【作者】 刘景圣；

【导师】 李玉；

【作者基本信息】 吉林农业大学 ， 作物栽培学与耕作学， 2005， 博士

【摘要】 蜜环菌(Armillaria spp.)属于担子菌门、伞菌目、白蘑科、蜜环菌属。在世界各大洲均有分布，在我国主要分布在黑龙江、吉林、辽宁、内蒙古等省区。蜜环菌是与传统中草药天麻(Gastrodia elata Blume)共生的著名食药兼用的真菌。多年来，经大量的试验研究表明，蜜环菌的菌丝体和发酵液均具有较强的催眠、抗惊厥、抗缺氧等神经调节功能和脑保护功能，还具有增加心、脑血管血流量，增强机体免疫调节功能等生理活性。尤其是在催眠、抗惊厥、抗缺氧和调节心、脑血管功能等方面具有与天麻相同的药理活性。因此，20世纪70年代开始，在对蜜环茵生理活性和化学成分研究，以及蜜环菌的深层发酵工艺和系列制剂的开发等方面引起了中国和世界许多国家的重视，取得了大量研究成果，开发生产出多种蜜环菌制剂。蜜环菌中含有丰富的化学成分，通过对蜜环菌菌丝体和发酵液的化学成分的系统研究，分离得到了蜜环菌多糖、蜜环菌素等倍半萜类化合物和嘌呤类化合物等多种生理活性物质，并对这些化合物的理化特性和生物学活性进行了较细致的研究。由于野生天麻在我国的资源有限，栽培天麻虽已获得成功，但因受到各种条件影响，其生长周期长，并且必须有蜜环菌作为萌发菌和营养供体，技术要求严格，生产成本高，价格贵，难以满足人们保健和临床用药的需求。以蜜环菌及其发酵产物替代天麻，来解决天麻供应不足，保护野生天麻资源等问题，是一条行之有效的资源开发新途径。多年来，经过科研工作者和医药生产企业的不懈努力，脑心舒口服液、蜜环菌片、蜜环菌糖浆等蜜环菌制剂，在调节睡眠和心、脑血管功能等方面收到了较好疗效。据不完全统计，目前在我国，仅脑心舒口服液的年产值就超过了6亿元以上。但是，脑心舒口服液等蜜环菌制剂的生产，主要以蜜环菌的菌丝体和发酵液为主要原料，没有明确有效部位和活性成分，只能属于三类药物。而且，用于发酵生产的蜜环菌菌种(菌株)还不统一，发酵工艺技术与控制不规范，发酵产物的产量不高，对蜜环菌发酵产物及其制剂的质量监测指标不明确。这些不利因素，影响了蜜环菌及其制剂的生产开发和质量、药效以及产品层次的进一步提高。本研究是作者主持的吉林省2001年科技发展计划项目“蜜环菌深层发酵工艺及其功能性食品研究”课题的重要组成部分。其主要内容和攻关目标是从长白山野生蜜环菌菌中分离生长性状良好的菌株，通过液体深层发酵特性和动物试验筛选具有催眠功能的优良蜜环菌菌株。在最适的培养基和摇床液体培养条件下扩大培养，将发酵产物利用分离提取等技术手段和动物试验进行有效部位活性跟踪试验，进一步明确蜜环菌发酵产物中催眠功能的有效部位，并以此有效部位的得率为主要指标，进一步优化蜜环菌液体深层发酵的生产工艺，以实现催眠功能提取部位有效得率的最大化。本研究的试验结果和研究结论包括以下几方面：1．从长白山不同地区采集到的天麻和蜜环菌的子实体中分离得到了16个蜜环菌菌株，分别为Ar-02，Ar-03，Ar-04，Ar-07，Ar-08，Ar-09，Ar-11，Ar-13，Ar-16，Ar-17，Ar-20，Ar-21，Ar-25，Ar-26，Ar-29，Ar-31。以目前“脑心舒”口服液生产所用的蜜环菌菌株(Ar-00)为对照，通过固体培养和液体培养，开展了各菌株的菌丝和菌索萌发时间及其形态特征、荧光特性、液体培养时菌丝球的形态、大小，发酵液色泽，荧光强度，产物的生物量等方面的试验研究。以各菌株的生长特性和生物量为主要指标，进一步筛选出生长性状良好的8个菌株Ar-02，Ar-03，Ar-07，Ar-08，Ar-09，Ar-11，Ar-13，Ar-31。2．经液体深层发酵试验研究，确定了8个生长性状良好的蜜环菌菌株各自的最适培养条件，在此基础上，对8个菌株进行了液体扩大培养，以“脑心舒”口服液生产菌株Ar-00为阳性对照菌株，将发酵产物(菌丝体和发酵液)进行催眠功能的动物试验，最终筛选出液体发酵生物量最高，催眠功能明显的优良菌株Ar-07。本研究还对Ar-07菌株进行了分子生物学鉴定，最终将Ar-07菌株确定为奥氏蜜环菌(Armillaria ostoyae Romagn)。通过单因素和正交试验，筛选确定了Ar-07菌株液体深层发酵的最适培养基和摇床扩大培养时的最适初始pH值、装液量、接种方法(菌丝球的破碎均质处理)和接种量、培养温度、摇床转速、乙醇添加量和最佳发酵时间。3．对Ar-07菌株的液体深层发酵产物(发酵液)进行有效部位的多层次分离、提取，并通过催眠功能的动物试验跟踪，确定了Ar-07菌株发酵液中具有明显催眠功能的有效部位。4．以发酵液中的催眠功能有效部位得率为主要指标，通过正交试验和发酵动力学研究，进一步优化了蜜环菌液体深层发酵的生产工艺，并与蜜环菌的普通发酵工艺相比较，开展了较大规模的中试研究。发酵产物中的菌丝体产量比普通发酵法提高了18.30％，发酵液中催眠功能有效部位得率比普通发酵法提高了21.64％。通过对中试结果分析检测和动物试验证明，本研究所确定的优化工艺是合理可靠的。作者首次从野生蜜环菌的采集、菌种分离、催眠活性菌株的优选、有效部位的确定到液体深层发酵工艺的优化，展开了全面、系统的研究工作。本论文具有以下创新点：1．通过大量的试验研究最终筛选出的蜜环菌Ar-07菌株为生长性状好、生物量高、活性明显、功效稳定的优良菌株，并利用分子生物学鉴定方法将该菌株确定为奥氏蜜环菌(Armillaria ostoyae Romagn)。可为蜜环菌制剂的开发生产提供稳定、可靠的优良菌种。2．通过对蜜环菌Ar-07菌株液体深层发酵产物的多层次分步提取和催眠功能动物试验跟踪，筛选确定了功能活性明显的有效部位，为蜜环菌深层发酵工艺的进一步优化，发酵产物的质量监控提供了可靠依据，为蜜环菌制剂的二次开发奠定了良好基础。3．以催眠活性有效部位的得率为主要指标，进一步优化了蜜环菌液体深层发酵的工艺条件和控制参数，并与普通发酵工艺相比较开展了中试生产研究。结果表明，采用优化工艺进行发酵与普通发酵工艺相比，菌丝体的生物量提高了18.30％，发酵液中有效部位的得率提高了21.64％。此项研究成果为今后蜜环菌液体深层发酵生产及其系列制剂的生产开发提供了可靠的工艺技术。4．本试验确定的破碎匀质接种法经蜜环菌中试生产验证可以提高发酵产物生物量，大量节省菌种的投放量(节省40％)。该法具有明显的创新性，经进一步完善后可以在食药用真菌的液体深层发酵生产中推广应用。本研究所取得的创新性成果为蜜环菌的发酵特性、发酵产物的化学成分分析和活性单体的提取分离研究以及蜜环菌系列产品的深层次开发利用研究奠定了一定基础，可以为蜜环菌液体深层发酵和系列产品的生产开发提供优良菌株和科学合理的工艺技术，对蜜环菌的标准化发酵生产和工艺质量监控具有良好的指导作用。在提高蜜环菌制剂质量和功效的稳定性，加快“脑心舒”口服液的二次开发和蜜环菌创新性药物的研究以及蜜环菌第三代功能性食品的研究与开发，促进食药用菌的产业化发展等方面具有良好的应用价值和现实意义。更多还原

【Abstract】 Armillaria spp., belongs to Basidiomycotina, Agaricales, Tricholomataceae, Armillaria. It is distributed all over the continents and mainly in HiLongjiang, JiLin, LiaoNing, NeiMenggu in China. Armillaria spp. is a kind of famous medicinal fungus symbiotic with Chinese herb "Gastrodia elata Blume"(Tianma). Many experiments showed that the mycelia and fermented broth of Armillaria spp. had the function of cerebrum protection and nerve regulation, such as hypnogenesis, anti-convulsion and anti-anoxia. It also has bioactivities such as increasing the blood flow rate in cerebral and cardiovascular vessel and strengthening immune regulation. So great attentions were paid to