蛹虫草中虫草素的提取及纯化工艺研究

目的:优化从蛹虫草中提取、纯化虫草素的方法并鉴定其纯度.方法和结果:通过3种提取方法的比较,确定了以恒温水浴法作为虫草素规模化提取方法,最佳提取条件为45℃、80％乙醇;同时,确定了ML-7大孔树脂纯化虫草素的工艺,采用该工艺虫草素纯度可达到92％以上,并对纯化虫草素和标准品进行了比较鉴定.结论:该方法提取、纯化的虫草素纯度高,适于虫草素的规模化生产.     

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%。     

人工培养蛹虫草

用柞蚕的活蛹 ,大米米饭培养基培养蛹虫草菌 ,控制好环境条件 ,严格操作环节 ,可培养出与天然蛹虫草形态相同的蛹虫草子座。用棉籽壳、玉米芯、葵花盘粉碎物培养蛹虫草菌 ,可以得到抑菌杀菌物质 ,成本低廉 ,可利用此法提取广谱抗菌物质。     

蛹虫草中虫草素、虫草多糖综合提取工艺研究

以蛹虫草为材料．采用超声波水提法、超声波醇提法、水热回流法和醇热回流法4种提取方法选择虫草素和虫草多糖的最优提取办法．并用正交试验方法考察水热回流法中提取温度、提取次数、提取时间、料液比4个因素对虫草素、虫草多精综合提取效率的影响．以确定从蛹虫草中综合提取虫草素、虫草多糖最佳工艺。结果表明．水热回流法是提取虫草多糖和虫草素的最优提取方法．其最佳工艺条件为：用10倍量的水于80℃热回流提取3次．每次90min。     

从蛹虫草小麦培养基残渣分离虫草素及提取物对肝癌细胞的抑制效果

从蛹虫草Cordyceps militaris小麦培养基残渣中分离和纯化虫草素,并进行了提取物体外人肝癌细胞Hep G2 (human hepatoma cell line,Hep G2)的抑制作用研究。以蛹虫草小麦培养基残渣为原料,热超声水提取虫草素,探究pH值、温度、浸提时间和超声功率对虫草素的提取效果,用苯乙烯型弱极性大孔吸附树脂(AB-8)柱层析分离、重结晶纯化虫草素,并用高效液相色谱法(high performance liquid chromatography,HPLC)、红外光谱法(infrared spectroscopy,IR)鉴定。虫草素处理肝癌细胞Hep G2后,倒置相差显微镜下观察肝癌细胞形态变化,并用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法测定细胞生长情况。结果表明：固液比1:50、pH值6.0、温度60 ℃、浸提时间3 h和超声功率300 W时提取效果好。经鉴定,分离提取物的虫草素纯度达到99%以上。不同浓度的虫草素对肝癌细胞Hep G2有明显的抑制作用,且与虫草素浓度和作用时间呈正相关,被抑制的细胞凝集、变圆、碎裂增加。本提取工艺得到的虫草素纯度高,工艺简单易扩大,且提取物对肝癌细胞增殖作用具有明显的抑制作用,为其在功能性食品添加剂方向的应用提供了数据基础。     

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。     

大孔吸附树脂分离纯化培养基残基中虫草素

采用大孔吸附树脂分离纯化人工蛹虫草培养基残基中的虫草素,以HPLC法测定样品中虫草素含量,筛选出了适宜的大孔树脂NKA-II。研究了pH、洗脱剂乙醇体积分数等因素对该树脂吸附性能及解析性能的影响。结果表明,NKA-II型树脂纯化虫草素的最佳吸附条件为pH9,吸附流速2BV/h,该树脂对虫草素的吸附量可达到16.5mg/g。洗脱工艺条件为2.5倍树脂柱体积的50%乙醇,NKA-II大孔树脂对虫草素的解析率可达到95%以上。     

