Intracellular Polysaccharide Content Research Articles

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.

Trehalose-6-phosphate synthase influences polysaccharide synthesis and cell wall components in Ganoderma lucidum.

Trehalose-6-phosphate synthase (TPS) is a key enzyme that participates in trehalose metabolism, which can synthesize trehalose in a two-step pathway with trehalose phosphatase, but its role in fungi is rarely studied, especially in large basidiomycetes. In this study, the tps gene of Ganoderma lucidum was cloned and named as gltps. And gltps-silenced strains were constructed by RNA interference. In this study, it is found that the extracellular polysaccharidecontent increased 1.6-2-fold, but there was no significant change on intracellular polysaccharidecontent in gltps-silenced strains compared with the wild-type (WT) strain. Furthermore, the cell wall compositions of the gltps-silenced strains were also altered, which showed that the chitin and β-1,3-glucan contents were significantly decreased. Compared with WT, the concentration of chitin decreased by 20%-50% and that of β-1, 3-glucan decreased by 15%-30%. The study found that the cells of gltps-silenced strains were more sensitive to cell wall stress, which might be due to changes in the compounds and structure of the cell wall. These results showed that gltps had an important effect on carbohydrate metabolism of G. lucidum cells.

Effect of the heme oxygenase gene on mycelial growth and polysaccharide synthesis in Ganoderma lucidum

The heme oxygenase gene has antioxidant and cytoprotective effects in organisms, but no related research has been conducted in Ganoderma lucidum. For the first time, we cloned the HMX1 gene in G. lucidum. The CDS is 1092 bp in length and encodes 363 amino acids. The HMX1 protein was prokaryotically expressed and purified, and the enzyme activity of the purified protein was measured. The value of Km was 0.699 μM, and Vm was 81.9 nmol BV h-1 nmol-1 protein. By constructing the silencing vector pAN7-dual-HMX1i, the transformants HMX1i1 and HMX1i2 were obtained. Compared with the wild-type (WT), the average growth rate of HMX1i1 and HMX1i2 decreased by 31% and 23%, respectively, and the mycelium biomass decreased by 53% and 48%, respectively. Compared with the WT, the extracellular polysaccharide content of HMX1i1 and HMX1i2 increased by 59% and 51%, and the intracellular polysaccharide content increased by 24% and 22%, respectively. These results indicate that the HMX1 gene affects mycelial growth and polysaccharide synthesis in G. lucidum.

Effect of sodium and calcium on polysaccharide production and the activities of enzymes involved in the polysaccharide synthesis of Lentinus edodes

Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca2+ and Na+ on L. edodes lentinan content were investigated. Metal ion concentrations and induction times were optimized according to mycelial biomass, and intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and total polysaccharide (TPS) content. The activities and gene expression of phospho-glucose isomerase (PGI), phosphoglucomutase (PGM), and UDP-glcpyrophosphorylase (UGP) were also measured. Ca2+ and Na+ concentration and induction time affected biomass, IPS, and EPS concentrations. Na+ increased EPS, IPS and TPS, while Ca2+ increased biomass, IPS, and TPS. During fermentation, mycelial biomass varied greatly under Ca2+ induction, while IPS, EPS and TPS varied greatly under Na+ induction. PGM and UGP activities increased in the presence of Na+, while PGI increased with Ca2+. Compared to control samples, pgi and pgm expression under Na+ was greater at days 45 and 60, respectively, while under Ca2+, ugp expression was greater at day 45. IPS content correlated significantly with enzyme activity, while EPS correlated with PGM activity. Our data contributes to better understanding how Na+ and Ca2+ affect mycelial growth and secondary metabolite production, and of polysaccharide biosynthesis mechanisms of L. edodes.

Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom Coprinopsis cinerea.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene

Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4mg/100mg dry weight and 0.83g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation.

Enhanced Production of Polysaccharide Through the Overexpression of Homologous Uridine Diphosphate Glucose Pyrophosphorylase Gene in a Submerged Culture of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes).

This study aimed to improve polysaccharide production by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of the homologous UDP glucose pyrophosphorylase (UGP) gene. The effects of UGP gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production, and transcription levels of 3 genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), UGP, and α-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the UGP gene were 24.32 mg/100 mg dry weight and 1.66 g/L, respectively, which were higher by 42% and 36% than those of the wild-type strain. The transcription levels of PGM, UGP, and GLS were up-regulated by 1.6, 2.6, and 2.4-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous α-phosphoglucomutase gene.

