Production of an anti-complement exo-polymer produced by Auricularia auricula-judae in submerged culture

Exo-polymer (EP) was produced at 1.2 g l(-1) in submerged culture of Auricular auricula-judae. Crude EP (AJ-0) has 70% anti-complementary activity (inhibition of total complementary hemolysis 50%; ITCH50). The activating pathway of the complement system occurred through both the classical and alternative pathways, though the major pathway was the classical one. Fractionation of AJ-0 using Sepharose CL-6B gel chromatography gave three major fractions (AJ-Fr-I, II and III) of which the first was the most active. The mycelial growth and EP production of A. auricula-judae were optimal at pH 6, 25 degrees C and pH 5, 25 degrees C, respectively.     

Hypolipidemic effect of an Exo-biopolymer produced from a submerged mycelial culture of Hericium erinaceus

The hypolipidemic effect of an exo-biopolymer produced from a submerged mycelial culture of Hericium erinaceus was investigated in dietary-induced hyperlipidemic rats. Hypolipidemic effects were proportionally increased with the increasing concentration of the exo-biopolymer for oral administration. The exo-biopolymer, at the dose of 200 mg/kg body weight, substantially reduced the plasma total cholesterol (32.9%), LDL cholesterol (45.4%), triglyceride (34.3%), phospholipid (18.9%), atherogenic index (58.7%), and hepatic HMG-CoA reductase activity (20.2%). It increased the plasma HDL cholesterol level (31.1%) as compared to the control group. The molecular mass of this exo-biopolymer measured by HPLC was under 40 kDa. Total sugar and protein contents were 91.2 and 8.8%, respectively. The sugar and amino acid compositions of the exo-biopolymer were analyzed in detail.     

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.     

Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

AIMS: The nutritional requirements for mycelial growth of Cordyceps sinensis in semi-synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus. METHODS AND RESULTS: Nutritional requirements, including 17 carbohydrates, 16 nitrogen compounds, nine vitamins, four macro-elements, four trace-elements and eight ratios of carbon to nitrogen, were studied for their effects on the mycelial growth in submerged cultures of C. sinensis by using one-factor-at-a-time and orthogonal matrix methods. Among these variables, sucrose, peptone, folic acid, calcium, zinc and a carbon to nitrogen ratio 12 : 1 were identified as the requirements for the optimum mycelial growth. The concentrations of sucrose, peptone and yeast extract were optimized and the effects of medium composition on mycelial growth were found to be in the order sucrose > yeast extract > peptone. The optimal concentration for mycelial growth was determined as 50 g l(-1) sucrose, 10 g l(-1) peptone and 3 g l(-1) yeast extract. CONCLUSIONS: Under optimal culture conditions, over 22 g l(-1) of mycelial biomass could be obtained after 40 days in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Cordyceps sinensis, one of the most valued medicinal fungi, is shown to grow in axenic culture. This is the first report on nutritional requirements and design of a simplified semi-synthetic medium for mycelial growth of this psychrophilic species, which grows slowly below 20 degrees C. The results of this study will facilitate research on mass production of the fungus under defined culture conditions.     

Formation of endomycorrhizas by an achlorophyllous orchid,Erythrorchis ochobiensis, andAuricularia polytricha

To test the mycorrhizal function of heterobasidiomycetous fungi on achlorophyllous orchids and to examine the symbiotic fungal range of a myco-heterotrophic orchid,Erythrorchis ochobiensis, synthetic cultures of the orchid seed were carried out withAuricularia polytricha isolates from Japan and Mexico. After three and a half mo of incubation, 57.0–70.7% of seeds germinated but none of them showed further growth. When cultured on peat moss at 25°C, the germination rate was 8.7% in the presence of Mexican isolate and 18.0% in the presence of Japanese isolate. Some germinated seeds developed into protocorms, and several seeds incubated with the Mexican isolate developed into plantlets after 5 mo. Pelotons were observed in the cells of protocorms and roots. The results indicated that some heterobasidiomycetous fungi could form endomycorrhizas with a myco-heterotrophic orchid. The results also showed that the symbiont ofE. ochobiensis extends, at least experimentally, to Heterobasidiomycetes. The variances of germination rate and seedling growth were suggested to be affected by the difference of isolates and culture conditions.     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Production and molecular characteristics of four groups of exopolysaccharides from submerged culture of Phellinus gilvus