study bioactivity and chemical components、develope series of preparation of Armillaria spp. and study the submerge fermentation technology since 1970s. And lots of achievements were obtained.There are abundant chemical components in Armillaria spp. Through the systemic study on the mycelia and fermented broth of Armillaria spp., many chemical compositions have been found, such as polysaccharide, sesquiterpenoids, purine and so on. The physical-chemical properties and bioactivities of those compounds were also studied. As wild TianMa resource is limited in our country. Although it had been cultivated successfully, it still needs Armillaria spp. as germination and nutrition donor. Tianma is hard to satisfy the need of healthcare and clinic use because of its selective techniques, high producing cost and price. It is an effective new way to use Armillaria spp. and its fermented products to replace "TianMa" in order to solve lack-supply and protect wild "TianMa" resource. With years of relentless efforts of scientists and medical enterprises, Armillaria spp. preparations as "NaoXinshu" Oral Liquid, Armillaria spp. pill and Armillaria spp. syrup work a comparatively good cure. Only "NaoXinshu" Oral Liquid one part was earned more than 600millions RMB annually in our country. The Armillaria spp. preparations, taking NaoXinshu Oral Liquid for example, just belongs to the Drug preparationⅢ, because they used Armillaria spp. mycelia and fermented broth as main materials, which did not have clear effective part and active components. What’s more, there are still several adverse factors: no unified strains of Armillaria spp. no regulated fermentation technology and control, low effective yield, no clear monitoring aims for products and preparation quality. Not only do all factors above put great impact on the producing, developing and preparation of Armillaria spp., but also restrain products advance.This paper was the most significant part of "Study on Armillaria spp. submerges fermentation technology and its functional food" which was a science and technology developing plan project of JiLin Province in 2001 conducted by the author. Its chief target was to screen strains with favorable growing characters and soporific function from wild Armillaria spp. in ChangBai Mountain through fermentation and animal trials. The target of this thesis is to clear effective pot with hypnogenesis function, and yield rate of it as chief index, in order to optimize the submerged fermentation technology to realized maximization of the yield rate. The research results as follows:1. 16 Armillaria spp. strains were separated from "TianMa" and the mycelia of Armillaria spp. collected from different areas in ChangBai Mountain, which were named Ar-02, Ar-03, Ar-04, Ar-07, Ar-08, Ar-09, Ar-11, Ar-13, Ar-16, Ar-17, Ar-20, Ar-21, Ar-25, Ar-26, Ar-29, Ar-31 after evaluation respectively. Taking the Armillaria spp. strain which was used to produce "NaoXinshu" Oral Liquid (At-00) as a control, many researches has been done to got information about sprouting time of mycelia and rhizomorph, morphological characters, the mycelia shape at liquid culture, the color and luster of the fermented broth, the fluorescence intensity, biomass of the products and so on. These ascertained the growing characters of the strains. And then 8 strains with favorable growing characters (Ar-02, Ar-03, Ar-07, Ar-08, Ar-09, Ar-11, Ar-13, Ar-31) were screened by the indexes, the strains’ growing character and biomass.2. The optimal culture conditions were screened respectively for the 8 Armillaria spp. strains through liquid submerged fermentation