Fungal Polysaccharides Research Articles

The effects of quorum sensing molecule farnesol on the yield and activity of extracellular polysaccharide from Grifola frondosa in liquid fermentation

A strategy by exogenous addition of quorum sensing molecule farnesol to improve the production, antioxidant activity and antitumor activity of extracellular polysaccharide (EPS) of Grifola frondosa by liquid fermentation was proposed in the study. The highest yield of EPS induced by farnesol was 1.25 g/L, which was 150% higher than that of the control. Four polysaccharides including EPS-C-0M, EPS-C-0.2M, EPS-F-0M and EPS-F-0.2M were extracted and purified under the conditions of control and farnesol respectively. The physicochemical properties, antioxidant activities and antitumor activities were studied. Their chemical composition differed in sugar, protein and uronic acid contents, and they were composed of six constituent monosaccharides with different ratios, with the average molecular weights of 1.12 × 103, 1.89 × 103, 1.41 × 103 and 2.02 × 103 kDa, respectively. They presented similar FT-IR spectra, but different surface morphology. Antioxidant experiments showed that they had strong scavenging activities on ABTS+, hydroxyl radical, O2− and DPPH radical. Antitumor experiments showed that they had strong inhibitory effects on human cervical cancer (HeLa) cells and human liver cancer cells (HepG2) cells. Among the four polysaccharides, EPS-F-0.2M showed the highest antioxidant and antitumor activities, indicating that farnesol could regulate the biological activity of EPS by affecting structure and properties. These results demonstrated that appropriate adjustment of culture conditions had potential application in the development of polysaccharides with high antioxidant and antitumor activity. It provided a new strategy to enhance the production and bioactivity of edible and medicinal fungal polysaccharides by using quorum sensing molecules.

Improving the biological properties of Fomes fomentarius MG835861 exopolysaccharide by bioincorporating selenium into its structure

Selenium (Se) is one of the essential elements for human health, where Se-containing compounds can prevent cancer and reduce mortality by regulating of the cell cycle and apoptosis. Exopolysaccharides from Fomes fomentarius have been used in traditional medicine to treat cancer. It appears that the combination of Se and fungal polysaccharides may be more effective. The effect of adding Na2SeO3 to the culture medium of the Iranian medicinal fungus F. fomentarius is investigated in this study. Adding 20 mg/L Na2SeO3 to the culture medium changed the F. fomentarius morphology and improved the structure of exopolysaccharides. Several methods, including FTIR, EDS, and ICP-OES, confirmed the presence of Se in the exopolysaccharides of F. fomentarius. The results demonstrated that the addition of Na2SeO3 increased the antibacterial activity against S.aureus (60%) and antioxidant activity (17.4%) significantly (p < 0.05). The cytotoxicity of these exopolysaccharides against 5637, A549, and KYSE30 cancer cells was also significantly increased (p < 0.05), and the viability of EPS-Se treated 5637 cells reached less than 10% after 72 h. Finally, F. fomentarius Se-polysaccharides can be used as a dietary supplement.

Dedicate to commemorate of Prof. Lina Zhang

Dedicate to commemorate of Prof. Lina Zhang

Computational and NMR Conformational Analysis of Galactofuranoside Cycles Presented in Bacterial and Fungal Polysaccharide Antigens.

Unlike pyranoside cycles which are generally characterized by strictly defined conformational preferences, furanosides are flexible and may adopt a wide range of available conformations. During our previous studies, conformational changes of galactofuranoside cycles upon total sulfation were described computationally, using a simple Hartree–Fock (HF) method, and principal conformers of the 5-membered galactose ring were revealed. However, in the case of more complex disaccharide structures, it was found that this method and the widely applied DFT-B3LYP produced results that deviated from experimental evidence. In this study, other DFT functionals (PBE0 and double hybrid B2PLYP) along with RI-MP2 are employed to study the conformational behavior of the galactofuranoside ring. Reinvestigation of galactofuranosides with a lactic acid substituent at O-3 revealed that changes in the orientation of lactic acid residue at O-3 might induce conformational changes of the furanoside cycle. Such findings are important for further modeling of carbohydrate–protein interaction.

Fungal α-1,3-Glucan as a New Pathogen-Associated Molecular Pattern in the Insect Model Host Galleria mellonella.

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to β-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as β-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.

