Selenium-Containing Exopolysaccharides Isolated from the Culture Medium of Lentinula edodes: Structure and Biological Activity.

In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, and immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5 × 106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure-activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells (HUVEC)) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10-100 µg/mL, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans, the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates, most likely due to a different mode of selenium binding and its higher degree of oxidation.

Relationship between flavor and energy status in shiitake mushroom (Lentinula edodes) harvested at different developmental stages.

To understand the relationship between flavor and energy, the flavor, energy, and enzyme activity related to energy metabolism in shiitake mushrooms harvested at different developmental stages were investigated. The results indicated that the adenosine triphosphate level increased significantly in developing mushrooms and was strongly correlated with the fresh weight. The levels of equivalent umami concentration (EUC), total aroma compounds, energy charge, adenosine triphosphatase, cytochrome c oxidase, and succinic dehydrogenase varied with maturity. In addition, a strong correlation was observed between aroma compounds, EUC, and energy status (p < 0.05). Our results suggest that the unique flavor of developing shiitake mushroom is closely related to energy. The findings may provide a new strategy to improve the flavor of mushrooms by regulating their energy levels. PRACTICAL APPLICATION: The unique flavor of shiitake mushroom, which has a significant impact on consumer preferences, is one of its key characteristics. This research paper provides a theoretical foundation for determining the optimal harvest period for shiitake mushrooms with high quality and a new strategy to improve the flavor of mushrooms by regulating their energy levels.

Combining the Anticancer and Immunomodulatory Effects of Astragalus and Shiitake as an Integrated Therapeutic Approach.

Astragalus root (Huang Qi) and Shiitake mushrooms (Lentinus edodes) are both considered medicinal foods and are frequently used in traditional Chinese medicine due to their anticancer and immunomodulating properties. Here, the scientific literatures describing evidence for the anticancer and immunogenic properties of Shiitake and Astragalus were reviewed. Based on our experimental data, the potential to develop medicinal food with combined bioactivities was assessed using Shiitake mushrooms grown over Astragalus beds in a proprietary manufacturing process, as a novel cancer prevention approach. Notably, our data suggest that this new manufacturing process can result in transfer and increased bioavailability of Astragalus polysaccharides with therapeutic potential into edible Shiitake. Further research efforts are required to validate the therapeutic potential of this new Hengshan Astragalus Shiitake medicinal food.

Antioxidant and Anti-Inflammatory Potential of Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes), Sporophores from Various Culture Conditions.

Lentinus edodes (= Lentinula edodes) is an edible mushroom grown and marketed for centuries due to its nutritional and medicinal properties. L. edodes has multiple pharmacological activities as an antioxidant and anti-inflammatory. Few studies were performed taking into account the influence of culture conditions to optimize the biological properties of L. edodes on human health. Our work focused on the comparison of antioxidant capacity and anti-inflammatory activity of L. edodes fruit bodies cultivated by three mushroom producers in the French Occitanie region using the same strain in various growing conditions (organic and nonorganic). Sequential extraction was performed on freeze-dried fungal materials. All extracts have a quantifiable but moderate antioxidant activity measured via DPPH and ORAC tests. The anti-inflammatory activity of the ethanol and aqueous extracts was evaluated on a model of inflammatory macrophages. The ethanol extracts inhibit NO production in a dose-dependent manner when the cells are pretreated for 4 h with a 24 h stimulation time.

Simultaneous Determination of 13 Organic Acids in Liquid Culture Media of Edible Fungi Using High-Performance Liquid Chromatography.

The study aimed at detecting 13 organic acids (oxalic acid, maleic acid, citric acid, tartaric acid, malic acid, quinic acid, succinic acid, fumaric acid, formic acid, acetic acid, propionic acid, isobutyric acid, and butyric acid) by establishing a high-performance liquid chromatography (HPLC). The analysis was performed using two sugar columns, i.e., SH1011 column and KC-811 column. The optimal conditions were as follows: 4 mmol/L HClO4 solution as the eluent with UV-visible detector (210 nm), a flow rate of 1 mL/min at the temperature of 60°C, and the injection volume at 10 µL. The results showed that all the calibration curves had excellent linearity (R 2 > 0.9991) within the test ranges. The RSD values of the thirteen analytes were lower than 2.94% at three levels, the recoveries were 91.9%-102.0%, the limit of detection (LOD) was between 0.05 and 10.63 µg/mL, and the quantification (LOQ) was between 0.10 and 19.53 µg/mL. Finally, the proposed methodology was successfully applied for the analysis of organic acids in the culture medium of edible fungi. In conclusion, the study findings proved that the method was sensitive, accurate, reproducible, and could be readily applied to analyze the organic acids in the samples.

