Optimization of Ganoderma lucidum Polysaccharides Fermentation Process for Large-Scale Production.

The objective of this study was to increase the intracellular polysaccharide yield of Ganoderma lucidum. The accordingly optimized fermentation medium by central composite design method contains glucose 40 g L-1, yeast powder 12 g L-1, potassium dihydrogen phosphate 3 g L-1, initial pH 5.5, and inoculum size 10 mL 100 mL-1. Under this condition, the predicted value of intracellular polysaccharide yield was 2.03 g L-1. Shake flask experiments confirmed that the average intracellular polysaccharide yield was 1.98 g L-1 similar to the predicted value. The yields of intracellular polysaccharides in the 5-L and 50-L fermentors were 2.59 g L-1 and 2.65 g L-1, respectively. The molecular weight distribution of intracellular and extracellular polysaccharides obtained was determined by HPSEC-MALLS-RI. The results showed that the weight-average molecular weight of component 1 in the intracellular crude polysaccharide was 4.695 × 106 Da and the mass fraction was 58%. The weight-average molecular weight of component 2 in the extracellular polysaccharide was 5.554 × 104 Da. The mass fraction was 94.9%. The liquid submerged fermentation process of G. lucidum mycelium obtained from this study has effectively increased the yield of intracellular polysaccharides. Its intracellular and extracellular polysaccharides have good immunological activity. Conceivably, the optimized process can be applied for the large-scale production.

Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 µm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Delimitation of some neotropical laccate Ganoderma (Ganodermataceae): molecular phylogeny and morphology.

Ganoderma includes species of great economic and ecological importance, but taxonomists judge the current nomenclatural situation as chaotic and poorly studied in the neotropics. From this perspective, phylogenetic analyses inferred from ribosomal DNA sequences have aided the clarification of the genus status. In this study, 14 specimens of Ganoderma and two of Tomophagus collected in Brazil were used for DNA extraction, amplification and sequencing of the ITS and LSU regions (rDNA). The phylogenetic delimitation of six neotropical taxa (G. chalceum, G. multiplicatum, G. orbiforme, G. parvulum, G. aff. oerstedtii and Tomophagus colossus) was determined based on these Brazilian specimens and found to be distinct from the laccate Ganoderma from Asia, Europe, North America and from some specimens from Argentina. Phylogenetic reconstructions confirmed that the laccate Ganoderna is distinct from Tomophagus, although they belong to the same group. The use of taxonomic synonyms Ganoderma subamboinense for G. multiplicatumnz, G. boninense for G. orbiforme and G. chalceum for G. cupreum was not confirmed. However, Ganoderma parvulum was confirmed as the correct name for specimens called G. stipitatu. Furthermore, the name G. hucidumn should be used only for European species. The use of valid published names is proposed according to the specimen geographical distribution, their morphological characteristics and rDNA analysis. 1208. Epub 2014 September 01.

Mycelial growth rate and macro- and micromorphological characteristics of medicinal species of genus Ganoderma (higher Basidiomycetes) from Iran.

Mycelial growth rate is a distinguishing quality that demonstrates continuous variation in different isolates collected from various hosts and locations. The objectives of this research were (1) to reinvestigate the previous identification of Iranian species, and (2) to recognize the best native isolate(s) for cultivation of different Ganoderma species. Of 78 samples collected from different hosts and sites, only 43 mycelia could be purified and examined for further study. Growth rate (GR; Δd/Δt) and growth coefficient (GC; dgh/t) were analyzed by growing isolate culture on 2% malt-extract agar medium (pH 5.5) incubated at 25°C. Macro- and micromorphological studies on mycelia and fruiting bodies such as basidiospore and cutis microcharacters as well as fruiting body quality were used for precise identification. Results revealed that samples belonged to 4 species: G. lucidum, G. applanatum, G. resinaceum, and G. australe. Among all samples, the isolate morphologically identified as G. applanatum showed the best GR (12 mm/day) and good GC (128 mm/day), followed by the 2 other isolates identified as G. resinaceum (GRs and GCs of 11 and 55 mm/day and 10.9 and 43.6 mm/day, respectively).

Delimitation of some neotropical laccate Ganoderma (Ganodermataceae): molecular phylogeny and morphology / Delimitación de algunos Ganoderma (Ganodermataceae) lacados neotropicales: filogenia molecular y morfología

Ganoderma includes species of great economic and ecological importance, but taxonomists judge the current nomenclatural situation as chaotic and poorly studied in the neotropics. From this perspective, phylogenetic analyses inferred from ribosomal DNA sequences have aided the clarification of the genus status. In this study, 14 specimens of Ganoderma and two of Tomophagus collected in Brazil were used for DNA extraction, amplification and sequencing of the ITS and LSU regions (rDNA). The phylogenetic delimitation of six neotropical taxa (G. chalceum, G. multiplicatum, G. orbiforme, G. parvulum, G. aff. oerstedtii and Tomophagus colossus) was determined based on these Brazilian specimens and found to be distinct from the laccate Ganoderma from Asia, Europe, North America and from some specimens from Argentina. Phylogenetic reconstructions confirmed that the laccate Ganoderma is distinct from Tomophagus, although they belong to the same group. The use of taxonomic synonyms Ganoderma subamboinense for G. multiplicatum, G. boninense for G. orbiforme and G. chalceum for G. cupreum was not confirmed. However, Ganoderma parvulum was confirmed as the correct name for specimens called G. stipitatum. Furthermore, the name G. lucidum should be used only for European species. The use of valid published names is proposed according to the specimen geographical distribution, their morphological characteristics and rDNA analysis. Rev. Biol. Trop. 62 (3): 1197-1208. Epub 2014 September 01.

Ganoderma incluye especies de gran importancia económica y ecológica, sin embargo, su nomenclatura actual es caótica y poco estudiada en el neotrópico. En este estudio se utilizaron 14 muestras de Ganoderma y dos de Tomophagus recolectados en Brasil para la extracción de ADN, amplificación y secuenciación de las regiones ITS y LSU. La delimitación filogenética de seis táxones neotropicales fue discutida con base en especímenes brasileños y secuencias del GenBank. Estas especies mostraron ser distintas de los Ganoderma lacados de Asia, Europa, América del Norte y de algunos ejemplares de Argentina. Las reconstrucciones filogenéticas confirman que los Ganoderma lacados son distintos de Tomophagus, aunque pertenecen al mismo grupo. No se confirman los sinónimos de G. subamboinense a G. multiplicatum, de G. boninense a G. orbiforme y G. chalceum a G. cupreum. G. parvulum se confirma como el nombre correcto para G. stipitatum. G. lucidum sólo se debe utilizar para especies europeas. Por lo tanto, se propone el uso de nombres publicados válidamente de acuerdo con la distribución geográfica de las muestras, características morfológicas y análisis de ADNr.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 µg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 µg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process.

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.