Preparation and structural analysis of fucomannogalactan and ß-1,6-glucan from Grifola frondosa mycelium.

Introduction: Polysaccharides, key components present in Grifola frondosa, can be divided into those derived from fruiting bodies, mycelium, and fermentation broth based on their source. The structure of G. frondosa fruiting body-derived polysaccharides has been fully characterized. However, the structure of G. frondosa mycelium-derived polysaccharides remains to be elucidated. Methods: In this study, we obtained mycelia from G. frondosa by liquid fermentation and extracted them with water and alkaline solution. Then, the mycelia were isolated and purified to obtain homogeneity and systematically characterized by methylation and FT infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Results and discussion: Structural analysis showed that two neutral fractions (WGFP-N-a and AGFP-N-a1) have a common backbone composed of α-1,6-D-Me-Galp and α-1,6-D-Galp that were substituted at O-2 by 1,2-Manp, α-1,3-L-Fucp, and α-T-D-Manp and thus are identified as fucomannogalactans. WGFP-A-a, AGFP-A-b, and AGFP-A-c are ß-1,6-glucans with different molecular weights and are branched with ß-1,3-D-Glcp and T-D-Glcp at the O-3 of Glc. Our results provide important structural information about G. frondosa mycelium-derived polysaccharides and provide the basis for their further development and application.

Insight into the effect of rice-straw ash on enhancing the anaerobic digestion performance of high salinity organic wastewater.

Anaerobic digestion is an economic method for treating high salinity organic wastewater (HSOW), but performance enhancement is needed because of the inhibitory effect of high salinity. In this study, rice-straw ash (RSA) was applied to alleviate the inhibitory effect during HSOW anaerobic digestion. The results showed that, when the NaCl content increased from 0% to 3.0%, the methane production decreased by 87.35%, and the TOC removal rate decreased to 34.12%. As a K+ and alkalinity source, RSA addition enhanced the anaerobic digestion performance, and the optimal dosage was 0.88 g/L. Under this dosage, the methane production increased by 221.60%, and TOC removal rate reached 66.42% at 3.0% salinity. The addition of RSA increased the proportion of living cells in the high salinity environment, and enhanced the activity of key enzymes and electron transfer efficiency in the anaerobic digestion process. The addition of RSA with a dosage of 0.88 g/L promoted the accumulation of acetoclastic methanogen Methanothrix. The abundance of substrate transporters, ion transporters and electron transfer related functional genes were enriched, which might be key for promoting HSOW anaerobic digestion performance. The results also showed that RSA addition played an important role in maintaining the stability of the anaerobic digestion system, and it could be a potential strategy for enhancing the anaerobic digestion performance under high salinity conditions.

Enhancement of methane production in anaerobic digestion of high salinity organic wastewater: The synergistic effect of nano-magnetite and potassium ions.

During high salinity organic wastewater (HSOW) anaerobic digestion treatment, the process of methanogenesis can be severely inhibited in the high salinity environment, and the accumulation of volatile organic acids (VFAs) leads to failure of the anaerobic reaction. In this study, nano-magnetite and KCl were adopted to alleviate the inhibitory effect of high salinity and enhance the HSOW anaerobic digestion performance. The result showed that, under the optimal dosage of 200 mg/L, nano-magnetite addition promoted the anaerobic digestion performance, and the methane production increased by 11.06%. When KCl was added with a dosage of 0.174%, the methane production increased by 98.37%. The simultaneous addition of nano-magnetite (200 mg/L) and KCl showed a synergistic effect on enhancing HSOW anaerobic digestion performance, and the methane production increased by 124.85%. The addition of nano-magnetite and KCl promoted the conversion of VFAs, especially accelerated the degradation of propionic acid and butyric acid, also it promoted the activity of acetate kinase, dehydrogenase and F420, and thereby enhanced the methanogenesis process. This study could provide a new method for enhancing the anaerobic digestion of HSOW.