蛹虫草菌生物合成虫草素的研究进展

蛹虫草Cordyceps militaris是我国传统的药用真菌,虫草素是蛹虫草的主要活性成分,具有抗癌、抗肿瘤、抗病毒等多种生理功能。蛹虫草菌液体发酵是最有希望实现高效生产虫草素的途径,但现阶段生产强度低,亟需应用发酵工程及代谢工程手段提高虫草素产量。文中对液体发酵体系中培养基组分(碳/氮源、前体物质、金属离子等)和培养条件(pH、溶氧量、光照等)对虫草素产量的影响进行了总结,并对虫草素的分离纯化、生物合成基因簇及合成代谢途径进行了阐述,最后探讨了实现虫草素高效生产的关键环节。     

产虫草素酿酒酵母工程菌株的构建与发酵优化

虫草素作为药用真菌蛹虫草的主要活性成分,具有抗肿瘤、抗病毒等多种生理功能。现阶段虫草素主要通过蛹虫草液体发酵生产,但发酵周期长、生产强度低,制约了其大规模开发利用。文中在酿酒酵母Saccharomyces cerevisiae S288C中异源表达虫草素合成关键基因ScCNS1和ScCNS2,成功构建了产虫草素的酵母工程菌SHC16,发酵240 h虫草素产量可达67.32 mg/L;基因表达分析显示,发酵后期磷酸戊糖途径、嘌呤代谢及虫草素合成途径关键酶编码基因ZWF1、PRS4、ADE4、ScCNS1及ScCNS2表达水平显著上调。进一步地,通过优化发酵培养基组成,确定以50 g/L葡萄糖为初始底物结合一次补料、添加5 mmol/L Cu2+和1.0 g/L腺嘌呤为最适培养基组成。基于此在5 L搅拌发酵罐中开展补料分批发酵,144 h虫草素产量达到137.27 mg/L,生产强度达0.95 mg/(L·h),较未优化发酵体系提高240%。     

大孔吸附树脂AB-8和十八烷基硅胶色谱分离纯化蛹虫草发酵液中虫草素工艺研究

主要研究了从蛹虫草发酵液中提取虫草素的工艺,确定了大孔吸附树脂AB-8及十八烷基键合硅胶反相层析柱对蛹虫草发酵液中虫草素的分离条件;吸附最佳工艺条件为上样液p H6,上样浓度0.4mg/m L,上样量5BV,吸附流速1.0BV/h;解吸最佳工艺条件包括解吸液20%乙醇,解吸体积15BV,解吸流速4BV/h。得到的解吸液进一步以十八烷基键合硅胶分离,条件为上样液p H6,梯度洗脱,流动相为p H6的水和p H6的乙醇。洗脱液通过结晶和重结晶得到精制虫草素。三步得到的虫草素纯度分别达到40%、90%和99%以上。     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Production of a COX-2 inhibitor, 2,4,5-trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata

AIMS: To investigate the active ingredient in fruiting bodies and to produce it with cultured mycelium in Antrodia camphorata (BCRC 35398). METHODS AND RESULTS: The volatile components from the fruiting bodies, the liquid cultured broth of A. camphorata and Cinnamomum kanehirae wood were separately isolated by steam distillation-solvent extraction and identified by gas chromatography-mass spectrometry. In the fruiting bodies, a COX-2 inhibitor 2,4,5-trimethoxybenzaldehyde (TMBA) was found to be the most abundant constituent, but was totally absent in its cultured broth and its natural host, C. kanehirae wood. On feeding with the acid-digested sawdust of C. kanehirae wood or vanillin to the broth for culture, TMBA was produced in both cultured broths. CONCLUSION: The TMBA identified in fruiting bodies was an active ingredient whose functions consisted with the reported experiences of this mushroom. Feeding vanillin to culture broth could produce TMBA containing mycelium product like its fruiting bodies did. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found an active ingredient in fruiting bodies of A. camphorata and elucidated this compound derived from digested sawdust of C. kanehirae wood. A feasible method was also developed to produce TMBA containing mycelium by feeding vanillin.     

