蛹虫草子实体中海藻糖含量的测定

为分析食用菌中海藻糖的含量,以蛹虫草子实体为材料,比较了提取溶剂、提取方式及提取时间等条件对海藻糖提取效果的影响,确定海藻糖分析的前处理方法为：1g子实体粉中加入100mL 90%乙醇热回流提取1h。采用高效液相色谱法测定海藻糖,优化后的色谱条件为：SUGAR SP0810柱（300mm×8mm）,超纯水洗脱,流速0.5mL/min,柱温70℃,示差折光检测器检测,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,对海藻糖标准品的检测特异性良好,适用于蛹虫草子实体中海藻糖含量的     

Cmku70基因敲除对蛹虫草生长发育的影响

ku70和ku80是非同源末端连接修复通路的关键基因,在一些丝状真菌中其基因敲除株可作为底盘菌株,提高同源重组效率和基因敲除效率.本研究从蛹虫草基因组中鉴定得到Cmku70 及 Cmku80基因,分别编码分子量为71.50kDa和80.96kDa的蛋白,均含有Ku core结构域,预测均定位于细胞核.系统进化分析表明K...     

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g⁻¹. The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.     

Enhancement of cordycepin production from Cordyceps militaris culture by epigenetic modification

Biotechnology Letters - Cordycepin (3′-deoxyadenosine) is a nucleoside analogue and biosynthesised by Cordyceps militaris, an entomopathogenic fungus. In this study, an epigenetic modifier...     

Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris

A fibrinolytic enzyme has been purified from the fruiting bodies of Korean Cordyceps militaris. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fibrin-zymography, and gel filtration chromatography. The 15 amino acid residues of the N-terminal sequence of the enzyme were APVEQCDAPVGLARL, which is dissimilar to those of fibrinolytic enzymes from other mushrooms. Optimal pH and temperature values of the enzyme were 7.0 and 40°C, respectively. The enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), TPCK, 1,10-phenanthroline, Cu(2+), and Ba(2+). It was also significantly inhibited by aprotinin, EDTA, and EGTA. The enzyme showed a higher specificity for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, exhibiting that it is a chymotrypsin-like serine metalloprotease. The enzyme preferentially hydrolyzed the fibrinogen Aα-, followed by the Bβ-chains and the γ-chain. The α, β, and γ-γ chains of fibrin were also degraded by the enzyme.     

Heat and light stresses affect metabolite production in the fruit body of the medicinal mushroom Cordyceps militaris

Applied Microbiology and Biotechnology - Cordyceps militaris is a highly valued edible and medicinal fungus due to its production of various metabolites, including adenosine, cordycepin,...     

蛹虫草原生质体紫外诱变选育胞外多糖高产菌株

蛹虫草是重要的药食兼用两用真菌,具有较高的医用及经济价值。本文通过单因素和正交试验的方法研究了不同酶系统、酶解温度、酶解时间、渗透压稳定剂、菌龄对蛹虫草原生质体形成的影响,并对蛹虫草原生质体进行紫外诱变,以生物量和胞外多糖产量为指标选育胞外多糖高产菌株。结果表明：在30℃、1％溶壁酶＋0．5％蜗牛酶＋0．5％纤维素酶条件下,以甘露醇为渗透压稳定剂对4日龄蛹虫草菌丝酶解2h,原生质体产量可达到9．2×10^6个/mL。从150株诱变株中筛选出1株最佳正诱变株,编号为44#,经深层培养其生物量比出发菌株提高10％,胞外多糖产量提高84．3％,继代培养10代后,遗传稳定性良好。     

冬虫夏草和蛹虫草发酵液抗菌活性研究

研究了冬虫夏草和蛹虫草发酵液的抑菌特性,结果表明:两种虫草的发酵液对金黄色葡萄球菌、大肠埃希菌、枯草芽胞杆菌、苏云金芽胞杆菌均有较好的抑制作用,且对前两者的抗菌作用强于后两者;热稳定性试验表明发酵液中的抑菌物质具有较强的热稳定性;虫草发酵液与25%糖液和10%盐液协同作用可以增加对参试细菌的抑菌作用;对比试验得出蛹虫草发酵液的抑菌活性较冬虫夏草发酵液的抑菌活性强.     

