Mycelial Polysaccharide Research Articles

Effect of extraction and purification on the structure and activity of Flammulina velutipes polysaccharides: a review

SummaryFlammulina velutipes (F. velutipes) is one of the most widely consumed edible mushrooms worldwide. The growth of F. velutipes involves two primary stages: the mycelium and fruiting body. Unique polysaccharides are produced in each stage; mycelial polysaccharides (FVMPs) are produced during the mycelial fermentation stage, while fruiting body polysaccharides (FVFBPs) are produced during the fruiting stage. These polysaccharides, the major bioactive components of F. velutipes, have garnered significant attention due to their various functions and activities. Notably, they exert functional activities by mediating gut flora, including antioxidant, anti‐inflammatory, and immunomodulatory properties, reduce blood sugar and lipid levels, and enhance cognitive performance. This study examined the variations in FVMPs and FVFBPs resulting from different extraction and purification methods, with a specific focus on delineating their distinct structural characteristics. This study further explored the impact of the structural composition of FVMPs and FVFBPs on their health‐promoting properties, focusing on the relationship between their structures and their functional and biological effects. Finally, this study outlines future research avenues designed to contribute to the ongoing research in the field of bioactive FVMPs and FVFBPs.

Structural characterization of red yeast rice-derived polysaccharide and its promotion of lipid metabolism and gut function in high-fat diet-induced mice

This study explored the structure and biological activities of a novel polysaccharide, PRY1–1, isolated from red yeast rice (RYR) through solid-state fermentation and biotransformation by Monascus purpureus. The structure of PRY1–1 was characterized by Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and other analytical techniques, revealing distinct differences from previously identified mycelial and extracellular polysaccharides. Functional assessments were performed on high-fat diet (HFD)-induced mice to evaluate the impact of PRY1–1 on lipid metabolism and gut function. The results demonstrated that PRY1–1 effectively ameliorated HFD-induced lipid metabolism disorders in the liver and epididymal white adipose tissue (eWAT) by regulating the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), leptin, adiponectin. Additionally, PRY1–1 protected gut function by enhancing gut barrier integrity, modulating gut microbiota composition, and regulating gut metabolite levels. This study offers new insights into the mechanisms by which RYR polysaccharides influence lipid metabolism, highlighting the potential of PRY1–1 as a functional component with significant health benefits.

A water-soluble mycelium polysaccharide from Monascus pilosus: Extraction, structural characterization, immunomodulatory effect and yield enhanced by overexpression of UGE gene

Mycelium polysaccharide (MPP) from Monascus pilosus with the compositions of glucose, galactose, mannose, glucosamine hydrochloride, rhamnose and arabinose, was obtained using alkaline extracting, and subsequently three purified components (MPP-0, MPP-0.1 and MPP-0.3) were separated. The purity and extraction volume of the MPP-0.1 fraction surpassed those of the other two groups, thus warranting its selection for subsequent experimental investigations. The sample MPP-0.1, with an average molecular weight of 3.7776 × 104 Da, exhibited exceptional thermal stability up to 170 °C. The main glycosidic linkage pattern of MPP-0.1 was structured as→[4)-α-D-Glcp-(1]6 → 4)-α-D-Glcp-(1 → [2)-α-D-Manp-(1]5 → 2)-α-D-Manp-(1 → 5)-β-D-Galf-(1 → 3)-β-D-Galf (1 → 3)-β-D-Galf-(1 → 3)-β-D-Galf-(1→, and branched Glcp, Manp, Galf fragments were connected with the main chain through →4, 6)-α-D-Glcp-(1→, →2, 6)-α-D-Manp-(1 → and →3, 6)-β-D-Galf-(1→. Besides, the up-regulated levels of Nitric oxide (NO), Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and other pro-inflammatory cytokines along with increased phagocytic activity revealed that MPP-0.1 has significant immunomodulatory effect, and can significantly enhance the proliferation and activation of RAW264.7 cells. Finally, the gene UGE (UDP-glucose 4-epimerase) was overexpressed in M. pilosus to increase the MPP production. Results showed that the biomass of the recombinant strain exhibited a remarkable increase of approximately 62.56 ± 1.50 % compared to that of the parental strain, and the extraction yield of MPP increased significantly by 83.19 ± 4.56 %.

