A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

不同培养条件和前体对蛹虫草液体发酵产虫草素的影响

蛹虫草能产生虫草素等多种活性物质。为考察不同液体发酵方式及添加前体物质对虫草素积累的影响,选用蛹虫草08Y1菌株,通过光照振荡、光照静置、黑暗振荡、黑暗静置四种培养条件和添加前体物质（腺嘌呤1g/L+甘氨酸16g/L）,发酵16d后检测虫草素和腺嘌呤含量。结果表明：08Y1菌株在黑暗振荡培养条件下,虫草素积累达1,015.0mg/L,腺嘌呤利用率达98.5%,说明黑暗振荡培养并添加前体物质是提高虫草素产量的有效方法。     

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.     

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

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.     

蓝光光照对蛹虫草子实体生长和主要活性成分的影响

光作为一种重要的环境因子,可影响真菌的生长发育、生理周期、形态变化及次级代谢产物的产生,对于蛹虫草而言,光照还是其子实体生长发育的必要条件。选择2株不同来源的蛹虫草菌株,研究了自然光照和蓝光光照条件下,其子实体生长、抗氧化酶活性和主要活性成分的变化。结果表明蓝光光照对于蛹虫草子实体产量没有明显的促进和抑制作用。自然光照条件下成熟子实体的超氧化物歧化酶(superoxide dismutase,SOD)活性显著高于蓝光光照,过氧化氢酶(catalase,CAT)活性没有明显差别,而过氧化物酶(peroxidase,POD)活性则表现出菌株差异。子实体主要活性成分方面,腺苷、甘露醇含量不受蓝光影响,类胡萝卜素在蓝光光照条件下含量极显著高于自然光光照,蓝光对子实体、虫草素、粗多糖含量的影响则存在菌株差异。研究为蛹虫草子实体的栽培条件优化,提高蛹虫草子实体质量提供依据。     

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high‐energy ion beam irradiation

Aims: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called ‘ion beam’. Methods and Results: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8‐azaadenine‐ and 28 classes of 8‐azaaguanine‐resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8‐azaadenine‐resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8‐azaaguanine‐resistant mutants, it was shown that mutant no. G81‐3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3·1 and 1·8 g l^?1 on 21‐day culture for mutant no. G81‐3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. Conclusions: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. Significance and Impact of the Study: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large‐scale production of cordycepin.     

培养基和栽培方式对蛹虫草子实体活性成分的影响

采用麦粒（W）、麦粒加蚕蛹粉（WW）、大米（R）、大米加蚕蛹粉（RW）的配料栽培方式,以及蚕蛹（CY）活体接种栽培方式培养蛹虫草子实体,比较蛹虫草子实体中多糖及核苷、游离糖醇和小分子糖类含量。结果表明：培养基和栽培方式影响蛹虫草多糖含量及其单糖组成和分子量分布,多糖含量最高的为蚕蛹活体接种培养的处理（CY）,多糖含量为6.19%,其他处理的多糖含量仅为CY的44.4%-62.8%;蚕蛹（CY）上培养的蛹虫草子实体中核苷类成分含量最高,在麦粒上培养获得的子实体中核苷含量显著高于在大米上的处理,并且麦粒添加蚕蛹粉后培养的子实体中尿苷、鸟苷、N⁶-(2-羟乙基)腺苷含量显著上升,大米添加蚕蛹粉后培养的子实体中尿苷、虫草素、N⁶-(2-羟乙基)腺苷含量显著上升;配料栽培的4个处理,其子实体中海藻糖含量为20.07%-23.40%,蚕蛹活体接种的处理（CY）海藻糖含量显著降低,为8.41%;添加蚕蛹粉后子实体中甘露醇含量显著降低。对各成分含量的数据进行归一化处理后进行蛹虫草品质的综合评价,在蚕蛹上培养的蛹虫草综合评分最高,麦粒为主要培养基培养的蛹虫草品质好于大米为主要培养基的,且添加蚕蛹粉的处理优于未添加的处理。     

冬虫夏草与韩芝液体发酵混合培养特性研究

平板培养基上比较了冬虫夏草、韩芝及树舌的菌丝生长速度和对淀粉的利用情况,结果显示三者的生长速度分别达到1.28、0.64和0.55 cm/d,且韩芝与树舌对淀粉的利用均优于冬虫夏草;混合培养结果得出菌种的最佳菌龄比为虫草:韩芝=3 d:5 d,最佳的接种比例为2:3;正交试验混合发酵最佳的培养基配方(%):葡萄糖1.0、淀粉2.0、黄豆粉2.0、酵母粉0.5,在此条件下,菌丝生物量达到最大值1.97 g/100 mL。     

Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris

The influence of initial pH value, various nitrogen sources, plant oils, and modes of propagation (shake-flask and static culture) on the production of biomass, exopolysaccharide (EPS), adenosine and, in particular, cordycepin, by Cordyceps militaris CCRC 32219 were investigated. Optimal conditions for mycelial growth, EPS and cordycepin production were observed at relatively low pH. Amongst organic sources, yeast extract (YE) was favorable for EPS and cordycepin production, while corn steep powder (CSP) was favorable for adenosine production. A lower C/N ratio was favorable for adenosine and cordycepin production; however, too low a C/N ratio led to diminished production. All plant oils tested stimulate mycelial growth and EPS production of C. militaris, but they did not show much effect on the adenosine and cordycepin production. A two-stage fermentation process by combining shake-flask fermentation with static culture significantly enhanced cordycepin production. A Box–Behnken experimental design was employed to optimize the production of cordycepin, which showed that the optimum conditions to produce cordycepin by C. militaris CCRC 32219 were at pH 6, YE concentration of 45 g/l and 8.0 day of the shake culture followed by 16 days of the static culture. Under the optimized conditions, the maximum production (2214.5 mg/l) of cordycepin was obtained, which is much higher than those reported up to date.     