study. On the base, the 8 Armillaria spp. strains were cultured enlarging, by using "NaoXinshu" Oral Liquid as a positive control and animal trail for soporific function. Favorable strain Ar-07 with definite soporific function and highest biomass was finally screened. Molecular biological identification was also done, and strain Ar-07 Was confirmed to be Armillaria ostoyae Romagn at last. The optimum culture conditions for Ar-07 was screened and determined through single factor experiment and orthogonal experiment, for example, optimal initial pH value, volume of culture, inoculating method, inoculating volume, cultural temperature, and time.3. After layers of separating and extracting, the extracting part with definite soporific function in Ar-07 fermented broth was ascertained through animal trials.4. Armillaria spp. liquid submerged fermentation technology was optimized through orthogonal experiment and kinetics study. The mycelia yield obtained by the optimized technique parameters was higher 18.30％than that of normal fermentation; the yield of soporific function extracting part was higher 21.64％. The optimum technology confirmed by this research was proved reasonable and reliable through analysis test and animal trial.Since collecting Armillaria spp. samples, separating the strains, screening favorable strains, ascertaining the extracting part and optimizing fermentation technology, the whole course was first systemically studied in this paper. Innovations as follows:1. Favourable strain Armillaria spp. Ar-07 was finally screened through lots of experiments for its better growing characters, high biomass, definite potency and steady activation. And this strain was confirmed to be Armillmraia ostoyae Romagn by molecular biological identification method. This provided steady and reliable strain for Armillaria spp. preparation production and exploration.2. The ascertain of Ar-07 extracting part by submerge fermentation and animal trials, provided reference to optimize fermentation technology and monitor the quality for fermented products, and laid foundation for the secondary exploration of Armillaria spp. preparations.3. The culture conditions and control parameter were optimized and expanding trials were done. The yield rate of mycelia raised 18.3％and the valid yield rate of the extracting part with soporific function in broth increased 21.64％. So reliable technology was prepared for the development of Armillaria spp. liquid submerged fermentation.4. The smashing and homogenizing inoculation method confirmed. It has proved that could raise the biomass and save the inoculation amount (save 40％). Obviously, this method is innovative. It can be practiced in the fungus liquid submerged fermentation after further perfecting.The innovative achievement obtained in this paper prepared the ground for the further study in Armillaria spp. fermented characters, chemical components analysis of fermented products, separating and extracting the effective part and active monomer and Armillaria spp. products applying. The research also provided scientific and proper technology and favorable Armillaria spp. strain for developing series of products. There was certain reference value to Armillaria spp. standard fermentation producing and technique monitoring. The results had great applying value and immediate significance in increasing the quality and stability of Armillaria spp. preparation. It could enhance the second development of"NaoXinshu" Oral Liquid and development of the Third Generation Functional Food, and improve the industrial secondary exploration of edible and pharmaceutical mushroom.更多还原