蛹虫草多糖和D-甘露醇深层发酵的非结构动力学模型

多糖和D-甘露醇是蛹虫草的重要药理活性成分。本文开展了蛹虫草在分批发酵过程中同时生产多糖和D-甘露醇的发酵动力学研究。利用Sigmoid函数构建了蛹虫草菌丝生长、糖基质消耗、多糖和D-甘露醇的非结构动力学模型,并根据Boltzmann方程拟合求解出各模型参数。结果显示,各模型的实测值和预测值拟合度较好。蛹虫草比生长速率在第1.0天达到最大值（μ_max）1.244d^-1;底物葡萄糖的比消耗速率在第0.6天达到最大值（q_{S, max}）2.163d^-1;多糖比合成速率在第2.0天达到最大值（q_{P, max}）51.852mg/(g·d);D-甘露醇比合成速率在第0.99天左右达到最大值（q_{D, max}）37.963mg/(g·d)。蛹虫草多糖的形成与菌丝细胞的生长呈现部分生长关联型,而D-甘露醇的形成与细胞生长呈现生长关联型关系。研究结果为利用分批发酵规模化同时发酵生产蛹虫草多糖和D-甘露醇提供了理论依据。     

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。     

适于蛹虫草遗传多样性研究的线粒体分子标记的筛选

摘要:【目的】从蛹虫草线粒体DNA中寻找适于遗传多样性研究的分子标记。【方法】通过PCR扩增和序列分析,比较了20个蛹虫草菌株在12个线粒体DNA片段和3个细胞核DNA片段上的序列变异。【结果】蛹虫草在线粒体DNA上的变异水平高于核DNA,主要表现为线粒体基因内含子的插入缺失多样性和较多的碱基变异位点。不同线粒体DNA片段的变异水平也有差异,而且内含子蛋白比外显子编码的蛋白质更易发生氨基酸的改变。增加使用的分子标记数目,其所揭示的遗传多样性程度也在逐渐提高。【结论】我们依 次推荐nad3-cox2、cox2-nad5、cox2、cox3、cob和cox1这6个线粒体DNA位点用于今后蛹虫草遗传多样性或群体遗传结构的分析。     

Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris

AIMS: The objective of the present study was to determine the optimal culture conditions for exo-biopolymer production by Cordyceps militaris in shake flask culture. METHODS AND RESULTS: The optimal temperature and initial pH for both mycelial growth and exo-biopolymer production by Cordyceps militaris in shake flask culture were found to be 20 degrees C and 6.0, respectively. Sucrose (40 g x l(-1)) and corn steep powder (10 g x l(-1)) were the most suitable carbon and nitrogen source for both mycelial growth and exo-biopolymer production. CONCLUSION: Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5-l jar fermenter indicated 10.3 g x l(-1), which was approximately three times higher than that in shake flask culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This process can have a significant impact on the industrial scale when sucrose and corn steep powder were used as carbon and nitrogen source.     

The anti-hyperglycemic activity of the fruiting body of Cordyceps in diabetic rats induced by nicotinamide and streptozotocin

Little scientific evidence exists to support the numerous herbs used to improve diabetes-related metabolic disorders. Cordyceps, a Chinese herbal medicine with fruiting body and carcass, has been proposed to have multiple medicinal activities. The objective of this study was to investigate the effects of fruiting body and carcass of Cordyceps on hyperglycemia. Male Wistar rats administered with placebo (STZ group), 1 g of fruiting body (FB group), 1 g of carcass (CC group), or 1g of fruiting body plus carcass (CF group) of Cordyceps for four weeks (d1 to d28) were injected with nicotinamide (200 mg/kg) and streptozotocin (65 mg/kg) on d15. Animals fed with placebo and injected with saline acted as the controls (CON group). The results showed that water intake (d15 to d29), changes in fasting blood glucose concentration (d15 to d26), and serum concentrations of fructosamine (d29) were significantly greater in the STZ, CC and CF groups than in the CON and FB groups (one-way ANOVA, P < 0.05). The diabetic rats had significantly lower weight gain and higher blood glucose response in oral glucose tolerance test than the control rats; and these changes were significantly reduced by administrating the fruiting body of Cordyceps. Our results revealed that fruiting body, not carcass, of Cordyceps attenuated the diabetes-induced weight loss, polydipsia and hyperglycemia, and these improvements suggest that fruiting body of Cordyceps has a potential to be the functional food for diabetes.     