Heterogalactan WPEP-N-b from Pleurotus eryngii enhances immunity in immunocompromised mice.

This study reports on in vivo immunomodulatory activities mediated by WPEP-N-b, a heterogalactan from Pleurotus eryngii. Using cyclophosphamide (CTX)-induced immunosuppressed mice, we demonstrate here that WPEP-N-b enhances immunity as determined by the immune organ index, peripheral blood immune cell content, splenocyte proliferation, NK cell activity and T lymphocyte subpopulations. WPEP-N-b prevented apoptosis of bone marrow cells induced by CTX. The level of cytokines (i.e. TNF-α, IL-6 and IL-1ß) and macrophage activity in these immunocompromised mice were restored upon treated with WPEP-N-b. Mechanistically, it appears that WPEP-N-b enhances splenocyte proliferation and NK cell activity might through the Toll-like receptor 4 (TLR4)-PKC signaling axis, and increases macrophage activity by activating JNK, p38 and NF-κB signaling pathways and Toll-like receptor 2 (TLR2) is the possible receptor of WPEP-N-b in macrophages. Our findings indicate that WPEP-N-b may function as a natural immune stimulant.

ß-1,6-Glucan From Pleurotus eryngii Modulates the Immunity and Gut Microbiota.

Polysaccharides from Pleurotus eryngii exhibit a variety of biological activities. Here, we obtained a homogeneous branched ß-1,6-glucan (APEP-A-b) from the fruiting bodies of P. eryngii and investigated its effect on immunity and gut microbiota. Our results showed that APEP-A-b significantly increases splenic lymphocyte proliferation, NK cell activity and phagocytic capacity of peritoneal cavity phagocytes. Furthermore, we found that the proportion of CD4+ and CD8+ T cells in lamina propria are significantly increased upon APEP-A-b treatment. Additionally, APEP-A-b supplementation demonstrated pronounced changes in microbiota reflected in promotion of relative abundances of species in the Lachnospiraceae and Rikenellaceae families. Consistently, APEP-A-b significantly increased the concentration of acetic and butyric acid in cecum contents. Overall, our results suggest that ß-1,6-glucan from P. eryngii might enhance immunity by modulating microbiota. These results are important for the processing and product development of P. eryngii derived polysaccharides.

Structure and antioxidant activity of six mushroom-derived heterogalactans.

Heterogalactans with weight-average molecular weights ~20 kDa were purified from several species of mushroom: Hypsizygus marmoreus, Pleurotus ostreatus, Pholiota nameko, Agrocybe cylindracea, Hygrophorus lucorum and Hericium erinaceus, and structurally characterized and assessed for antioxidant activity in vitro. Methylation analysis, combined with NMR spectral analysis, indicates that these glycans have a common backbone composed of (1 â†’ 6)-linked-α-D-galactopyranosyl residues that are substituted at O-2. The (1 â†’ 6)-α-D-galactans, branched primarily with ß-D-mannopyranosyl (Manp) or α-L-fucopyranosyl (Fucp) residues, have been assigned to mannogalactans or fucogalactans, respectively, as well as to ß-D-Manp and α-L-Fucp residues attached in tandem to the main chain as fucomannogalactans. In addition, 3-O-methylated-α-D-galactopyranosyl (3-O-Me-Galp) residues within the mannogalactan chains, exhibit strong reducing power and radical scavenging activity suggesting that this sugar moiety functions as an antioxidant. Our results provide important structural information on mushroom heterogalactans and prompt further investigations into their structure-activity relationships.

Bee Pollen Polysaccharide From Rosa rugosa Thunb. (Rosaceae) Promotes Pancreatic ß-Cell Proliferation and Insulin Secretion.