This study aimed to improve the production of polysaccharide by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of α-phosphoglucomutase (PGM) gene. PGM is responsible for the linkage between sugar catabolism and sugar anabolism. The effects of PGM gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including PGM, UDP-glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the PGM gene were 23.67 mg/100 mg dry weight and 1.76 g/L, respectively, which were higher by 40.5 and 44.3% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were upregulated by 4.77-, 1.51- and 1.53-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Optimization of fermentation process for the production of intracellular polysaccharide from Paecilomyces cicadae and the immuno-stimulating activity of intracellular polysaccharide

Optimization of fermentation process for the production of intracellular polysaccharide (IPS) from the fungus Paecilomyces cicadae and the immuno-stimulating activity of IPS were carried out. The quantitative effects of initial pH, fermentation temperature and time on the yield of IPS content produced by P. cicadae in submerged fermentation were investigated separately using response surface methodology (RSM). The three factors chosen for the present investigation were based on the results of a previous Plackett-Burman (PB) design. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. RSM analysis showed good correspondence between experimental and predicted values. It was found that three parameters represented significant effect. Probability value (p < 0.0001) demonstrated a very high significance for the regression model. By solving the regression equation and analyzing the response surface contour plots, the optimal process parameters were determined, i.e. fermentation temperature 24.53 °C, initial pH 7.46 and fermentation time 73.9 h. The maximum predicted yield of IPS was 356.02 μg/ml under the optimal conditions. Meanwhile, IPS from P. cicadae was found to have direct immuno-stimulating activity in vitro on murine macrophage RAW264.7 proliferative response and to stimulate nitric oxide generation in a dose-dependent manner.

Medium modification to enhance the formation of bioactive metabolites in shake flask cultures of Antrodia cinnamomea by adding citrus peel extract

Antrodia cinnamomea has recently become a well-known medicinal mushroom in Taiwan. Bioactive compounds found in A. cinnamomea include: polysaccharide, sesquiterpene lactone, steroids and triterpenoids. The aim of this study was to evaluate the feasibility of adding citrus peel extract to enhance the formation of bioactive metabolites in the submerged culture of A. cinnamomea. With the exception of grapefruit, citrus peel extracts tested were proved to be beneficial to mycelial growth and to the production of intracellular polysaccharide. Lemon was the most effective for enhancing bioactive metabolite production. With an addition of 2% (v/v), the mycelium biomass concentration and intracellular polysaccharide content rose from 11.96g DW/L of the control and 123.6mg/g DW to 21.96g DW/L and 230.8mg/g DW, respectively, on day 8. The production of triterpenoids also increased from 86.7 to 282.9mg/L. Moreover, this study also demonstrates that although the addition of peel extract could cause the lengthening of the exponential phase and reduce the specific growth rate, the production rate of biomass, intracellular polysaccharide and triterpenoids was still enhanced significantly.

Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea

Effects of carbon sources, nitrogen sources, plants oils and oxygen supply on the cell growth and production of bioactive metabolites such as exopolysaccharide (EPS), intracellular polysaccharide (IPS) and triterpenoid in the submerged culture of Antrodia cinnamomea CCRC36716 were studied in detail. Malt extract (ME), yeast extract (YE) and corn steep powder (CSP) were favorable nitrogen sources to the mycelial growth. The highest cell growth (12.52 ± 0.03 g/l cell dry weight), EPS production (1861 ± 62 mg/l) and IPS content (41 ± 12 mg/g DW) can be obtained on day 10 of cultivation in the medium containing 3% CSP, 3% YE, and 3% ME, respectively. The highest overall triterpenoid production (30 mg/g DW) was obtained in 3% CSP medium after 14 d of cultivation. Amongst five carbon sources examined, maltose and glucose yielded relatively high mycelial biomass; high cell densities of 8.29 ± 0.05 and 8.69 ± 0.05 g DW cells/l were obtained after 10 d of cultivation when 4% of maltose and glucose was used, respectively. Pronounced production of EPS, 1482 ± 63 and 1318 ± 48 mg/l, was obtained after 10 d of cultivation when 4% of lactose and sucrose was used, respectively. The maximum IPS content (49 ± 9 mg/g DW) was achieved in 4% glucose medium after 10 d of cultivation; the highest overall triterpenoid production (31 mg/g DW) was obtained in 2% glucose medium after 14 d of cultivation. All plant oils tested stimulated cell growth of A. cinnamomea, enhanced the production of IPS, but inhibited the triterpenoid production. EPS production was slightly inhibited with soy oil but enhanced by the other oils tested, and the maximal EPS production (1147 ± 47 mg/l) was obtained when 0.5% of peanut oil was supplemented. The high O 2 supply in the A. cinnamomea culture was favorable for cell growth and polysaccharide production, but was inhibitory on the triterpenoid production. The results obtained are useful in regulation and optimization of A. cinnamomea culture for efficient production of cell mass and bioactive metabolites such as EPS, IPS and tripterpenoids in the submerged culture.

