Polysaccharides as Quality Marker to Rapid Profile for Ophiocordyceps sinensis by PXRD.

BACKGROUND: Ophiocordyceps sinensis has long been recognized as a mysterious and valuable traditional Chinese medicine but there has been little research on quality markers for O. sinensis. PURPOSE: This study looked into the potential of using powder X-ray diffractometry (PXRD) to analyze polysaccharides as a quality marker for O. sinensis. STUDY DESIGN: There were 16 different habitats of O. sinensis collected in Qinghai, Gansu, Sichuan, Yunnan, and Tibet. In addition, five different types of Cordyceps species were collected. The characteristic diffraction peaks of O. sinensis were determined and then matched with the characteristic diffraction peaks of intracellular polysaccharides obtained from O. sinensis to determine the attribution relationship of the characteristic diffraction peaks. METHODS: O. sinensis powder's X-ray diffraction pattern is determined by its composition, microcrystalline crystal structure, intramolecular bonding mechanism, and molecular configuration. After fractionation and alcohol precipitation of crude intracellular polysaccharide, mycelium crude intracellular polysaccharide (MCP) and fruiting body crude intracellular polysaccharide (FCP) were obtained and the fingerprint of O. sinensis was identified by the specific characteristic peaks of the X-ray diffraction pattern from intracellular polysaccharide. RESULTS: The results indicated that the PXRD patterns of different populations of O. sinensis were overlaid well with 18 characteristic diffraction peaks obtained by microcrystalline diffraction. Moreover, the powder diffractograms as a fingerprint provided a practical identification of O. sinensis from other Cordyceps species. In addition, we detected that the powder diffractograms of intracellular polysaccharide MCP and MCP75 could be coupled with the PXRD of O. sinensis. Specifically, 18 characteristic diffraction peaks were identified as coming from MCP and MCP75 according to those interplanar crystal spacing, which matched well with those of PXRD of O. sinensis. CONCLUSIONS: PXRD spectra combined with an updated multivariable discriminant model were found to be an efficient and sensitive method for O. sinensis quality control. According to the findings of this study, PXRD should be further investigated for quality control assessments and plant extract selection trials.

Cloning and characterization of phosphoglucose isomerase in Lentinula edodes.

Phosphoglucose isomerase (PGI) is a key enzyme that participates in polysaccharide synthesis, which is responsible for the interconversion of glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P), but there is little research focusing on its role in fungi, especially in higher basidiomycetes. The pgi gene was cloned from Lentinula edodes and named lepgi. Then, the lepgi-silenced strains were constructed by RNA interference. In this study, we found that lepgi-silenced strains had significantly less biomass than the wild-type (WT) strain. Furthermore, the extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) levels increased 1.5- to 3-fold and 1.5-fold, respectively, in lepgi-silenced strains. Moreover, the cell wall integrity in the silenced strains was also altered, which might be due to changes in the compounds and structure of the cell wall. The results showed that compared to WT, silencing lepgi led to a significant decrease of approximately 40% in the ß-1,3-glucan content, and there was a significant increase of 2-3-fold in the chitin content. These findings provide support for studying the biological functions of lepgi in L. edodes.

Effects of supplementation of sea buckthorn press cake on mycelium growth and polysaccharides of Inonotus obliquus in submerged cultivation.

AIMS: Investigation of the influence of cultivation time and sea buckthorn press cake (Hippophaë rhamnoides) dosage on mycelium yield of Inonotus obliquus in submerged cultivation and on the yield, monomer composition, and macromolecular properties of the exopolysaccharides (EPS) from culture media and intracellular polysaccharides (IPS) extracted from mycelia. METHODS AND RESULTS: Supplementation at 5 g l-1 combined with cultivation time of 250 h granted highest yield increase in mycelia (by 122%). The supplementation reduced extraction yield and decreased the molecular weight of the main IPS population. The supplementation increased production and molecular weight of EPS. The relative content of arabinose and rhamnose in EPS positively correlated with dosage of the press cake. The press cake supplementation increased the content of galacturonic acid in IPS, but not in EPS. CONCLUSION: Sea buckthorn press cake is a food industry fibrous side stream with high oil content. It increases the cultivation yield of Inonotus obliquus mycelium and influences the produced polysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycelium is a resource of bioactive polysaccharides, attracting the interest of nutraceutical companies. Sea buckthorn press cake is a promising supplement for increasing mycelium production. The utilization of this agricultural side stream would therefore favour circular economy.

