冬虫夏草与产地植物及土壤的重金属含量测定

本文测定了野生采集和人工培育冬虫夏草、人工培育冬虫夏草子实体、冬虫夏草发酵菌液、冬虫夏草寄主蝙蝠蛾昆虫食物(蕨麻Potentilla anserine和珠芽蓼Polygonum viviparum),以及野生冬虫夏草产地土壤中5种重金属元素(铜Cu、铅Pb、砷As、镉Cd、汞Hg)的含量。结果表明,人工培育冬虫夏草、人工培育冬虫夏草子实体、冬虫夏草发酵菌液、蕨麻块根、珠芽蓼种子的5种重金属元素含量符合《中医药-中药材重金属限量》ISO国际标准;而野生冬虫夏草,除砷含量(6.170 mg/kg)超标外,铜、铅、镉、汞元素含量均符合要求。按国家标准《土壤环境质量农用地土壤污染风险管控标准(试行)》(GB 1516-2018),四川产地土壤(KDX)与青海产地土壤(GD、HN)的5种重金属含量均低于农用土壤污染风险筛选值;而四川产地土壤(KDT),除镉含量(0.923 mg/kg)高于风险筛选值而低于风险管制值外,其余4种元素含量均低于风险筛选值。结果为人工培育冬虫夏草的质量控制提供了参考。     

人工冬虫夏草的氨基酸含量及其营养价值评价

目的为明确人工和野生冬虫夏草在氨基酸方面的差异,并为进一步开发利用人工冬虫夏草提供理论依据,对室内全人工培殖冬虫夏草和产自四川康定的野生冬虫夏草的氨基酸营养价值进行分析评价。方法用日立L-8800型全自动分析仪检测氨基酸含量并根据FAO/WHO的氨基酸评分标准模式和鸡蛋蛋白模式进行氨基酸营养价值评价。结果人工冬虫夏草氨基酸种类齐全,其全草氨基酸总量高于产自康定的野生冬虫夏草;其全草的鲜味氨基酸高于产自康定的野生冬虫夏草;其全草必需氨基酸总量低于产自康定的野生冬虫夏草。人工冬虫夏草必需氨基酸与氨基酸总量的比值高于WHO/FAO评分模式而低于鸡蛋蛋白模式。结论人工冬虫夏草氨基酸不仅种类组成合理,而且具有较高的营养价值。这一结果为人工冬虫夏草的进一步开发利用提供了理论基础。     

冬虫夏草(Cordyceps sinensis)菌丝体固体发酵粉化学成分的分析

本研究对一种冬虫夏草(Cordyceps sinensis)菌丝体固体发酵粉的化学成份进行了分析,包括粗成份,生物活性成份,脂肪酸,氨基酸,金属元素等。冬虫夏草菌丝体固体发酵粉中3’-脱氧腺苷含量40．81μg/g,腺苷含量745．4μg/g;甘露醇的含量为165．8mg／g,多糖含量为157．3mg／g;谷氨酸,天门冬氨酸,苯丙氨酸是三大类主要氨基酸;不饱和脂肪酸是优势脂肪酸,以亚油酸,油酸为主。和文献记录的结果相比,虫草发酵粉中3’-脱氧腺苷,甘露醇的含量明显高于冬虫夏草子实体中相应成份的含量;各种氨基酸的含量和水平与冬虫夏草子实体中相应氨基酸的含量和水平相当;虫草发酵粉中的其它化学成份和冬虫夏草子实体中相应成份基本一致。     

伏毛铁棒锤不同器官生物碱含量动态变化研究

采用紫外分光光度法,对宁夏六盘山地区不同海拔、不同月份、野生和人工栽培的伏毛铁棒锤总生物碱含量进行分析.结果显示:(1)总生物碱在伏毛铁棒锤各器官中均有分布,其中以块根中含量最高(9.07±0.92 mg/g),叶的含量(3.2±1.7 mg/g)最低.(2)块根中的总生物碱含量随生长发育期的变化而不同,其中:6～11月份块根中总生物碱含量随生长期延长而逐渐升高,10月份块根含量达最高(9.07±0.92 mg/g).(3)在同一地区,总生物碱含量随海拔高度的升高而增加,在海拔高度为2 800 m时块根中含量达到10.04±0.51 mg/g,其他器官中总生物碱的含量变化基本相似.(4)在相同海拔高度下,野生伏毛铁棒锤总生物碱含量略高于人工栽培的,但差异不显著(P>0.05).研究表明,人工栽培的伏毛铁棒锤块根可以替代野生种做为药用,且在六盘山区人工种植的块根最佳采收期为10月,在选择人工种植区应考虑选择较高的海拔地区.     

