201139663 六、發明說明： 【發明所屬之技術領域】 本發明係提供一種培育北冬蟲夏草之方法，特定言之，係關於 以發光二極體（LED)燈作為光源，培育北冬蟲夏草（Cor办cepi militaris)之方法。 【先前技術】 『新華本草綱目』上記載：冬蟲夏草性味甘平入肺、腎二經， 具有「補而不峻、滋而不腻j之特性，對於虛不受補的體質亦可 取代人參、當歸等藥材，集天地陰陽之精氣於一身，既可滋陰、 亦可助陽，是極為高貴細緻的珍品，各種體質均可適用，所以古 人云：「寧要蟲草一把，不要金玉滿堂」，在傳統中藥中，冬蟲夏 草被譽為漢方中之王者，日本人更稱之為終極漢方，其珍貴由此 可見一斑。 根據研究，冬蟲夏草之前述珍貴特性，主要係來自所包含之蟲 草素（Cordycepin)與多醋（polysaccharide)。其中，蟲草素為一 種腺嗓吟（adenine )的類似物，具有清除自由基、抗菌性、抗原 蟲性、抗腫瘤、抗病毒等功效，且可防止脂質、蛋白質、低密度 脂蛋白（low density lipoprotein，LDL)之氧化，進而降低動脈硬 化（arteriosclerosis)、冠狀動脈心臟病（coronary heart disease) 等疾病的發生。多醣則為一種由葡萄糖（glucose)為基本單位所 形成之葡聚糖（glucan)，無法藉由人工方式合成；多餹對於人體 細胞具有特殊之活化作用（例如活化巨噬細胞），且具有增強免疫 系統、抗老化、清除自由基、解毒、降低膽固醇、調整血壓、降 低紫外線傷害、以及促進腸道内益生菌生長等功效。而在冬蟲夏 201139663 草400多種品系中，又以北冬蟲夏草（（：〇咖哪磁·㈣，又名 蛹蟲草）中蟲草素之含量最高、也最為珍貴。 北冬蟲夏草為一種蟲生真菌，在生物學上的分類為：真菌界、 真菌群、子囊菌亞群、核菌綱、球殼菌目、麥角科、冬蟲夏草屬。 在夏天時，成熟的北冬蟲夏草會釋放出真菌孢子，感染蝙蝠蛾之 幼蟲並寄生於其體内，以蟲體為營養來源持續生長；到冬天時， 真菌於幼蟲體内生長成為菌絲體（myceHa)，逐漸佈滿蟲體，因而 導致幼蟲枯死，但幼蟲之外殼完整無損，仍呈蟲體之外形，故此 ♦階段被稱為「冬蟲」。到翌年春夏時，幼蟲體内之菌絲體生長成熟 時，受環境因子之刺激，即自枯死幼蟲的頭部發芽，長出棒狀之 子實體（hymenophore ’ fruit body )並穿出地面，此階段即稱為「夏 草」。於北冬蟲夏草中，蟲草素與多醣主要存在於子實體部分北 冬蟲夏草之下端則為枯死幼蟲，其體内為餘存之菌絲體。 野生之北冬蟲夏草必須在夏至前後積雪融化前採摘因枯死幼 蟲體内的菌絲體會開始分解退化，子實體所含之蟲草素與多醣亦 φ 會逐漸流失。然而，由於野生之北冬蟲夏草均為隨機發現、難以 掌握採摘時間，因此，目前野生之北冬蟲夏草現貨的有效成分品 質不一。此外，北冬蟲夏草之年產量受環境變遷明顯逐年減少， 目前已不敷市場的需求。 目前已開發出各種人工培育北冬蟲夏草之技術’以得到有效成 分（即，蟲草素與多醣）品質穩定之北冬蟲夏草，諸如：固態洋 菜靜置培養、液態旋轉震盪培養、液相靜置培養及深層液態發酵 培養等等，該等培養技術之相關細節可參考，例如，台灣專利公 告第1231825號所揭露者；此外，台灣專利公開第2〇〇833836號 201139663 及台灣專利公開第200600006號亦有提及北冬蟲夏草之培育方 法，主要涉及暗室（避光）培養與光照培養，前述專利文獻之内 容均併於此處以供參考。 已知之北冬蟲夏草之培育方法中，幾乎皆使用日光燈或太陽光 作為培育光源。惟於利用太陽光時，會大幅受限於難以控制的自 然因素，不易提供穩定的光照環境；而當使用日光燈時則會有 耗電量大、產生大量熱能、光度弱及使用壽命短等缺點。因此， 亟待進一步改良現有之北冬蟲夏草之培育方法。 【發明内容】 本發明之一目的在於提供一種培育北冬蟲夏草之方法，其係包 含讓含有北冬蟲夏草之菌種的培養基經歷以下二階段：a)暗處培育 階段，以及b)照光培育階段’其中該照光階段係以LED燈為光源， 該LED燈係選自以下群組：白光LED燈、藍光LED燈綠光LED 燈、及其組合。 本發明之詳細技術及較佳實施態樣，將描述於以下内容中，以 供本發明所屬領域具有通常知識者具以明瞭本發明之特徵。 【實施方式】 本發明方法所涉之含 有北冬蟲夏草之菌種的培養基 ，可以任何 一斜面菌種取出北冬蟲夏 20°C至約30°C之溫度與約 已知之合宜方法製備。舉例言之，先自 草之菌種，並於一液體培養基中，在約 _啊至約綱啊之條件下，進行液態培育約4至約5天，再 將所㈣種接種至-_料基上，接著進行a)暗處培養階段。 可用之液體培養基之配方並無任何特殊的限制 ，通常係包含碳 源（例如澱粉、葡萄糖、果糖、麥芽糖等）、說源、（例如硝酸鹽、 201139663 玉米漿、黃豆餅粉、花生餅粉、酵母粉、蛋白睐、尿素等）、無 機鹽與微量元素（例如碳、氫、氧、氮、磷、鉀等）及水等，其 中，液體培養基之水分含量係佔培養基總重量之約80重量％至約 90重量％且其碳氮比為約3··1至約10:1。舉例言之，可如下製備 一合宜之液體培養基：先混合馬鈴薯醣粉、酵母抽出物（yeast extract )、硫酸鎮（MgS〇4 )及填酸二氫钟（KH2P〇4 )，再以去離 子水溶解所得混合物以得到一水溶液，接著於一高壓滅菌釜 (autoclave )中對該水溶液進行消菌程序，提供所欲之液體培養 φ 基。 至於可用之固體培養基，其成份與碳氮比大致上與上述液體培 養基相同，但所含之水分的比例則相對較低，約為50重量％至約 60重量％。一可用於本發明方法中之固體培養基，例如可藉由混 合澱粉（如白米）及上述液體培養液，再以高壓滅菌釜進行消菌 而得。 於將液體培養基所培育之北冬蟲夏草之菌種接種到固態培養基 之後，接著對該含有北冬蟲夏草之菌種的固態培養基進行a)暗處 ^ 培育步驟。咸知如培育溫度、周圍環境的相對溼度、培養基之pH 值等皆會影響北冬蟲夏草之菌體生長，尤其北冬蟲夏草喜好低溫 及濕潤之環境。根據本發明方法之一具體實施態樣，於a)暗處培 育階段中，所採用之培育溫度約18°C至約25°C，較佳約20°C至約 22°C ;相對濕度約60%至約80%，較佳約65%至約75% ;且固體 培養基之pH值約5至約7，較佳約5.5至約6\5。 