沪农灵芝一号生长过程中子实体各部位糖醇和海藻糖积累及其代谢酶的表达变化

食用菌子实体通常会在生长过程中积累较高含量的糖醇及海藻糖,这些碳水化合物的积累能够促进食用菌的生长,而在灵芝中的同类研究较少,本研究通过高效阴离子-脉冲安培法对沪农灵芝一号子实体发育过程中不同部位的糖类成分的含量变化进行分析,发现灵芝子实体中主要的可溶性糖类成分是阿拉伯糖醇、甘露醇和海藻糖,甘露醇在子实体成熟时的菌盖中的含量达到最高值,阿拉伯糖醇在产孢子期的子实体中含量较高,两种糖醇的含量呈现相反的变化趋势,一种糖醇积累的同时会消耗利用另一种糖醇,而海藻糖在灵芝子实体的整个生长过程中含量处于较低水平,仅在子实体初期的菌基部位检测到较高的含量;同时通过qRT-PCR技术检测灵芝子实体不同部位中这几种糖类的主要代谢酶基因的表达变化,发现这些代谢酶在子实体的菌基部位的表达水平相对其他部位较高,且随着子实体生长这一差异更加显著,这一结果表明灵芝中的糖醇和海藻糖分布差异可能是先由菌基的菌丝体中合成产物并转运到子实体不同部位,再经过一段时间的积累和代谢之后产生。     

HPLC指纹图谱技术在灵芝组织分离试验中的应用

利用 HPLC 指纹图谱技术研究从同一灵芝子实体不同部位组织分离获得的菌株三萜化合物的差异。用常规组织分离方法获得不同菌株,在 A、B、C、D 四种培养基上进行出菇试验,运用HPLC指纹图谱技术获得各子实体三萜提取物 HPLC 指纹图谱,计算图谱间的相似度,分析三萜指纹的差异。结果表明,从子实体不同部位分离获得的四个菌株在同一培养基相同条件下培养获得的灵芝子实体的三萜指纹图谱相似度均大于0.99;同一部位分离获得的菌株在不同培养基相同培养条件下获得的子实体,其粗三萜 HPLC 图谱相似度均大于0.96;不     

HPLC指纹图谱技术在灵芝组织分离试验中的应用

利用HPLC指纹图谱技术研究从同一灵芝子实体不同部位组织分离获得的菌株三萜化合物的差异。用常规组织分离方法获得不同菌株,在A、B、C、D四种培养基上进行出菇试验,运用HPLC指纹图谱技术获得各子实体三萜提取物HPLC指纹图谱,计算图谱间的相似度,分析三萜指纹的差异。结果表明,从子实体不同部位分离获得的四个菌株在同一培养基相同条件下培养获得的灵芝子实体的三萜指纹图谱相似度均大于0.99;同一部位分离获得的菌株在不同培养基相同培养条件下获得的子实体,其粗三萜HPLC图谱相似度均大于0.96;不同的组织分离部位和不同的培养基对灵芝菌株三萜指纹图谱的影响均不显著。18号菌株在C培养基上培养获得的子实体三萜含量最高,上层菌肉为最优组织分离部位,C培养基为最优培养基。灵芝的三萜组成不受生长环境的影响,而生长环境会对其三萜化合物含量产生一定的影响。本文首次应用HPLC指纹图谱方法对灵芝组织分离获得的菌株的差异性进行了研究。     

Fruiting body and soil rDNA sampling detects complementary assemblage of Agaricomycotina (Basidiomycota, Fungi) in a hemlock-dominated forest plot in southern Ontario

