Evaluation of ergosterol composition and esterification rate in fungi isolated from mangrove soil, long-term storage of broken spores, and two soils.

Ergosterol is an important fungal-specific biomarker, but its use for fungal biomass estimation is still varied. It is important to distinguish between free and esterified ergosterols, which are mainly located on the plasma membrane and the cytosolic lipid particles, respectively. The present study analyzes free and esterified ergosterol contents in: (1) the fifty-nine strains of culturable fungi isolated from mangrove soil, (2) the broken spores of the fungus Ganoderma lucidum stored in capsule for more than 12 years, and (3) the mangrove soil and nearby campus wood soil samples by high performance liquid chromatography (HPLC). The results show that the contents of free and esterified ergosterols varied greatly in fifty-nine strains of fungi after 5 days of growth, indicating the diversity of ergosterol composition in fungi. The average contents of free and total ergosterols from the fifty-nine strains of fungi are 4.4 ± 1.5 mg/g and 6.1 ± 1.9 mg/g dry mycelia, respectively, with an average ergosterol esterification rate of 27.4%. The present study suggests that the fungi might be divided into two classes, one is fungi with high esterification rates (e.g., more than 27%) such as Nectria spp. and Fusarium spp., and the other is fungi with low esterification rates (e.g., less than 27%) such as Penicillium spp. and Trichoderma spp. Moreover, the ergosterol esterification rate in the spores of G. lucidum is 91.4% with a very small amount of free ergosterol (0.015 mg/g), compared with 41.9% with a higher level of free ergosterol (0.499 mg/g) reported in our previous study in 2007, indicating that free ergosterol degrades more rapidly than esterified ergosterol. In addition, the ergosterol esterification rates in mangrove soil and nearby campus wood soil samples range from 0 to 39.0%, compared with 80% in an old soil organic matter reported in a previous study, indicating the potential relationship between aging degree of fungi or soil and esterification rate. The present study proposes that both free and esterified ergosterols should be analyzed for fungal biomass estimation. When the ergosterol esterification rates in soils are higher, free ergosterol might be a better marker for fungal biomass. It is speculated that the ergosterol esterification rate in soils might contain some important information, such as the age of old-growth forests over time scales of centuries to millennia, besides the senescence degree of fungal mycelia in soils. KEY POINTS: • Fungi might be divided into two classes depending on ergosterol esterification rates. • Ergosterol esterification rate of broken spores stored for long time raised evidently. • Both free and esterified ergosterols should be analyzed for fungal biomass estimate. • Free ergosterol is a better marker for fungal biomass with a high esterification rate.

Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with HPLC and Artificial Neural Network Analysis for Ganoderma lucidum.

Ganoderma lucidum is widely used in traditional Chinese medicine (TCM). Ganoderic acid A and D are the main bioactive components with anticancer effects in G. lucidum. To obtain the maximum content of two compounds from G. lucidum, a novel extraction method, an ionic liquid-based ultrasonic-assisted method (ILUAE) was established. Ionic liquids (ILs) of different types and parameters, including the concentration of ILs, ultrasonic power, ultrasonic time, rotational speed, solid-liquid ratio, were optimized by the orthogonal experiment and variance analysis. Under these optimal conditions, the total extraction yield of the two compounds in G. lucidum was 3.31 mg/g, which is 36.21% higher than that of the traditional solvent extraction method. Subsequently, an artificial neural network (ANN) was developed to model the performance of the total extraction yield. The Levenberg-Marquardt back propagation algorithm with the sigmoid transfer function (logsig) at the hidden layer and a linear transfer function (purelin) at the output layer were used. Results showed that single hidden layer with 9 neurons presented the best values for the mean squared error (MSE) and the correlation coefficient (R), with respectively corresponding values of 0.09622 and 0.93332.

Penalized discriminant analysis for the detection of wild-grown and cultivated Ganoderma lucidum using Fourier transform infrared spectroscopy.

An effective and simple analytical method using Fourier transform infrared (FTIR) spectroscopy to distinguish wild-grown high-quality Ganoderma lucidum (G. lucidum) from cultivated one is of essential importance for its quality assurance and medicinal value estimation. Commonly used chemical and analytical methods using full spectrum are not so effective for the detection and interpretation due to the complex system of the herbal medicine. In this study, two penalized discriminant analysis models, penalized linear discriminant analysis (PLDA) and elastic net (Elnet),using FTIR spectroscopy have been explored for the purpose of discrimination and interpretation. The classification performances of the two penalized models have been compared with two widely used multivariate methods, principal component discriminant analysis (PCDA) and partial least squares discriminant analysis (PLSDA). The Elnet model involving a combination of L1 and L2 norm penalties enabled an automatic selection of a small number of informative spectral absorption bands and gave an excellent classification accuracy of 99% for discrimination between spectra of wild-grown and cultivated G. lucidum. Its classification performance was superior to that of the PLDA model in a pure L1 setting and outperformed the PCDA and PLSDA models using full wavelength. The well-performed selection of informative spectral features leads to substantial reduction in model complexity and improvement of classification accuracy, and it is particularly helpful for the quantitative interpretations of the major chemical constituents of G. lucidum regarding its anti-cancer effects.