Mycelial biomass estimation and metabolic quotient of Lentinula edodes using species-specific qPCR.

Lentinula edodes, commonly known as shiitake, is an edible mushroom that is cultivated and consumed around the globe, especially in Asia. Monitoring mycelial growth inside a woody substrate is difficult, but it is essential for effective management of mushroom cultivation. Mycelial biomass also affects the rate of wood decomposition under natural conditions and must be known to determine the metabolic quotient, an important ecophysiological parameter of fungal growth. Therefore, developing a method to measure it inside a substrate would be very useful. In this study, as the first step in understanding species-specific rates of fungal decomposition of wood, we developed species-specific primers and qPCR procedures for L. edodes. We tested primer specificity using strains of L. edodes from Japan and Southeast Asia, as well as related species of fungi and plant species for cultivation of L. edodes, and generated a calibration curve for quantification of mycelial biomass in wood dust inoculated with L. edodes. The qPCR procedure we developed can specifically detect L. edodes and allowed us to quantify the increase in L. edodes biomass in wood dust substrate and calculate the metabolic quotient based on the mycelial biomass and respiration rate. Development of a species-specific method for biomass quantification will be useful for both estimation of mycelial biomass and determining the kinetics of fungal growth in decomposition processes.

Characterization of brown film formed by Lentinula edodes.

Lentinula edodes is a widely-produced mushroom in China that forms a brown film via pigment accumulation on mature mycelial surfaces to ensure high-quantity and high-quality fruiting body formation. Here, ultraviolet-visible, infrared spectra, and elemental analyses predicted that the pigment in the brown film was melanin. Electron microscopy revealed the size, morphological characteristics, accumulation, and morphogenesis of electron-dense material, which were similar to those of melanin, as well as subcellular structural changes during brown film formation. The electron-dense material appeared as granules, vesicles, and polymers. The accumulation of electron-dense materials on the cell wall was followed plasmolysis, plasma membrane disruption, electron-dense material accumulation in the interstitial space, and gradual accumulation on the outer cell wall. Dolipore septa degradation and morphogenetic cell death occurred during browning. In the final stage of browning, the dolipore septum disappeared and the cell was nearly empty. This study provides a cytological foundation for evaluating the regulation of brown film formation in L. edodes.

Physicochemical properties and bioactivities of Lentinula edodes polysaccharides at different development stages.

Lentinula edodes polysaccharides from at four different development stages (referred to L1, L2, L3 and L4, respectively) were extracted by hot water method, and graded ethanol precipitation to final concentration of 20%, 50% and 70%, then12 crude polysaccharide fractions (referred to L1P20, L2P20, L3P20; L4P20, L1P50, L2P50, L3P50, L4P50 and L1P70, L2P70, L3P70, L4P70, respectively) were obtained. Physicochemical properties and exoteric bioactivities of the crude polysaccharide fractions were measured. The results of physicochemical properties revealed that extraction yields of P20 fractions were significantly higher than those of P50 and P70 fractions, and the contents of polysaccharide and ß-glucan in L3P50 fractions were higher, and the viscosity-average molecular weight reached a maximum at L2, and high molecular weight polysaccharides could be obtained at a low alcohol concentration in P20 fractions, and the glycosidic bonds were found to exist in all crude polysaccharide fractions. These crude polysaccharide fractions showed different bioactivities, wherein the polysaccharides of higher molecular weight in P20 fractions had greater bioactivity. These results showed that immature stage of Lentinula edodes was the optimal harvest time for obtaining higher bioactivity of crude polysaccharides.

HS-SPME/GC-MS Assisted Analysis of Volatile Constituents in Different Strains of Shiitake Culinary- Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

The shiitake culinary-medicinal mushroom Lentinus edodes is one of the most consumed species worldwide because it has nutritional, medicinal, and palatable properties. Its organoleptic characteristics contribute substantially to its high popularity. The pleasant aromas result from the presence of volatile compounds. The objective of the present work was to study the profiles of volatile constituents of fresh fruiting bodies of five strains of L. edodes. The volatile compounds were extracted by solid phase microextraction method and analyzed by gas chromatography and mass spectrometry. The aromatic profiles of the strains revealed variability. Both alcohols and sulfides were the most abundant volatile compounds. LE6 strain presented the highest number of volatile compounds with predominance of sulfides (dimethyl pentasulfide, s-tetrathiane) and for LE2, the aldehydes were the most representative chemical class with the main volatile being (E)-2-octen-1-al.