[Study of mycelial polysaccharide from Paraisaria dubia of Ophiocordyceps gracilis asexual].

In order to provide a foundation for the development and application of Ophiocordyceps gracilis and increase the new resources of cordyceps,an asexual Paraisaria dubia was isolated from an O. gracilis fruit body. After 10 days of liquid fermentation,white globular mycelium and clear transparent fermentation were produced. The mycelium was extracted by hot water and precipitated with ethanol to obtain intracellular crude polysaccharide. The protein was deproteinized to obtain deproteinized polysaccharide. The intracellular pure polysaccharide was purified by Sepharose 4 B column chromatography and were analyzed by UV,IR,1 H-NMR,and13 CNMR data,as well as GC and HPLC. The results showed that the intracellular polysaccharide of P. dubia was composed of glucose,galactose and mannose with a molar ratio of 25. 54 ∶2 ∶1. It was a ß-configuration glycosylic bond,containing pyranoside. The initial connection of polysaccharide was ß(1→2)(1→4)(1→6) connection. This experiment provides a theoretical basis for the development and application of P. dubia.

Ganoderma lucidum phosphoglucomutase is required for hyphal growth, polysaccharide production, and cell wall integrity.

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of ß-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.

Structure of rhamnoglucan, an unexpected alkali-stable polysaccharide extracted from Streptococcus mutans cell wall.

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.

Intracellular Polysaccharides of Eurotium cristatum Exhibited Anticolitis Effects in Association with Gut Tryptophan Metabolism.

This study is to investigate the protective effects of Eurotium cristatum intracellular polysaccharides (ECIP) on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC). The oral administration of ECIP could downregulate the disease activity index (DAI) and ameliorate the colonic shortening, immune stress, and damage caused by DSS. In addition, ECIP treatment increased the colonic contents of SCFAs including acetic, propionic, and butyric acids in UC mice. Targeted and untargeted metabolic analysis suggested that ECIP dramatically altered the tryptophan metabolism in the feces of UC mice and promoted the conversion of tryptophan into indole metabolites including indolepyruvate and indole-3-acetic acid (IAA) and indolealdehyde (IAId). Moreover, ECIP observably increased the content of colonic IL-22 and stimulated the relative concentration and relative expression of tight junction molecules in mRNA and proteins levels. Conclusively, consumption of ECIP can improve colon damage and its related effects of UC by promoting the production of IAA and IAId to reinforce intestinal barriers.

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.

Preparation and Characterization of Intracellular and Exopolysaccharides during Cycle Cultivation of Spirulina platensis.

The dried cell weight (DCW) of Spirulina platensis gradually decreased from 1.52 g/L to 1.18 g/L after five cultivation cycles. Intracellular polysaccharide (IPS) and exopolysaccharide (EPS) content both increased with increased cycle number and duration. IPS content was higher than EPS content. Maximum IPS yield (60.61 mg/g) using thermal high-pressure homogenization was achieved after three homogenization cycles at 60 MPa and an S/I ratio of 1:30. IPS showed a more fibrous, porous, and looser structure, and had a higher glucose content and Mw (272.85 kDa) compared with EPS, which may be indicative of IPS's higher viscosity and water holding capacity. Although both carbohydrates were acidic, EPS had stronger acidity and thermal stability than IPS; this was accompanied by differences in monosaccharide. IPS exhibited the highest DPPH (EC50 = 1.77 mg/mL) and ABTS (EC50 = 0.12 mg/mL) radical scavenging capacity, in line with IPS's higher total phenol content, while simultaneously showing the lowest HO• scavenging and ferrous ion chelating capacities; thus characterizing IPS as a superior antioxidant and EPS as a stronger metal ion chelator.

Physicochemical characterization, digestion profile and gut microbiota regulation activity of intracellular polysaccharides from Chlorella zofingiensis.

