Ganoderma lucidum Spore Polysaccharide Inhibits the Growth of Hepatocellular Carcinoma Cells by Altering Macrophage Polarity and Induction of Apoptosis.

BACKGROUND: Ganoderma lucidum has certain components with known pharmacological effects, including strengthening immunity and anti-inflammatory activity. G. lucidum seeds inherit all its biological characteristics. G. lucidum spore polysaccharide (GLSP) is the main active ingredient to enhance these effects. However, its specific biological mechanisms are not exact. Our research is aimed at revealing the specific biological mechanism of GLSP to enhance immunity and inhibit the growth of H22 hepatocellular carcinoma cells. METHODS: We extracted primary macrophages (Mø) from BALB/c mice and treated them with GLSP (800 µg/mL, 400 µg/mL, and 200 µg/mL) to observe its effects on macrophage polarization and cytokine secretion. We used GLSP and GLSP-intervened macrophage supernatant to treat H22 tumor cells and observed their effects using MTT and flow cytometry. Moreover, real-time fluorescent quantitative PCR and western blotting were used to observe the effect of GLSP-intervened macrophage supernatant on the PI3K/AKT and mitochondrial apoptosis pathways. RESULTS: In this study, GLSP promoted the polarization of primary macrophages to M1 type and the upregulation of some cytokines such as TNF-α, IL-1ß, IL-6, and TGF-ß1. The MTT assay revealed that GLSP+Mø at 400 µg/mL and 800 µg/mL significantly inhibited H22 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that GLSP+Mø induced apoptosis and cell cycle arrest at the G2/M phase, associated with the expression of critical genes and proteins (PI3K, p-AKT, BCL-2, BAX, and caspase-9) that regulate the PI3K/AKT pathway and apoptosis. GLSP reshapes the tumor microenvironment by activating macrophages, promotes the polarization of primary macrophages to M1 type, and promotes the secretion of various inflammatory factors and cytokines. CONCLUSION: Therefore, as a natural nutrient, GLSP is a potential agent in hepatocellular carcinoma cell treatment and induction of apoptosis.

Distribution of Zinc in Mycelial Cells and Antioxidant and Anti-Inflammatory Activities of Mycelia Zinc Polysaccharides from Thelephora ganbajun TG-01.

This study demonstrates that Thelephora ganbajun had a strong ability to absorb zinc, and zinc can be compartmentally stored in the small vesicles and mainly accumulated in the form of zinc-enriched polysaccharides (zinc content was 25.0 ± 1.27 mg/g). Mycelia zinc polysaccharides (MZPS) and its fractions were isolated. The main fraction (MZPS-2) with the highest antioxidant activity in vitro was composed of mannose : galacturonic acid : glucose : galactose in a molar ratio of 61.19 : 1 : 39.67 : 48.67, with a weight-averaged molecular weight of 5.118 × 105 Da. MZPS-2 had both α-pyranose and ß-pyranose configuration and had a triple helical conformation. By establishing zebrafish models, we found that MZPS-2 can significantly scavenge free radicals, reduce the generation of reactive oxygen species caused by inflammation, and inhibit the recruitment of neutrophils toward the injury site. Therefore, MZPS-2 exhibited antioxidant and anti-inflammatory effects and can be used as a zinc supplement with specific biological activities to alleviate zinc deficiency complications, such as chronic oxidative stress or inflammation.

Effect of Carbon Source on Properties and Bioactivities of Exopolysaccharide Produced by Trametes ochracea (Agaricomycetes).