氮源对蛹虫草生长及类胡萝卜素产生的影响

【背景】蛹虫草是一种珍稀食药用菌,类胡萝卜素不仅为其重要活性成分,而且影响子实体的外观品相,但是类胡萝卜素产生的影响因素不明。【目的】揭示氮源对蛹虫草生长和类胡萝卜素产生的影响。【方法】测定不同氮源培养基中菌株生长速度、分生孢子产生及类胡萝卜含量,筛选菌株生长的最适氮源,进一步研究不同浓度的氮源对蛹虫草生长及类胡萝卜素产生的影响,并测定不同光照条件下氮源浓度对蛹虫草子实体类胡萝卜素产生的影响。【结果】蛹虫草在不同的氮源培养基中菌落形态和类胡萝卜素产生存在明显差异。麦麸和黄豆粉培养基中菌株生长速度最快,但是菌落稀疏,正面分别呈现荧光黄色和极微弱红色;蛋白胨和酵母提取物培养基中菌落致密,产孢量极显著高于其它氮源培养基(P0.01),菌落正面为橙黄色;甘氨酸和柠檬酸为氮源时完全没有色素产生,其它无机和氨基酸氮源培养基平板背面有微量色素产生。固体和液体静置培养条件下均发现蛋白胨浓度在0-3%的范围内,随着浓度的增加,类胡萝卜素含量增加;子实体栽培中不同的氮源浓度均表现为蓝光光照条件下类胡萝卜素含量显著高于白光;而在白光和蓝光光照条件下均表现为蛋白胨浓度为1%时类胡萝卜素含量最高,分别为2 809.38±386.24μg/g和4 093.75±518.37μg/g。【结论】氮源种类和浓度显著影响蛹虫草类胡萝卜素的产生,蓝光光照和1%蛋白胨浓度为子实体类胡萝卜素产生的最佳条件,这为栽培富含类胡萝卜素的蛹虫草子实体提供了试验依据。     

Chiral inversion of 2-phenylpropionic acid by Cordyceps militaris

Y.-F. HUNG, M.J. THOMASON, W. RHYS-WILLIAMS, A.W. LLOYD AND G.W. HANLON. 1996. Fifty-nine microbial cultures were screened for their ability to carry out the chiral inversion of (R,S) and (R)-2-phenylpropionic acid under both growing and non-growing conditions. While a number of these micro-organisms metabolized the substrate, only Verticillium lecanii IMI 68689 and Cordyceps militaris CBS 128.25 were able to invert the (R)-enantiomer to its (S)-antipode with no other biotransformation occurring. The rate of inversion was faster for C. militaris but in both cases the reaction was independent of growth and reached an equilibrium after 5 d incubation. Evidence suggests that the (R) to (S) inversion is inhibited in the presence of excess (S)-enantiomer which may account for the equilibrium enantiomeric ratios (S:R) of 90:10 and 70:30 for C. militaris and V. lecanii , respectively. Optimization studies revealed that the fastest rates of reaction for C. militaris were found at lower substrate concentrations (100 μg ml^-1) whilst the greatest yield of (S)-2-PPA was obtained at 250 μg ml^-1. Optimum inversion was found to occur using Sørensen's phosphate buffer at pH 5.5. These results may have implications for the use of micro-organisms in the production of pure enantiomers from racemic mixtures and, in addition, provide an in vitro model system for the study of a unique and interesting biotransformation.     

低能离子束修饰蛹虫草菌株高产虫草素

虫草素具有抗肿瘤、免疫调节、抗炎等多种功效。为了更好地开发蛹虫草资源,选择合适剂量的低能离子束注入蛹虫草,优化虫草素的提取工艺条件,采用紫外分光光度法检测注入前后菌株中虫草素的含量。结果表明:最佳注入剂量为2.60×1015ions/cm2,虫草素最佳的微波-超声波提取工艺为:乙醇浓度70%,提取功率200W,提取时间110s,料液比1:240。选育出虫草素含量较高的15株菌株,最高含量达(11.924±0.063)mg/g,比原始菌株增长了近30%。     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

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。