Insufficient pancreatic ß-cell or insulin-producing ß-cell are implicated in all types of diabetes mellitus. Our previous studies showed bee pollen polysaccharide RBPP-P improves insulin resistance in type 2 diabetic mice by inhibiting liver fat deposition. However, its potential of regulating ß-cell function and integrity is not fully known. Herein, we observed that ß-cell proliferation (n = 10), insulin synthesis (n = 5, p = 0.01684) and insulin incretion (n = 5, p = 0.02115) were intensely activated in MIN6 cells when treatment with RBPP-P. In alloxan-induced diabetic mice, oral administration of RBPP-P (n = 10) effectively decreased the blood glucose (p = 0.0326), drink intake (p < 0.001) and urine (p < 0.001). It directly stimulated phosphorylation of p38 (p = 0.00439), ERK (p = 0.02951) and AKT (p = 0.0072) to maintain the islet function and mass. Thus, our data suggest that RBPP-P is a natural compound to regulate ß-cell proliferation and function, indicating it might have therapeutic potential against type 1 diabetes.

Structural analysis and macrophage activation of a novel ß­glucan isolated from Cantharellus cibarius.

The present study was designed to investigate the structure and immunomodulatory activity of a polysaccharide. A novel acidic ß­glucan (WCCP­A­b; molecular weight, 7.3 kDa) was purified from the fruiting bodies of the edible mushroom Cantharellus cibarius, which possesses high nutritional values. WCCP­A­b was composed primarily of glucose (89.7%) and glucuronic acid (8.8%). Methylation and nuclear magnetic resonance analysis suggested that WCCP­A­b contained ß­D­1,6­glucan as its main chain, which was substituted at O­3 by ß­1,3­D­Glcp oligosaccharides or a single­unit of ß­Glcp residues. Minor ß­1,4­D­GlcpA residues may also be present in the side chains. The degree of branching was ~20.9%. Moreover, WCCP­A­b possessed a macrophage activating effect by promoting the secretion of nitric oxide, TNF­α and IL­6 in a dose­dependent manner. At a cellular mechanistic level, WCCP­A­b activated macrophages via the MAPK signaling pathway. The present results provided useful information for supporting further investigations on the structure­activity association of polysaccharides from C. cibarius, and indicated that the novel ß­glucan may be a potent natural immunomodulator, thus promoting the application of C. cibarius as a valuable source for functional food.

Hypoglycemic effects of polysaccharides from Gomphidiaceae rutilus fruiting bodies and their mechanisms.

Insulin resistance is the main cause of type 2 diabetes, fatty liver and obesity. Our previous study found that mushroom polysaccharides improved insulin resistance in vitro, but the underlying mechanisms were still unknown. Thus, we investigate the hypoglycemic effects of polysaccharides from Gomphidiaceae rutilus fruiting bodies and their mechanisms. The total polysaccharides (AGRP) from Gomphidiaceae rutilus fruiting bodies and the neutral polysaccharide (AGRP-N) fraction both enhance insulin-stimulated glucose uptake in an autophagy-dependent manner in high glucose and fatty acid-treated hepatic cells, but not the acidic polysaccharide (AGRP-A) fraction. Further, we elucidate the oral hypoglycemic effects of polysaccharides on ob/ob mice. AGRP and AGRP-N lower blood glucose and improve insulin sensitivity. They inhibit liver lipid deposition, not only by activating AMPK to increase autophagy but also by increasing the expressions of PPARα and CPT-1a to enhance lipolysis. Our results provide a basis for the development of polysaccharides from Gomphidiaceae rutilus as a hypoglycemic healthy food.

Galactan isolated from Cantharellus cibarius modulates antitumor immune response by converting tumor-associated macrophages toward M1-like phenotype.