Isolation, purification, and structural elucidation of a water-soluble polysaccharide derived from Phellinus baumii Pilát mycelia

This study involved the successful production of mycelial polysaccharides by optimizing submerged culture conditions of Phellinus baumii Pilát. Then, the investigation focused on the composition and chemical structure of the water-soluble polysaccharide from P. baumii Pilát mycelia (PBMP) by extraction and purification. Specifically, this study indicated that a water-soluble PBMP fraction 1 (PBMP1) was isolated from PBMP. Moreover, this study discovered a PBMP1 isolated from PBMP, which was found to have the monosaccharide compositions comprised of fucose (Fuc), glucose (Glc), and galactose (Gal) despite the absence of proteins. Subsequently, the composition and structure of PBMP1 were characterized using Fourier transform infrared (FT-IR) spectroscopy, periodate oxidation, Smith degradation, methylation reaction, gas chromatography-mass spectrometer (GC-MS), and Nuclear magnetic resonance (NMR). The composition and structure of PBMP1 were characterized in this study, revealing an α-glycosidic bond conformational linkage with 1,4-Glc residues and 1,6-Gal residues forming the backbone. Additionally, a highly branched hetero-polysaccharide was identified with a non-reducing terminus of Fuc containing 1,3,4- and 1,4,6-Glc branching. The findings of this study offer valuable insights and information that can be utilized by researchers, manufacturers, and other stakeholders to advance the field of P. baumii Pilát product development. Moreover, these results have significant implications for future large-scale mass production and functional applications of PBMP1 products.

Revealing the regulatory impact of nutrient on the production of (R)-2-(4-Hydroxyphenoxy)propanoic acid by Beauveria bassiana biofilms through comparative transcriptomics analyse.

Carbon and nitrogen play a fundamental role in the architecture of fungal biofilm morphology and metabolite production. However, the regulatory mechanism of nutrients remains to be fully understood. In this study, the formation of Beauveria bassiana biofilm and the production of (R)-2-(4-Hydroxyphenoxy)propanoic acid in two media with different carbon and nitrogen sources (GY: Glucose as a carbon source and yeast extract as a nitrogen source, MT: Mannitol as a carbon source and tryptone as a nitrogen source) were compared. R-HPPA production increased 2.85-fold in media MT than in media GY. Different fungal biofilm morphology and architecture were discovered in media GY and MT. Comparative transcriptomics revealed up-regulation of mitogen-activated protein kinase (MAPK) pathway and polysaccharides degradation genes affecting mycelial morphology and polysaccharides yield of the extracellular polymeric substances (EPS) in MT medium biofilms. Upregulation of genes related to NADH synthesis (carbon metabolism, amino acid metabolism, glutamate cycle) causes NADH accumulation and triggers an increase in R-HPPA production. These data provide a valuable basis for future studies on regulating fungal biofilm morphology and improving the production of high-value compounds.

Utilization of mycelial polysaccharide from Schizophyllum commune for the formation of highly stable o/w emulsion