於進行完a)暗處培育階段之後，接著進行b)照光培育階段。相 同地，培育溫度、周圍環境的相對溼度、培養基之pH值等因素， 201139663 亦會影響照光培育階段之北冬蟲夏草之菌體生長。根據本發明方 法之一具體實施態樣，係於b)照光培育階段採用如下培育條件： 培育溫度約18°C至約25°C ’較佳約20。(：至約22°C ;相對濕度約 60%至約80%，較佳約65%至75%;以及固體培養基之pIi值約5 至約7，較佳約5.5至約6.5。 除培育溫度、周圍環境的相對溼度、以及培養基之pH值等因素 之外，咸知光照條件亦會影響b)照光培育階段下北冬蟲夏草的生 長。特定言之’在「夏草」階段之北冬蟲夏草，係需要一光照充 足的生長環境。於本發明方法中’係以LED燈作為光源來進行光 照培育’通常可視需要來選用LED燈的光源色。於本發明方法之 部分具體實施態樣中’係選用白光LED燈、藍光LED燈、、綠光 LED燈、及其組合，較佳係選用白光LED燈。其中，每日照光時 間約15至約20小時，以約17至約19小時較佳；且照明度係控 制為約300 Lux至約500 Lux，以約350 Lux至約450 Lux較佳， 以約380 Lux至約420 Lux最佳。於此光照條件之範圍下，可培育 出相對而言令人滿意之北冬蟲夏草，例如具有厚實、健康的子實 體。 於本發明之培育方法中，a)暗處培育階段之培育時間一般約⑺ 天至約20天，以約13天至約15天較佳；b)照光培育階段之培育 則通常歷時約50天至約70天’以約55天至約&工私从 〇月 ' J 〇：)天較佳。—般 而言，培育時間越長可增加北冬蟲夏草之乾重，但延長培育時間 並無法保證可相對提高活性成分之含量，反提高培育北冬蟲夏草 之成本；另-方δ，若培育時間太短，則可能無法提供令人滿意 之北冬蟲夏草之產量。 （' 201139663 本發明方法以LED燈取代以往慣用之日光燈作為培育北冬蟲夏 草之光源，可降低耗電量、產生較少的熱能、光度夠強且使用壽 命較長。此外，本案發明人亦發現，利用LED燈進行北冬蟲夏草 之照光培育時，更可提高北冬蟲夏草之產量並同時增加其中之有 效成分，即蟲草素及多聽，如下文中實施例所示。 茲以下列具體實施態樣以進一步例示說明本發明。其中該些實 施態樣僅提供作為說明，而非用以限制本發明之範疇。 實施例 φ 實施例1 :利用LED燈培育北冬蟲夏草 取12公克馬鈴薯醣粉、2公克酵母抽出物、1公克硫酸鎂及2 公克磷酸二氫鉀，溶於1公升去離子水中，於121°C下利用高壓滅 菌釜殺菌20分鐘後加以冷卻，製得一液體培養基。取20公克台 梗九號白米及30毫升所製得之液體培養液，於121°C下利用高壓 滅菌爸殺菌20分鐘後加以冷卻，製得一固體培養基。 取12公克馬鈐薯醣粉、2公克酵母抽出物、20公克瓊脂、1公 克硫酸鎂及2公克磷酸二氩鉀，溶於1公升去離子水中，於121 °(：下利用高壓滅菌釜殺菌20分鐘後，分裝至玻璃試管中，並傾斜 置放至冷卻凝固，製得固態之斜面培養基。之後於該斜面培養基 上培育北冬蟲夏草菌種（寄存編號：BCRC930126)，得一斜面菌 種(slant culture )。 自該斜面菌種取出北冬蟲夏草之菌種，並接種於所製得之液體 培養基中，在25°C之溫度及150 rpm之振盪條件下，培育7天， 再接種至所製得之固體培養基上，在20°C至22°C之溫度與60%至 80%之相對濕度下於暗室中培育14天；接著於相同條件下，以照 201139663 明度為400 Lux之LED燈（分別使用白光LED燈、綠光LED燈 及藍光LED燈，其中LED燈照射裝置係如第丨圖所示）作為光源， 每曰照光18小時’持續照光培育約6〇天。所培育之北冬蟲夏草 係如第2圖所示。 比較實施例1 :利用曰光燈培育北冬蟲夏草 重複實施例1之步驟，惟於照光培育階段改用日光燈作為培育 光源。所培育之北冬蟲夏草係如第2圖所示。 結果分析： (1) 產量： 將所培育之北冬蟲夏草之子實體直接進行秤重，並計算每單位 培養面積之子實體重量，如表1中所示。 (2) 蟲草素含量： 將所培育之北冬蟲夏草之子實體在4(TC下乾燥並磨成粉末，精 秤10公克之該粉末並添加100毫升水，再以60〇c水萃取丨小時， 重複萃取至萃取液無色為止，合併萃取液並真空濃縮至完全乾 燥，定量秤取萃取物。 接著’利用一高效能液相層析儀（HPLC) (Hitachi系統，包含 Pump : Hitachi L-7100、Detector: Hitachi L-7420、Recorder: Hitachi D_7000、Autosampler : Hitachi L-7200、以及 Hitachi D-7000 HSM software )分析每公克北冬蟲夏草之子實體粉末所含之蟲草素含 量，如表1所示。 201139663 表1 光源 產量a) (公克/平方公分） 蟲草素含量b) (毫克/公克） 曰光燈 0.37 3.584 土 0.463 白光LED燈 0.41 26.390 ± 1.376 綠光LED燈 0.46 24.949 ± 0.738 藍光LED燈 0.50 21.238 ± 0.180 a)數值為40組培養瓶之平均值 b>數值為三組樣品之平均值 由表1可知，利用藍、綠、白光LED燈所培育之北冬蟲夏草， 其產量約為利用日光燈所培育之北冬蟲夏草的1丨至丨35倍蟲 草素3量約為5.92至7.36倍。根據以上結果，可證明利用藍、綠、 以及白光LED燈培育北冬蟲夏草，其產量及蟲草素含量均明顯増 加0 (3)多醣含量 A.葡萄糖標準曲線之繪製 取0.25、0.50、〇·75、κοο及125毫升之葡萄糖標準溶液分 別置於U)毫升之刻度試管中，各自添加水至Μ毫升，另取一含 = 1.5毫升諸水之刻度試f作為—空白對照組。每個刻度試管各 ^加2毫升之5重量/_苯祕液及6 5毫升之％重量％濃 置於机水浴中歷時2G分鐘’再取出放置至室溫下，搖 晃均勻後，於490奈米處測定吸光值，求 歸方程式。 I出，糖標準曲線回 201139663 Β·北冬蟲夏草之多醣純品的製備 取25公克比較實施例丨所培育之北冬蟲夏草之子實體粉末添 加250毫升乙輕’置於一索氏提取器（s〇xhiet extract〇r)中，迴 流抽取1小時進行脫脂，將脫脂後之殘渣中的乙醚溶劑揮發移除 後，添加250毫升之80重量❶/。