Quantitative response of cell growth and polysaccharide biosynthesis by the medicinal mushroom Phellinus linteus to NaCl in the medium

The effect of NaCl on cell growth and polysaccharide biosynthesis in the medicinal mushroom Phellinus linteus was studied. With the increase of NaCl concentration between 1 g/l and 7 g/l in the culture medium, the cell growth and intracellular polysaccharide (IPS) accumulation were decreased; extracellular polysaccharide (EPS) concentration was enhanced, with an increase of NaCl concentration from 1 g/l to 3 g/l. Under the optimum NaCl concentration of 3 g/l, the maximum EPS and IPS production reached 2.2±0.15 g/l and 53.6±2.45 mg/g DW on day 12, which improved 32.27% and decreased 16.89% compared to the control, respectively. Both EPS and IPS showed new polysaccharide components by fractionation with DEAE-cellulose ion exchange chromatography compared to the control. The results presented in this study are considered helpful for further investigation on the diversity of polysaccharide biosynthesis of this medicinal fungus under NaCl environments.     

Inhibition of cytochrome P450 isozymes in rat liver microsomes by polysaccharides derived from Phellinus linteus

Polysaccharides (0.5, 1 and 3 mg ml^–1) from cultured broth and mycelia of Phellinus linteus inhibited cytochrome P450 (CYP) 1A1, CYP 1A2, CYP 2B1, and CYP 2E1 activities in rat liver microsomes. The polysaccharides from the broth of Phellinus linteus grown with 5% (v/v) mulberry extract had highest inhibitory potency for CYP 1A1, 1A2 and 2B1 activities. The most potent inhibitor of CYP 2E1 activity were the polysaccharides from the broth of Phellinus linteusgrown with 10% (v/v) mulberry extract.     

pH control strategy in a shaken minibioreactor for polysaccharide production by medicinal mushroom Phellinus linteus and its anti-hyperlipemia activity

A process at various pH values ranging from 4.5 to 7.5 for production of mycelia and extracellular polysaccharide (EPS) by P. linteus fermentation in pH-controlled shaken bioreactor was investigated. A two-stage pH control strategy in which pH value was kept at 6.5 for the first 24 h, and then switched to 4.5 was developed successfully to enhance simultaneously the cell growth and EPS production. The maximum cell density and EPS production reached 15.13 ± 0.1 g/l on day 6 and 6.74 ± 0.1 g/l on day 4, respectively. The anti- hyperlipemia effect of EPS and intracellular polysaccharide (IPS) extracted from mycelia were observed that both EPS and IPS can obviously reduce the serum triglyceride (TG), the blood cholesterol (TC) and serum low density lipoprotein (LDL) level, and increase the high density lipoprotein (HDL) level of the hyperlipemia mice. Polysaccharides from submerged cultivation of medicinal fungus P. linteus have favorable potency to develop anti-hyperlipermia drugs.     

Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus

Mycelial growth and extracellular polysaccharide production of Phellinus linteus were optimal at pH 5 and 25 °C. Maximum biomass production (14.2 g l^–1) was after 15 d of cultivation, whereas, extracellular polysaccharide was maximal (3.5 g l^–1) after 21 d. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, decreased plasma glucose, total cholesterol and triacylglycerol concentrations by 49%, 32%, and 28%, respectively, and aspartate aminotransferase activity by 20%. The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats

Extracts of Phellinus linteus (EPB), grown on germinated brown rice, protected rats from liver injury induced by carbon tetrachloride (CCl4). Peroxidation products in the liver were decreased to 10% by EPB. Catalase and superoxide dismutase activities were significantly decreased to 55% and 39% by CCl4 administration, but EPB blocked this effect, resulting in enzyme activities at control levels. Expression of cytochromeP450 2E1 (CYP2E1) protein was significantly decreased to 88% in CCl4-treated rats but remained at control levels when EPB was also administered. EPB did not affect the altered fatty acid composition induced by CCl4. The hepatoprotective effect of EPB may be mediated by EPB's prevention of CCl4-induced CYP2E1 degradation.     

Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus

A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC₅₀ of 6 μM and also showed antibacterial activity against S. aureus and MRSA.     

Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+ DC and type I helper T cell-dominant immune state

Dendritic cells (DC) are known to not only induce the activation of T cells, but are also associated with the polarization of T cells. This study investigated whether or not proteoglycan (PG) isolated from Phellinus linteus induces the phenotypic and functional maturation of CD11c+ DC in vitro and in vivo. PG was found to induce the phenotypic and functional maturation of bone marrow-derived DC via Toll-like receptors (TLR) 2 and 4 in vitro. Administration of PG in vivo strongly inhibited the MCA-102 tumor growth and increase in vivo. The ratio of CD8+ DC to CD8- DC increased, and PG enhanced IL-12 and IFN-gamma production, and expression of surface molecules including major histocompatibility complexes (MHC) classes I, MHC II, CD80, and CD86 in MCA-102-challenged mice. PG also caused a marked increase in the production of Th (helper T cells)-1 cytokine (IFN-gamma) and a decrease in the production of Th-2 cytokine (IL-4) by splenic cells and inguinal lymph node cells in MCA-102 tumor-bearing mice. Furthermore, PG stimulated the proliferation of CD4+ and CD8+ T cells. In addition, a combination of PG and tumor lysate-pulsed DC inhibited completely the growth of MCA-102 cells in tumor-bearing mice. These results indicate that the administration of PG inhibited the tumor growth through a mechanism leading to a Th-1 dominant immune state and the activation of CD11cCD8+ DC.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Medium optimization of antifungal lipopeptide,iturin A,production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in solid-state fermentation by response surface methodology

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 μg/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.     

<Emphasis Type="BoldItalic">Phellinus linteus</Emphasis> grown on germinated brown rice suppresses IgE production by the modulation of Th1/Th2 balance in murine mesenteric lymph node lymphocytes

We compared the immunomodulating effects of Phellinus linteus (PL), germinated brown rice (BR) and P.␣linteus grown on germinated brown rice (PB) on IgE production in murine mesenteric lymph node (MLN) lymphocytes. All extracts decreased IgE concentrations by 43–65% compared to control mice in both serum and MLN lymphocytes. In addition, PL and PB increased the proportion of CD4⁺ T cells by␣9% and 12% in MLN lymphocytes. IFN-γ concentration, Th1 cytokine, was significantly increased by 44–67%, whereas IL-4 and IL-10 concentrations, Th2 cytokine, significantly decreased by 30–60% in the three treated groups compared to control group. These results suggest that PB suppresses IgE production through the modulation of Th1/Th2 balance to down-regulate Th2 response in MLN lymphocytes, even though a synergistic effect of PB was not found.     

Bioreactor for solid-state cultivation of <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

Phlebiopsis gigantea fungus used in biological control of root rot is currently cultivated commercially in disposable, sterilizable plastic bags. A novel packed bed bioreactor was designed for cultivating P. gigantea and compared to the plastic bag method and to a tray bioreactor. The spore viability of 5.4 × 10⁶ c.f.u./g obtained with the packed bed bioreactor was of the same order of magnitude as the viabilities obtained with the other cultivation methods. Furthermore, the packed bed bioreactor was less time and space consuming and easier to operate than the tray bioreactor.     

Enhancement glucose oxidase production by solid-state fermentation of Aspergillus niger on polyurethane foams using mussel processing wastewaters

In the present work, studies of glucose oxidase (GOD) production in solid-state fermentation on polyurethane foams, using mussel processing wastewaters (MPW) as culture media, were carried out. Initially, the results generated in this experimental modality were compared with those that were obtained in conventional submerged fermentation. Later on, cultures were tested in solid state with fed-batch performance, defining suitable conditions of operation. These conditions were finally corroborated in experiments with a bioreactor of own design, in which GOD production was improved 13 times regarding to the initial cultures.     

Production of rhamnolipids in solid-state cultivation: Characterization,downstream processing and application in the cleaning of contaminated soils

Pseudomonas aeruginosa UFPEDA 614 produced a rhamnolipid biosurfactant when grown on sugarcane bagasse impregnated with a solution containing glycerol. Biosurfactant levels reached 40 g of rhamnolipid per kilogram of dry initial substrate after 12 days. On the basis of the volume of liquid used, the biosurfactant levels were similar to those obtained in submerged liquid culture of a medium identical to the impregnating solution. The properties of the biosurfactant were very similar to those obtained with rhamnolipids produced in submerged culture, with a critical micelle concentration of 46.8 mg/L and an emulsification index at 24 h of over 90% against gasoline. The surface properties were maintained after autoclaving of the fermented solids, meaning that it is possible to minimize safety risks by killing the producing organism with a heat treatment of the solids prior to product extraction. The biosurfactant was used in the washing of soils contaminated with gasoline. An aqueous biosurfactant solution was 3.2-fold more efficient than water in leaching organic material from the soil, demonstrating the viability of application of rhamnolipids in the bioremediation of soils contaminated with gasoline.     

