201200143 ' 六、發明說明： 【發明所屬之技術領域】 -種可提高_草有效成份之_、培養基成份及方法，利用 培養過程中添加動物性蛋白、植物性蛋白、益生g㈣液或中藥 萃取物來培育職草’加上適當之培養環境，已_縮短培育時 間、提升蜗蟲草子實體的產能及提高有效成分蟲草素(c〇rdycepin) 含量。 • 【先前技術】 蟲草是我_-種名貴野生帽材，具有補虛損、益精氣、 保肺、益腎、止血化痰、滋補強壯等功效，蟲草與人參、鹿茸列 為中國三大補藥。如西元i757年《本草從新》中就有「冬蟲夏草 甘平保肺’益腎’補精趙，止血化痰，已勞咳，治腸症皆良」的 記載。中醫認為，蟲草入肺腎二經，既能補肺陰，又能補腎陽， 主治腎虛，陽萎遺精，腰膝酸痛，病後虛弱，久咳虛弱，勞咳瘦 鲁血’自汗盜汗等’是唯一的一種能同時平衡、調節陰陽的中藥。 全世界已知蟲草類有餘種，在中國大陸目前有62種，但最為 叩貴的只有冬蟲草和北蟲草。古語曰：寧要蟲草一把，不要金玉 滿車；民間則稱之為「活黃金」。 在本草攸新」及「本草網目拾遺」中記載的冬蟲夏草 你/咖聊心咖如就是野生蟲草的一種，有記载的蟲草屬約 略有300多種，但迄西元1993年止已報導的中國產蟲草僅約6〇 餘種。冬蟲夏草是—齡貴的_真菌，具有較高的醫療、保健 201200143 及經濟價值，因此在中國大陸許多中醫研究所、藥學研究所、蠶 業科學研究所、大學及醫院…等，紛紛投入有關蟲草的研究，成 果豐碩，而研究培育出來的蟲草，便稱為人工蟲草。目前這些人 工蟲草大致分為四類： (一） 冬蟲夏草野生冬蟲夏草的寄主為蝙 竭i^Uiepialus oblifurous chuet Wang)，無也馆故良今 華被毛孢菌57ν^/?57·5·)。人工培育者多數為 各種蟲草菌的菌絲體及其發酵產物，其成份和藥理作用雖 與野生冬蟲夏草相類似，但其間差異仍大。 (二） 蛹蟲草印S奶·w&ey:蛹蟲草的寄主為柞蠶蛹， %ipascil〇myces militarae) 〇 草菌接種在柞蠶蛹上，所形成之蛹蟲草子實體，稱為踊蟲 草（即蠶蛹蟲草）。 (三） 古尼蟲草汾雄聊卿切：是—種寄生蝙酬幼蟲的大 型蟲草。子座從寄主頭部生出，柄白色，長1至9公分， 粗0.5至0.6公分，頂部一般灰色至灰黑色，長印圓形至 柱狀，單生，二叉分枝或成簇著生，一般大小約8至22公 厘x5至8公厘，成熟時與柄的界限分明’無不孕頂端。子 囊殼擬卵形或安瓿瓶形，〇· 7至〇. 9公厘χ 〇 2至〇 3公 厘，埋生’成熟時孔口外露。寄生於土中蝙蝠蛾科 (Hepiaidae)昆蟲的幼蟲上。古尼蟲草菌為古尼擬青霉 201200143 . (卵SCJ」〇WCeS卽朋以），可在一般培養基中形成子囊殼的 子實體。 Λ (四）蟬草:又名蟬花或蟬茸寄主 為蟬的幼蟲體’子座單生或2至3個分又，從寄主頭部前 端生出，長2至6公分，棍棒狀或鹿角狀，初灰白色，後 淡黃色至黑褐色。全草入藥，有明目之效。我國江南各省 均有分佈。該草系利用蟬草菌在蛋白質等成份之培養基上 • 生長培育’目前已能工業化生產。 蛹蟲草（Cordyc^s militaris)與冬蟲夏草（c〇rdyceps sinensis)同是子囊菌門、核菌綱、肉座菌目、麥角菌科、蟲草屬 的真菌’又稱北蟲草或是北方蟲草、蜗草、蠶踊蟲草、鱗蜗蟲草、 北冬蟲夏草，是寄生於昆蟲幼體或蛹體上所形成的—種假菌核， 學名為加細/賊7/如如世界性分佈天然資源數量很少。蛹 蟲草型,4上及培養方式與冬蟲夏草有相#的差異，真正的冬蟲夏 »草均為野生，無法以人工培養’冬蟲夏草僅可寄主於編竭蛾幼蟲， 蛹蟲草的寄主範圍較為廣泛，蜎蟲草的孢子一般會飛散在葉片或 土壤中’當翻目的幼蟲在取食時將孢子食人，隨後孢子發芽， 菌絲纏繞諸，當蟲體化猶，蟲體會社，子座即從財長出， 因此传名蛹蟲草。竭蟲草一般為單生子座或數個從寄主頭部生 出也有蟲體節。p長出，為橘紅色或橘黃色，頂部呈棒狀的子座， 人工培養蛹蟲草的外型與天然踊蟲草相似，但長度與寬度略大， 一般長度平均為5至7公分。早於时年，―响棚扣^ 201200143 等學者率先於蛹蟲草中分離出有效成分蟲草素201200143 ' VI. Description of the invention: [Technical field to which the invention belongs] - A method for improving the active ingredient of the grass, the medium composition and the method, and adding animal protein, vegetable protein, probiotic g (four) liquid or Chinese medicine extract during the cultivation process In order to cultivate the job, the appropriate cultivation environment has shortened the cultivation time, increased the productivity of the wormwood body and increased the content of the active ingredient c〇rdycepin. • [Prior Art] Cordyceps is my _-a kind of wild wild cap material, which has the effects of supplementing deficiency, benefiting essence, protecting lung, tonifying kidney, stopping bleeding and removing phlegm, nourishing and strengthening. Cordyceps and ginseng and antler are listed as the top three in China. tonic. For example, in the i757 "Materia Medica from New Year", there is a record of "Winter Cordyceps sinensis Ganping Baofei" Yishen 'supplementing Zhao, stopping bleeding and removing phlegm, has been coughing, and treating intestinal diseases are good." Chinese medicine believes that Cordyceps into the lung and kidney, can not only fill the lung yin, but also kidney yang, attending kidney deficiency, impotence and nocturnal emission, waist and knee pain, weakness after illness, chronic cough and weakness, labor cough and thin blood 'self-sweat, night sweats, etc.' It is the only traditional Chinese medicine that can balance and regulate yin and yang at the same time. There are some species of Cordyceps known in the world. There are currently 62 species in mainland China, but the most valuable ones are Cordyceps and Cordyceps. The old saying goes: I would rather have a cordyceps, not a golden jade full of cars; the folks call it "living gold." The Cordyceps sinensis recorded in the "Materia Medica" and "Materia Medica" is a kind of wild Cordyceps. There are about 300 species of Cordyceps recorded, but the Chinese products have been reported since 1993. Cordyceps only about 6 〇. Cordyceps sinensis is an expensive _ fungus with high medical and health care 201200143 and economic value. Therefore, many Chinese medicine research institutes, pharmaceutical research institutes, sericulture scientific research institutes, universities and hospitals in China have invested in