A number of studies have shown that the polysaccharides from microalgae exhibit diverse biological activities, however, little is known about their digestibility and impact on human gut microbiota. In this study, a simulating digestion and fermentation system were established to investigate the digestibility and fermentation of intracellular polysaccharides from Chlorella zofingiensis (CZIP-S3). The results indicated that CZIP-S3 is a macromolecular polysaccharide composed of mannose, glucose, galactose and rhamnose, consisting of a main chain and two branched repeating units. CZIP-S3 could not be digested in the upper gastrointestinal tract. However, CZIP-S3 could be metabolized into smaller molecules by the gut microbiota. The pH values continuously decrease during fermentation, whereas, the amount of short-chain fatty acids steadily increase. Furthermore, CZIP-S3 could modulate the composition of gut microbiota, via lowering the ratio of Firmicutes/Bacteroidetes and increasing the relative abundance of Bacteroides, Bifidobacterium and Akkermansia. The data suggested that CZIP-S3 could potentially be used as an ingredient for functional foods or prebiotics to improve human health by promoting the relative abundances of beneficial bacteria.

Mycelial biomass and intracellular polysaccharides production, characterization, and activities in Auricularia auricula-judae cultured with different carbon sources.

The carbon source, an essential factor for submerged culture, affects fungal polysaccharides production, structures, and activities. This study investigated the impact of carbon sources, including glucose, fructose, sucrose, and mannose, on mycelial biomass and the production, structural characterization, and bioactivities of intracellular polysaccharides (IPS) produced by submerged culture of Auricularia auricula-judae. Results showed that mycelial biomass and IPS production varied with different carbon sources, where using glucose as the carbon source produced the highest mycelial biomass (17.22 ± 0.29 g/L) and IPS (1.62 ± 0.04 g/L). Additionally, carbon sources were found to affect the molecular weight (Mw) distributions, monosaccharide compositions, structural characterization, and activities of IPSs. IPS produced with glucose as the carbon source exhibited the best in vitro antioxidant activities and had the strongest protection against alloxan-damaged islet cells. Correlation analysis revealed that Mw correlated positively with mycelial biomass (r = 0.97) and IPS yield (r = 1.00), while IPS antioxidant activities correlated positively with Mw and negatively with mannose content; the protective activity of IPS was positively related to its reducing power. These findings indicate a critical structure-function relationship for IPS and lay the foundation for utilizing liquid-fermented A. aruicula-judae mycelia and the IPS in functional food production.

Immunomodulatory effect of intracellular polysaccharide from mycelia of Agaricus bitorquis (QuéL.) Sacc. Chaidam by TLR4-mediated MyD88 dependent signaling pathway.

Agaricus bitorquis (QuéL.) Sacc. Chaidam is a valuable edible fungus in Qinghai-Tibet plateau and ABSP is a novel intracellular polysaccharide from its mycelia. GC and NMR analysis determined ABSP is galactoglucomannan-like polysaccharide that may have immunomodulatory effect. This study used RAW264.7 as model cell to determine immunomodulatory effect of ABSP. After ABSP treatment, viability and phagocytic ability promoted, and NO, ROS, TNF-α levels also raised which proved ABSP had immune regulation to RAW264.7. WB and qRT-PCR determined the key proteins and genes expression of TLR4, MyD88, TRAF-6 and NF-κB significantly increased while protein and gene expression of TRAM had no significant increase. Also, TNF-α level extremely decreased by adding inhibitors of TLR4 and MyD88 which confirmed ABSP could immunologically regulate RAW264.7 byTLR4-MyD88 dependent pathway. This study would provide theoretical basis for further study on ABSP and be helpful for development of beneficial functionally foods and exploitation of this resource.

Structural characterization and immunomodulatory activity of intracellular polysaccharide from the mycelium of Paecilomyces cicadae TJJ1213.