The effects of carbon source on properties and bioactivities of exopolysaccharide (EPS) produced by Trametes ochracea were investigated in this study. The results indicated that EPS production varied with five different carbon sources. After a fermentation period of 8 days, sucrose was the most suitable carbon source for biomass and EPS production. The predominant carbohydrate compositions in EPSs identified were glucose and mannose. The EPS fermented by sucrose has the highest glucose content. Then, FT-IR spectral analysis revealed prominent characteristic groups in EPSs. Each particular EPS possessed the specific bands at 808-809 cm-1 and 914-922 cm-1, indicating both α- and ß-configurations of the sugar units. Furthermore, thermogravimetric analysis (TGA) indicated the EPS with sucrose and glucose as carbon source showed different degradation behavior compared with the other three EPSs. The variation also affects antioxidant and antihyperlipemia activities investigated using hydroxyl and DPPH radical scavenging assay, and in hyperlipemia mice. Sucrose was the best carbon source from the viewpoint of OH and DPPH radical scavenging activities, and antihyperlipemia activity, probably due to the relatively high glucose content in EPS.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J·cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Transcriptome analysis of three cultivars of Poria cocos reveals genes related to the biosynthesis of polysaccharides.

Poria cocos (P. cocos) polysaccharides (PCPs) are used to improve immunity and possess antitumor activities. We compared three cultivars of P. cocos (5.78, XJ 28 and JHYH) PCP contents. Then we determined that malZ, galA, SORD, gnl and bglX are key enzymes within the PCP biosynthetic pathway by using HiSeq2500 transcriptome and qRT-PCR validation. Our results provide more detailed information about the PCP biosynthesis pathway at the molecular level in P. cocos and establish the functions for the molecular breeding to produce polysaccharides in general for therapeutic use in Chinese medicinal plants.

Triterpenes and Soluble Polysaccharide Changes in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), During Fruiting Growth.

We analyzed the changes in triterpenes and soluble polysaccharides in Ganoderma lucidum strain G0119 during 4 growth phases in 3 regions of the fruiting bodies using reversed-phase high-performance liquid chromatography, and we also analyzed the soluble polysaccharides using high-performance size-exclusion chroma-tography-multiple-angle laser-light scattering refractive index analysis. The strong polar triterpenes decreased while weak polar triterpenes increased during the growth cycle of G. lucidum. The highest contents of ganoderic acid B, ganoderic acid A, and ganoderenic acid B were detected in the stipe during phase II, and ganoderic acid S, ganoderic acid T, and ganoderiol B peaked in the base during phase IV. The total content of soluble polysaccharides in samples decreased after the primordium developed into a fruiting body. Two high-molecular-weight fractions were detected in the soluble polysaccharide samples: α-l,4-glucan and ß-l,3-glucan, respectively. They were primarily distributed in the pileus during phase II, and both decreased after this phase. These results led us to select a more suitable growth phase and region for harvesting to obtain extracts with higher contents of triterpenes and soluble polysaccharides.

A novel exopolysaccharide elicitor from endophytic fungus Gilmaniella sp. AL12 on volatile oils accumulation in Atractylodes lancea.

Endophytes and plants can establish specific long-term symbiosis through the accumulation of secondary metabolites. Previous studies have shown that the endophytic fungus Gilmaniella sp. AL12 can stimulate Atractylodes lancea to produce volatile oils. The purpose of this report is to investigate key factors involved in the stimulation of A. lancea by AL12 and reveal the mechanism. We identified the active component from AL12 as an extracellular mannan with a polymerization degree of 26-42. Differential membrane proteomics of A. lancea was performed by 2D electrophoresis. The results showed that there were significant differences in the expression of 83 proteins. Based on these results, we conclude that AL12 secreted mannan contributes to the antagonistic balance seen in interactions between AL12 and A. lancea. One portion of the mannan was degraded to mannose for hexokinase activation, promoting photosynthesis and energy metabolism, with a potential metabolic fluxes flowing towards terpenoid biosynthesis. The other portion of the mannan directly enhanced autoimmunity of A. lancea through G protein-mediated signal transduction and the mannan-binding lectin pathway. Volatile oil accumulation was ultimately promoted in subsequent defense reactions. This study provides a new perspective on the regulation of secondary metabolites by endophytic fungal elicitors in medicinal plants.

Optimization of the Production of Extracellular Polysaccharide from the Shiitake Medicinal Mushroom Lentinus edodes (Agaricomycetes) Using Mutation and a Genetic Algorithm-Coupled Artificial Neural Network (GA-ANN).