This is the first study to assess the diversity and community structure of the Agaricomycotina in an ectotrophic forest using above-ground fruiting body surveys as well as soil rDNA sampling. We recovered 132 molecular operational taxonomic units, or 'species', from fruiting bodies and 66 from soil, with little overlap. Fruiting body sampling primarily recovered fungi from the Agaricales, Russulales, Boletales and Cantharellales. Many of these species are ectomycorrhizal and form large fruiting bodies. Soil rDNA sampling recovered fungi from these groups in addition to taxa overlooked during the fruiting body survey from the Atheliales, Trechisporales and Sebacinales. Species from these groups form inconspicuous, resupinate and corticioid fruiting bodies. Soil sampling also detected fungi from the Hysterangiales that form fruiting bodies underground. Generally, fruiting body and soil rDNA samples recover a largely different assemblage of fungi at the species level; however, both methods identify the same dominant fungi at the genus-order level and ectomycorrhizal fungi as the prevailing type. Richness, abundance, and phylogenetic diversity (PD) identify the Agaricales as the dominant fungal group above- and below-ground; however, we find that molecularly highly divergent lineages may account for a greater proportion of total diversity using the PD measure compared with richness and abundance. Unless an exhaustive inventory is required, the rapidity and versatility of DNA-based sampling may be sufficient for a first assessment of the dominant taxonomic and ecological groups of fungi in forest soil.     

基于改良RAPD标记的花梅与果梅品种的遗传关系分析

为了探讨梅(Prunus mume Sieb.et Zucc.)的2种类型--花梅与果梅的遗传多样性,利用23条长度为11bp的随机引物以及严格筛选退火温度的改良RAPD-PCR优化体系对25个花梅和21个果梅品种的基因组总DNA进行了分析,并在此基础上采用UPGMA聚类方法对46个花梅与果梅品种间的遗传关系进行了分析.结果显示:使用重复性好、条带清晰且多态性强的23条随机引物,共从46个花梅和果梅品种基因组总DNA中扩增出131条带,其中多态性条带107条,多态性条带百分率达81.7%,说明花梅和果梅各品种间具有丰富的遗传多样性.聚类分析结果显示:46个花梅和果梅品种的遗传相似系数为0.63～0.83;在遗传相似系数0.66处,供试的46个品种被分为5组.A组包含6个果梅品种和11个花梅品种;B组仅包含4个花梅品种;C组包含16个品种,其中15个品种为果梅;D组仅包含4个花梅品种;E组也仅包含5个花梅品种.研究结果表明:花梅和果梅具有相近的亲缘关系,遗传背景相似.     

Reproducible and controllable light induction of in vitro fruiting of the white-rot basidiomycete Pleurotus ostreatus

Fruiting is a crucial developmental process in basidiomycetes yet the genetic and molecular factors that control it are not yet fully understood. The search for fruiting inducers is of major relevance for both basic research and for their use in industrial applications. In this paper, an efficient and reproducible protocol for controlled fruiting induction of Pleurotus ostreatus growing on synthetic medium is described. The protocol is based on the control of light intensity and photoperiod and permits the life cycle for this fungus to be completed in less than two weeks. The fruiting bodies produced by this method release fertile spores after 4-5 d of culture. Our results indicate that fruiting induction is solely dependent on the illumination regime and that it occurs long before the available nutrients are depleted in the culture. This protocol will greatly facilitate molecular and developmental biology research in this fungus as it avoids the need for complex culture media based on lignocellulosic materials or the use of chemical inducers.     

Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains

By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.     

桑黄菌丝体和子实体中次级代谢产物及其活性的比较

研究桑黄发酵菌丝体次级代谢产物及活性与子实体的差异性,探讨其替代子实体的可能性。研究通过高效液相色谱分析和化学法比较菌丝体和子实体石油醚、氯仿、乙酸乙酯和正丁醇4个萃取相中的成分差异,以二苯基三硝基苯肼自由基（DPPH）清除率和Trolox当量抗氧化能力（TEAC）作为抗氧化活性的指标、HepG2和MCF-7癌细胞的抑制率作为抑制肿瘤细胞生长的指标,比较其活性差异。结果表明,菌丝体和子实体4个萃取相在化学成分上存在差异;在活性方面,菌丝体各萃取相的抗氧化活性高于子实体,而子实体抗肿瘤活性优于菌丝体。菌丝体醇提取的总黄酮含量高于子实体醇提物,抗氧化活性和总黄酮含量有显著相关性,发酵菌丝体在抗氧化活性方面具有替代子实体的可行性。