This study investigated the emulsifying properties of a hot water extracted polysaccharide (SCMP) derived from liquid fermentation mycelium of Schizophyllum commune in stabilizing oil-in-water (o/w) emulsions. SCMP demonstrated the ability to stabilize o/w emulsions (referred to as SCMPe) for extended periods (>21 days) even at low concentrations (2 g/L) and increased the viscosity and elasticity of the emulsion system. Through confocal laser-scanning microscope visualization, it was observed that SCMP created a physical barrier on the surface of the oil droplets, contributing to emulsion stability. Furthermore, SCMPe demonstrated excellent tolerance to varying concentrations of Na+ (0.1–0.5 mol/L) and Ca2+ (0.01–0.05 mol/L) as well as a wide pH range (3.0–9.0). Moreover, increasing SCMP concentration effectively inhibited lipid oxidation and improved the stability of β-carotene and curcumin in the emulsion during storage. A detailed analysis of SCMP's composition and structure was done to better understand its emulsifier properties, revealing SCMP to be composed of two β-glucans with a rigid chain and high molecular weight. These results suggest that SCMP holds great potential an efficient stabilizer in the food industry, particularly in enhancing emulsion stability.

The study on in vitro antioxidant properties, cellular immune activities and mice anti-fatigue effects of Sanghuangporus vanninii mycelium polysaccharides

Sanghuang mycelial polysaccharides (SVMPs) were studied using poplar sanghuang as raw material (sanghuangporus vanninii, SV). The bioactivity of SVMPs was evaluated through in vitro antioxidant and cellular immunity activity assays, and the acute oral toxicity and anti-fatigue ability of SVMPs in mice were determined using mice as an experimental animal model. The results showed that SVMPs have a good scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+) radicals, with a slightly weaker reducing power. SVMPs promote, to some extent, the secretion of cytokines Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6), and the release of NO from mouse macrophage RAW264.7. In mouse experiments, the mouse tolerance of SVMPs was more greater than 18.2 g/kg BW, which was 2724 times the daily dosage of humans, and it was a non-toxic substance; the high dose group of SVMPs could significantly prolong the swimming time of weight-bearing mice. Thus, the results of this study clearly demonstrate the biological activity of SVMPs and provide a valuable basis for the potential application of SVMPs as antioxidants, immunomodulators, and functional foods.

Mechanistic and Functional Studies on the Microbial Induction of Wolfiporia cocos Liquid Fermentation Products.

Liquid fermentation is an efficient culture for obtaining polysaccharides from edible mushrooms. In this study, the polysaccharide content and biomass were examined by introducing microorganisms into the Wolfiporia cocos fermentation system. Three edible mushroom co-fermentation systems were established, among which the Wolfiporia cocos-Ganoderma lucidum co-fermentation system significantly increased the mycelial biomass of the system by 57.71% compared to Wolfiporia cocos alone and 91.22% compared to Ganoderma lucidum alone, and the intracellular polysaccharide content was significantly increased. Physiological activities of polysaccharides showed that mycelial polysaccharides in the Wolfiporia cocos-Ganoderma lucidum system had stronger anti-tumor cell value-adding and anti-tumor cell migration activities compared with Wolfiporia cocos and Ganoderma lucidum fermentation alone. The transcriptomic study of Wolfiporia cocos mycelium induced by exogenous substances suggested that the exogenous substances could enhance the intracellular polysaccharide content of Wolfiporia cocos through the upregulation of the expression of α-glycosyltransferase encoded by ALG10 and the downregulation of α-glycosidases encoded by MAN1B in the glycolytic metabolism of Wolfiporia cocos. This study provides a new direction for the transformation of polysaccharides from Wolfiporia cocos and Ganoderma lucidum into functional foods and new product development, and provides an experimental basis.

Tolypocladium sinense Mycelium Polysaccharide Alleviates Obesity, Lipid Metabolism Disorder, and Inflammation Caused by High Fat Diet via Improving Intestinal Barrier and Modulating Gut Microbiota.