Fungal endophytes from medicinal plant Bletilla striata (Thunb.) Reichb. F. promote the host plant growth and phenolic accumulation

Endophytic fungi are capable of increasing the growth and secondary metabolite production of medicinal plants in an ecofriendly and sustainable manner. Although these responses have been demonstrated in many plant species, they are not well understood in the medicinal plant Bletilla striata (Thunb.) Reichb.F.. Co-culture of B.striata with three endofungal isolates was carried out to evaluate the effects of endophytic fungal interaction on the plant growth and accumulation of phenols. The plant growth-promoting traits of isolated fungi were investigated, and the isolates had the ability to produce indole-3-acetic acid (IAA), siderophore and cellulolytic enzyme. The fungal strain Byssochlamys spectabilis 1-N2 co-cultured with plantlets of B. striata significantly increased certain growth properties, including shoot length by 71.40%, pseudobulbs dry weight by 52.58%, number of new roots by 5.25-fold, pseudobulbs diameter by 30.94% and improved the total phenolic content of the medicinal part by 25.41%. The fungal polysaccharide elicitor prepared from B.spectabilis 1-N2 mycelium also altered the accumulation of total phenolic content at different time points. Polysaccharide elicitor treatment significantly enhanced the accumulation of nitric oxide (NO) and salicylic acid (SA), which are involved in the plant defence response and significantly upregulated the activities of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and tyrosine ammonia lyase (TAL). The findings of this study contribute to our understanding of B. striata and endophytic fungi interactions and provide vital information for the use of endophytic fungi to improve plant growth and active component accumulation in B.striata cultivation.

Immunomodulatory Effects of Different Edible Fungi Polysaccharide Complex on RAW264.7 Macrophages

Objective: The present study was carried on to assess the immunoregulatory effects of complexes obtained by any 3 polysaccharides from five kinds of different edible fungi polysaccharides, including Tremella fuciformis polysaccharide, Poriacocos polysaccharide, Lentinan, Hericium edodes polysaccharide and Dictyophora indusiata polysaccharide. Methods: Effects of different concentrations of complexes on the proliferation of RAW264.7 were explored using cell counting kit (CCK)-8, the levels of tumor necrosis factor (TNF)-α in the culture medium were detected after complexes treatment by enzyme-linked immunosorbent assay (ELISA). In addition, flow cytometry was employed to analyze the effect of complexes on RAW 264.7 phagocytic capacity. Results: Compared with normal control group, ten kinds of complexes exerted better positive effects on the proliferation of macrophages. Meanwhile, the complexes obviously enhanced phagocytic capacity as well as increased the TNF-α level. Furthermore, 10 µg/mL complex of Tremella fuciformis polysaccharide, Poriacocos polysaccharide and Dictyophora indusiata polysaccharide exhibited the best effect on the proliferation of macrophages, 160 µg/mL complex of Tremella fuciformis polysaccharide, Poriacocos polysaccharide and Lentinan showed the best effect on enhancing both secretion of TNF-α and phagocytic ability of RAW264.7. Conclusion: The complexes of polysaccharides from these five kinds of edible fungi displayed excellent effect on regulating immunity of macrophage RAW 264.7. The results could provide data reference for the development of immunomodulatory polysaccharide products.

Chitin-rich heteroglycan from Sporothrix schenckii sensu stricto potentiates fungal clearance in a mouse model of sporotrichosis and promotes macrophages phagocytosis

BackgroundFungal cell wall polysaccharides maintain the integrity of fungi and interact with host immune cells. The immunomodulation of fungal polysaccharides has been demonstrated in previous studies. However, the effect of chitin-rich heteroglycan extracted from Sporothrix schenckiisensu stricto on the immune response has not been investigated.ResultsIn this study, chitin-rich heteroglycan was extracted from S. schenckiisensu stricto, and immunomodulation was investigated via histopathological analysis of skin lesions in a mouse model of sporotrichosis and evaluation of the phagocytic function and cytokine secretion of macrophages in vitro. The results showed that the skin lesions regressed and granulomatous inflammation was reduced in infected mice within 5 weeks. Moreover, heteroglycan promoted the fungal phagocytosis by macrophages and modulated the cytokine secretion. Heteroglycan upregulated TNF-α expression early at 24 h and IL-12 expression late at 72 h after incubation, which might result from moderate activation of macrophages and contribute to the subsequent adaptive immune response.ConclusionsChitin-rich heteroglycan extracted from S. schenckiisensu stricto potentiated fungal clearance in a mouse model of sporotrichosis. Moreover, chitin-rich heteroglycan promoted fungus phagocytosis by macrophages and modulated cytokines secretion. These results might indicate that chitin-rich heteroglycan could be considered as an immunomodulator used in the treatment of sporotrichosis.

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin-carbohydrate complexes during fungal degradation of lignocellulose.

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin-carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.