Cordyceps. The research has yielded fruitful results, and the Cordyceps sinensis researched and developed is called artificial cordyceps. At present, these workers and insects are roughly divided into four categories: (1) The host of Cordyceps sinensis in the wild Cordyceps sinensis is ^ i i ^ Uiepialus oblifurous chuet Wang), no yin yin yin yin yin yinensis 57ν^/?57·5·). Most of the artificial breeders are mycelia of various Cordyceps and their fermentation products. Although their composition and pharmacological effects are similar to those of wild Cordyceps sinensis, the difference is still large. (2) Locust grass print S milk · w & ey: the host of Cordyceps militaris is tussah pupa, %ipascil 〇 myces militarae) 〇 菌 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种 接种Silkworm cocoon). (3) Gurney Cordyceps sinensis is a large-scale Cordyceps sinensis. The sub-seat is born from the head of the host. The handle is white, 1 to 9 cm long, 0.5 to 0.6 cm thick. The top is generally gray to grayish black. The long mark is round to column, solitary, bifurcated or clustered. Generally, the size is about 8 to 22 mm x 5 to 8 mm, and the boundary between the stem and the stem is clear when there is no infertility. The capsule is imitation or ampule-shaped, 〇·7 to 〇. 9 mm χ 〇 2 to 〇 3 mm, buried at the time of ripening. Parasitic on the larvae of the Hepiaidae insects in the soil. The Cordyceps militaris is Paecilomyces genus 201200143. (egg SCJ" 〇WCeS卽), which can form the fruiting bodies of the ascospores in general medium. Λ (4) 蝉草: Also known as 蝉花 or 蝉 寄 寄 蝉 蝉 蝉 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' Shape, initially grayish white, then pale yellow to dark brown. The whole herb is used as a medicine, which has the effect of eyesight. China's Jiangnan provinces are distributed. This grass system utilizes valerian in the medium of protein and other ingredients. • Growth and cultivation is now industrially produced. Cordyc^s militaris and Cordyceps sinensis are fungi of Ascomycetes, Nucleobacter, Pleurotus, Trichoderma, Cordyceps, also known as Cordyceps militaris or Cordyceps militaris. Coleoptera, silkworm, Cordyceps militaris, Cordyceps militaris, Cordyceps sinensis, is a parasitic sclerotium formed by parasitic insect larvae or carcass. The scientific name is fine / thief 7 / such as the distribution of natural resources in the world less. The Cordyceps militaris type, the 4th and the culture method and the Cordyceps sinensis have the difference of #, the real Cordyceps sinensis»grass are wild, can not be artificially cultured. 'The Cordyceps sinensis can only be hosted in the larvae of the moth, and the host range of the Cordyceps militaris is more extensive. The spores of Cordyceps militaris will generally scatter in the leaves or soil. 'When the larvae are turned, the spores are eaten, and then the spores are germinated, the hyphae are entangled, and when the worms are still in place, the worms are in the community, and the scorpions are Growing up, hence the name 蛹 Cordyceps. Cordyceps is usually a solitary or several from the head of the host. p grows out in orange-orange or orange-yellow, with a rod-shaped sub-seat at the top. The artificially cultivated Cordyceps is similar in appearance to natural Cordyceps, but has a slightly longer length and width, and the average length is 5 to 7 cm. In the early years, the shackle buckles ^ 201200143 and other scholars took the lead in separating the active ingredient cordycepin from Cordyceps militaris.
Cordycepin 〔3'deoxyadenosine〕’在西元1997年後的許多研究中，蛹蟲草 被學者證實具有與冬蟲夏草相_功效，有抗顧、活化、調節 免疫系、.績改善呼吸系統、腎功能、心血管、肝臟及性功能等功 效。提高人體免疫調節作时增強了單核巨嗟細胞的吞嗟能力、 增強體液免疫功能、對細胞免疫起免疫作用及增強自然殺手 細胞（natural killer cell)的活性。Cordycepin [3'deoxyadenosine]' In many studies after 1997, Cordyceps sinensis has been confirmed by scholars to have efficacy with Cordyceps sinensis. It has anti-care, activation, regulation of immune system, improvement of respiratory system, renal function, cardiovascular , liver and sexual function. Increasing the immune regulation of the human body enhances the swallowing ability of mononuclear giant scorpion cells, enhances humoral immunity, immunizes cellular immunity, and enhances the activity of natural killer cells.
