Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method.

The CRISPR/Cas9 system has become a popular approach to genome editing. Compared with the plasmid-dependent CRISPR system, the ribonucleoprotein (RNP) complex formed by the in vitro assembly of Cas9 and single-guide RNA (sgRNA) has many advantages. However, only a few examples have been reported and the editing efficiency has been relatively low. In this study, we developed and optimized an RNP-mediated CRISPR/Cas9 genome editing system for the monokaryotic strain L1 from the Ganoderma lucidum cultivar 'Hunong No. 1'. On selective media containing 5-fluoroorotic acid (5-FOA), the targeting efficiency of the genomic editing reached 100%. The editing efficiency of the orotidine 5'-monophosphate decarboxylase gene (ura3) was greater than 35 mutants/107 protoplasts, surpassing the previously reported G. lucidum CRISPR systems. Through insertion or substitution, 35 mutants introduced new sequences of 10-569 bp near the cleavage site of ura3 in the L1 genome, and the introduced sequences of 22 mutants (62.9%) were derived from the L1 genome itself. Among the 90 mutants, 85 mutants (94.4%) repaired DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), and five mutants (5.6%) through microhomology-mediated end joining (MMEJ). This study revealed the repair characteristics of DSBs induced by RNA-programmed nuclease Cas9. Moreover, the G. lucidum genes cyp512a3 and cyp5359n1 have been edited using this system. This study is of significant importance for the targeted breeding and synthetic metabolic regulation of G. lucidum.

Polysaccharide extracted from cultivated Sanghuangporous vaninii spores using three-phase partitioning with enzyme/ultrasound pretreatment: Physicochemical characteristics and its biological activity in vitro.

Sanghuangporous vaninii, as a valuable dietary supplement and medicinal ingredient, contains abundant bioactive polysaccharides that have health-promoting effects. In the present study, four polysaccharides (SVSPs-C, SVSPs-E, SVSPs-U, and SVSPs-E/U) were extracted for the first time from S. vaninii spores by three-phase partitioning (TPP), enzyme pretreatment before TPP (E-TPP), ultrasonic pretreatment before TPP (U-TPP), and enzyme pretreatment followed by ultrasonic before TPP (E/U-TPP) methods, respectively. Their physicochemical characteristics and in vitro pharmacological functions were determined and compared. Results showed that four TPP-based extraction methods had remarkable impacts on the extraction yield, chemical properties, monosaccharide compositions, and molecular weights (Mw) of SVSPs. Specifically, SVSPs-E/U obtained by E/U-TPP showed the highest extraction yield (25.40 %), carbohydrate content (88.50 %), and the lowest protein content (0.86 %). The four SVSPs had high-Mw (183.8-329.1 kDa) and low-Mw (23.0-156.4 kDa) fractions and mainly consisted of galactose, glucose, and mannose with different contents. In vitro bioactivities assays indicated that SVSPs-E/U possessed stronger antioxidant, hypoglycemic, hypouricemic, immunostimulatory, and antitumor activities than those of SVSPs-C, SVSPs-E, and SVSPs-U. Therefore, our results provide an efficient and promising extraction technique for bioactive polysaccharides from S. vaninii spores, as well as SVSPs had the potential to be applied in functional food, pharmaceutical, and cosmetics fields.

Recent Advances in the Preparation, Structure, and Biological Activities of ß-Glucan from Ganoderma Species: A Review.

Ganoderma has served as a valuable food supplement and medicinal ingredient with outstanding active compounds that are essential for human protection against chronic diseases. Modern pharmacology studies have proven that Ganoderma ß-d-glucan exhibits versatile biological activities, such as immunomodulatory, antitumor, antioxidant, and antiviral properties, as well as gut microbiota regulation. As a promising polysaccharide, ß-d-glucan is widely used in the prevention and treatment of various diseases. In recent years, the extraction, purification, structural characterization, and pharmacological activities of polysaccharides from the fruiting bodies, mycelia, spores, and fermentation broth of Ganoderma species have received wide attention from scholars globally. Unfortunately, comprehensive studies on the preparation, structure and bioactivity, toxicology, and utilization of ß-d-glucans from Ganoderma species still need to be further explored, which may result in limitations in future sustainable industrial applications of ß-d-glucans. Thus, this review summarizes the research progress in recent years on the physicochemical properties, structural characteristics, and bioactivity mechanisms of Ganoderma ß-d-glucan, as well as its toxicological assessment and applications. This review is intended to provide a theoretical basis and reference for the development and application of ß-d-glucan in the fields of pharmaceuticals, functional foods, and cosmetics.