Effects of GGT and C-S Lyase on the Generation of Endogenous Formaldehyde in Lentinula edodes at Different Growth Stages.

Endogenous formaldehyde is generated as a normal metabolite via bio-catalysis of γ-glutamyl transpeptidase (GGT) and L-cysteine sulfoxide lyase (C-S lyase) during the growth and development of Lentinula edodes. In this study, we investigated the mRNA and protein expression levels, the activities of GGT and C-S lyase, and the endogenous formaldehyde content in L. edodes at different growth stages. With the growth of L. edodes, a decrease was found in the mRNA and protein expression levels of GGT, while an increase was observed in the mRNA and protein expression levels of C-S lyase as well as the activities of GGT and C-S lyase. Our results revealed for the first time a positive relationship of formaldehyde content with the expression levels of Csl (encoding Lecsl) and Lecsl (C-S lyase protein of Lentinula edodes) as well as the enzyme activities of C-S lyase and GGT during the growth of L. edodes. This research provided a molecular basis for understanding and controlling the endogenous formaldehyde formation in Lentinula edodes in the process of growth.

Modulation of T Regulatory and Dendritic Cell Phenotypes Following Ingestion of Bifidobacterium longum, AHCC® and Azithromycin in Healthy Individuals.

The probiotic Bifidus BB536 (BB536), which contains Bifidobacterium longum, has been shown to have enhanced probiotic effects when given together with a standardized extract of cultured Lentinula edodes mycelia (AHCC®, Amino Up Co. Ltd., Sapporo, Japan). BB536 and AHCC® may modulate T cell and dendritic cell (DC) phenotypes, and cytokine profiles to favour anti-inflammatory responses following antibiotic ingestion. We tested the hypothesis that orally administered BB536 and/or AHCC®, results in modulation of immune effector cells with polarisation towards anti-inflammatory responses following antibiotic usage. Forty healthy male volunteers divided into 4 equal groups were randomised to receive either placebo, BB536, AHCC® or a combination for 12 days in a double-blind manner. After 7 days volunteers also received 250 mg azithromycin for 5 days. Cytokine profiles from purified CD3+ T cells stimulated with PDB-ionomycin were assessed. CD4+ CD25+ forkhead box P3 (Foxp3) expression and peripheral blood DC subsets were assessed prior to treatment and subsequently at 7 and 13 days. There was no difference in cytokine secretion from stimulated CD3+ T cells between treatment groups. Compared with baseline, Foxp3 expression (0.45 ± 0.1 vs. 1.3 ± 0.4; p = 0.002) and interferon-gamma/interleukin-4 (IFN-γ/IL-4) ratios were increased post-treatment in volunteers receiving BB536 (p = 0.031), although differences between groups were not significant. For volunteers receiving combination BB536 and AHCC®, there was an increase in myeloid dendritic cells (mDC) compared with plasmacytoid DC (pDC) counts (80% vs. 61%; p = 0.006) at post treatment time points. mDC2 phenotypes were more prevalent, compared with baseline, following combination treatment (0.16% vs. 0.05%; p = 0.002). Oral intake of AHCC® and BB536 may modulate T regulatory and DC phenotypes to favour anti-inflammatory responses following antibiotic usage.

GC⁻MS-Based Nontargeted and Targeted Metabolic Profiling Identifies Changes in the Lentinula edodes Mycelial Metabolome under High-Temperature Stress.

To clarify the physiological mechanism of the Lentinula edodes (L. edodes) response to high-temperature stress, two strains of L. edodes with different tolerances were tested at different durations of high temperature, and the results showed that there were significant changes in their phenotypes and physiology. To further explore the response mechanism, we established a targeted GC-MS-based metabolomics workflow comprising a standardized experimental setup for growth, treatment and sampling of L. edodes mycelia, and subsequent GC-MS analysis followed by data processing and evaluation of quality control (QC) measures using tailored statistical and bioinformatic tools. This study identified changes in the L. edodes mycelial metabolome following different time treatments at high temperature based on nontargeted metabolites with GC-MS and further adopted targeted metabolomics to verify the results of the analysis. After multiple statistical analyses were carried out using SIMCA software, 74 and 108 differential metabolites were obtained, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the metabolic pathways with significant changes included those related to the following: amino acid metabolism, the glycolysis pathway, the tricarboxylic acid (TCA) cycle, and sugar metabolism. Most amino acids and carbohydrates enriched in these metabolic pathways were upregulated in strain 18, downregulated in strain 18N44, or the synthesis in strain 18 was higher than that in strain 18N44. This result was consistent with the physiological phenotypic characteristics of the two strains under high-temperature stress and revealed the reason why strain 18N44 was more heat-sensitive. At the same time, under high temperature, the decrease of intermediate products in glycolysis and the TCA cycle resulted in carbon starvation and insufficient energy metabolism, thus inhibiting the growth of L. edodes. In addition, the results also showed that the metabolites produced by different L. edodes strains under high-temperature stress were basically the same. However, different strains had species specificity, so the changes in the content of metabolites involved in the response to high-temperature stress were different. This provides a theoretical basis for further understanding the mechanism of the L. edodes response to high temperature and can be used to establish an evaluation system of high-temperature-resistant strains and lay the foundation for molecular breeding of new L. edodes strains resistant to high temperature.