目則生物學家纽_生物科技方法將蛹蟲賴接種至家蠢 或柞蠢蛹進彳了人工大量培育，藥效、藥理與野生種相祕至更好。 由於蛹蟲草與我們家喻戶曉的冬蟲夏草⑹咖聊同 屬蟲草屬’因其為蟲草屬的赋種，為_外學者普遍接受。野 生蜗蟲草是―種具有_、滋補功能的珍貴中騎，自西元讓 年以來’吉林省蠢業研究所和長春中藥廠，從野生蜗蟲草（亦稱蜗 j)子座巾分離_草8，人王麵職蛹上而軸的—種人工择 :的蟲草。由於_草的寄主廣泛，野外採集之蛹蟲草往往合有° η主或雜品系不_問題產生，故以人碎培的蛹蟲 化的&株進行感染，故具有 ' 和系列㈣，糊繼 :隨著科技的進步，其栽培、應用前景十分：開闢： ==菌接種在液體培養基中，從培養基中可分離出蟲草有:者 里為25錢/公升。在_培養射可得噼㈣，射可分 201200143 -離離胚脂醇、麥角固醇、卜甘露醇(5. 37%)、蟲草素(〇.瞧）、 腺嗓吟(0. 007%)、腺苦(〇. 016%)、尚有氨基酸、微量元素等。 北冬蟲夏草（蜗蟲草）所含蟲草素及生命元素碼均遠遠高於其 他野生蟲草’並且含豐富的麥角_、多較氨基轉多種營養 成份。研錢示仙抗_、抗縣、抗絲、治療心律失常、 調節脂類代謝、增強免疫機能等方面都有_效果情人體的泌 尿系統、免疫纽、呼吸系統、造血纽、心血衫統、代謝系 •統、令樞神經系統的調節具有十大藥理作用而初露鋒芒。在高科 技培養的觀裡，鮮的生長顧衫自祕不鄕件的影響， 有效成份不受損失，所以品質優良且較為穩定。經檢測人工培養 蟲草的主要#養與_成分均與野生蟲草的含量相當甚至更高。 現代科學驗證触草不僅具树殊的營養價值，*且有明顯的藥 用價值。其中尤以蟲草酸、蟲草素、蟲草多酶和s〇d等多種生物 活性物質的藥用價值最為顯著。蟲草酸(甘露醇）可以顯著地降低 鲁顱壓’促進身體聽代謝使·血和腦血栓病症得到緩解。 蟲草素是-種具有抗g活性的核_純，對核錄料酸聚合 酶有很強的抑製作用。在舰轉錄·A過程中使祕成熟障礙， 抑制癌細胞長。並有降血糖的作用。蟲草多糖是—種高度分 枝的半乳甘露雜，它能促進淋巴細胞，提高血清㈣的抗 體含量和機體的免疫功能，增強機體自身抗癌抑癌的能力。 SOD(超氧化物歧化酶)可叫__超氧自由基，具有抗衰老、 抗癌抑癌的作用。此外，竭蟲草還含有豐富的砸㈣，這麵量 201200143 .70素已被絲公認是人體必需的微量元素，是麵胱甘肽(GSH)的活 性中心’⑽半胱氨酸的形式連接在酶蛋白的肽鏈上，保護細胞 膜的穩定性的正常的通透性，並刺激免疫球蛋白和抗體的產生， 增強機體免疫和抗氧化能力。同時，大量的科學實__可以 明顯地抑制癌細胞的生長。 有研究學者對_蟲草進行藥化、藥理、毒理分析測定，指 出人工培育的蠶蜗蟲草，不但無毒，且具有明顯的鎮靜、抗疲勞、 •抗腫瘤、抑制癌細胞及雄性激素等作用，可增強機體免疫功能等。 另有學者針對細蟲草的藥化、藥理研究顯示，具有抗疲勞、抗 惽厥作用’可提高淋巴細胞轉化率，增強小鼠免疫能力，對 肺癌有抑製作用，體外實驗證明對喉癌細胞有殺滅作用，並能使 大鼠血漿睪細含量增高，使大鼠體重及包皮腺、缝、前列腺 的重1顯著增加，有明顯的雄性激素作用。 有關蟲草的研究正蓬勃發展’射研究好者為冬蟲夏草與 _蛹蟲草。冬蟲夏草讲&.職蝴是由鮮时生在編福 蛾幼蟲體内，生長發月而成，而蛹蟲草⑹，办鄉則 是_蟲草S寄生在昆蟲_上發“成。冬蟲夏草的寄主專丄 性非常高，而補蟲草則可感染較廣泛的寄主。冬蟲夏草已被學者 證明具有抗腫瘤、活化或調節免疫系統、改善呼吸系統、腎功能、 心血官、肝臟及性功能等功效，並對冬蟲夏草傳統療效如滋肺、 補腎狀強精力等深入探討；蛹蟲草則在西元膽年後漸漸被學 者证實具有與冬蟲夏草姻的功效，且根據研究結麵示，蜗蟲 201200143 '草之菌絲體與冬蟲夏草菌絲體之成份頗為相似。然而野生的冬蟲 夏草其產里有限’採集不易’加上人們瘋狂搜尋挖掘冬蟲夏草， 導致生態魏的破壞，_與冬蟲夏草齊名細鮮在國際上日 盈受到人們_注。研究學者對於野生冬蟲夏草化學成分的研究 較夕而對人工蜗蟲草化學成分研究較少。加上研究結果證明菌 絲體僅具有-些雜’其功效或主要成分仍與子倾大不相同， 且菌絲體品質往往隨著培養基之組成份、培養條件或培養方式改 籲，’造成中藥界在__以_體取代實際的蟲草，故生物學 豕直希望月匕以人工培養方式培養出與野生種相似的冬蟲夏草及 蜗蟲草。 蛹触草（北冬蟲夏草菌）是一種較嗜低溫的真菌，高溫會抑制 其生長。由於自齡產的資源有限，年產量明顯逐年減少，目前 已不敷市場的需求。因此，如何發展人卫培養技術，以滿足市場 的品求，事貫上存在著一個極大的商機。 d存在於蜗蟲草子貝體中之蟲草素含量遠較菌絲體為高， 但培養之瓶齡於生成子實體之咖較長（約需二则），子 之出解低且變異率高，若駄幅提高子實體之育鮮與子實 中之蟲草素含量，則具有極高產業應用價值。 【發明内容】 有鑑於此，為解決上述問題，本發明提供一種提高竭蟲 能及有效成份之固態培養基組合物及方法，其特徵在於利用 動物性蛋白（牛奶、肉汁萃取物、胎牛血清、魚粉、魚精、蠢蜗11 9 LS] 201200143 紅㈣）、祕性蛋白、起_賴⑽ =菌種源軸一 ^中進行暗培養與光培養_蟲草子實體之固體培養方法，進而 升蛹蛾草子倾的錢、触培養時狀提高有效成分蟲草素The project is based on the biologist's method of inoculation of locusts to the home stupidity or stupidity, and artificial cultivation, the efficacy, pharmacology and wild species are more secret. Because Cordyceps sinensis and our well-known Cordyceps sinensis (6) are the same as Cordyceps genus, because it is a species of Cordyceps, it is generally accepted by foreign scholars. Wild wormwood is a rare medium-riding cultivar with _, nourishing function. Since the year of the dynasty, 'Jilin Province Stupid Industry Research Institute and Changchun Chinese Medicine Factory, separated from the wild worm (also known as worm j) sub-seat _ grass 8 The king's face is on the axis and the axis is a kind of artificial choice: the Cordyceps. Due to the wide range of _ grass hosts, the Cordyceps sinensis collected in the field is often combined with η main or miscellaneous strains, so it is infected with the mites of the worms, so it has 'and series (four), paste Following: With the advancement of science and technology, its cultivation and application prospects are very: open up: == bacteria inoculated in liquid medium, the cordyceps can be separated from the medium: 25 money / liter. In the _ culture shot can be obtained 四 (four), shot can be divided into 201200143 - separated from the embryonic alcohol, ergosterol, mannitol (5. 37%), cordycepin (〇.瞧), adenine (0. 