Tumor-associated macrophages (TAMs) with an M2-like phenotype have been linked to the proliferation, invasion and metastasis of tumor cells. Resetting tumor-associated macrophages represents an attractive target for an effective cancer immunotherapy. WCCP-N-b, a novel linear 3-O-methylated galactan, isolated from Cantharellus cibarius, can convert tumor-promoting M2-like macrophages to tumor-inhibiting M1-like phenotype. On a cellular mechanistic level, WCCP-N-b inhibited M2-like macrophages polarization through suppression of STAT6 activation. Furthermore, WCCP-N-b increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and degradation of IκB-α through targeting Toll-like receptor 2 (TLR2). The activation of MAPKs and degradation of IκB-α were responsible for converting M2-like macrophages to M1-like macrophages. Importantly, cell culture supernatants of WCCP-N-b-treated M2-like macrophages could inhibit the cell viability of B16F1 and B16F10. Our findings provide a potential natural and harmless polysaccharide for macrophage-based tumor immunotherapy.

A novel linear 3-O-methylated galactan isolated from Cantharellus cibarius activates macrophages.

A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was isolated and purified from the fruiting bodies of Cantharellus cibarius. Monosaccharide composition, methylation analysis and NMR spectra indicated that WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b could significantly increase macrophage phagocytosis, release of NO and secretion of TNF-α, IL-6 and IL-1ß. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid. Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were significantly decreased along with the molecular weight decrease. Collectively, WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its molecular weight.

Analyses of active antioxidant polysaccharides from four edible mushrooms.

Four water-soluble polysaccharides were extracted from Pleurotus eryngii, Flammulina velutipes, Pleurotus ostreatus and white Hypsizygus marmoreus. Using anion exchange and gel permeation chromatography, a neutral and an acidic fraction were purified from each water-soluble polysaccharide. Their molecular weights were all around 20 kDa except that the acidic polysaccharide from Pleurotus ostreatus (named WPOPA) had a lower molecular weight of 5 kDa. Four neutral polysaccharides were mainly composed of galactose (42.7%-69.1%), followed by Man (19.4%-39.3%) and Glc (1.1%-15.9%). Four acidic polysaccharides contained glucose (59.0%-81.8%) as major sugar and minor glucuronic acid (4.5%-9.5%). Acidic polysaccharides exhibited stronger antioxidant activities than neutral fractions, and WPOPA showed the best antioxidant effects. Structural analysis indicated WPOPA had ß-(1 → 6)-glucan backbone branched at O-3 by ß-1,3-d-Glcp, t-ß-d-Glcp and t-ß-d-GlcpA. This investigation would be useful for screening natural antioxidants and significant in developing mushroom polysaccharides as functional foods.

Structure elucidation and immunomodulatory activity of a ß-glucan derived from the fruiting bodies of Amillariella mellea.

A novel polysaccharide AAMP-A70 (5.6kDa) has been purified from the fruiting bodies of Amillariella mellea. Compositional analysis and 1H and 13C NMR spectra indicate that AAMP-A70 is a branched ß-glucan with a main chain that consists of ß-d-(1→6) linked Glcρ residues substituted at O-3 by ß-Glcρ or α-d-(1→6)-linked Galρ side chains. AAMP-A70 increases macrophage phagocytosis and secretion of NO, ROS, TNF-α, IL-6 and IL-1ß. Mechanistically, AAMP-A70 promotes degradation of IκB-α and nuclear translocation of the NF-κB p65 subunit, and enhances phosphorylation of MAPKs. In particular, the function blocking antibody to TLR2 substantially suppresses TNF-α and IL-6 production. Our data demonstrate that AAMP-A70 activates macrophages via NF-κB/MAPK signaling pathways and the TLR2 receptor. Overall, AAMP-A70 may serve as a good food supplement to enhance immunity.

Layer-by-Layer self-assembled acetylcholinesterase/PAMAM-Au on CNTs modified electrode for sensing pesticides.

In this paper, an acetylcholinesterase (AChE)/dendrimers polyamidoamine (PAMAM)-Au/Carbon nanotubes (CNTs) multilayer modified electrode based on LbL self-assembled technique was employed in the detection of carbofuran in samples. The configuration of the nanostructure on the electrode provided a favorable environment to the immobilization of AChE. The modified films also improved the electrocatalytic characteristics and electron transfer speed between the films and the surface of electrode. The PAMAM-Au nanoparticles were characterized by SEM and UV-VIS methods. A set of experimental conditions were also optimized for the detection of the pesticides. A linear response over carbofuran concentration in the range of 4.8x10(-9)M to 0.9x10(-7)M was exhibited with a detection limit of 4.0x10(-9)M. The biosensor showed high sensitivity, good stability and reproducibility with promising application.