Natural microbial polysaccharides as effective factors for modification of the catalytic properties of fungal cellobiose dehydrogenase

Polysaccharides are biopolymers composed of simple sugars like glucose, galactose, mannose, fructose, etc. The major natural sources for the production of polysaccharides include plants and microorganisms. In the present work, four bacterial and two fungal polysaccharides (PS or EPS) were used for the modification and preservation of Pycnoporus sanguineus cellobiose dehydrogenase (CDH) activity. It was found that the presence of polysaccharide preparations clearly enhanced the stability of cellobiose dehydrogenase compared to the control value (4 °C). The highest stabilization effect was observed for CDH modified with Rh110EPS. Changes in the optimum pH in the samples of CDH incubated with the chosen polysaccharide modifiers were evidenced as well. The most significant effect was observed for Rh24EPS and Cu139PS (pH 3.5). Cyclic voltammetry used for the analysis of electrochemical parameters of modified CDH showed the highest peak values after 30 days of incubation with polysaccharides at 4 °C. In summary, natural polysaccharides seem to be an effective biotechnological tool for the modification of CDH activity to increase the possibilities of its practical applications in many fields of industry.

Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

BackgroundLytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry.ResultsWe have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes.ConclusionThe work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

Derivation of an occupational exposure limit for β-glucans

β-Glucans are abundant bacterial, yeast, and fungal cell wall polysaccharides that have been shown to activate the immune system. Establishment of an occupational exposure limit (OEL) for β-glucan exposure is critical to the protection of worker health, as these exposures have been linked to immunosuppressive and inflammatory reactions and possibly the development of respiratory diseases. Detectable concentrations of β-glucans have been identified in common occupational inhalation exposure scenarios, such as in the agricultural and waste management sectors. However, no published exposure benchmarks for inhalation of β-glucans are available for workers or the general population. Thus, a health-based OEL for inhalation exposure of workers to β-glucans was derived based on consideration of human and non-human effect data for this class of compounds and contemporary risk assessment methods. The weight of the evidence indicated that the available data in humans showed significant methodological limitations, such as lack of a representative study size, appropriate control population, and clear dose-response relationship. Thus, an OEL of 150 ng/m3 was derived for β-glucans based on the most relevant nonclinical study. This OEL provides an input to the occupational risk assessment process, allows for comparisons to worker exposure, and can guide risk management and exposure control decisions.

Vitamin D and Beta-Glucans Synergically Stimulate Human Macrophage Activity.

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.

Isolation, structure characteristics and antioxidant activity of two water-soluble polysaccharides from Lenzites betulina

BackgroundFungal polysaccharides belong to a very important class of biological macromolecules in nature, and have complex monosaccharide composition and structure. These studies on structure and biological activity of fungal polysaccharides have become one of the research hotspots of scholars at home and abroad.ResultsThis study was performed in order to understand the structural characteristics and antioxidant activity of polysaccharides from Lenzites betulina (LBPs). The LBPs were deproteinized using sevag method, and further purified by DEAE cellulose-52 column and Sephadex G-100 column chromatographies, then the two refined polysaccharides were obtained and named LBPs-5 and LBPs-6. Fourier transform infrared spectrometry (FT-IR) showed that LBPs-5 and LBPs-6 are typical β-pyranose with characteristic peaks of polysaccharides. The molecular weight of the two water-soluble polysaccharides were estimated to be 3.235 × 103 Da and 6.196 × 103 Da by HPGPC, respectively. HPLC with PMP derivatization analysis indicated that the monosaccharide compositions of LBPs-5 were mannose, glucuronic acid, glucose, and galactose in a molar ratio of 0.05:0.15:0.76:0.04. The monosaccharide compositions of LBPs-6 were mannose, glucuronic acid, and glucose, in a molar ratio of 0.04:0.17:0.79. Furthermore, the two water-soluble polysaccharides demonstrated strong scavenging effects on DPPH·, ABTS·+, ·OH and weak total reducing power, especially LBPs-6 was significantly stronger in scavenging rate than that of LBPs-5.ConclusionsThe outcome of the study indicated that LBPs had good potential as medicine and food.

Comparison of Polysaccharides Extracted from Cultivated Mycelium of Inonotus obliquus with Polysaccharide Fractions Obtained from Sterile Conk (Chaga) and Birch Heart Rot.

The polysaccharides of the sterile conk of Inonotus obliquus (Chaga) have demonstrated multiple bioactivities. The mycelium of this basidiomycete, obtained after submerged cultivation, has been considered a feasible alternative to the sterile conk for the production of polysaccharides. However, previous research has paid little attention to the differences in the structures of polymers obtained from the different resources. Moreover, the birch wood colonized by I. obliquus has never been investigated as a source of bioactive polysaccharides. In the present study, polysaccharide fractions produced from cultivated mycelium, sterile conks of different geographical origins, and birch heart rot were investigated. High amounts of phenolic compounds, possibly lignans, were bound to the sterile conk polysaccharides. Mycelial polysaccharides were rich in α- and β-glucans and had high (105 Da) and low (104 Da) molecular weight populations. On the other hand, sterile conk polysaccharides were mainly β-glucan of lower and monodispersed molecular weight (103 Da). Heart rot polysaccharides were comprised mainly of low molecular weight (103 Da) hemicelluloses. Nevertheless, fungal polysaccharides were identified in the extracts. The differences in structure and molecular properties among the polysaccharide fractions of mycelium, heart rot, and sterile conk are likely associated with differences in bioactivities and, therefore, in nutraceutical potential.