007 %), adenine (〇. 016%), still have amino acids, trace elements and so on. Cordyceps sinensis (Worm Cordyceps) contains much higher levels of cordycepin and life elements than other wild Cordyceps and is rich in ergot horns. Researching money shows immortality _, anti-county, anti-silk, treatment of arrhythmia, regulation of lipid metabolism, enhancement of immune function, etc. _ effect of the human body's urinary system, immune nucleus, respiratory system, hematopoietic neoplasm, blood-stained, Metabolic system, the regulation of the central nervous system has ten major pharmacological effects and is initially exposed. In the concept of high-tech training, the fresh growth of the shirt is not affected by the secrets, and the effective ingredients are not lost, so the quality is good and stable. The main #养和_ ingredients of the artificially cultured Cordyceps were tested to be comparable or even higher than the wild Cordyceps. Modern science has proven that it is not only a nutritional value, but also has obvious pharmaceutical value. Among them, medicinal value of various biologically active substances such as cordycepic acid, cordycepin, cordyceps sinensis and s〇d is the most significant. Cordycepic acid (mannitol) can significantly reduce the pressure of the cerebral thrombus, which promotes the body's auditory metabolism and relieves blood and cerebral thrombosis. Cordycepin is a kind of nucleus with anti-g activity, which has a strong inhibitory effect on the nuclear acid-polymerase. In the process of transcription and A of the ship, the secret of maturation is inhibited, and the cancer cells are inhibited. And have the effect of lowering blood sugar. Cordyceps polysaccharide is a highly branched galactomannan, which can promote lymphocytes, improve the serum (IV) anti-body content and immune function of the body, and enhance the body's own anti-cancer and anti-cancer ability. SOD (superoxide dismutase) can be called _ superoxide free radical, which has anti-aging, anti-cancer and anti-cancer effects. In addition, Cordyceps militaris is also rich in strontium (IV). This amount of 201200143.70 has been recognized as an essential trace element in human body. It is the active center of gSH (GSH) and is connected in the form of cysteine. The peptide chain of the enzyme protein protects the normal permeability of the cell membrane and stimulates the production of immunoglobulins and antibodies, enhancing the body's immunity and antioxidant capacity. At the same time, a large number of scientific __ can significantly inhibit the growth of cancer cells. Some researchers have conducted pharmacological, pharmacological and toxicological analysis on wormwood, pointing out that artificially cultivated silkworm, Cordyceps militaris, is not only non-toxic, but also has obvious sedative, anti-fatigue, anti-tumor, inhibition of cancer cells and androgen. Can enhance the body's immune function. Other scholars have shown that anti-fatigue and anti-caries effects can improve the lymphocyte transformation rate and enhance the immunity of mice, and have an inhibitory effect on lung cancer. In vitro experiments have shown that there are The killing effect can increase the plasma sputum content of rats, and significantly increase the body weight and the weight of the foreskin, sulcus and prostate of the rats, and have obvious androgenic effects. Research on Cordyceps is booming. The best researchers are Cordyceps sinensis and Cordyceps sinensis. Cordyceps sinensis &. is the birth of the larvae of the larvae, which grows in the larvae of the larvae, and grows into the moon, while the locusts (6), the township is _ wormwood S parasitic in the insect _ on the hair. The host of Cordyceps sinensis The specificity is very high, and Cordyceps can infect a wider range of hosts. Cordyceps has been proven by scholars to have anti-tumor, activate or regulate the immune system, improve the respiratory system, kidney function, blood, blood, liver and sexual function, and The traditional curative effect of Cordyceps sinensis, such as nourishing lung and nourishing kidney, was discussed in depth. Cordyceps sinensis was gradually confirmed by scholars to have the effect of winter worms, and according to the research results, worms 201200143 'grass The silk body is quite similar to the mycelium of Cordyceps sinensis. However, the wild Cordyceps sinensis has limited