Chemical Components of Volatile Oil Extracted from the Fermentation Broth of Ganoderma lingzhi (Agaricomycetes) Coupled with Its Antitumor and Antioxidant Activities In Vitro.

Volatile oil extracted from fermentation broth of Ganoderma lingzhi by hydrodistillation was analyzed based on gas chromatography-mass spectrometry (GC-MS). Its antitumor activity was tested on K562, SW620, A549, HepG2 cells in vitro. In addition, the antioxidant activity of the oil was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. In total, 16 constituents were identified accounting for about 99.99% of the total volatile oil in the fermentation broth of G. lingzhi. Among these components, 1-propanol (33.33%), phenylacetaldehyde (24.24%), 2-hexyl-1-decanol (12.12%) were found to be the major constituents. The antitumor results showed that the IC50 of the inhibition to the proliferation of K562, SW620, A549, HepG2 cells were 32.2, 78.9, 96.4, 99.0 µg/mL, respectively. And the oil could inhibit the proliferation of K562 cells by apoptosis induction and cell cycle arrest at S phase. Moreover, the oil exhibited radical scavenging activity (IC50 = 0.1469 mg/mL) in DPPH assay.

Multiple Fingerprint-Activity Relationship Assessment of Immunomodulatory Polysaccharides from Ganoderma lucidum Based on Chemometric Methods.

Polysaccharides with molecular weights ranging from 1.75 × 103 to 1.14 × 104 g/mol were obtained from the fruit bodies of Ganoderma lucidum. The multiple fingerprints and macrophage immunostimulatory activity of these fractions were analyzed as well as the fingerprint-activity relationship. The correlation analysis of molecular weight and immune activity demonstrated that polysaccharides with molecular weights of 4.27 × 103~5.27 × 103 and 1 × 104~1.14 × 104 g/mol were the main active fractions. Moreover, the results showed that galactose, mannose, and glucuronic acid were positively related to immunostimulatory activity. Additionally, partial least-squares regression and grey correlation degree analyses indicated that three peaks (P2, P3, P8) in the oligosaccharide fragment fingerprint significantly affected the immune activity of the polysaccharides. Hence, these ingredients associated with activity could be considered as markers to assess Ganoderma lucidum polysaccharides and their related products, and the study also provides a reference for research on the spectrum-effect relationship of polysaccharides in the future.

The key technologies of Ganoderma lucidum liquid spawn preparation and scale expansion.

Ganoderma lucidum possesses a variety of valuable pharmacological activities, and it has long been used to prevent and treat various human diseases. Up to now, far too little attention has been paid to the liquid spawn of G. lucidum, and the development of the G. lucidum industry is constrained by them. This work aimed to study the key technologies and scale-up preparation of G. lucidum liquid spawn, to achieve large-scale preparation of liquid spawn and solve the problem of unstable quality of G. lucidum. The plate culture, primary shake flask culture, shake flask preparation, and fermentor preparation of G. lucidum liquid spawn were explored in the process of liquid fermentation. The results showed that plate broth volume significantly affected mycelial growth rate. Biomass in the primary shake flask culture is significantly influenced by the picking position of plate mycelium. An artificial neural network coupled with a genetic algorithm was used for carbon and nitrogen sources concentration optimization to increase biomass and substrate utilization. The optimized parameter combination is as follows: glucose, 14.5 g L-1; yeast extract powder, 8.5 g L-1. Under this condition, the biomass (9.82 g L-1) and biomass on reducing sugar (0.79 g g-1) increased by 18.03% and 27.41% compared to the control, respectively. The metabolic activity of liquid spawn prepared by different fermentation scales was diverse, and the liquid spawn prepared by the fermentor has better activity. Conceivably, the liquid spawn process can more conducive be applied to large-scale industrial production.

A Novel Strain Breeding of Ganoderma lucidum UV119 (Agaricomycetes) with High Spores Yield and Strong Resistant Ability to Other Microbes' Invasions.