AIMS: The objective of the present study was to determine the optimal culture conditions for the production of four groups of exopolysaccharides (EPSs) in Phellinus gilvus by submerged culture and to investigate their molecular properties by multi-angle laser-light scattering (MALLS) analysis. METHODS AND RESULTS: The optimal temperature and initial pH for the production of both mycelial biomass and EPSs in P. gilvus by submerged flask cultures were found to be 30 degrees C and pH 9.0, respectively. Glucose and corn steep powder were the most suitable carbon and nitrogen source for both mycelial biomass and EPS production. Optimal medium composition was determined to be glucose 30 g l-1, corn steep powder 5 g l-1, MgSO4 1.23 g l-1, KH2PO4 0.68 g l-1, and K2HPO4 0.87 g l-1. Four groups of EPSs (Fr-I, II, III, and IV) were obtained from the culture filtrates by gel filtration chromatography on Sepharose CL-4B and characterized by size exclusion chromatography (SEC) coupled with MALLS. The weight average molar mass (Mw) of Fr-I, Fr-II, Fr-III and Fr-IV were determined to be 8.628 x 106 (+/-129 420), 1.045 x 106 (+/-19 855), 61.09 x 104 (+/-1244), and 33.55 x 104 (+/-134) g mol-1, respectively. CONCLUSIONS: Under optimal culture conditions, the maximum EPS production in a 5-l stirred fermenter indicated 5.3 g l-1 after 11 days of fermentation. The SEC/MALLS analysis revealed that Fr-I, which has extremely high molecular weight, was presumably an aggregate of complex polysaccharides forming a compact globular shape; whereas Fr-II was nearly spherical, Fr-III and Fr-IV were rod-like chains in an aqueous solution. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of high amounts of EPSs from liquid-culture of the basidiomycete, P. gilvus. The SEC/MALLS approach used in this study could be useful in providing greater insight into the characterization of the mushroom polysaccharides without carrying out elaborate fractionation procedures prior to analysis.     

Species composition of food-spoiling mycelial fungi

We investigated the composition of the microflora that spoils foodstuffs (the surface of hard cheeses and sausages) at agribusiness factories. Mycelial fungi, mostly ascomycetes of the order Eurotiales belonging to the genus Penicillium play the main role in spoiling food. Most representatives of these fungi are mesophiles and possess the capacity for utilizing nutrient substrates in surface and submerged cultures.     

Isolation and characterization of the exopolysaccharide produced by Daedalea quercina

The production of exopolysaccharide (EPS) by a strain of the basidiomycete Daedalea quercina was investigated. Of seven different carbon sources, glucose and dextrins gave the highest crude polysaccharide yield (4.7–5 g l^–1, 55–60% carbohydrate content) in shake-flask cultures, at 14 days of fermentation. Experiments carried out in a 10 l fermenter, at two different agitation speeds, gave the best results at 300 rpm, resulting in 12–14 g l^–1 of crude exopolysaccharide in 9–11 days. Fractionation of the EPS samples, carried out by tangential flow ultrafiltration, evidenced a single EPS fraction (MW >30 000 Da) in samples from glucose, while two fractions (MW > 30 000 Da and 30 000 > MW > 10 000 Da) were present in samples from dextrins. Fractions characterization by HPLC and proton NMR spectroscopy revealed diversity in composition and structure in the obtained EPS: from glucose mainly an -linked mannan, and from dextrins mainly an - and -linked glucan.     

Hypoglycemic effect of a Lentinus edodes exo-polymer produced from a submerged mycelial culture

The hypoglycemic effect of an exo-polymer produced from a submerged mycelial culture of Lentinus edodes was investigated in streptozotocin-induced diabetic rats. The administration of the exo-polymer (200 mg/kg BW) reduced the plasma glucose level by as much as 21.5%, and increased plasma insulin by 22.1% as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively. Gel chromatography of the exo-polymer revealed a single peak which is likely to have been a glycoprotein with a molecular weight of 52 kDa and was found to contain 83.5% carbohydrate and 16.5% protein. The Sugar and amino acid compositions of the exo-polymer were analyzed in detail.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct observation of biopolymers produced from submerged culture of edible mushrooms by atomic force microscopy

Two biopolymers produced from submerged culture of edible mushrooms were directly observed by atomic force microscopy. Biopolymers were deposited on mica from dilute aqueous solution and imaged in air through a thin layer of adsorbed water and their hydrated structures were observed by a tapping mode. A single biopolymer molecule obtained from Cordyceps militaris was typical of a rod-like structure with bending point, which can form intra- and inter-molecular supercoils. In contrast, the image for low molecular weight biopolymer from Paecilomyces sinclarii is typical of a branched structure in which more extensive interaction leads to the formation of network-like matrix.     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Hypoglycemic effect of polysaccharides produced by submerged mycelial culture of Laetiporus sulphureus on streptozotocininduced diabetic rats