Tolypocladium sinense is a fungus isolated from Cordyceps. Cordyceps has some medicinal value and is also a daily health care product. This study explores the preventive effects of T. sinense mycelium polysaccharide (TSMP) on high-fat diet-induced obesity and chronic inflammation in mice. Here, the study establishes an obese mouse model induced by high-fat diet. In this study, the mice are administered TSMP daily basis to evaluate its effect on alleviating obesity. The results show that TSMP can significantly inhibit obesity and alleviate dyslipidemia by regulating the expression of lipid metabolism-related genes such as liver kinase B1 (LKB1), phosphorylated AMP-activated protein kinase (pAMPK), peroxisome proliferator activated receptor α (PPARα),fatty acid synthase (FAS), and hydroxymethylglutaryl-CoA reductase (HMGCR) in the liver. TSMP can increase the protein expression of zona occludens-1 (ZO-1), Occludin, and Claudin-1 in the colon, improve the intestinal barrier dysfunction, and reduce the level of serum LPS, thereby reducing the inflammatory response. 16S rDNA sequencing shows that TSMP alters the intestinal microbiota by increasing the relative abundance of Akkermansia, Lactobacillus, and Prevotellaceae_NK3B31_group, while decreasing the relative abundance of Faecalibaculum. The findings show that TSMP can inhibit obesity and alleviates obesity-related lipid metabolism disorders, inflammatory responses, and oxidative stress by modulating the gut microbiota and improving intestinal barrier.

Extraction and Chemical Composition Analyses of Intracellular and Extracellular Polysaccharides from Trametes lactinea Liquid Fermentation

Trametes lactinea is a macrofungus commonly found on broadleaf tree felling piles with rich bioactivity, and its main active substances are polysaccharides, which have a certain medicinal value. Few studies have been conducted on the simultaneous study of intracellular and extracellular polysaccharides of fermented mycelia in liquid culture. In this study, T. lactinea collected from Eucalyptus stumps was subjected to liquid fermentation culturing and the extraction of intracellular and extracellular polysaccharides from the mycelium was optimized using unidirectional and orthogonal test methods. The intracellular and extracellular crude polysaccharides were isolated and purified, and polysaccharide fractions were obtained and assayed for their chemical composition. The main findings of this study are as follows: (1) The optimal conditions for the extraction of intracellular polysaccharides from T. lactinea mycelium were a material–liquid ratio of 1:40, an extraction temperature of 100 °C, a time of 5 h, and four repeats, wherein the polysaccharide extraction rate reached 5.1%. The optimal extraction conditions for the extracellular polysaccharides were a concentration ratio of 5:1, a concentration temperature of 70 °C, an ethanol concentration of 100.0%, and an alcohol settling time of 12 h. The polysaccharides could thus be extracted up to 0.63 mg/mL. (2) The intracellular and extracellular crude polysaccharides of T. lactinea mycelium were successfully isolated and purified, resulting in three homogeneous fractions of intracellular polysaccharides (IP-1, IP-2, and IP-3) and two homogeneous fractions of extracellular polysaccharides (EP-1 and EP-2). (3) The polysaccharide chemical composition resulted in the highest total sugar content of EP-1. IP-3 had the highest uronic acid content. The results of the monosaccharide composition analysis showed that the mycelial intracellular polysaccharides IP-1, IP-2, and IP-3 were all highest in glucose. Both IP-2 and IP-3 contained guluronic acid and glucuronic acid. Mannose was the most abundant extracellular polysaccharide in both EP-1 and EP-2. This study provides theoretical and technical bases for the comprehensive development and utilization of T. lactinea polysaccharides.

Effects of Medium Additives on the Mycelial Growth and Polysaccharide Biosynthesis in Submerged Culture of Bjerkandera fumosa.