The spore powder of Ganoderma lucidum (G. lucidum) has been proven to have a variety of pharmacological activities, and it has become a new resource for the development of health products and pharmaceuticals. However, the scarcity of natural resources, strict growth conditions and difficulty in controlling the stable yield, and quality of different culture batches seriously limit the development and utilization of G. lucidum spore powder. In the present study, the strain with the highest spore powder yield, G0109, was selected as the original strain to generate mutants of G. lucidum using ultraviolet ray irradiation. A total of 165 mutagenic strains were obtained, and fifty-five strains were chosen for the cultivation test. Importantly, one mutagenic strain with high spore powder yield and strong resistance to undesired microorganisms was acquired and named strain UV119. More cultivations demonstrated that the fruiting body and basidiospore yields from UV119 were, respectively, 8.67% and 19.27% higher than those of the parent (G0109), and the basidiospore yield was 20.56% higher than that of the current main cultivar "Longzhi No.1". In conclusion, this study suggested that ultraviolet ray irradiation is an efficient and practical method for Ganoderma strain improvement and thus provided a basis for the development and application of G. lucidum spore production and outstanding contributions to the rapid development of the G. lucidum industry.

Effects of molecular weight on intestinal anti-inflammatory activities of ß-D-glucan from Ganoderma lucidum.

To investigate the influence of molecular weight (M w) on the anti-inflammatory activity of ß-D-glucan from Ganoderma lucidum, ultrasonic irradiation was applied to treat the ß-D-glucan (GLP, 2.42 × 106 g/mol) solution to obtain two degraded fractions with molecular weight of 6.53 × 105 g/mol (GLPC) and 3.49 × 104 g/mol (GLPN). Structural analysis proved that the degraded fractions possessed similar repeated units with the original ß-D-glucan. The in vitro anti-inflammatory activity studies showed that all fractions could significantly inhibit LPS-induced expression of cytokines including TNF-α, IL-8, MIF and MCP-1 in Caco-2 cells at certain concentrations. Moreover, GLPC and GLPN exhibited better anti-inflammatory activity than GLPC. The intestinal anti-inflammatory activity evaluated by dextran sulfate sodium (DSS)-induced colitis mice model showed that intragastric administration of GLPN (lower M w fraction) could significantly recover inflamed tissues of mice. Compared with GLP and GLPC, GLPN exhibited stronger ability to inhibit the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). The results revealed that M w of ß-D-glucan influenced its anti-inflammatory activity and decreasing of M w would improve the activity, which provided evidence for the potential use of ß-D-glucan from G. lucidum as anti-colitis ingredients.

Effects of Oleic Acid Addition Methods on the Metabolic Flux Distribution of Ganoderic Acids R, S and T's Biosynthesis.

The effects of oleic acid addition methods on the metabolic flux distribution of ganoderic acids R, S and T's biosynthesis from Ganoderma lucidum were investigated. The results showed that adding filter-sterilized oleic acid in the process of submerged fermentation and static culture is of benefit to the synthesis of ganoderic acids R, S and T. The metabolic fluxes were increased by 97.48%, 78.42% and 43.39%, respectively. The content of ganoderic acids R, S and T were 3.11 times, 5.19 times and 1.44 times higher, respectively, than they were in the control group, which was without additional oleic acid. Ganoderic acids R, S and T's synthesis pathways (GAP), tricarboxylic acid cycles (TCA), pentose phosphate pathways (PP) and glycolysis pathways (EMP) were all enhanced in the process. Therefore, additional oleic acid can strengthen the overall metabolic flux distribution of G. lucidum in a submerged fermentation-static culture and it can reduce the accumulation of the by-product mycosterol. This study has laid an important foundation for improving the production of triterpenes in the submerged fermentation of G. lucidum.

Integrative Analysis of Selected Metabolites and the Fungal Transcriptome during the Developmental Cycle of Ganoderma lucidum Strain G0119 Correlates Lignocellulose Degradation with Carbohydrate and Triterpenoid Metabolism.

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum. The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum.

Development and Optimization of the Triterpenoid and Sterol Production Process with Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum Strain G0017 (Agaricomycetes), in Liquid Submerged Fermentation at Large Scale.

Ganoderma lucidum mycelia are rich in active substances such as triterpenoids and sterols. However, reports on the development of effective submerged fermentation processes are lacking and the resulting total triterpene and sterol yield is still quite low. In this study, a new G. lucidum strain G0017 mycelium isolated by screening was studied in a 3-L fermenter to investigate the effect of aeration rate in liquid submerged fermentation production of triterpenoids and sterols. By fitting the specific mycelial growth rate and the specific production rate of the triterpenoid and sterol model, an effective multistage aeration rate control process for triterpenoid and sterol fermentation production was developed. This process was validated and proven in 3-L and 50-L fermenters. The resulting yields of triterpenoids and sterols were 3.34 and 3.46 g/L, respectively, which were 69.54% and 75.63% higher than the fixed aeration rate of 1.50 volume of air per volume of liquid per minute. This optimized fermentation production process conceivably could be applied to larger-scale industrial production and perhaps also to improve liquid submerged fermentation processes with relevant edible and medicinal mushrooms.