MRI visualization of shiitake mycelium growing in woodchip blocks used for shiitake mushroom cultivation.

In order to eliminate woodchip blocks where unwanted fungi have grown and select only blocks where shiitake mycelium are growing well, there is a need to develop a visualization technique for shiitake mycelium growing in woodchip blocks, and MRI is an obvious candidate technique. From the results of measurements of the woodchip bed in a small bottle (26 mm inside diameter) where shiitake mycelium was growing, the T1 relaxation time constant immediately after inoculation was 77.9 ±â€¯5.5 ms, and the value after about 10 to 20 days increased to 135.0 ±â€¯9.8 ms (the increase rate was 73%). The T1 maps of the wood-chip block (130 mm length, 75 mm height and 55 mm thickness) in which shiitake mycelium grew were calculated from T1 weighted images measured by changing TR from 28 to 400 ms. From the T1 maps of time series, it was found that the shiitake mycelium extended from the right-hand side to the left-hand side of the woodchip block in a planar manner. Furthermore, in a woodchip block in which penicillium was generated, since the T1 relaxation time constant of only the shiitake mycelium became longer, it was possible to visualize the shiitake mycelium distinctly from penicillium.

Overexpression and repression of the tyrosinase gene in Lentinula edodes using the pChG vector.

Tyrosinase is an industrially useful enzyme, however, it causes gill browning of Lentinula edodes fruiting bodies during preservation. In this study, we constructed two vectors, pChG-gTs and pChG-gTa, expressing sense and antisense tyrosinase gene of L. edodes, respectively, using promoters derived from the glyceraldehyde-3-phosphate dehydrogenase gene. The host strain SR-1 of L. edodes was selected because of its fast growth, high protoplast yield, and high regeneration rate. Upon transformation of the host strain SR-1 with the pChG-gTs vector, a clone with 3.6-fold and 14.5-fold higher tyrosinase activity in vegetative mycelia and in fresh gills, respectively, than that of the host strain was obtained from nine transformants. Similarly, two clones containing the pChG-gTa vector with effectively repressed tyrosinase gene expression in vegetative mycelia and gills during the late stage of post-harvest preservation of fruiting bodies were obtained from 10 transformants. However, it remained unclear whether repression of the tyrosinase gene prevented gill browning, as the host strain also showed less browning than a commercial strain. Thus, this study highlights the usefulness of the pChG vector in expressing homologous enzyme coding genes in the vegetative mycelia and fruiting bodies of L. edodes.

Energy-efficient substrate pasteurisation for combined production of shiitake mushroom (Lentinula edodes) and bioethanol.

Hot-air (75-100 °C) pasteurisation (HAP) of birch-wood-based substrate was compared to conventional autoclaving (steam at 121 °C) with regard to shiitake growth and yield, chemical composition of heat-pretreated material and spent mushroom substrate (SMS), enzymatic digestibility of glucan in SMS, and theoretical bioethanol yield. Compared to autoclaving, HAP resulted in faster mycelial growth, earlier fructification, and higher or comparable fruit-body yield. The heat pretreatment methods did not differ regarding the fractions of carbohydrate and lignin in pretreated material and SMS, but HAP typically resulted in lower fractions of extractives. Shiitake cultivation, which reduced the mass fraction of lignin to less than half of the initial without having any major impact on the mass fraction of glucan, enhanced enzymatic hydrolysis of glucan about four-fold. The choice of heating method did not affect enzymatic digestibility. Thus, HAP could substitute autoclaving and facilitate combined shiitake mushroom and bioethanol production.

Review of Bioactive Molecules Production, Biomass, and Basidiomata of Shiitake Culinary-Medicinal Mushrooms, Lentinus edodes (Agaricomycetes).