Exopolysaccharide (EPS) production by a strain of Lentinus edodes was studied via the effects of treatments with ultraviolet (UV) irradiation and acridine orange. Furthermore, optimization of EPS production was studied using a genetic algorithm coupled with an artificial neural network in submerged fermentation. Exposure to irradiation and acridine orange resulted in improved EPS production (2.783 and 5.548 g/L, respectively) when compared with the wild strain (1.044 g/L), whereas optimization led to improved productivity (23.21 g/L). The EPS produced by various strains also demonstrated good DPPH scavenging activities of 45.40-88.90%, and also inhibited the growth of Escherichia coli and Klebsiella pneumoniae. This study shows that multistep optimization schemes involving physical-chemical mutation and media optimization can be an attractive strategy for improving the yield of bioactives from medicinal mushrooms. To the best of our knowledge, this report presents the first reference of a multistep approach to optimizing EPS production in L. edodes.

High-Level Production of Exopolysaccharides by a Cosmic Radiation-Induced Mutant M270 of the Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

A new Grifola frondosa mutant, M270, was successfully isolated for high production of exopolysaccharides (EPSs) using cosmic radiation-induced mutagenesis. We found that the mutant M270 had a clearer and thicker EPS layer (~10 µm) adhering to mycelia than those of its parent strain 265 after Congo red staining. In the 20-L batch fermentation for M270, 10.3 g/L of EPS and 17.9 g/L of dry mycelia biomass were obtained after 204 hours of fermentation. Furthermore, a main water-soluble fraction (EP1) in the EPS was purified from M270 and then confirmed to be heteroglycan-protein complex with 91% (w/w) total carbohydrates and 9% (w/w) total proteins. Four kinds of monosaccharide-D-mannose, D-glucosamine, D-glucose, and D-xylose-were detected in EP1 with a molar ratio of 17.6:1.8:100:2.5. The molecular mass of the main component in EP1 was 8.9 kDa. The EPS from M270 significantly inhibited the growth of sarcoma 180 solid tumors in mice. This G. frondosa M270 mutant could serve as a better candidate strain for polysaccharide production.

Application of residual polysaccharide-degrading enzymes in dried shiitake mushrooms as an enzyme preparation in food processing.

OBJECTIVE: To examine the activities of residual enzymes in dried shiitake mushrooms, which are a traditional foodstuff in Japanese cuisine, for possible applications in food processing. RESULTS: Polysaccharide-degrading enzymes remained intact in dried shiitake mushrooms and the activities of amylase, ß-glucosidase and pectinase were high. A potato digestion was tested using dried shiitake powder. The enzymes reacted with potato tuber specimens to solubilize sugars even under a heterogeneous solid-state condition and that their reaction modes were different at 38 and 50 °C. CONCLUSION: Dried shiitake mushrooms have a potential use in food processing as an enzyme preparation.

Uranium Binding Mechanisms of the Acid-Tolerant Fungus Coniochaeta fodinicola.

The uptake and binding of uranium [as (UO2)(2+)] by a moderately acidophilic fungus, Coniochaeta fodinicola, recently isolated from a uranium mine site, is examined in this work in order to better understand the potential impact of organisms such as this on uranium sequestration in hydrometallurgical systems. Our results show that the viability of the fungal biomass is critical to their capacity to remove uranium from solution. Indeed, live biomass (viable cells based on vital staining) were capable of removing ∼16 mg U/g dry weight in contrast with dead biomass (autoclaved) which removed ∼45 mg U/g dry weight after 2 h. Furthermore, the uranium binds with different strength, with a fraction ranging from ∼20-50% being easily leached from the exposed biomass by a 10 min acid wash. Results from X-ray absorption spectroscopy measurements show that the strength of uranium binding is strongly influenced by cell viability, with live cells showing a more well-ordered uranium bonding environment, while the distance to carbon or phosphorus second neighbors is similar in all samples. When coupled with time-resolved laser fluorescence and Fourier transformed infrared measurements, the importance of organic acids, phosphates, and polysaccharides, likely released with fungal cell death, appear to be the primary determinants of uranium binding in this system. These results provide an important progression to our understanding with regard to uranium sequestration in hydrometallurgical applications with implications to the unwanted retention of uranium in biofilms and/or its mobility in a remediation context.

