclassified Lachnospiraceae increased. In linear discriminant analysis effect size (LEfSe) analysis, the characteristic bacteria in CC, CL, CM, and CH groups showed significant differences. In addition, some characteristic bacteria significantly correlated with related energy metabolism indicators. Conclusion: The preventive effect of G. frondosa on obesity is related to changing the structure of intestinal content microbiota and promoting the growth of SCFAs. While excessive intake of G. frondosa may not be conducive to the antioxidant capacity and energy metabolism.

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.

Anti-Diabetic and Anti-Nephritic Activities of Grifola frondosa Mycelium Polysaccharides in Diet-Streptozotocin-Induced Diabetic Rats Via Modulation on Oxidative Stress.

Grifola frondosa is an edible fungus with a variety of potential pharmacological activities. This study investigates the hypoglycemic, anti-diabetic nephritic, and antioxidant properties of G. frondosa polysaccharides in diet-streptozotocin-induced diabetic rats. After a 4-week treatment with 100 mg/kg of metformin and 200 mg/kg of one of four different G. frondosa polysaccharide mixtures (especially GFPS3 and GFPS4), diabetic rats had enhanced body weight and suppressed plasma glucose, indicating the hypoglycemic activities of the G. frondosa polysaccharides. G. frondosa polysaccharides regulated the level of serum creatinine, blood urea nitrogen, N-acetyl-ß-D-glucosaminidase, and albuminuria; inhibited the serum levels of interleukin (IL)-2, IL-6, and TNF-α; and enhanced the serum levels of matrix metalloproteinase 9 and interferon-α, confirming their anti-diabetic nephritic activities. G. frondosa polysaccharides ameliorated the pathological alterations in the kidneys of diabetic rats. Moreover, G. frondosa polysaccharides modulated the serum levels of oxidant factors such as superoxide dismutase, glutathione peroxidase, catalase, malondialdehyde, and reactive oxygen species, revealing their antioxidant properties. Furthermore, the administration of G. frondosa polysaccharides inhibited nuclear factor kappa B activities in the serum and kidneys. All of the data revealed that the activation of nuclear factor kappa B plays a central role in G. frondosa polysaccharide-mediated anti-diabetic and anti-nephritic activities.

Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc.

BACKGROUND: Mushroom showed pellet, clump and/or filamentous mycelial morphologies during submerged fermentation. Addition of microparticles including Talc (magnesium silicate), aluminum oxide and titanium oxide could control mycelial morphologies to improve mycelia growth and secondary metabolites production. Here, effect of microparticle Talc (45 µm) addition on the mycelial morphology, fermentation performance, monosaccharide compositions of polysaccharides and enzymes activities associated with polysaccharide synthesis in G. frondosa was well investigated to find a clue of the relationship between polysaccharide biosynthesis and morphological changes. RESULTS: Addition of Talc decreased the diameter of the pellets and increased the percentage of S-fraction mycelia. Talc gave the maximum mycelial biomass of 19.25 g/L and exo-polysaccharide of 3.12 g/L at 6.0 g/L of Talc, and mycelial polysaccharide of 0.24 g/g at 3.0 g/L of Talc. Talc altered the monosaccharide compositions/percentages in G. frondosa mycelial polysaccharide with highest mannose percentage of 62.76 % and lowest glucose percentage of 15.22 % followed with the corresponding changes of polysaccharide-synthesis associated enzymes including lowest UDP-glucose pyrophosphorylase (UGP) activity of 91.18 mU/mg and highest UDP-glucose dehydrogenase (UGDG) and GDP-mannose pyrophosphorylase (GMPPB) activities of 81.45 mU/mg and 93.15 mU/mg. CONCLUSION: Our findings revealed that the presence of Talc significantly changed the polysaccharide production and sugar compositions/percentages in mycelial and exo-polysaccharides by affecting mycelial morphology and polysaccharide-biosynthesis related enzymes activities of G. frondosa.

Rice bran polysaccharides and oligosaccharides modified by Grifola frondosa fermentation: Antioxidant activities and effects on the production of NO.

Rice bran polysaccharides (RBPSs) are valuable compounds with many biological activities. In this work, a fungus called Grifola frondosa, was selected to ferment defatted rice bran water extracts and modify the RBPSs, which were then isolated by ethanol precipitation and deproteinization. GC analysis of fermented products suggested they are composed of glucose, arabinose, galactose, mannose, and xylose at a molar ratio of 9:5:8:2:5, which was 32:4:6:2:5 before fermentation. HPLC analysis revealed that the molecular weight of unfermented RBPS was distributed mainly from 103 to 104Da, and it changed to 102 to 103Da after fermentation. Antioxidant activities and effects on the production of NO were analyzed and it indicated that the scavenging ratios of hydroxyl and DPPH radicals by the fermented products were significantly enhanced compared to the unfermented ones, and also the products fermented for 9days exhibited two-way adjusting effects on the production of NO in macrophages.