Wound healing activity of an aqueous extract of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

The Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes) is popular because of its health-promoting properties. The effects of G. lucidum extract on cancer, hypertension, hypercholesterolemia, and hepatitis have been reported by many researchers. This investigation was undertaken to evaluate the healing efficacy of an aqueous lyophilized extract of G. lucidum from the Indian Himalayan region on dermal excision wound in experimental rats. The extract used in the study was found to be rich in total polyphenol and flavonoid contents. The healing efficacy was comparatively assessed with a reference povidone-iodine ointment. The G. lucidum extract showed significant enhanced healing activity, evidenced by an increase in wound contraction, collagen accumulation (hydroxyproline), hexosamine, and total protein contents. Histopathological findings further supported the biochemical indices. The results suggest that aqueous lyophilized extract of G. lucidum possesses significant wound-healing activity.

Syntheses of new rare earth complexes with carboxymethylated polysaccharides and evaluation of their in vitro antifungal activities.

In the present paper, La, Eu and Yb were selected to represent light, middle and heavy rare earths to form complexes with polysaccharides through chelating coordination of carboxyl groups, which were added into polysaccharide chains by means of carboxymethylation. Their antifungal activities against plant pathogenic fungi were evaluated using growth rate method. These rare earth complexes exhibited various antifungal activities against the tested fungi, depending on rare earth elements, polysaccharide types and fungal species. Among these three metal elements (i.e. La, Eu and Yb), Yb formed the complexes with the most effective antifungal properties. Furthermore, the results showed that ligands of carboxymethylated polysaccharides played a key role in promoting cytotoxicity of the rare earth complexes. Carboxymethylated Ganoderma applanatum polysaccharide (CGAP) was found to be the most effective ligand to form complexes with antifungal activities, followed by carboxymethylated lentinan (CLNT) and carboxymethylated Momordica charantia polysaccharide (CMCP).

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

Optimization of exopolysaccharides production from a novel strain of Ganoderma lucidum CAU5501 in submerged culture

This study aimed at optimizing the medium of a new Ganoderma lucidum strain CAU5501 to enhance the yield of exopolysaccharides (EPS) and mycelial growth. Firstly, the suitable level of glucose, magnesium, phosphate and C/N ratio was determined by single factor experiment. Subsequently, the optimum concentrations of these medium components were investigated using the orthogonal matrix method. The results indicated that the higher levels of EPS were correlated with the level of cell growth when glucose concentration was studied (data no show). The optimum medium for EPS yield was found to be 70 g/l glucose, 5 C/N ratio, 2.5 g/l KH2PO4, 0.75 g/l MgSO4·7H2O, and for mycelial growth was 50 g/l glucose, 5 C/N ratio, 1.5 g/l KH2PO4, 0.5 g/l MgSO4·7H2O. When cultivated in the obtained optimal media in 3 L shake flask, compared to the basal medium, the EPS yield increased markedly from 1.003 to 1.723 g/l, and the mycelium formation was also markedly improved from 2.028 to 7.235 g/l. Results obtained in this study are beneficial to further study for enhancing the production of Ganoderma lucidum polysaccharides in large scale commercialized production.

Efecto de nutrientes sobre la producción de biomasa del hongo medicinal Ganoderma lucidum / Effect of nutrients in the biomass production of the medicinal mushroom Ganoderma lucidum

El hongo Ganoderma lucidum, en los constituyentes de su biomasa, tiene compuestos con propiedades benéficas para la salud; es por esto que el conocimiento de las condiciones nutricionales adecuadas para su crecimiento permitirá su producción industrial y a bajo costo. En este trabajo se evaluó a nivel de matraz el efecto de la relación C/N, y la presencia de diferentes fuentes de carbono, nitrógeno y micronutrientes sobre la producción de biomasa. Empleando glucosa y peptona como fuentes de carbono y nitrógeno, respectivamente, se encontró una relación C/N óptima de 16,7:1 para la cual la máxima producción de biomasa fue de 25 g/L. Manteniendo esta relación C/N, y sustituyendo la glucosa por lactosa o harina de cebada y la peptona por extracto de levadura, la producción de biomasa se incrementó a 35 g/L. En presencia de harina de cebada la adición al medio de cultivo de sales de Mg y K, y de tiamina, no generó un mayor incremento en la producción de biomasa. La producción de biomasa de G. lucidum se ve favorecida por la presencia en el medio de cultivo de relaciones C/N cercanas a las reportadas conforme a la composición típica de los hongos, así como por la presencia de sustratos complejos como la harina de cebada que le aportan además de la fuente de carbono micronutrientes necesarios para su desarrollo.