Implementation of glycogen accumulating bacteria in treating nutrient-deficient wastewater

Activated sludge treatment of nutrient-deficient wastes may lead to severe slime formation and consequent biomass separation difficulties. The purpose of this paper has been to show that bioreactor arrangement essentially influences the manner of biological excess carbon removal. In a comparative lab-scale experiment two differently arranged activated sludge systems were operated simultaneously: an aerated CSTR with an aerobic selector and an aerated CSTR with an anaerobic selector. The seed derived from an anaerobic/aerobic activated sludge plant of a winery. The model wastewater contained wine, sugar and acetic acid as organic carbon sources and lacked nutrients regarding both N and P, similarly to the influent of the full-scale plant. During the 52 days of the experiment the SVI values of the fully aerated system increased up to 600-800 cm3 g(-1) whereas those of the anaerobic/aerobic system remained below 250 cm3 g(-1) The SVI values showed a strict correlation with the amount of extracellular polysaccharides. In the anaerobic/aerobic experimental system, the high (40% of MLSS) intracellular polysaccharide content of the seed could be maintained. Besides the analytical data, also the microscopic observations of the biomass structure referred to the presence of glycogen accumulating organisms in both the lab- and full-scale anaerobic/aerobic systems.

Respiratory activity, molar growth yields, and ATP content in an Arthrobacter sp. ammonium-limited chemostat culture

Arthrobacter fluorescens was grown in chemostat culture under ammonium-limited conditions and respiratory activity; molar growth yields and ATP content were determined over a wide range of dilution rates. Within a range of dilution rates between 0.10 h−1 and 0.20 h−1, morphological transition occurred, the ratio of cocci to rods appearing inversely related to growth rate. Molar growth yields for both glucose and ammonium decreased with increasing dilution rates because of the higher intracellular polysaccharide content at the lower growth rates. Different metabolic activities were shown in cocci, in rods, and during morphogenesis. A sharp decrease in in situ and potential oxygen quotient (Q(O2)) and in ATP content was observed in the range of dilution rates in which morphological changes occurred.

The influence of cultural conditions on polysaccharide production by Aerobacter aerogenes.

SUMMARY: The polysaccharide content of Aerobacter aerogenes surface cultures grown on defined media was estimated by the anthrone method and related to the amount of growth as measured by the non-dialysable nitrogen content. Observations after different periods of incubation at 35° showed that on a medium containing excess sugar, growth was complete after 24 hr. and polysaccharide production was complete after 4 days. While most of the polysaccharide was produced after the cessation of growth, the rate of production per cell was greatest in the logarithmic phase. The polysaccharide: nitrogen ratio was low in 4-day cultures on media wherein cessation of growth was due to exhaustion of sugar (the sole carbon and energy source) and also was low in growths limited by anaerobic conditions or by the development of acidity. It was increased 10–20-fold in cultures wherein cessation of growth was due to exhaustion of limited nitrogen, phosphorus or sulphur sources, so that an excess of sugar remained available for extra polysaccharide synthesis. This great increase in the polysaccharide: nitrogen ratio of the culture was paralleled by similar increases in the degree of mucoidness as measured by the wet weight: nitrogen ratio, in the intracellular polysaccharide content as measured by periodate-Schiff staining, in capsular polysaccharide as measured by capsule diameter, and in loose slime polysaccharide as measured chemically in the supernatant after centrifugation of the culture. After 4 days at 35° the loose slime accounted for about half of the total polysaccharide in the highly mucoid cultures and about a fifth of that in the non-mucoid cultures.