Lentinus edodes (shiitake) is a basidiomycete that has been consumed for more than 2000 years because of its nutritional value and health benefits. It has a low lipid content, high fiber content, and a considerable amount of proteins; it also contains B vitamins and minerals in addition to a wide range of functional metabolites including polysaccharides, polysaccharopeptides, lectins, and secondary metabolites with bioactivity, e.g., lentinan, a ß-(1-3)-glucan with immunomodulatory activity, among others. Extracts and pure compounds of shiitake exhibit antibacterial, antifungal, cytostatic, antioxidant, anticancer, and immunomodulatory activity. Because of these attributes, different products derived from shiitake are on the market and are sold as dietary supplements. The traditional substrate for shiitake production is oak wood, yet the search for unconventional substrates has intensified over the past three decades. In particular, submerged cultivation of medicinal mushrooms has attracted great interest because it enables greater control of different fermentation factors to obtain products of interest. However, it is necessary to perform in vivo studies to determine the appropriate doses, side effects, and action spectrum of different bioactive compounds and fractions as well as to improve their production in liquid media and to potentiate their activity. We present an updated review of existing studies on the production of biomass and bioactive compounds of L. edodes in liquid culture and on solid fermentation for obtaining secondary mycelia and basidiomata.

The heat shock protein 40 LeDnaJ regulates stress resistance and indole-3-acetic acid biosynthesis in Lentinula edodes.

DnaJ proteins, termed heat shock proteins based on their molecular weight, function as molecular chaperones that play critical roles in regulating organism growth and development as well as adaptation to the environment. However, little has been reported on their gene function in higher basidiomycetes. Here, the heat shock protein 40 (LeDnaJ) gene was cloned and characterized from Lentinula edodes. RNA interference was used to explore the function of LeDnaJ in response to heat stress and Trichoderma atroviride. Integration of the target gene into the L. edodes genome was confirmed by Southern blot analysis, and the silence efficiency of LeDnaJ was analyzed by qRT-PCR. The results revealed that LeDnaJ silence caused defects in mycelial growth and resistance to heat stress and T. atroviride, but increased the mycelial density compared with the wild type (WT) strain S606. Additionally, the IAA content showed a more than 10-fold increase in the WT after heat stress, but an about two-fold increase in the two LeDnaJ RNAi transfortants (LeDnaJ-i-6 and LeDnaJ-i-8). Previous study has shown that enhanced IAA (indole-3-acetic acid) content enhanced the thermotolerance of the heat-sensitive strain YS3357. In this study, it was documented that IAA amendments could partly restore the resistance to T. atroviride and thermotolerance of the two LeDnaJ RNAi transformants. Overall, LeDnaJ is nvolved in fungal growth, T. atroviride resistance, and thermotolerance by regulating the IAA biosynthesis in L. edodes.

Diversity of A mating type in Lentinula edodes and mating type preference in the cultivated strains.

Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5' region of HD2-intergenic region-5' region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.

Bioavailability of Compounds Susceptible to Enzymatic Oxidation Enhances Growth of Shiitake Medicinal Mushroom (Lentinus edodes) in Solid-State Fermentation with Vineyard Prunings.

Grapes are widely produced in northwestern Mexico, generating many wood trimmings (vineyard prunings) that have no further local use. This makes vineyard prunings a very attractive alternative for the cultivation of white-rot medicinal mushrooms such as Lentinus edodes. This type of wood can also offer a model for the evaluation of oxidative enzyme production during the fermentation process. We tested the effect of wood from vineyard prunings on the vegetative growth of and production of ligninolytic enzymes in L. edodes in solid-state fermentation and with wheat straw as the control substrate. The specific growth rate of the fungus was 2-fold higher on vineyard pruning culture (µM = 0.95 day-1) than on wheat straw culture (µM = 0.47 day-1). Laccase-specific production was 4 times higher in the vineyard prunings culture than on wheat straw (0.34 and 0.08 mU · mg protein-1 · ppm CO2-1, respectively), and manganese peroxidase production was 3.7 times higher on wheat straw culture than on vineyard prunings (2.21 and 0.60 mU · mg protein-1 · ppm CO2-1, respectively). To explain accurately these differences in growth and ligninolytic enzyme activity, methanol extracts were obtained from each substrate and characterized. Resveratrol and catechins were the main compounds identified in vineyard prunings, whereas epigallocatechin was the only one detected in wheat straw. Compounds susceptible to enzymatic oxidation are more bioavailable in vineyard prunings than in wheat straw, and thus the highest L. edodes growth rate is associated with the presence of these compounds.

Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying.

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.