Medium additives have been shown to affect the synthesis of active products in fungi. This study investigated the effects of corn stalk, poplar sawdust, Tween-80, and oleic acid on mycelial biomass and physicochemical properties, as well as the bioactivity of polysaccharides, including exopolysaccharides (EPS) and intracellular polysaccharides (IPS), in the submerged culture of Bjerkandera fumosa. Results showed that the addition of corn stalk or poplar sawdust increased the production of EPS but decreased the production of IPS; Tween-80 had less effect on the production of EPS and IPS; and oleic acid stimulated polysaccharide production significantly. Polysaccharide property analysis showed that the addition of corn stalk or poplar sawdust promoted the production of high-molecular-weight components in polysaccharides and changed the monosaccharide composition of polysaccharides, as well as increased the mannose, glucuronic acid, and xylose contents of IPS. Tween-80 and oleic acid also changed the molecular weight distribution of polysaccharides but only slightly affected the composition of monosaccharides. The bioactivity assay indicated that the polysaccharides obtained by adding corn stalk possessed high hydroxyl radical scavenging and antitumor activities. The effect of poplar sawdust was slightly weaker than that of corn stalk. EPS and IPS obtained from a culture with Tween-80 and oleic acid possessed low antioxidant activity. Moreover, their antitumor activity was improved and lost, respectively. The results obtained in this work are useful for improving the understanding of the optimization and regulation of bioactive polysaccharide production in the submerged culture of B. fumosa.

Antioxidation and Hepatoprotection of Selenium Mycelium Polysaccharides Against Alcoholic Liver Diseases from the Cultivated Morel Mushroom Morchella esculenta (Ascomycota).

The liver was regarded as the most important metabolic and detoxification organ in vivo, and Morchella esculenta had been reported as the admittedly rare edible fungus belonging to Ascomycetes contributing to the abundant bioactivities. The objective of this study aimed to confirm the potential antioxidant activities of selenium mycelium polysaccharides (Se-MIP) from M. esculenta against alcoholic liver diseases (ALD) in mice. The results indicated that a selenium concentration of 25 μg/mL exhibited potential in vitro antioxidant capacities of Se-MIP. The in vivo mice results demonstrated that Se-MIP showed potential anti-ALD effects by improving the antioxidant activities and alleviating the hepatic dysfunctions. The present conclusions suggested that Se-MIP could be used as a candidate on improving ALD and its complications for further clinical investigations.

Anti-inflammatory mechanism and application of mycelial polysaccharides

Studies have shown that mycelial polysaccharides can regulate the body's resistance to inflammation in different ways, this paper briefly describes how the extraction of mycelial polysaccharides can reduce the impact on the performance of mycelium, as well as some of the mycelial polysaccharides on the mechanism of inflammation resistance. In order to provide a theoretical basis for helping mycelial polysaccharides to become good anti-inflammatory drugs.

Optimization of Se- and Zn-Enriched Mycelium of Lentinula edodes (Berk.) Pegler as a Dietary Supplement with Immunostimulatory Activity.

Mycelial cultures of Lentinula edodes, an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current attempts to obtain a new preparation containing both selenium and zinc, two micronutrients necessary for the functioning of the immune system, extended our interest in the simultaneous accumulation of these elements by mycelia growing in media enriched with selenite and zinc(II) ions. Subsequently, we have studied the effects of new L. edodes mycelium water extracts with different concentrations of selenium and zinc on the activation of T cell fraction in human peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis was used to measure the expression of activation markers on human CD4+ and CD8+ T cells stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs). It was demonstrated that statistically significant changes were observed for PD-1 and CD25 antigens on CD8+ T cells. The selenium and zinc content in the examined preparations modified the immunomodulatory activity of mycelial polysaccharides; however, the mechanisms of action of various active ingredients in the mycelial extracts seem to be different.

Necessity of Different Lignocellulose on Exopolysaccharide Synthesis and Its Hypoglycemic Activity In Vitro of Inonotus obliquus.