Two intracellular polysaccharide fractions (IPS1 and IPS2) were obtained from the mycelium of Paecilomyces cicadae TJJ1213, and the structures were conducted. Results showed that they were homogenous with the average molecular weight of 2.40 × 106 Da and 6.79 × 105 Da. Two fractions were composed of mannose, glucose and galactose with molar ratios of 1.35: 6.93: 1.0 and 2.04: 1.0: 1.87, respectively. The backbone of IPS1 was â†’ 4)-α-D-Glcp (1 â†’ and â†’ 3,4)-α-D-Manp (1 â†’ residues with a side chain consisted of T-α-D-Galp. IPS2 was consisted of â†’ 4)-α-D-Glcp-(1→, →3,4)-α-D-Manp-(1 â†’ and â†’ 2,6)-α-D-Manp-(1 â†’ residues and the branches were also consisted of T-α-D-Galp. In addition, the scanning electron microscope and atomic force microscope images presented different features of IPS1 and IPS2, respectively. Furthermore, two fractions exhibited better immunomodulatory effects. They could markedly promote the proliferation of RAW264.7 cells and enhance phagocytosis, nitric oxide release and cytokines production. These results indicated that IPS1 and IPS2 had potential to enhance immune responses.

Optimisation of biomass, exopolysaccharide and intracellular polysaccharide production from the mycelium of an identified Ganoderma lucidum strain QRS 5120 using response surface methodology.

Wild-cultivated medicinal mushroom Ganoderma lucidum was morphologically identified and sequenced using phylogenetic software. In submerged-liquid fermentation (SLF), biomass, exopolysaccharide (EPS) and intracellular polysaccharide (IPS) production of the identified G. lucidum was optimised based on initial pH, starting glucose concentration and agitation rate parameters using response surface methodology (RSM). Molecularly, the G. lucidum strain QRS 5120 generated 637 base pairs, which was commensurate with related Ganoderma species. In RSM, by applying central composite design (CCD), a polynomial model was fitted to the experimental data and was found to be significant in all parameters investigated. The strongest effect (p < 0.0001) was observed for initial pH for biomass, EPS and IPS production, while agitation showed a significant value (p < 0.005) for biomass. By applying the optimized conditions, the model was validated and generated 5.12 g/L of biomass (initial pH 4.01, 32.09 g/L of glucose and 102 rpm), 2.49 g/L EPS (initial pH 4, 24.25 g/L of glucose and 110 rpm) and 1.52 g/L of IPS (and initial pH 4, 40.43 g/L of glucose, 103 rpm) in 500 mL shake flask fermentation. The optimized parameters can be upscaled for efficient biomass, EPS and IPS production using G. lucidum.

Effects of Tween 80 on the liquid fermentation of Lentinus edodes.

This paper explored the effects of Tween 80 on the biomass, intracellular polysaccharide (IPS) content, fermentation parameters, the pellets size of mycelium, and the antioxidant activity of IPS in Lentinus edodes liquid fermentation. With adding to Tween 80, the outputs of biomass and IPS increased during the L. edodes fermentation, respectively, while the reducing sugar content was decreased, as well as, the time courses of pH value were different. It was also shown that the addition of Tween 80 could protect the intact of pellets from breaking down. The effects of Tween 80 on the main structure of IPS were no obvious, and the IPS were revealed similar infrared spectrum, as was indicated by the infrared spectrum analysis. Improvements in the scavenging capacity of DPPH radicals of IPS were observed in Tween 80 treated group compared with the control group. Tween 80 exerts impacts on the liquid fermentation of L. edodes.

Characterization and antioxidant activities of extracellular and intracellular polysaccharides from Fomitopsis pinicola.

Fomitopsis pinicola (F. pinicola) is a kind of medicinal fungi, and few studies has been carried out on F. pinicola polysaccharides from liquid submerged cultivation. The characterization and antioxidant activities of extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) isolated from F. pinicola were investigated. The results showed that the molecular weight of EPS was 2.30×10(4)Da, and EPS was composed of mannose, rhamnose, xylose and galactose with the molar ratio of 0.1:1.0:0.3:0.5. The molecular weight of IPS was 4.07×10(5)Da, and the monosaccharide compositions included glucose, mannose, rhamnose, xylose and galactose with the molar ratio of 1.0:0.9:0.9:0.8:1.1. Antioxidant activities of both EPS and IPS including in vitro scavenging activities on 1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals, cellular protective effects on yeast cells from ultraviolet (UV) radiation and H2O2 oxidative damage were tested. Both EPS and IPS showed antioxidant activities in a dose dependent manner, and IPS had higher antioxidant activity than EPS. So EPS and IPS could be potential novel antioxidants for functional food.