Regioselective sulfation of ß-glucan from Ganoderma lucidum and structure-anticoagulant activity relationship of sulfated derivatives.

In the present study, regioselective sulfation of ß-glucan (GLP) from Ganoderma lucidum were firstly established by using 4,4'-dimethoxytrityl chloride and hexamethyldisilazane as protecting precursor. 2,4,6-O-sulfated, 6-O-sulfated and 2,4-O-sulfated GLP derivatives were prepared and the molecular weights (Mw) of derivatives were determined to range from 0.94 × 104 to 6.27 × 104 g/mol, while the degrees of sulfation (DS) were calculated to vary from 0.83 to 1.74. The regioselective sulfation of GLP was confirmed by FT-IR, 13C NMR spectroscopy and methylation analysis. Results indicated that the sulfated substitution sites were predominantly at C-6 in 6-O-sulfated GLP (S6-OGLP) and C-4 in 2,4-O-sulfated GLP (S2,4-OGLP), respectively. Clotting assays (APTT, PT and TT) in vitro showed that sulfate groups were essential for anticoagulant activity and S6-OGLP exhibited much higher than others. Meanwhile, sulfated GLP with higher DS and Mw showed stronger anticoagulant activity in the case of the same condition.

A Review on Linking the Medicinal Functions of Mushroom Prebiotics with Gut Microbiota.

In recent years, gut microbiota have been linked to prevention and treatment of human diseases. Mushrooms are a source of potentially useful prebiotics because they contain polysaccharides, terpenoids, and other bioactive compounds. In the present review, we have summarized the prebiotic effects of mushrooms on gut microbiota in the context of immunological, metabolic, neurological, and cancer-related diseases in the last five years. We propose that mushrooms can not only change the composition of gut microbiota, but also promote secretion of beneficial metabolites. In addition, we point to the effects of host mRNA expression in gut microbiota as a direction of further study. Overall, these provide a background for further studies on the mechanisms of regulation of gut microbiota by mushrooms.

Increased Inhibition Effect of Antrodin C from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes), on A549 through Crosstalk between Apoptosis and Autophagy.

Antrodin C was obtained from Taiwanofungus camphoratus mycelia. The inhibition effect of antrodin C on A549 lung adenocarcinoma cells was evaluated by plate clone formation, wound healing, cell cycle, activated caspase-3, Bax, P53, Bcl-2, and RAPR activities as well as reactive oxygen species release. Plate clone formation assay revealed that antrodin C could significantly inhibit the viability of A549 cells in vitro. Wound healing assay revealed that cell migration was inhibited by exposure to antrodin C at concentrations of 50 and 80 µg/mL. Flow cytometry revealed that antrodin C increased the percentages of cells in the G0/G1 phase at concentrations of 50 and 80 µg/mL and the apoptosis was related to upregulation of caspase-3, Bax, P53 expression, downregulation of Bcl-2, RAPR expression, and the release of reactive oxygen species in the A549 cells. CQ or RAPA could significantly promote or inhibit the inhibition effect on A549 proliferation induced by antrodin C, which suggests that the autophagy played a cytoprotective role on inhibition proliferation of A549 induced by antrodin C. These results indicated that the combination of pro-apoptosis agents and anti-autophagy agents may be a useful strategy in enhancing the anticancer efficacy in non-small cell lung cancer.

Antrodin C, an NADPH Dependent Metabolism, Encourages Crosstalk between Autophagy and Apoptosis in Lung Carcinoma Cells by Use of an AMPK Inhibition-Independent Blockade of the Akt/mTOR Pathway.

The current study aims to explore the possible anti-lung carcinoma activity of ADC as well as the underlying mechanisms by which ADC exerts its actions in NSCLC. Findings showed that ADC potently inhibited the viability of SPCA-1, induced apoptosis triggered by ROS, and arrested the cell cycle at the G2/M phase via a P53 signaling pathway. Interestingly, phenomena such as autophagosomes accumulation, conversion of the LC3-I to LC3-II, etc., indicated that autophagy could be activated by ADC. The blockage of autophagy-augmented ADC induced inhibition of cell proliferation, while autophagy activation restored cell death, indicating that autophagy had a protective effect against cell death which was induced by ADC treatment. Meanwhile, ADC treatment suppressed both the Akt/mTOR and AMPK signaling pathways. The joint action of both ADC and the autophagy inhibitor significantly increased the death of SPCA-1. An in vitro phase I metabolic stability assay showed that ADC was highly metabolized in SD rat liver microsomes and moderately metabolized in human liver microsomes, which will assist in predicting the outcomes of clinical pharmacokinetics and toxicity studies. These findings imply that blocking the Akt/mTOR signaling pathway, which was independent of AMPK inhibition, could activate ADC-induced protective autophagy in non-small-cell lung cancer cells.