A novel biosensor based on photoelectro-synergistic catalysis for flow-injection analysis system/amperometric detection of organophosphorous pesticides.

In this study, a highly sensitive amperometric biosensor based on photoelectro-synergistic catalysis for detecting organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system has been developed. The acetylcholinesterase enzyme (AChE) was immobilized by adsorption into the nanostructured PbO2/TiO2/Ti, which also acted as the working electrode. This strategy was found to catalyze the oxidative reaction of thiocholine effectively, make the AChE/PbO2/TiO2/Ti biosensor detect the substrate at 0.30 V (vs. SCE), hundreds milli-volts lower than others reported. PbO2/TiO2/Ti and TiO2/Ti electrodes were prepared and investigated with atomic force microscope (AFM). Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, and stable response. A value of 1.34 mM for the apparent Michaelis-Menten constant (K(M)(app)) was obtained. A wide linear inhibition response for trichlorfon was observed in the range of 0.01-20 microM with the detection limit of 0.1 nM. The results using this biosensor agreed very well with chromatographic method and we also examined the real samples successfully in this work.

Au-TiO2/Chit modified sensor for electrochemical detection of trace organophosphates insecticides.

In this paper, Au-TiO2/Chit modified electrode was prepared with Au-TiO2 nanocomposite (Au-TiO2) and Chitosan (Chit) as a conjunct. The Au-TiO2 nanocomposite and the films were characterized by electrochemical and spectroscopy methods. A set of experimental conditions was also optimized for the film's fabrication. The electrochemical and electrocatalytic behaviors of Au-TiO2/Chit modified electrode to trace organophosphates (OPs) insecticides such as parathion were discussed in this work. By differential pulse voltammetry (DPV) measurement, the current responses of Au-TiO2/Chit modified electrode were linear with parathion concentration ranging from 1.0 ng/ml to 7.0 x 10(3)ng/ml with the detection limit of 0.5 ng/ml. In order to evaluate the performance of the detection system, we also examined the real samples successfully in this work. It exhibited a sensitive, rapid and easy-to-use method for the fast determination of trace OPs insecticides.

Electrochemistry and electrocatalytic properties of hemoglobin in layer-by-layer films of SiO2 with vapor-surface sol-gel deposition.

In this paper, layer-by-layer {Hb/SiO2}n films assembled by alternate adsorption of positively charged hemoglobin (Hb) and vapor-surface sol-gel deposition of silica at 50 degrees C onto a glassy carbon electrode were reported. The result films were characterized with cyclic voltametery, electrochemical impedance spectroscopy, UV-vis spectroscopy, and SEM, and the direct electrochemical and electrocatalytic properties of Hb in these layer-by-layer films were investigated. A pair of well-defined quasi-reversible cyclic voltammetric peaks were observed, and the formal potential of the heme FeIII/FeII redox couple was found to be -0.330 V(vs SCE). The electron-transfer behavior of Hb in {Hb/SiO2}n films was dependent on the vapor temperature, the number of layers, and the pH of the Hb solution, based on which a set of optimized conditions for film fabrication was inferred. The hemoglobin in{Hb/SiO2}n films displayed good electrocatalytic activity to the reduction of hydrogen peroxide, and H2O2 had linear current response from 1.0 x 10(-6) to 2.0 x 10(-4) M with a detection limit of 5.0 x 10(-7) M (S/N = 3). The apparent heterogeneous electron-transfer rate constant (ks) was 1.02 +/- 0.03 s(-1), and the apparent Michaeli-Menten constant (Kmapp) was 0.155 mM, indicating a potential application in the third-generation biosensor.