production and is not easy to collect. People add crazy to search for Cordyceps sinensis, which leads to the destruction of ecological Wei, and _ with the same name of Cordyceps sinensis in the world. Received by people. Note: Researchers have studied the chemical constituents of wild Cordyceps sinensis and have less research on the chemical constituents of artificial Cordyceps sinensis. The results show that the mycelium has only a few impurities, its efficacy or main components are still different from the sub-dip, and the quality of the mycelium often changes with the composition of the medium, culture conditions or culture methods, resulting in the Chinese medicine community. In the __ to replace the actual Cordyceps with _ body, it is hoped that the scorpion will cultivate the Cordyceps sinensis and the Cordyceps sinensis similar to the wild species by artificial culture. The stalk of the grass (Northern Cordyceps sinensis) is a fungus with lower temperature. High temperature will inhibit its growth. Due to the limited resources of self-aged production, the annual output is obviously decreasing year by year, and it is no longer enough for the market. Therefore, how to develop human health culture technology to meet the market demand, there is a A great business opportunity. d The content of cordycepin in the shellfish of the wormwood is much higher than that of the mycelium, but the age of the cultured bottle is longer in the generation of the fruiting body (about two). It is low and has a high mutation rate. If the width of the fruiting body improves the content of the cordycepsin in the fruiting and fruiting of the fruiting body, it has a very high industrial application value. [Invention] In view of this, in order to solve the above problems, The invention provides a solid medium composition and method for improving deworming ability and active ingredient, which is characterized by using animal protein (milk, gravy extract, fetal bovine serum, fish meal, fish essence, stupid volcano 11 9 LS) 201200143 red (four) ), secret protein, _ _ _ (10) = strain source axis - ^ in the dark culture and light culture _ Cordyceps fruit body solid culture method, and then raise the moth grass money, touch the culture time to improve the effective component Cordyceps Prime
Cconlycepin)含量。 本發明亦提供-種蛹蟲草_培養方法，包含菌種活化、踊 蟲草液態培養基製備、接聽配方製備、蛹蟲草_'培養基製備、 與光/暗靜置培養等步驟。 另-方面’本發明亦提供一種最佳踊蟲草固態培養環境條 件’其條件為培養溫度攝氏18至25度、培養渔度7〇至9〇百分 比。 【實施方式】 有關本發明的特徵與實作，茲以最佳實施例詳細說明 # 如下： 1.材料製備 1.1蜗蟲卓菌種活化 將存放攝氏負80度(。〇的蛹蟲草（購自食品工業研 九所BCRC32219)冷束小管取出，置入水浴槽中以攝氏 22度(°C)水浴解凍；於無菌操作台，以接種環將冷凍小 管中的菌絲液接種到PCA培養基，並置入培養箱中以攝 氏22度(°〇培養7至14天。 201200143 1. 2蜗蟲草液態培養基製備 秤取胰化蛋白腺(Tryptone)5. 0公克(g)、酵母萃取 物（Yeast extract)2. 5公克（g)、右旋葡萄糖 (Dextrose)l. 〇 公克(g)、蒸餾水(Distilled water)l. 0 公升(L)，以滅菌釜滅菌攝氏121度(。〇，20分鐘，放置 24小時以上，確定滅菌完全後備用。 1. 3添加物製備： (1) 動物/植物性蛋白：將牛奶、肉汁萃取物（如·· Beef extract)、胎牛血清（FBS)、魚粉、魚精、蠶蛹、 活體紅蚯蚓（幻出、豆類植物、植物等 洗淨、過濾（〇· 45/zm)後’進行冷凍乾燥並細化研 磨。 (2) 姐生菌發酵液:如乳酸菌發酵液、真菌發酵液等。 (3) 中藥材萃取液。 1.4蟲草接菌液製備： 接菌液製備分為： ()PCB接菌液.將已活化之蜎蟲草菌接種於⑽Cconlycepin) content. The invention also provides a method for cultivating Cordyceps militaris, comprising the steps of strain activation, preparation of liquid culture medium of Cordyceps militaris, preparation of answering formula, preparation of Cordyceps militaris, preparation of medium, and incubation with light/dark static. In addition, the present invention also provides an optimum condition for solid-state culture of Cordyceps militaris. The conditions are a culture temperature of 18 to 25 degrees Celsius and a culture degree of 7 to 9 percent. [Embodiment] The features and implementations of the present invention are described in detail in the preferred embodiments. The following are the following: 1. Preparation of materials 1.1 Activation of the species of the worms will store minus 80 degrees Celsius (. 〇 蛹 Cordyceps (purchased from Food Industry Research Institute 9 BCRC32219) cold bundle tube was taken out, placed in a water bath and thawed in a 22 ° C (water) bath; on the aseptic rack, the mycelial liquid in the cryotube was inoculated into PCA medium with an inoculating loop, and Place in the incubator at 22 degrees Celsius (°〇 culture for 7 to 14 days. 201200143 1. 