人工培养蛹虫草与野生冬虫夏草氨基酸含量的比较

对人工培养蛹虫草与青海产野生冬虫夏草的氨基酸含量进行测定,结果表明:野生冬虫夏草中所含有的各种氨基酸,在人工培养虫草的子实体及菌丝体中都有存在;且后者的精氨酸及脯氨酸含量明显高于野生冬虫夏草。由此为鉴定人工培养虫草的质量,进一步为工业规模开发与利用人工培养虫草,提供了科学理论依据。     

额济纳绿洲胡杨（Populus euphratica）酚类物质含量和分布及其与土壤水分的关系

采用分光光度法测定了额济纳绿洲胡杨(Populus euphratica)的披针形叶、卵圆形叶、嫩枝、枝(D《5 mm)、枝(5～10 mm)、主干树皮、根(D《2 mm)、根(2～5 mm)和根(5～10 mm)9类器官中的总酚、黄酮和缩合单宁含量.结果表明,总酚含量较高的器官为皮(27.93 mg/s),叶、根、枝中总酚含量分别为17.64 mg/g(两类叶均值)、16.72mg/g(三类根均值)、12.19 mg/g(三类枝均值);黄酮含量较高的器官为皮(51.30 mg/g),叶、根、枝中黄酮含量分别为28.45 mg/g(两类叶均值)、39.99 mg/g(三类根均值)、23.67 mg/g(三类枝均值);根中缩合单宁含量较高,三类根均值为22.10 mg/g,皮、叶、枝中缩合单宁含量分别为8.41 mg/g、4.03 mg/g(两类叶均值)、4.47 mg/g(三类枝均值).披针形叶和卵圆形叶中酚类物质含量没有显著性差异(P》0.05);随着枝不断成熟,嫩枝、枝(D《5mm)、枝(5～10mm)中酚类物质逐渐减少;随着根直径减少,根中缩合单宁逐渐增加,细根(D《2mm)中的缩合单宁含量最高(25.95 mg/g).分析胡杨各器官中酚类物质含量与土壤水分的关系,结果表明卵圆形叶中酚类物质含量与土壤水分含量成显著负相关关系(P《0.05,总酚:r=-0.949;黄酮:r=-0.923;缩合单宁:r=-0.944).研究揭示了极端干旱地区胡杨各器官中酚类物质的变化规律,及其与环境因子的相互作用关系.     

野生及人工养殖中华鲟幼鱼肌肉营养成分的比较

对野生及人工养殖中华鲟幼鱼的肌肉营养成分和营养品质进行了分析比较.结果表明:野生中华鲟幼鱼肌肉中水分、粗蛋白和粗灰分含量均显著高于人工养殖中华鲟(P＜0.05),而粗脂肪含量显著低于人工养殖中华鲟(P＜0.05).野生和人工养殖中华鲟幼鱼的氨基酸组成基本一致,均含有18种氨基酸,必需氨基酸指数(EAAI)分别为72.02、66.21,其构成比例符合联合国粮农组织/世界卫生组织(FAO/WHO)的标准.脂肪酸中二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的含量野生中华鲟显著高于人工养殖中华鲟(P＜0.05),分别为22.99%、7.15%.矿物质含量丰富,微量元素含量野生中华鲟明显高于人工养殖中华鲟[动物学报 53(3):502-510,2007].     

蛹虫草子实体中甘露醇含量的测定

建立了HPLC法测定蛹虫草子实体中甘露醇含量的方法。通过比较提取溶剂、提取方式及提取时间等条件对甘露醇提取效果的影响,确定甘露醇分析的前处理方法为:1g子实体粉中加入150 mL 90%乙醇热回流提取1h。采用SUGAR SP0810柱(300 mm×8 mm)为分析柱,超纯水为流动相,流速1.0 mL/min,柱温70℃,示差折光检测器检测,进样量10μL。甘露醇在0.04-9.9 mg/mL范围内线性关系良好,回归方程为y=103860x+2183.9(r=0.9999),平均回收率为104.27%,RSD=2.19%(n=9)。本方法准确度高,稳定性、精密度、重现性好,适用于蛹虫草子实体中甘露醇含量的分析。     