乙醇浸泡過夜，隔天迴流抽取2小 時進仃脫脂’重複2次，韻、後得到—舰A與殘渣A。添加_ 毫升水至㈣A中’於8代下搜拌提取3次，每次2小時，過渡 後得到一濾液B及殘渣B，合併濾液A與濾液B。201139663 VI. Description of the Invention: [Technical Field] The present invention provides a method for cultivating Cordyceps militaris, in particular, for cultivating Cordyceps militaris with a light-emitting diode (LED) lamp as a light source (Cor cepi militaris) ) method. [Prior Art] "Xinhua Compendium of Materia Medica" records that: Cordyceps sinensis has a sweet taste and enters the lungs and kidneys. It has the characteristics of "complementing no stagnation, nourishing but not greasy j. It can also replace ginseng for imaginary physique." Angelica and other medicinal materials, the essence of yin and yang in the world, can not only nourish yin, but also help the yang, is a very noble and meticulous treasure, all kinds of physique can be applied, so the ancients said: "Ning want a Cordyceps, do not gold jade Full of traditional Chinese medicine, Cordyceps sinensis is known as the king of the Han Dynasty, and the Japanese are more called the ultimate Han Fang, and its preciousness can be seen. According to the research, the aforementioned precious characteristics of Cordyceps sinensis mainly come from Cordycepin and polysaccharide contained. Among them, cordycepin is an adenine analog, which has the functions of scavenging free radicals, antibacterial, antiprotozoal, antitumor, antiviral, etc., and can prevent lipid, protein, low density lipoprotein (low density). Oxidation of lipoprotein, LDL), thereby reducing the occurrence of arteriosclerosis, coronary heart disease and other diseases. Polysaccharide is a glucan formed by glucose as the basic unit and cannot be synthesized by artificial means; polysaccharides have special activation effects on human cells (such as activated macrophages) and have enhanced Immune system, anti-aging, scavenging free radicals, detoxification, lowering cholesterol, adjusting blood pressure, reducing UV damage, and promoting the growth of probiotics in the intestine. Among the more than 400 strains of Cordyceps sinensis 201139663, the Cordyceps sinensis is the highest and most valuable in Cordyceps militaris ((: 〇 哪 哪 磁 ( ( 又 又 又 又 又 。 。 。 