2 Preparation of liquid culture medium of Cordyceps militaris. Tryptone 5. 0 g (g), yeast extract (Yeast extract 2, 5 grams (g), dextrose (dextrose) l. gram (g), distilled water (Distilled water) l. 0 liters (L), sterilized in a sterilization pot 121 degrees Celsius (. 〇, 20 minutes, Leave for more than 24 hours, make sure that the sterilization is complete and use. 1. 3 Additive preparation: (1) Animal/plant protein: milk, gravy extract (such as Beef extract), fetal bovine serum (FBS), fish meal, Fish essence, silkworm cocoons, living red cockroaches (magic, bean plants, plants, etc.) After filtration (〇·45/zm), freeze-drying and refining are carried out. (2) Fermentation liquid for sister bacteria: such as lactic acid bacteria fermentation broth, fungal fermentation broth, etc. (3) Chinese herbal medicine extract. 1.4 Cordyceps sinensis Preparation: The preparation of the inoculum is divided into: () PCB inoculum. Inoculate the activated Cordyceps sinensis (10)
Count Broth)培養基中，並培養於攝氏2〇至24度環 境中，以每分鐘90轉(rpm)進行搖瓶培養3至7 即可作為接菌液使用； (二）BR-PCB接菌液：將各種添加物之乾燥粉末秤取⑴ 公克(g)加到500毫升(ml) PCB培養液中以均質機 201200143 ' PCB培養射的動触蛋自均自打散，轉種環勾取已 活化之踊鮮菌種源菌絲體，置入動物性蛋白接菌液 中’將已接種動物性蛋白的翻液置人培養箱，以攝 氏20至24度’每分鐘90轉(rpm)進行搖瓶培養3至7 天後即可作為接菌液使用。 1.5蛹蟲草固態培養基製備： 固態培養基有可分為三種： • ㈠穀物培養基.枰取30至公克(g)的穀物培養基（白米 或糙米或燕麥等’以白米尤佳）置入玻输中，加入30 至40宅升(ml)的R. 0.水，輕晃瓶身，使R 〇水完全浸 渔榖物培養基’蓋上玻璃瓶瓶蓋，以高壓滅菌方法（攝 氏121度(°C)’30分鐘）進行培養基滅菌。滅菌完成後， _態培養基溫度低於攝氏5〇度(。〇前將玻璃瓶的瓶 蓋完全鎖緊後冷卻至f溫。麵後織培養基高度約為 # 2. 5至3· 5公分，水分為5〇至7〇百分比(％)。 (二）固態BR穀物培養基：秤取30至4〇公克(g)的穀物培養 基（白米或糙米或燕麥等，以白米尤佳）置入玻璃瓶中， 加入30至40毫升(ml)的R. 〇.水，輕晃瓶身，使R. 〇. 水完全浸溼穀物培養基，蓋上玻璃瓶瓶蓋，以高壓滅菌 方法（攝氏121度（°C)，30分鐘）進行培養基滅菌。滅 菌完成後，於固態培養基溫度低於攝氏5〇度（。〇）前將玻 璃甑的瓶蓋完全鎖緊後冷卻至常溫。滅菌後穀物培養基 12 201200143 - 高度約為2. 5至3. 5公分，水分為50至70百分比（％)。 於榖物培養基中均勻加入〇· 1至lg動物性蛋白質粉末備 用。 (三）液態BR榖物培養基：科取3〇至40公克(g)的榖物培養 基（白米或糙米或燕麥等，以白米尤佳）置入玻璃瓶中， 加入30至40亳升(mi)的R.〇.水，輕晃瓶身，使尺〇. 水完全浸溼穀物培養基，蓋上玻璃瓶瓶蓋，以高壓滅菌 • 方法（攝氏121度(。〇，30分鐘）進行培養基滅菌。滅 菌完成後’於固態培養基溫度低於攝氏5〇度（。〇)前將玻 璃瓶的瓶蓋完全鎖緊後冷卻至常溫。滅菌後穀物培養基 尚度約為2. 5至3_ 5公分，水分為50至70百分比(％)。 於榖物培養基中均勻加入濃度〇. 〇1%至〇. 5%動物性蛋 白質製備液備用。 鲁 2.固知培養基接種觀察生長情形並分析蟲草子實體所含蟲草 素之差異情形： 2.1固態培養基接種之生長情形 吸取已培養3至7天之蟲草接菌液至5毫升(ml)均勻 加入培養絲面。將已接種翻液之_、培養基移入暗培養 至，以溫度攝氏18至25度（。〇，溼度70至90百分比 進行培養5至7天，待_培養基表面長出白色菌絲體，且 菌絲體約佔m態培養基表面積2/3時，即將_鱗移放到 13 201200143 有照光設備的培養環境，進行光照培養。進行照光培養，其 光照強度至少500勒克斯（lux)以上，且照光時間約8至15 小時/天，將培養ί哀境控制為溫度攝氏18至25产d):呈产 70至90百分比（％)。 ’又 本實驗之實驗組分別為： (一） 對照組：穀物培養基+蛹蟲草培養液 (二） 實驗組：PCB-BR穀物培養基+蛹蟲草培養液 上述實驗組鋪照組，以_培養魏及培養條件進行 培養，以鮮子實體長度為7公分為培養終點，觀察兩組於 暗培養階段、照光培概段柯軸之生長_，並分析蟲 草子實體所含蟲草素之差異情形。 兩組於暗培養階段，觀察_、體_分佈情形，如圖1Α 所示實驗組_體幾乎佈滿培養基，但圖1Β所示對照組約只 生長2/3培養基表面。 兩組於光培養階段，觀察菌絲體菌絲分佈情形，於第2 天實驗組白色菌絲轉為黃色’且菌絲已幾乎長滿穀物培養基 中’如圖2Α所示’但對照組白色菌絲轉為黃色，但仍也部份 穀物培養基尚未長滿菌絲，如圖2β所示；第8天實驗組菌絲 顏^呈金η色並已有出兹現象，如圖％所示’對照組菌絲顏 色壬鵝黃色且尚未有紐，如圖2D所示；第18天實驗把多 數出^ ’如®I 2E所示，對照組僅部分祕，如圖2ρ所示. 第26天實驗_對照組如圖2G、H所示；第5{)天實組蟲草 14 201200143 對照組蟲草子實體長 子貫體長度已達7公分，如圖21所示 度尚未達7公分，如圖2J所示。 添力^物性蛋白之實驗組（此例為活體_粉），於固離 培養子貫體生鱗間較賴組短。子魏紐平均7公分， 實驗組所需培養日销㈣天,照_需時_〇天。 2. 2子實體重量及蟲草素含量Count Broth) medium and cultured in a temperature range of 2 to 24 degrees Celsius, shake flask culture at 3 revolutions per minute (rpm) 3 to 7 can be used as a contact solution; (2) BR-PCB inoculant : Weigh the dry powder of various additives (1) g (g) into 500 ml (ml) PCB culture solution to homogenize the machine 201200143 'PCB culture shots are self-dispersing, and the ring is activated. The provenance mycelium of the fresh bacterium is placed in the animal protein bacterium. 'The lysate that has been vaccinated with animal protein is placed in a human incubator and shaken at 90 rpm (rpm) at 20 to 24 degrees Celsius. After 3 to 7 days of bottle culture, it can be used as a contact solution. 