自然干燥对冬虫夏草寄主蝠蛾卵孵化的影响

研究在室内自然空气湿度下放置的时间长短对冬虫夏草(Cordyceps)寄主昆虫贡嘎蝠蛾Hepialus gonggaensis Fu et Huang卵孵化率的影响。卵早期的研究结果为:第1批、第2批和第3批卵于室内自然空气湿度下保存的时间达26,11和16h后再保湿都可以正常孵化并且孵化率与对照无显著性差异,所孵化幼虫在饲养初期的成活率分别达62.0%,41.4%和43.4%,与对照无显著性差异。卵中期干燥放置36h的孵化率为66.7%,所孵化幼虫在饲养初期的成活率为50.0%,孵化率和成活率都与对照无显著性差异。卵晚期干燥放置24h的孵化率为70.0%,所孵化幼虫在饲养初期的成活率为49.0%,孵化率和成活率都与对照无显著性差异。以上结果表明,经历一定时间的干燥不会对卵的正常孵化有影响。     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

不同培养基对冬虫夏草菌丝生长的影响

不同培养基对3个来源不同的冬虫夏草菌菌丝生长影响的研究结果表明,在不同培养基上,各菌株之间在菌丝萌发时间、40d菌落直径和菌落形态上表现出一定的差异。同时筛选到的最优培养基比已报道培养基的菌落生长速度快2～3倍,且菌丝为白色、粗壮,菌丝质量较优。     

冬虫夏草菌侵染及寄生阶段的生长发育研究

冬虫夏草菌侵染进入寄主幼虫血体腔后,断裂形成长梭形的菌体。菌体以顶端出芽的方式增殖,分隔分化,互相质配。质配菌体生长出菌丝充满寄主幼虫血体腔,使幼虫死亡并形成僵虫形的菌核,菌核萌生子座即形成冬虫夏草。     

Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells

Cordyceps sinensiscontains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis(CS) and thesignaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximun at 25 μg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis. J. Cell. Biochem. 69:483–489, 1998. © 1998 Wiley-Liss, Inc.     

磷酸氢二铵对糙皮侧耳菌丝生长及基质降解酶活性的影响

氮源种类和浓度均会对糙皮侧耳Pleurotus ostreatus菌丝长势、生长速率以及代谢产生影响,而氮匮乏与氮过量积累都会对糙皮侧耳的生长产生不利影响。为了研究氮源对糙皮侧耳菌丝生长和基质降解酶活性的影响,本研究选取磷酸氢二铵[(NH4)2HPO4]作为唯一氮源,通过在培养基中添加不同浓度的磷酸氢二铵检测糙皮侧耳菌丝生长速率和木质纤维素降解酶的活性,筛选出糙皮侧耳生长的最适磷酸氢二铵浓度。结果表明,当磷酸氢二铵的添加浓度区间为10–20 mmol/L时菌丝长势最好。以缺氮时的酶活作为对照,当磷酸氢二铵的添加浓度为10 mmol/L时,羧甲基纤维素酶的活力最强,显著高于对照组(P<0.05);当添加浓度为5 mmol/L时,滤纸纤维素酶的活力最强,显著高于对照组(P<0.05),而10 mmol/L浓度下的酶活力比5 mmol/L时稍有下降,但二者之间无显著性差异(P>0.05);当添加浓度为40 mmol/L时,木聚糖酶的活力最强,显著高于对照组(P<0.05);当添加浓度为10mmol/L时,漆酶的活力最强,显著高于对照组(P<0.05)。基质降解酶...     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

高速逆流色谱分离制备蛹虫草中虫草素和N6-(2-羟乙基)-腺苷

采用高速逆流色谱（HSCCC）技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N⁶-(2-羟乙基)-腺苷。利用高效液相色谱（HPLC）测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N⁶-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水（1:4:5,V/V/V）为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N⁶-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N⁶-(2-羟乙基)-腺苷的大量制备建立了基础。     

维生素B_1、B_6和生长激素2,4‐D对蛹虫草液体发酵虫草素产量的影响

分别研究了不同添加浓度的维生素B1、B6和2,4‐D(2,4‐二氯苯氧乙酸)3种微量物质对蛹虫草Cordyceps militaris液体发酵中虫草素产量的影响。结果表明在设置的几个添加浓度梯度中维生素B1、B6和2,4‐D均能提高虫草的产量,其中维生素B1和B6的最佳添加浓度均为0.83g/L,2,4‐D的最佳添加浓度为0.015mg/mL。此外,高温灭菌对维生素B1、B6和2,4‐D对蛹虫草发酵液中虫草素产量有影响:高温灭菌可使部分维生素B1分解,高温灭菌前添加维生素B1比灭菌后添加更有利于虫草素产量的增加,虫草素的产量提高了19.9%,表明维生素B1的分解产物具有更好的促进虫草素合成的作用。     

冬虫夏草成熟过程中冬虫夏草菌及其突变基因型在子座和僵虫体中的差异表达

文献报道了从冬虫夏草(Cs)中同时检出蝙蝠蛾拟青霉(Ph)、中华被毛孢(Hs)和冬虫夏草菌(Os)的多个基因型;Cs的成熟伴有化学成分的改变和僵虫体中Hs竞争性菌落形成能力的下降.同时检验了Cs成熟过程中Os突变基因型生物量的变化.应用Southern杂交检验发现:Cs的成熟伴有Ph和2组Os基因型生物量的显著增加.o...