Antioxidant and hepatoprotective activities of intracellular polysaccharide from Pleurotus eryngii SI-04.

In present study, the intracellular polysaccharide (IPS) and its two fractions of IPS-1 and IPS-2 were obtained and purified by DEAE-52 cellulose chromatography from Pleurotus eryngii SI-04 mycelia, and their hepatoprotective effects were also investigated. The results showed that the IPS-2 had superior hepatoprotective effects by increasing the serum enzyme activities and bilirubin (BIL) levels, decreasing the serum albumin (ALB) and triglyceride (TG) levels, improving the hepatic antioxidant status, and ameliorating the hepatic structure damage. Furthermore, the monosaccharide composition and main bond types were also analyzed. These conclusions demonstrated that the both IPS and its fractions might be suitable for functional foods and natural drugs in preventing the acute liver damage.

Properties of a glycogen like polysaccharide produced by a mutant of Escherichia coli lacking glycogen synthase and maltodextrin phosphorylase.

Escherichia coli mutant TBP38 lacks glycogen synthase (GlgA) and maltodextrin phosphorylase (MalP). When grown on maltose in fed-batch fermentation TBP38 accumulated more than 50-fold higher glycogen-type polysaccharide than its parental strain. The polysaccharides were extracted at different growth stages and migrated as one peak in size-exclusion chromatography. TBP38 produced polysaccharides ranging 2.6 × 10(6)-4.6 × 10(6)Da. A ratio of short side-chains (DP ≦ 12) in the polysaccharides was greater than 50%, and number-average degree of polymerization varied from 9.8 to 8.4. The polysaccharides showed 70-290 times greater water-solubility than amylopectin. Km values using porcine and human pancreatic α-amylases with polysaccharides were 2- to 4-fold larger than that of amylopectin. kcat values were similar for both α-amylases. The TBP38 polysaccharides had 40-60% lower digestibility to amyloglucosidase than amylopectin. Intriguingly, the polysaccharides showed strong immunostimulating effects on mouse macrophage cell comparable to lipopolysaccharides. The lipopolysaccharide contamination levels were too low to account for this effect.

Enzymatic and acidic degradation effect on intracellular polysaccharide of Flammulina velutipes SF-08.

The intracellular polysaccharide (IPS) from Flammulina velutipes SF-08 mycelia was isolated and degraded by enzyme and acid. IPS and its derivative were purified by DEAE-52 cellulose chromatography, and five fractions were obtained. The structural features and antioxidant activities in vitro of the isolated fractions were evaluated. On the basis of chemical composition and antioxidant ability analyses, rhamnose as the main monosaccharide might contribute to the strongest antioxidant capacity. The in vivo results showed that IPS significantly enhanced the activities of anti-aging enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and reduced the content of lipid peroxidantion (LPO). These results suggested that IPS should be a potent natural polymer and can be developed to be novel functional food.

Study of mycelial polysaccharide from Paraisaria dubia of Ophiocordyceps gracilis asexual / 中国中药杂志

In order to provide a foundation for the development and application of Ophiocordyceps gracilis and increase the new resources of cordyceps,an asexual Paraisaria dubia was isolated from an O. gracilis fruit body. After 10 days of liquid fermentation,white globular mycelium and clear transparent fermentation were produced. The mycelium was extracted by hot water and precipitated with ethanol to obtain intracellular crude polysaccharide. The protein was deproteinized to obtain deproteinized polysaccharide. The intracellular pure polysaccharide was purified by Sepharose 4 B column chromatography and were analyzed by UV,IR,1 H-NMR,and13 CNMR data,as well as GC and HPLC. The results showed that the intracellular polysaccharide of P. dubia was composed of glucose,galactose and mannose with a molar ratio of 25. 54 ∶2 ∶1. It was a β-configuration glycosylic bond,containing pyranoside. The initial connection of polysaccharide was β(1→2)(1→4)(1→6) connection. This experiment provides a theoretical basis for the development and application of P. dubia.