1.5 Preparation of Cordyceps sinensis solid medium: There are three types of solid medium: • (1) Grain medium. Take 30 to g (g) of the grain medium (white rice or brown rice or oats, etc., especially white rice). Add 30 to 40 liters (ml) of R. 0. water, shake the bottle, and let R water completely immerse in the sputum culture medium' cover with a glass bottle cap for autoclaving (121 ° C (°C) ) '30 minutes) The medium was sterilized. After the sterilization is completed, the temperature of the medium is less than 5 degrees Celsius (the bottle cap of the glass bottle is completely locked and then cooled to f temperature. The height of the back-weaving medium is about #2.5 to 3.5 cm, The water is divided into 5〇 to 7〇% (%). (2) Solid BR grain medium: 30 to 4 gram (g) of grain medium (white rice or brown rice or oats, especially white rice) placed in glass bottles Add 30 to 40 ml (ml) of R. 〇. water, shake the bottle, and let R. 〇. Water completely wet the grain medium, cover the glass bottle cap, and autoclave (121 ° C ( °C), 30 minutes) The medium is sterilized. After the sterilization is completed, the bottle cap of the glass crucible is completely locked and cooled to normal temperature before the temperature of the solid medium is lower than 5 degrees Celsius (.〇). The sterilized grain medium 12 201200143 - The height is about 2.5 to 3.5 centimeters, and the water content is 50 to 70% (%). Add 〇·1 to lg animal protein powder evenly in the mash medium. (3) Liquid BR sputum medium: Take 3 to 40 grams (g) of sputum medium (white rice or brown rice or oats, etc., white Miyoujia) Put in a glass bottle, add 30 to 40 liters of (R) R. 〇. water, shake the bottle to make the ruler. Water completely wet the grain medium, cover the glass bottle cap, Autoclaving • Method (sterilization of the medium at 121 ° C. (〇, 30 min). After the sterilization is completed, the cap of the glass bottle is completely locked and cooled until the solid medium temperature is below 5 ° C (.〇). Normal temperature. After sterilization, the grain medium is about 2.5 to 3 5 cm, and the water is 50 to 70% (%). The concentration is uniformly added to the mash medium. 〇1% to 〇. 5% animal protein preparation Lu 2. Fixation medium inoculation to observe the growth situation and analyze the difference of cordycepin contained in Cordyceps sinensis fruit body: 2.1 Growth condition of solid medium inoculation: Pipette the inoculum liquid that has been cultured for 3 to 7 days to 5 ml (ml The cultured silk surface is uniformly added. The culture medium has been inoculated into the dark culture to a temperature of 18 to 25 degrees Celsius (〇, humidity 70 to 90% for 5 to 7 days, and the surface of the medium is grown. White mycelium, and mycelium accounts for about m-state medium When the surface area is 2/3, the _ scale is moved to 13 201200143. The culture environment of the illuminating equipment is used for light culture. The illuminating intensity is at least 500 lux and the illumination time is about 8 to 15 hours/day. The culture grief is controlled to a temperature of 18 to 25 degrees Celsius. d): 70 to 90% (%) of production. 'The experimental group of this experiment is: (1) Control group: grain medium + Cordyceps militaris culture solution (2) Experimental group: PCB-BR grain medium + Cordyceps militaris culture group The above experimental group was incubated with _ culture and culture conditions, and the length of the fresh fruit body was 7 cm. In the culture stage, the growth of the Ke axis in the illuminating section, and the analysis of the difference in cordycepin contained in the Cordyceps fruiting body. In the dark culture stage, the two groups were observed, and the body_distribution was observed. As shown in Fig. 1Α, the experimental group was almost covered with the medium, but the control group shown in Fig. 1 only grew about 2/3 of the medium surface. In the light culture stage, the mycelial mycelial distribution was observed. On the second day, the white hyphae of the experimental group turned yellow, and the hyphae were almost filled with the grain medium, as shown in Fig. 2Α, but the control group was white. The hyphae turned yellow, but some of the grain medium was not yet full of hyphae, as shown in Figure 2β. On the 8th day, the mycelial color of the experimental group was gold n-color and had a phenomenon, as shown in Figure 'The color of the control hyphae is yellow and there is no