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.

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.

Enhancement of wheat grain antioxidant activity by solid state fermentation with Grifola spp.

Grifola frondosa, Grifola gargal, and Grifola sordulenta are edible and medicinal mushrooms with antioxidant properties. To obtain wheat flour (Wf ) with a higher antioxidant activity than the one exhibited by regular Wf, solid state fermentation (SSF) of wheat grains with mycelia of those Grifola spp. was used to obtain biotransformed wheat grain (BWG) flour. The methanolic extract of control Wf and BWG flour of G. gargal, G. sordulenta, and G. frondosa (GfWG, GgWG, and GsWG, respectively) were studied for their radical scavenging (RS) activity against 2,2-diphenyl-1-picrylhydracyl (DPPH) and their Fe(III) reducing power (RP). The values for RS-EC50 decreased in BWG flour, therefore presenting a higher antioxidant activity: GgWG (0.56 mg/mL), GfWG (0.81 mg/mL), and GsWG (5.80 mg/mL) in comparison to Wf (57.60 mg/mL). The antioxidant content for this RS activity in terms of ascorbic acid content (RS-EQAA) was highest in GfWG, followed by GgWG and GsWG (71.73, 14.46, and 3.02 mg/g, respectively) and lowest in Wf (0.25 mg/g). The RP-EC50 values in GgWG, GfWG, and GsWG were low (0.55, 0.64, and 4.20 mg/mL, respectively) with respect to Wf (55.00 mg/mL). Compared with Wf (0.56 mg/g), the RP capacity in terms of ascorbic acid content (RP-EQAA) was very high in GfWG (193.67 mg/g) followed by GgWG and GsWG (31.42 and 8.74 mg/g, respectively). The high content in gallic acid equivalents was consistent with RS-EQ(AA) and RP-EQ(AA) contents. TLC revealed that antioxidant activity in BWG could be related to the presence of phenolic compounds. Thus, a valuable food alternative can easily be obtained with wheat grains, that is, by markedly increasing their antioxidant value through SSF with Grifola spp.

Purification and characterization of a novel small-molecule polysaccharide from the Maitake medicinal mushroom Grifola frondosa (higher Basidiomycetes).

A novel small-molecule polysaccharide with a molecular mass of 2.6 kDa, was isolated from the culinary-medicinal Maitake mushroom Grifola frondosa. GFPS is composed of fucose, rhamnose, galactose, glucose, and mannose; galactose, glucose, and mannose were the dominant monosaccharides. Absorption peaks at 1077 cm-1, 1024 cm-1, and 873 cm-1, as revealed by infrared spectrum, suggesting that GFPS consists of pyranoside. GFPS significantly enhanced the production of nitric oxide and secretion of cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-δ) from macrophages in vitro. These results indicate that this novel small-molecule polysaccharide might be beneficial for immune defense.

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.

Determination of trace elements in three mushroom samples of basidiomycetes from Shandong, China.

We have determined the trace element composition of three mushrooms of Basidiomycetes, used in traditional Chinese medicine using atomic absorption spectrophotometer (AAS). Metal concentrations in mushrooms were 203-401 mg/kg for iron, 22-51 mg/kg for manganese, 84-116 mg/kg for zinc, 24.1-41.3 mg/kg for copper, 1.6-5.6 mg/kg for lead, 3.3-4.4 mg/kg for chromium, 9.3-11.5 mg/kg for nickel, 0 mg/kg for vanadium, and 55.3-71 mg/kg for magnesium. The trace metal concentrations in mushrooms are hardly affected by the ecosystem and soil where they grew, as well as by the mushroom species and trace metal species. The results can be used to set new standards to control the quality of the three mushrooms of Basidiomycetes-Ganoderma lucidum, Coprinus comatus, and Grifola frondosa.

Biological activities of Fructus arctii fermented with the basidiomycete Grifola frondosa.

Fructus arctii extract containing phenolic glycosides was cultured with Grifola frondosa mycelia to produce ß-glucosidase and its biological activities were studied. This ß-glucosidase converted the glycosides (arctiin and caffeic acid derivatives) into aglycones (arctigenin and caffeic acid). Fermented Fructus arctii extract (G-FAE) with G. frondosa had antioxidant and 5-lipoxygenase inhibitory activities. The photoprotective potential of G-FAE was tested in human dermal fibroblasts (HDF) exposed to ultra-violet A (UVA). It was revealed that G-FAE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF. The treatment of UVA-irradiated HDF with G-FAE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. G-FAE also showed notable stimulation of collagen biosynthetic activity for fibroblasts. These diverse functionalities suggest that G-FAE could be a promising cosmetic ingredient.