Ganoderma lucidum fungus has some biomass components with beneficial health properties. The knowledge about its nutritionals requirements for growing will favor its industrial production at lower cost. In this work, the effect of C/N ratio, the presence of different carbon, nitrogen and micronutrients sources, on fungal biomass production, were evaluated. Using glucose and peptone as carbon and nitrogen sources, respectively, an optimal C/N ratio of 16,7:1 was found, for which the maximal biomass production was 25 g/L. Replacing glucose by lactose or barley flour and peptone by yeast extract at the same C/N ratio, the biomass production was enhanced to 35 g/L. With barley flour in the culture medium, the presence of Mg and K salts and thiamine did not turn out into a major increase of biomass. The G. lucidum biomass production is promoted by C/N ratios in the culture medium nearly equivalent to that found in the fungus, as well as the presence of complex substrates as barley flour which, additionally, contributes with important micronutrients along with the carbon source.

Quality control and original discrimination of Ganoderma lucidum based on high-performance liquid chromatographic fingerprints and combined chemometrics methods.

In this paper, the feasibility and advantages of employing high-performance liquid chromatographic (HPLC) fingerprints combined with chemometrics methods for quality control of the cultured fruiting bodies of Ganoderma lucidum were investigated and demonstrated for the first time. In order to compare the HPLC fingerprints chromatograms between G. lucidum from different origins, the similarities of all the 60 samples and relative peak areas of 19 characteristic compounds were firstly calculated respectively. Then different pattern recognition procedures, including hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA) and soft independent modeling of class analogy (SIMCA) were applied to classify the G. lucidum samples according to their cultivated origins. Consistent results were obtained to show that G. lucidum samples could be successfully grouped in accordance with the province of origin. Furthermore, four marker constituents were screened out to be the most discriminant variables, which could be applied to accurate discrimination and quality control of G. lucidum by quantitative analysis. Finally, the chemical properties of those samples were also investigated to find out the differences of quality between them. Ranked in decreasing order, the quality of the G. lucidum can be arranged as Jinzhai/Huangshan, Shandong followed by Zhejiang samples. Our results revealed that the developed method has potential perspective for the original discrimination and quality control of G. lucidum.

Discrimination of Ganoderma lucidum according to geographical origin with near infrared diffuse reflectance spectroscopy and pattern recognition techniques.

A rapid and nondestructive near infrared (NIR) method combined with chemometrics was used to discriminate Ganoderma lucidum according to cultivation area. Raw, first, and second derivative NIR spectra were compared to develop a robust classification rule. The chemical properties of G. lucidum samples were also investigated to find out the difference between samples from six varied origins. It could be found that the amount of polysaccharides and triterpenoid saponins in G. lucidum samples was considerably different based on cultivation area. These differences make NIR spectroscopic method viable. Principal component analysis (PCA), discriminant partial least-squares (DPLS) and discriminant analysis (DA) were applied to classify the geographical origins of those samples. The results showed that excellent classification could be obtained after optimizing spectral pre-treatment. For the discriminating of samples from three different provinces, DPLS provided 100% correct classifications. Moreover, for samples from six different locations, the correct classifications of the calibration as well as the validation data set were 96.6% using the DA method after the SNV first derivative spectral pre-treatment. Overall, NIR diffuse reflectance spectroscopy using pattern recognition was shown to have significant potential as a rapid and accurate method for the identification of herbal medicines.

Ganoderma lucidum: a cause of pseudoparasitosis.

We report a pseudoparasitosis case due to Ganoderma lucidum, (lingzhi or reishi mushroom); we believe this to be a first reported case in Thailand. A 49-year-old male patient with non-Hodgkins lymphoma presented with chronic watery diarrhea. He had a history of consumption of powdered lingzhi extract as a dietary supplement and herbal medicine. Stool examination demonstrated many spores of G. lucidum, which must be differentiated from intestinal helminth ova and coccidia. After discontinuation of mushroom spores ingestion, the diarrheal symptoms improved and fecal examination subsequently showed no Ganoderma spores. Many artifacts in the stool may be confused with parasites. Differentiation of parasites from artifacts depends on characterization of the size, shape, structure, and reactivity with common stains.

Possible mode of action of antiherpetic activities of a proteoglycan isolated from the mycelia of Ganoderma lucidum in vitro.

A bioactive fraction (GLPG) was extracted and purified from the mycelia of Ganoderma lucidum by EtOH precipitation and DEAE-cellulose column chromatography. GLPG was a proteoglycan and had a carbohydrate:protein ratio of 10.4:1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated by the cytopathic effect (CPE) inhibition assay in cell culture. This kind of polysaccharide inhibited the development of the cytopathic effect in dose-dependent manner in HSV-infected cells, moreover did not show any cytotoxic effects on cells even when a concentration was as high as 2000 microg/ml. In order to study the possible mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during and after infection, and the viral titers in the supernatant of cell culture 48 h post-infection were tested by TCID(50) assay. The antiviral effects in pre-treated and treated during virus infection with GLPG were more remarkable than the treatment of post-infection. Although the precise mechanism has yet to be defined, our work suggested that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan seems to be a potential candidate for anti-HSV agents.