Current work proposes elm sawdust, poplar sawdust, pine sawdust, and cotton straw with different lignocellulose compositions and structures as the research objects to investigate the relationship between the hypoglycemic activity of mycelium polysaccharides from Inonotus obliquus and lignocellulose biodegradation. Four kinds of lignocellulosic materials could significantly increase the exopolysaccharide content and α-glucosidase inhibition rate and advance the occurrence time of α-glucosidase inhibition activity. Among all groups, the polysaccharide synthesis promotion effect of the cotton straw group was the best, which exopolysaccharide yield was 92.05% higher than that of the control group after 11-day fermentation. Meanwhile, the highest α-glucosidase inhibitory activity was found in the elm sawdust group on the 11th day (30.99%, which was 137.47% higher than control), and the exopolysaccharide in the elm sawdust group showed its effectiveness on glucose consumption of insulin resistant HepG2 cells at the concentration of 20 µg/mL, significantly higher than that of the metformin group (P < 0.05). The cellulose in the non-crystalline region of elm and pine and the hemicellulose of poplar were mainly used in the fermentation of I. obliquus, while the cellulose in the crystalline zone and amorphous zone of cotton straw was degraded to improve the exopolysaccharide content of I. obliquus. This paper revealed the necessity of different kinds of lignocellulose for the synthesis of active polysaccharide from I. obliquus and provided a new idea for the regulation of polysaccharide synthesis pathway.

Study on Extraction, Physicochemical Properties, and Bacterio-Static Activity of Polysaccharides from Phellinus linteus

We optimized an ultrasound-assisted extraction process of Phellinus linteus mycelium polysaccharides (PLPs) and studied their monosaccharide composition and bacteriostatic properties. Based on a single-factor experiment, a three-factor, three-level Box–Behnken design was used to optimize the ultrasound-assisted extraction process of PLP, using the yield of PLP as the index. The chemical composition and monosaccharide composition of PLP were determined by chemical analysis and HPLC analysis, respectively. Microscopic morphological analysis of the surface of PLP was performed via swept-surface electron microscopy. The bacteriostatic properties of PLP were determined using the spectrophotometric turbidimetric method. The results showed that the best extraction process of PLP with ultrasonic assistance achieved a result of 1:42 g/mL. In this method, the ultrasonic temperature was 60 °C, ultrasonic extraction was performed for 20 min, and the yield of PLP was 12.98%. The monosaccharide composition of PLP mainly contains glucose (Glc), mannose (Man), galactose (Gal), and glucuronic acid (GlcA). The intracellular polysaccharide of Phellinus igniarius Mycelia (PIP) is an irregular spherical accumulation, the surface is rough and not smooth, and the extracellular polysaccharide (PEP) is a crumbly accumulation. PIP has a stronger inhibitory ability for S. aureus and E. coli and a slightly weaker inhibitory effect for B. subtilis; the inhibitory effect of PEP on S. aureus, E. coli, and B. subtilis is slightly inferior to that of PIP.

Rheological properties and thickening effect of high molecular weight exopolysaccharide and intracellular polysaccharide from Schizophyllum commune

Edible and medicinal fungal polysaccharides with excellent biological activity and high viscosity are potential thickeners for food applications. In this study, the rheological properties of aqueous solutions of exopolysaccharide or fermentation broth polysaccharide (SCFP) and intracellular polysaccharide or mycelial polysaccharide (SCMP) obtained from liquid submerged fermentation of the fungus Schizophyllum commune (S. commune), and their thickening effects on coffee and fruit tea were investigated. The polysaccharide aqueous solutions behaved as pseudoplastic fluids, and their viscosity was concentration-dependent but not significantly affected by temperature (10–70 °C). The critical concentrations (c*) of SCFP and SCMP were 0.275 g/L and 0.356 g/L, respectively. The intensity of the thixotropic properties of the polysaccharide aqueous solutions was positively correlated with polysaccharide concentration. Both polysaccharide solutions exhibited solid-like viscoelastic behavior and formed weak gels, with enhanced gel strength at high concentrations and low temperatures. The viscosity at a shear rate of 50 s−1 (η50) of the 5 g/L SCFP- and SCMP-thickened coffee was 0.07 Pa·s, while that of SCFP- and SCMP-thickened fruit tea was 0.1 Pa·s and 0.08Pa·s, respectively, which were significantly higher than that of polysaccharide aqueous solutions (0.05 Pa·s for SCFP and SCMP). The yield stress values increased with increasing polysaccharide concentration, suggesting a potential role of for these polysaccharides in dysphagia management. Overall, SCFP and SCMP have significant potential for application in the food industry.