Bioaccumulation and bioavailability of copper and zinc on mineral-enriched mycelium of Grifola frondosa.

Grifola frondosa is an edible and medicinal mushroom. The bioaccumulation and potential biovailability of Cu and Zn were studied to obtain mycelium with potential properties as a food dietary supplement. Mycelia grown in the presence of nonmycotoxic concentrations of 100 and 200 ppm of Cu or 25 and 50 ppm of Zn accumulated 200-322 ppm and 267-510 ppm of Cu or Zn, respectively. When these enriched metal mycelia were subjected in vitro to a simulated gastrointestinal digestion, the solubility in these digestive fluids was 642-669 ppm and 102-530 ppm, which represent 32-33% and 0.7-3.5% of the recommended daily intake (RDI) for Cu and Zn, respectively, in 1 g of mycelium. These results are discussed in relation to the RDI values exhibited by two commercial supplements, and arguments are given on the potential use of these mineral-enriched mycelia in capsules (in the case of Cu-enriched mycelia), in food preparations, and also as a component of cosmetic mixtures.

Augmentation of sebaceous lipogenesis by an ethanol extract of Grifola frondosa (Maitake mushroom) in hamsters in vivo and in vitro.

Grifola frondosa (Maitake mushroom) is an edible and medicinal mushroom with versatile effects such as antitumor and immunomodulating actions. Here, we demonstrated that an ethanol extract of G. frondosa fruiting body (Maitake extract) augmented intracellular lipid droplet formation and the production of triacylglycerols (TG), a major component of sebum, along with the activation of diacylglycerol acyltransferase, a rate-limiting enzyme of TG synthesis in cultured hamster sebocytes. The topical treatment of Maitake extract on the skin of hamster auricles augmented sebum accumulation in sebaceous glands and ducts. However, in comparison with the Maitake extract, another ethanol extract prepared from Agaricus blazei Murill showed less activity in sebaceous lipogenesis in hamsters in vivo and in vitro. These results provide novel evidence that Maitake extract augments sebaceous lipogenesis in hamsters in vivo and in vitro. Thus, Maitake extract is likely to be a unique agent leading to the remission of dry skin.

Solid-state cultivation of Grifola frondosa (Dicks: Fr) S.F. Gray biomass and immunostimulatory effects of fungal intra- and extracellular beta-polysaccharides.

Grifola frondosa strain GF3, was cultivated on solid-state substrate consisting of milled whole corn plant (Zea mays) and olive press cake supplemented with mineral additives and olive oil. Maintenance of the moisture content in the solid substrate is of crucial importance. Moistures higher than 70% promote growth of G. frondosa mycelium and polysaccharide production. Four fractions of pure extracellular beta-D-glucans with total mass 127.2mg and four fractions of intracellular polysaccharides with total mass 47.2mg were isolated. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. Isolated polysaccharide fractions from fungal mycelium proved to induce moderate amounts of TNF-alpha in PBMC cells in vitro. The extent of TNF-alpha induction was up to 322pgmL(-1) at a polysaccharide concentration of 200microgmL(-1) for the intracellular fraction. The TNF-alpha inducing activity is comparable to romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.

Induction of apoptosis in SGC-7901 cells by polysaccharide-peptide GFPS1b from the cultured mycelia of Grifola frondosa GF9801.

The biological function of GFPPS1b, a novel polysaccharide-peptide isolated from cultured mycelia of Grifola frondosa GF9801, was well investigated. GFPS1b has anti-tumor activity and can significantly inhibit the proliferation of SGC-7901 cells, whereas slightly influences the growth of human normal liver cell line L-02. When treated with GFPS1b, SGC-7901 cells showed typical apoptotic morphological features such as the loss of villus and appearance of apoptotic bodies on the cell surface, volume reduction, and chromatin condensation, by scanning electron microscopy (SEM) and fluorescent microscopy (Hoechst 33342). The results of flow cytometry analysis and annexin V-PI assay showed that the SGC-7901 cell cycle was arrested in the G(2)/M phase, the subdiploid peak of DNA characteristic of apoptotic was also observed, and the apoptosis ratio was about 15.08%. DNA isolated from SGC-7901 cells cultured with GFPS1b showed a typical DNA 'ladders' of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of SGC-7901 induced by GFPS1b was associated with drop in mitochondrial trans-membrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3. Our finding suggests that GFPS1b could suppress SGC-7901 cell growth and reduce cell survival via arresting cell cycle and inducing apoptosis of tumor cells.