Biobleaching of banana fibre pulp using <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C O1 xylanase produced from wheat bran under solid-state cultivation

A cellulase-free xylanase produced by Bacillus subtilis C 01 from wheat bran under solid-state cultivation was tested for its efficacy in biobleaching of raw banana fibre and banana pulp obtained through a mechanical pulping process. Banana pulp samples treated with crude xylanase (450 nkat g⁻¹ pulp) resulted in a 19.6% increase in the brightness as compared to untreated pulp. The presence of chromophores, hydrophobic compounds and an increased reducing sugar (10.79 mg g⁻¹ pulp) quantity in the bleached solution after enzymatic treatment indicated the removal of materials that were absorbed at 237 nm from the banana pulp.     

Factors affecting gibberellic acid production by Fusarium moniliforme in solid-state cultivation on starch

The production of gibberellic acid (GA₃) by Fusarium moniliforme M-7121 in solid-state culture was evaluated in flask cultures as well as in 3-I horizontal rotary reactors. The highest production rate of GA₃ was with 80% (w/v) maize flour mixed with wheat bran. The optimum initial moisture content was inversely dependent on the ambient relative humidity. The initial water activity range for optimal growth and GA₃ accumulation was about 0.98 to 0.99, which is unusually high for a filamentous fungus. A low O₂ concentration resulted in a much decreased GA₃ yield and the appearance of a yellow to reddish pigmentation in the mycelium. The lag phase was short and rapid growth continued for up to 2 days in the rotary reactor, with a maximum specific growth rate of 0.12 h^–1. The maximum rate of GA₃ production occurred during the subsequent 3 to 10 days of incubation and the final GA₃ concentration reached was 18.7 mg to 19.3 mg/g dry culture. The point of maximum GA₃ accumulation after 10 to 12 days of incubation was usually marked by a sharp increase in pH.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, 9300 Bioemfontein, Republic of South Africa     

The effect of urea on growth of moulds and biomass yield in solid-state cultivation

Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergillus oryzae and Rhizopus oligosporus, led to a higher concentration of NH₄ ⁺ and pH but a decrease in biomass production.The authors are with the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia.     

Changes in quality of Phellinus gilvus mushroom by different drying methods

This study was conducted to investigate the changes in characteristics of the Phellinus gilvus mushroom as influenced by drying methods after harvest. The lowest weight loss rate of P. gilvus mushroom was 75.8% with drying in the shade and 80% by dryer (60°C). The size loss rate of pileus was 19.3% of that in a hot air dryer (60°C). The hardness of dried material context using a hot air dryer (60°C) was the lowest (20 kg/cm²), and that by a dry oven (60°C) was the highest (457 kg/m²). For ΔE value, 4.9 of context and 2.6 of tubes using drying in the shade (20°C) were found to be the lowest. The survival rate of sarcoma 180 treated with P. gilvus dried in the sun was the lowest (51.8%), and this was considered the most effective method for antitumor activity against sarcoma 180.     

Enhancing arachidonic acid production by <Emphasis Type="Italic">Mortierella alpina</Emphasis> ME-1 using improved mycelium aging technology

Traditional mycelium aging technology was improved to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1. Filtration step was skipped and additional carbon and nitrogen sources were fed during aging. The levels of the significant factors (time, temperature, ethanol, and KNO₃) affecting ARA production during improved aging process were also optimized by applying response surface methodology (RSM), and the maximum ARA yield of 19.02 g/l was achieved in a 5 l fermentor at 5.6 days, temperature 13.7 °C, ethanol 42.44 g/l, and KNO₃ 2.62 g/l. This yield was 1.55 times higher than that of traditional aging technology. The improved mycelium aging technology is considered to be a useful strategy for enhancing ARA production.     

Puroindoline A-gene expression is involved in association of puroindolines to starch

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars (Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.Communicated by H.F. Linskens     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.