蝙蝠蛾拟青霉菌丝体化学成分的研究

应用梯度提取、反复重结晶及硅胶色谱法分离和纯化,采用NMR等谱学方法鉴定结构对蝙蝠蛾拟青霉(Paecilomyces hepiali)菌丝体进行了化学成分的分析。从蝙蝠蛾拟青霉菌丝体中分离得到6个化合物,分别鉴定为4,6,8(14),22(23)-四烯-3-酮-麦角甾烷(1)、麦角甾醇(2)、β-谷甾醇(3)、麦角甾醇过氧化物(4)、啤酒甾醇(5)、甘露醇(6)和1-油酰基-2-亚油酸-3-棕榈酸甘油(7),并对其石油醚提取物中的油状组分进行GC-MS分析,结果表明其中主要为亚油酸(9,12-十八碳二烯酸,含量41.949%)、反油酸(反9-十八碳烯酸,含量为27.696%);同时采用高效液相色谱法测定其中的麦角甾醇的含量,与海鲜菇、杏鲍菇、香菇、金针菇、滑子蘑、平菇等食用菌子实体比较,结果蝙蝠蛾拟青霉菌丝体中麦角甾醇含量为15.65 mg/g,平菇为21.43 mg/g,杏鲍菇为10.16 mg/g,香菇为13.34 mg/g,金针菇为9.48 mg/g,滑子蘑为10.43 mg/g,海鲜菇为14.42 mg/g。     

基于双酶级联协调表达策略高效催化合成D-甘露醇北大核心CSCD

D-甘露醇(D-mannitol)作为合成抗肿瘤药和免疫刺激剂的重要前体被广泛应用于制药和医疗等行业,酶法合成D-甘露醇反应成本昂贵无法满足工业化生产。本研究首先筛选关键酶获得较优性能的甘露醇脱氢酶Lp MDH和用于辅因子NADH再生的葡萄糖脱氢酶Ba GDH,在大肠杆菌(Escherichia coli)BL21(DE3)中共表达,实现了基于双酶级联反应催化底物D-果糖合成D-甘露醇,D-甘露醇的初步摩尔转化率为59.7%。针对双酶级联催化反应中辅酶再生用酶与催化用酶表达量不协调的问题,通过增加Bagdh拷贝量来提高辅因子循环能力,获得了双酶催化速率平衡的重组大肠杆菌E.coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh。进一步对重组菌的全细胞转化条件进行优化,确定了最适转化条件为反应温度30℃,初始pH值6.5,菌体量OD600=30,底物D-果糖100.0 g/L,辅底物葡萄糖与底物1︰1摩尔当量。于最优转化条件下5 L发酵罐转化24 h,D-甘露醇的最高产量为81.9g/L,摩尔转化率为81.9%。本研究提供了一种绿色、高效生物催化生产D-甘露醇的方法,为实现其规模化生产奠定了基础,同时也对其他相关稀有糖醇的研究具有指导意义。     

人工发酵古尼虫草中甘露醇的测定

利用比色法简便、准确和快速地测定发酵生产的古尼虫草菌丝中甘露醇含量。结果表明 ,甘露醇的最大特征吸收峰在 41 2nm处 ,质量浓度在 1 0～ 5 0mg/L范围内线性较好 ,其回归方程 ρ=-0 842 +97 3 2 9A,r=0 9992 ;平均回收率 1 0 0 2 4% ,RSD =0 5 4% (n=5 )。样品中甘露醇采用水提取法 ,提取时间 2h。甘露醇显色后 2 0min内测定对结果影响不大 ,在测定时要注意排除果糖对测定结果的影响。用此法测得发酵古尼虫草菌丝体中甘露醇的含量为 7.4% ,RSD =0 .2 4% (n =6)。     

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.     