New Zealand, as shown in Figure 2D; on the 18th day, the experiment showed that most of them were as shown by ® I 2E, and the control group was only partially secret, as shown in Figure 2ρ. Day experiment _ control group shown in Figure 2G, H; 5th {) day group Cordyceps 14 201200143 control group Cordyceps sinensis body length of the child has reached 7 cm, as shown in Figure 21 has not reached 7 cm, as shown 2J shows. The experimental group of the addition of the physical protein (in this case, the living body _ powder) was shorter in the spheroids than in the spheroids. Zi Wei New average 7 cm, the experimental group needs to cultivate daily sales (four) days, according to _ time _ 〇 day. 2. 2 fruit body weight and cordycepin content
曰依據培養_為蟲草子實體長為7公分進行子實體重 量及蟲草素含量分析，添加動物性蛋白之實，於每瓶固 態培養瓶的子實體重量㈣驗重。實驗崎朗態培養子 實體重量（溼重）平均為1G.62±1.31公克(趣，對照組每 瓶固態培養子實體重量（㈣）平均為8.咖肩公克⑻/ 瓶；添加動物性蛋白之實驗組，蛹蟲草子實體的蟲草素 (cordycepin)較對照組高。實驗組子實體之乾品蟲草素為〇· 8 百分比（％)，對照組子實體之乾品蟲草素為〇. 5百分比， 兩組於重量上具有統計顯著差異㈣._)，結果如圖請 TJx ° 雖然已說明且描述了本發明之實施例，但是熟悉此項 技術者可作各種錢及改良。衫意欲將本發明限制於如 所說明之特殊形式，且所有不背離本發明之精神及範圍的 修改都屬於如隨附之申請專利範圍辛所界定之範圍内。 综觀上述，本發明以其整體之組合與特徵而言，既未 201200143 •曾見諸於同類產品中，申請前亦未公開，誠已符合專利法 之法定要件，依法提出發明專利之申請。 【圖式簡單說明】 圖1A實驗組於暗培養階段之生長情形； 圖1B對照組於暗培養階段之生長情形； 圖2A照光培養階段第2天實驗組之生長情形； 圖2B照光培養階段第2天對照組之生長情形； ® 圖2C照光培養階段第8天實驗組之生長情形； 圖2D照光培養階段第8天對照組之生長情形； 圖2E照光培養階段第18天實驗組之生長情形； 圖2F照光培養階段第18天對照組之生長情形； 圖2G照光培養階段第26天實驗組之生長情形； 圖2H照光培養階段第26天對照組之生長情形； 圖2G照光培養階段第50天實驗組之生長情形； φ 圖2H照光培養階段第50天對照組之生長情形；以及 圖3蟲草子實體重量統計數據。 【主要元件符號說明】 益 * »»、 [S] 16曰 According to the culture _ for the body weight of the Cordyceps sinensis, the body weight and the cordycepin content were analyzed, and the animal protein was added, and the weight of the fruit body of each bottle of the solid culture bottle was measured (4). The average weight (wet weight) of the cultured fruit body in the experimental state was 1G.62±1.31 g (the interest, the weight of the solid body culture body per bottle in the control group ((4)) averaged 8. coffee shoulder (8) / bottle; animal protein added In the experimental group, the cordycepin of the Cordyceps militaris fruiting body was higher than the control group. The dried cordycepin of the fruiting body of the experimental group was 〇·8 percentage (%), and the dried cordycepin of the control group was 〇. Percentage, the two groups have statistically significant differences in weight (IV)._), and the results are as shown in the figure. TJx ° Although the embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by those skilled in the art. The invention is intended to be limited to the particular forms of the invention, and the scope of the invention as defined by the appended claims. Looking at the above, the present invention, in terms of its overall combination and characteristics, is neither 201200143. It has been seen in similar products and has not been disclosed before the application. Cheng has already complied with the statutory requirements of the Patent Law and has filed an application for invention patents according to law. Figure 1A shows the growth of the experimental group in the dark culture stage; Figure 1B shows the growth of the control group in the dark culture stage; Figure 2A shows the growth of the experimental group on the second day of the light culture stage; 2 days of growth of the control group; ® Figure 2C Growth of the experimental group on the 8th day of the light culture stage; Figure 2D Growth of the control group on the 8th day of the light culture stage; Figure 2E Growth of the experimental group on the 18th day of the light culture stage Figure 2F Growth of the control group on the 18th day of the light culture stage; Figure 2G Growth of the experimental group on the 26th day of the light culture stage; Figure 2H Growth of the control group on the 26th day of the light culture stage; Figure 2G50 of the light culture stage Days of the experimental group; φ Figure 2H growth of the control group on the 50th day of the light culture stage; and Figure 3 the weight data of Cordyceps sinensis. [Main component symbol description] Benefit * »», [S] 16