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.

Investigation of lignocellulolytic enzymes during different growth phases of Ganoderma lucidum strain G0119 using genomic, transcriptomic and secretomic analyses.

Ganoderma lucidum is a medicinal mushroom that is well known for its ability to enhance human health, and products made from this fungus have been highly profitable. The substrate-degrading ability of G. lucidum could be related to its growth. CAZy proteins were more abundant in its genome than in the other white rot fungi models. Among these CAZy proteins, changes in lignocellulolytic enzymes during growth have not been well studied. Using genomic, transcriptomic and secretomic analyses, this study focuses on the lignocellulolytic enzymes of G. lucidum strain G0119 to determine which of these degradative enzymes contribute to its growth. From the genome sequencing data, genes belonging to CAZy protein families, especially genes involved in lignocellulose degradation, were investigated. The gene expression, protein abundance and enzymatic activity of lignocellulolytic enzymes in mycelia over a growth cycle were analysed. The overall expression cellulase was higher than that of hemicellulase and lignin-modifying enzymes, particularly during the development of fruiting bodies. The cellulase and hemicellulase abundances and activities increased after the fruiting bodies matured, when basidiospores were produced in massive quantities till the end of the growth cycle. Additionally, the protein abundances of the lignin-modifying enzymes and the expression of their corresponding genes, including laccases and lignin-degrading heme peroxidases, were highest when the mycelia fully spread in the compost bag. Type I cellobiohydrolase was observed to be the most abundant extracellular lignocellulolytic enzyme produced by the G. lucidum strain G0119. The AA2 family haem peroxidases were the dominant lignin-modifying enzyme expressed during the mycelial growth phase, and several laccases might play roles during the formation of the primordium. This study provides insight into the changes in the lignocellulose degradation ability of G. lucidum during its growth and will facilitate the discovery of new approaches to accelerate the growth of G. lucidum in culture.

One single standard substance for the simultaneous determination of 17 triterpenes in Ganoderma lingzhi and its related species using high-performance liquid chromatography.

Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150mm×4.6mm I.D., 2.6µm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252nm), at a flow rate of 1mL/min. The results showed that all calibration curves had good linearity (r2>0.9999) within test ranges. The LOD and LOQ were lower than 2.52ng and 6.43ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4-103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma. In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes.

Characterization of Polysaccharides from the Fruiting Bodies of Two Species of Genus Ganoderma (Agaricomycetes) and Determination of Water-Soluble ß-D-Glucan Using High-Performance Liquid Chromatography.

Molecular weight (Mw) distributions of polysaccharides from the fruiting bodies of different Ganoderma lucidum strains and G. sinense were investigated and compared using high-pressure size exclusion chromatography/multiangle laser light scattering/refractive index analysis. Results showed that there were big differences in the Mw distributions and characteristics of polysaccharides from 2 species of Ganoderma. All tested G. lucidum materials exhibited similar polysaccharide distributions and similar characteristics for each fraction. The fraction with highest Mw (peak 1) was identified as ß-(1→3)-linked D-glucan with (1→6)-ß-D-glucopyranosyl side branches. G. sinense fruiting bodies did not include the ß-D-glucan when compared with G. lucidum. A high-pressure size exclusion chromatography method was developed and applied to determine the amount of high-Mw ß-D-glucan in G. lucidum fruiting bodies. Results indicated that there was no obvious relationship between ß-D-glucan content and the genetic similarity of G. lucidum. The strain labeled "Longzhi no. 2" was determined to possess the largest amount of ß-D-glucan: 8.2 mg/mL based on the dry weight of fruiting bodies. The ß-D-glucan content in the hot water extract of Longzhi no. 2 reached 17.05%. For the "Hunong no. 1" strain, the ß-D-glucan content in log-cultivated fruiting bodies was much higher than that in bag-cultivated ones. This method could be used to improve quality control of polysaccharides in G. lucidum.