Can fungal zoospores be the source of energy for the rumen protozoa Eudiplodinium maggii?

Results of our earlier studies showed the ability of ciliates Eudiplodinium maggii to digest and metabolize commercial chitin. The natural source of this polysaccharide in the rumen are fungi. The objectives of present research were to determine the effect of fungal zoospores on the survival and population density of E. maggii to quantify the concentration of chitin in the cells of protozoa and to examine the ability of E. maggii, to ferment chitin of fungal zoospores. The cultivation experiment showed that the survival of protozoa was shorter than 4 days when the culture medium was composed of buffer solution and lyophilized fungal spores. An enrichment of this medium with wheat gluten prolonged the survival of ciliates up to 8 days. The supplementation of the last medium with meadow hay enabled the protozoa to survive for 28 days but a positive effect was observed only during the last 8 days of experiment. The chitin content was 0.27 ng and 0.21-0.35 ng per single zoospore and ciliate, respectively. An increase in the concentration of volatile fatty acids (VFA) was found when protozoa were incubated with zoospores. The production rate of VFA was 46.3 pM/protozoan per h whereas the endogenous production did not exceed 31 pM/protozoan per h. The molar proportion of acetic acid was 77.7% and these of butyric and propionic acids-12.2 and 11.0%, respectively. The obtained results make it evident that carbohydrates present in fungal zoospores were utilized by protozoa in energy yielding processes.

Production of extracellular polysaccharides by the medicinal mushroom Trametes trogii (higher Basidiomycetes) in stirred-tank and airlift reactors.

Extracellular polysaccharides (EPSs) produced by submerged culture of Trametes trogii exhibit antioxidant and antitumor activities. In this study, mycelial growth and EPS production of T. trogii were investigated using optimal culture conditions (maltose [53.12 g/L] and polypeptone [4.21 g/L] in distilled water) in a 5-L jar fermenter. Maximum biomass growth (10.81 g/L) occurred after 5 days of cultivation, whereas maximal EPS yield (1.86 g/L) was achieved after 5 days in a 5-L stirred-tank reactor. Furthermore, the morphological parameters (i.e., mean diameter, circularity, roughness, and compactness) of the pellets and the viscosity of the broth were characterized. It was proved that the compactness of the pellets were significantly positively correlated with EPS content.

Polysaccharide production by submerged and solid-state cultures from several medicinal higher Basidiomycetes.

Polysaccharides produced by microorganisms represent an industrially unexploited market. An important number of polysaccharides have been isolated from fungi, especially mushrooms, with many interesting biological functions, such as antitumor, hypoglycemic, and immunostimulating activities. In the search of new sources of fungal polysaccharides, the main goal of this research was to test the ability of several species of basidiomycetes, among them various edible mushrooms, to produce both extracellular polysaccharides (EPSs) and intracellular polysaccharides (IPSs). Among 10 species screened for production of EPSs in submerged cultures with glucose, soy oil, and yeast extract, the best results were obtained with Ganoderma lucidum (0.79 g/L EPS) and Pleurotus ostreatus (0.75 g/L EPS). Agitation strongly improved EPS production in most of the studied strains. Eight of 10 species assayed successfully developed basidiomes during synthetic "bag-log" cultivation on a substrate consisting of oak sawdust and corn bran. This work describes for the first time the environmental factors required for fruiting of 4 species under such conditions: Schizophyllum commune, Ganoderma applanatum, Trametes versicolor, and T. trogii. IPSs were extracted from the carpophores. The IPS content of the carpophores varied from 1.4% (G. applanatum) up to 5.5% and 6% in G. lucidum and Grifola frondosa, respectively.