Enhanced α-glucosidase inhibition activity of exopolysaccharides fractions from mycelium of Inonotus obliquus under addition of birch sawdust lignocellulose component

The addition of lignocellulose in the submerged cultivation of Inonotus obliquus has been considered a feasible alternative to improvement the bioactivity of exopolysaccharides. To investigate the key components of lignocellulose in the synthesis of active polysaccharide by I. obliquus, cellulose and hemicellulose polysaccharides were isolated from birch sawdust and their effects on the production and activity of exopolysaccharides from I. obliquus were evaluated in the present study. Results showed that α-glucosidase inhibition rates of exopolysaccharides in two cellulose polysaccharide groups (C-36 and C-48) and water extracted hemicellulose polysaccharide group (H-0) were significantly increased by 3.44–4.09 times compared with control group. Three polysaccharides fractions (DPC36–1, DPC48–1 and DPH0–1) in above three groups were isolated from I. obliquus using DEAE-52 cellulose chromatography, which were composed of glucose with molecular weight distribution ranging from 13.94 kDa to 74.48 kDa. These results might stimulate the development of mycelium polysaccharides from I. oblqiuus as alternative anti-diabetes agents and provide important insights into the relationship of structure-bioactivity of fungal polysaccharides with cellulose and hemicellulose as carbon source donors.

MicroRNA-like RNA Functions Are Required for the Biosynthesis of Active Compounds in the Medicinal Fungus Sanghuangporus vaninii.

miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.

Enhanced Effects of Iron on Mycelial Growth, Metabolism and In Vitro Antioxidant Activity of Polysaccharides from Lentinula edodes.

The polysaccharides found in Lentinula edodes have a variety of medicinal properties, such as anti-tumor and anti-viral effects, but their content in L. edodes sporophores is very low. In this study, Fe2+ was added to the liquid fermentation medium of L. edodes to analyze its effects on mycelial growth, polysaccharide and enzyme production, gene expression, and the activities of enzymes involved in polysaccharide biosynthesis, and in vitro antioxidation of polysaccharides. The results showed that when 200 mg/L of Fe2+ was added, with 7 days of shaking at 150 rpm and 3 days of static culture, the biomass reached its highest value (0.28 mg/50 mL) 50 days after the addition of Fe2+. Besides, Fe2+ addition also enhanced intracellular polysaccharide (IPS) and exopolysaccharide (EPS) productions, the levels of which were 2.98- and 1.79-fold higher than the control. The activities of the enzymes involved in polysaccharides biosynthesis, including phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), and UDPG-pyrophosphorylase (UGP) were also increased under Fe2+ addition. Maximum PGI activity reached 1525.20 U/mg 30 days after Fe2+ addition, whereas PGM and UGP activities reached 3607.05 U/mg and 3823.27 U/mg 60 days after Fe2+ addition, respectively. The Pearson correlation coefficient showed a strong correlation (p < 0.01) between IPS production and PGM and UGP activities. The corresponding coding genes of the three enzymes were also upregulated. When evaluating the in vitro antioxidant activities of polysaccharides, EPS from all Fe2+-treated cultures exhibited significantly better capacity (p < 0.05) for scavenging -OH radicals. The results of the two-way ANOVA indicated that the abilities of polysaccharides to scavenge O2− radicals were significantly (p < 0.01) affected by Fe2+ concentration and incubation time. These results indicated that the addition of iron provided a good way to achieve desirable biomass, polysaccharide production, and the in vitro antioxidation of polysaccharides from L. edodes.