Fungal Polysaccharides Promote Protective Immunity.

Fungal pathogens represent a rising threat against immunocompromised patients. By using Aspergillus fumigatus, Briard et al. showed that the cell wall galactosaminogalactan (GAG) triggers macrophage inflammasome activation, promoting protective immunity. This provides new insights into the role of GAG in host-pathogen interactions and also perspectives for developing GAG-based anti-inflammatory therapeutics.

Investigation of the possible mechanism of polysaccharides extracted from Leucocalocybe mongolica in exerting antitumor effects in H22 tumor-bearing mice.

A response surface method was used to optimize the extraction of polysaccharides from Leucocalocybe mongolica. Moreover, the preliminary structural characteristics and antitumor activity of L. mongolica polysaccharide (LMP) were investigated. The results showed that the optimized extraction technological parameters for LMP were 93°C extraction temperature, 5hr extraction time, and 30ml/g liquid-to-solid ratio. The LMP content extracted under the optimal conditions was 6.64%. LC-MS/MS results indicated that LMP is a neutral polysaccharide composed of d-fructose, d-mannose, dextrose anhydrate, d-xylose, trehalose, and galactose. The tumor inhibition rate was significantly improved by LMP treatment. LMP had minimal toxicity based on the significant decrease in AST and BUN levels; VEGF protein levels were also significantly decreased. In contrast, the levels of IFN-γ, IL-2, IL-6, and TNF-α were improved. The results of ELISA, H&E staining, TUNEL assay, immunohistochemistry, and western blotting indicated that the LMP exhibited antitumor activity in vivo by promoting apoptosis, mediating inflammatory responses, and inhibiting angiogenesis. PRACTICAL APPLICATIONS: As one of the main bioactive components, fungal polysaccharide has always been a hot research topic. Fungal polysaccharides are carbohydrate polymers composed of monosaccharide units bound together by glycosidic linkages, which have been found to be involved in many biological processes. In this research, the LMP structure was analyzed, and the immunohistochemical and western blot analysis confirmed that, LMP could effectively reduce the generation of tumor angiogenesis, promote apoptosis of tumor cell sand inhibit tumor growth. The results of this study can effectively provide a basis for clinical research and development of antitumor drugs, and lay a foundation for the study of the antitumor effects of wild edible and medicinal fungi.

Characterization of biomass-degrading enzymes using neutron diffraction and scattering

Efficient enzymatic degradation of cellulose is essential to the low-cost production of cellulosic ethanol and high-value-added chemicals. Fungal lytic polysaccharide monooxygenases (LPMOs), which ...

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single ‘priming’ electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.

Enzymatic-fingerprinting workflow of polysaccharides in Hericium erinaceus fruiting bodies: From HILIC-ESI−-MS screening to targeted MIM profiling

In this study, an uncommon enzymatic-fingerprinting workflow, was proposed for characterization and discrimination of mushroom polysaccharides (MPs) by hydrophilic interaction liquid chromatography-negative electrospray mass spectrometry (HILIC-ESI–-MS). Firstly, the HILIC-ESI–-MS was used to screen and identify the enzymatic digestion products of MPs using HILIC-Orbitrap based on full scan and MS/MS modes. Secondly, a targeted structural-fingerprinting of polysaccharides (SFP) was built in a multiple-ion monitoring (MIM) mode using the same HILIC separation with a triple quadrupole MS. Thirdly, a case study of polysaccharides in Hericium erinaceus fruiting bodies (HEP) was performed to obtain the expected SFP based on dextranase digestion that allows for visual discrimination of polysaccharides from other five edible mushrooms attributed to Agrocybe cylindracea, Arimillaria mellea, Flammulina velutipes, Pleurotus eryngii, and Lentinula edodes. Furthermore, a major structural backbone of HEP was unveiled by occurrence of → 6(Hex)1 → along with multiple possible substitutions including of terminal GalA, Fuc, acetyl, → 4Hex1 →, and → 3Hex1 →. Finally, the similarity analysis, hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) were performed to visualize various MPs. As a result, the enzymatic-fingerprinting workflow presents an effective example for quality evaluation of fungi polysaccharides using a SFP strategy.