蝉拟青霉菌丝体与天然蝉花中化学成分的比较分析

以天然蝉花Cordyceps cicadae做对比,对一株蝉拟青霉Paecilomyces cicadae菌株发酵菌丝体的化学成分进行了分析,包括粗成分、生物活性成分、无机元素、氨基酸、脂肪酸等。结果表明:蝉拟青霉菌丝体中虫草多糖的含量为33.2mg/g,甘露醇的含量为78.9mg/g,麦角甾醇的含量为0.6256mg/g,腺苷的含量为1.0620mg/g,钙、铁、锌、硒和锰微量元素的含量分别为5187.59μg/g、207.23μg/g、41.93μg/g、0.087μg/g和28.24μg/g,18种氨基酸齐全,不饱和脂肪酸是优势脂肪酸,以亚麻油酸、油酸为主,其相对含量分别为53.91%和20.63%,与天然蝉花相比,发酵菌丝体中虫草多糖、甘露醇、麦角甾醇、腺苷、氨基酸总量、必需氨基酸总量和不饱和脂肪酸的含量明显高于后者,其它化学成分和天然蝉花中相应成分基本一致。     

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.     

中国弯颈霉培养物的化学成份分析

中国颈霉T.sinense是与冬虫夏草无性型密切相关的真菌。对中国弯颈霉ZN923菌株培养物进行测试,证明其所含化学成分如甘露醇、氨基酸、生物碱、有机酸及甾醇类等与天然冬虫夏草基本相同。红外、紫外吸收图谱也表明两者所含的各类化学成份极为相似。     

Induction of HL-60 apoptosis by ethyl acetate extract of Cordyceps sinensis fungal mycelium

The cultivated mycelium of a Cordyceps sinensis (Cs) fungus was sequentially extracted by petroleum ether, ethyl acetate (EtOAc), ethanol and water. The EtOAc extract showed the most potent cytotoxic effect against the proliferation of human premyelocytic leukemia cell HL-60, with an ED50 < or = 25 microg/ml for 2-day treatment. The EtOAc extract induced the characteristic apoptotic symptoms in the HL-60 cells, DNA fragmentation and chromatin condensation, occurring within 6-8 h of treatment at a dose of 200 microg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly ADP-ribose polymerase were detected during the course of apoptosis induction. These results suggest that the Cs mycelium extract inhibited the cancer cell proliferation by inducing cell apoptosis.     

Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S rDNA

The anamorph determination of Cordyceps sinensis remains problematic due to the lack of clear links between the sexual and conidial forms of the fungus. In this study, we applied molecular approaches to analyze the genetic variation of Cordyceps sinensis and its allies to identify the anamorph-teleomorph connection. The sequences of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA gene of Cordyceps sinensis (teleomorph) collected from Qingzang plateau (altitude over 4000m), Tibet and several related asexual conidial forms were determined. The sequence comparison showed that Cordyceps sinensis was most closely related to Hirsutella sinensis, and was clearly divergent from Paecilomyces sinensis, Stachybotrys sp. or Tolypocladium sp.; distance values, estimated according to Kimura two-parameter models between Cordyceps sinensis and Hirsutella sinensis, were extremely low (<0.02), whereas distance values between Cordyceps sinensis and Paecilomyces sinensis, Stachybotrys sp. and Tolypocladium sp. were 0.34, 0.21 and 0.25, respectively. Taken together, Hirsutella sinensis and Cordyceps sinensis are the different stages of the life cycle stages of the same organism. Hirsutella sinensis is therefore the anamorph of Cordyceps sinensis, rather than Paecilomyces sinensis or other species. The possible reasons as to why different taxa can be obtained when culturing Cordyceps sinensis are also discussed.     

利用原生质体紫外诱变技术选育可常温发酵的中国被毛孢新菌株

中国被毛孢(Hirsutella sinensis)是天然冬虫夏草[Cordyceps sinensis(Berk)Sacc.]的真正无性型,其生长温度低(低于20℃),生长缓慢(发酵周期长达20～30d),对人工发酵的技术条件要求很高,因而大大增加了工业化发酵生产的成本和技术难度。为了解决上述问题,实验中从天然冬虫夏草子座中分离到中国被毛孢菌株BS-1,并对其进行了原生质体紫外诱变处理。经过粗筛和精筛,从中选出一株可常温下(25～28℃)发酵、生长迅速(发酵周期4～5 d)、有效成分(甘露醇和腺苷)含量显著高于原始菌株的诱变株BS-5962。通过连续10代PDA斜面继代培养和摇瓶试验,以及连续3批的1t罐的中试试验,检查结果表明所得诱变株性状优异而稳定。可为降低中国被毛孢的液体深层发酵成本和技术难度提供一种有效的解决方案。