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北 北The biological classification is: fungal community, fungal group, ascomycete subgroup, nucleobacteria, Phytophthora, ergotaceae, Cordyceps genus. In summer, mature Cordyceps sinensis will release fungal spores, infection The larvae of the bat moth are parasitic in the body and continue to grow with the worm as a nutrient source; in winter, the fungus grows into a mycelium (myceHa) in the larva, gradually filling the worm, causing the larva to die, but The outer shell of the larva is intact and still looks like a worm. Therefore, the ♦ stage is called “winter worm.” When the mycelium grows mature in the spring and summer of the next year, it is stimulated by environmental factors, that is, it is dead. The larva's head sprouts, grows a hymenophore 'fruit body and wears out the ground. This stage is called "summer grass." In Cordyceps militaris, cordycepin and polysaccharide mainly exist. The lower part of the fruiting body of Cordyceps militaris is a dead larva, which is the remaining mycelium. The wild Cordyceps sinensis must be harvested before the summer solstice is melted. The mycelium in the dead larvae will begin to decompose and degenerate. The cordycepin and polysaccharide contained in the polysaccharide will gradually lose. However, since the wild Cordyceps sinensis is randomly found and difficult to grasp the picking time, the current active ingredients of the wild Cordyceps militaris are different. In addition, the Cordyceps militaris The annual output is obviously reduced year by year, and the demand of the market is not enough. At present, various techniques for artificially cultivating Cordyceps militaris have been developed to obtain the stable quality of Cordyceps sinensis, such as solid state. Acacia static culture, liquid rotary shock culture, liquid phase static culture and deep liquid fermentation culture, etc., the details of such culture techniques can be referred to, for example, as disclosed in Taiwan Patent Publication No. 1231825; Publication No. 2, No. 833836, 201139663 and Taiwan Patent Disclosure The method of cultivating Cordyceps militaris is also mentioned in 200600006, mainly involving darkroom (light-proof) culture and light culture. The contents of the aforementioned patent documents are hereby incorporated by reference. It is known that almost all of the cultivation methods of Cordyceps militaris use fluorescent lamps. Or sunlight as a source of light. However, when using sunlight, it is greatly limited by natural factors that are difficult to control, and it is difficult to provide a stable lighting environment. When using fluorescent lamps, it consumes a large amount of electricity, generates a large amount of heat, and produces luminosity. The invention has the disadvantages of weakening and short service life. Therefore, it is urgent to further improve the existing cultivation method of Cordyceps militaris. [Invention] It is an object of the present invention