The hypoglycemic effect of the crude extracellular polysaccharides (EPS) produced from submerged mycelial culture of an edible mushroom Laetiporus sulphureus var. miniatus in streptozotocin (STZ)-induced diabetic rat was investigated. Hypoglycemic effect of EPS was evaluated in STZ-induced diabetic rats, and its possible mechanism was suggested by the results of western blot analysis and immunohistochemical staining. The results revealed that orally administrated EPS, when given 48 h after STZ treatment exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in EPS-fed rats to 43.5% of STZ-treated rats. The plasma levels of total cholesterol and triglyceride were significantly increased upon STZ treatment and they were markedly reduced by oral administration of EPS to near-normal levels. The results of immunohistochemical staining of the pancreatic tissues showed that EPS treatment considerably increased the insulin antigenesity of diabetic islet β-cells, suggesting the possibility of β-cell proliferation or regeneration by EPS therapy. Moreover, immunoblotting study revealed that protein levels of iNOS was increased and SOD2, catalase, GPx were significantly increased after EPS treatments, suggesting alleviated oxidative stress mediated by STZ. Orally administrated EPS exhibited considerable hypoglycemic effect in STZ-induced diabetic rats and that these EPS may be useful for the management of diabetes mellitus.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

Dilution rate as a determinant of mycelial morphology in continuous culture

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched "colonial" variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h(-1). The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.     

Mycelial yield of<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> using thinned apple,pear, and peach on submerged culture

The effect of thinned fruits, apple, pear and peach, on the mycelial growth of mushrooms was investigated. The growth of mycelia with the addition of thinned fruit was clearly better than that in the control for all the tested mushrooms. The growth rate ofPleurotus ostreatus was faster than any other mushroom. The optimal concentrations of thinned apple, pear, and peach in a solid culture were 1.0%, 1.0%, and 3%, respectively, while in a liquid culture the optimal concentrations were 5,0%, 3.0%, and 5.0%, respectively. WhenPleurotus ostreatus was incubated in a 20-L pilot scale fermenter with 10 L of a liquid medium containing 3% thinned fruit at 25°C and 6 vvm for 10 days, the mass-production of mycelia was 74.2 g/10 L (apple), 96.2 g/10 L (pear), and 86.3 g/10 L (peach). The mycelial yield ofPleurotus ostreatus in a medium containing thinned fruit was 2≈3 times higher than that in the control.     

Production ofbeauveria bassiana [Deuteromycotina. Hyphomycetes] sympoduloconidia in submerged culture

Submerged conidiation of the entomogenous HyphomyceteBeauveria bassiana is reported. Conidiogenous cells produce sympoduloconidia on conidiogenous cells in liquid shaker culture; hyphal bodies and mycelium fragments are also produced. The morphology of these fungal structures is discussed and illustrated. Several simple liquid media are tested for the production of conidia and hyphal bodies. Maximum yields of conidia (170×10⁶ conidia/ml) are produced in a medium consisting of sucrose (2%) — yeast extract (0.5%) and basal salts, and maximum yields of hyphal bodies (740×10⁶ hyphal bodies/ml) in a sucrose (2.5%) — yeast extract (2.5%) medium.      

Nutritional environment of soil and roots in and around mycelial blocks of an ectomycorrhizal fungusTricholoma bakamatsutake in an evergreen Fagaceae forest

In the evergreen Fagaceae forests of Japan, an ectomycorrhizal fungusTricholoma bakamatsutake forms shiros or developing mycelial blocks. To determine the physiological characteristics of the mycelial blocks, organic acids of the soil and major nutrient elements of the soil and roots were compared at three types of sites: presently colonized mycelial blocks, previously colonized sites behind the blocks, and uncolonized sites in front of the blocks. The upper part of the mycelial blocks showed the following features compared with the uncolonized site: lower pH (5.1), higher concentrations of oxalic and gluconic acids, lower content of total nitrogen, a similar amount of total carbon, reduced total and available phosphorus, higher content of total calcium and lower content of exchangeable calcium. These findings suggested that the activity of the fungus led to soil acidification by the organic acids, an increase in C/N ratio, depletion of phosphorus and accumulation of calcium.     

Sporulation ofStreptomyces roseosporus in submerged culture

Summary A soil isolate ofStreptomyces roseosporus was found to produce spores in stirred submerged culture. Both biological mass and respiratory activity increased during the sporulation process. Contrary to other reports, the differentiation process was not purposefully initiated by critical manipulation of either nutritional or environmental conditions.