Production of biomass and polysaccharides of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), by submerged cultivation.

Submerged batch and repeated fed-batch cultivation techniques were used for mycelia cultivation and polysaccharide production of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum. Although most publications use various Asiatic G. lucidum strains, the growth of the strain Ga.l 4 (Biotechnical Faculty Strain Collection, Ljubljana, Slovenia), originally isolated from the Slovenian forest, is much faster. The results between the batch and repeated fed-batch cultivation are compared with the polysaccharide production in batch cultivation. From the aspect of biomass production, the best results were obtained in repeated fed-batch after 44 days, where 12.4 g/L of dry fungal biomass was obtained.

Trametes versicolor extract modifies human fecal microbiota composition in vitro.

Trametes versicolor is a mushroom used as a traditional Chinese medicine (Yun-zhi) for a wide array of seemingly disparate conditions. We hypothesized that many of its multiple purported activities could be mediated through stimulation of beneficial mutualist components of the microbiota. Human fecal microbiota was cultured anaerobically to determine its ability to ferment a common extract of T. versicolor, designated polysaccharide peptide (PSP), and the ability of PSP to alter the composition of the microbial community. The presence of PSP and fructooligosaccharides (FOS, a common prebiotic) in the medium, but not cellulose, significantly increased levels of Bifidobacterium spp. PSP also elevated Lactobacillus spp., while reducing Clostridium spp., Staphylococcus spp. and Enterococcus spp. Levels of Streptococcus spp., Bacteroides spp. and Escherichia did not significantly change. Fermentation of PSP increased the concentration of organic acids (lactate and short-chain fatty acids), decreased the pH, and induced ß-galactosidase and ß-glucosidase activities. The genera of the human microbiota that are promoted by FOS and other prebiotics are also stimulated by the Trametes versicolor extract, PSP. Thus, Trametes versicolor, a common East Asian botanical, contains putative prebiotic agents that alter human gut microbiota and pH. This prebiotic-like activity may help explain some of the plethora of the health benefits attributed to this traditional Chinese medicine.

Production, purfication, molecular characterization and bioactivities of exopolysaccharides produced by the wine cap culinary-medicinal mushroom, Stropharia rugosoannulata 2# (higher Basidiomycetes).

The optimal culture conditions of exopolysaccharides (EPS) production in submerged culture medium by Stropharia rugosoannulata 2# were determined using the orthogonal matrix method. The optimal defined medium (per liter) was 60.0 g sucrose, 6.0 g tryptone, 5 mM KH2PO4, and initial pH 7.0 at 28°C. In the optimal culture medium, the maximum EPS production was 9.967 g/L in shake-flask culture. One fraction of EPS was purified from the culture filtrates by size exclusion chromatography (SEC), and the molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The weight-average molar masses and the polydispersity ratio of the EPS fraction were determined to be 5.305 × 103 g/mol and 2.014, respectively. FTIR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. The obvious characteristic absorption at 884.3 cm-1 revealed the existence of ß configuration. Furthermore, the experiments in vitro indicated that S. rugosoannulata 2# EPS exhibit high antitumor and antioxidative effects.

Production of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), biomass and polysaccharides by solid state cultivation.

Solid state cultivation of Ganoderma lucidum biomass, strain BFWS Gal 4, originally isolated from the Slovenian forest, was studied in a horizontal stirred tank reactor. Periodic mixing of N = 80 rpm, 2 min/day was used. Production of fungal polysaccharides and fungal biomass on solid substrate based on beech sawdust, olive oil, and mineral salts was studied. Optimal moisture of the solid matrix was in the range of 80% to 74%. When the moisture content dropped below 57%, the growth of the mycelium and polysaccharide production stopped, but it revived when wet air was applied in further processing. Final concentration of biomass was 0.68 mg/g of solid substrate, while proportions of extracellular and intracellular polysaccharides were 4.5 mg/g and 1.05 mg/g, respectively.