to provide a method for cultivating Cordyceps militaris, which comprises a strain containing Cordyceps militaris. The medium undergoes the following two stages: a) dark incubation stage, and b) illumination development stage, where the illumination stage is based on LED light, the LED light is selected from the group consisting of white LED light, blue LED light green light. LED lights, and combinations thereof. The detailed description of the present invention and the preferred embodiments thereof will be described in the following description of the present invention. [Embodiment] The culture medium containing the strain of Cordyceps militaris according to the method of the present invention can be prepared by taking a temperature of 20 ° C to about 30 ° C of any of the stalk species and any suitable method. For example, the grass seed is firstly cultivated in a liquid medium under conditions of about _ah to about 6.5, and the liquid is incubated for about 4 to about 5 days, and then the (four) species are inoculated to the - On the basis, a) dark culture stage is followed. The formulation of the liquid medium that can be used is not subject to any particular limitation, and usually includes a carbon source (such as starch, glucose, fructose, maltose, etc.), a source, (for example, nitrate, 201139663 corn syrup, soybean cake powder, peanut cake powder, Yeast powder, protein favor, urea, etc.), inorganic salts and trace elements (such as carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium, etc.) and water, etc., wherein the liquid medium has a moisture content of about 80% by weight of the total weight of the medium. % to about 90% by weight and having a carbon to nitrogen ratio of from about 3·1 to about 10:1. For example, a suitable liquid medium can be prepared by first mixing potato sugar powder, yeast extract, sulfuric acid (MgS〇4), and dihydrogen clock (KH2P〇4), followed by deionization. The resulting mixture is dissolved in water to obtain an aqueous solution, which is then subjected to a sterilization procedure in an autoclave to provide the desired liquid culture φ group. As for the solid medium which can be used, the composition has a carbon to nitrogen ratio substantially the same as that of the above liquid medium, but the ratio of the water contained therein is relatively low, from about 50% by weight to about 60% by weight. A solid medium which can be used in the method of the present invention can be obtained, for example, by mixing starch (e.g., white rice) and the above liquid culture solution, followed by sterilization in an autoclave. After inoculating the strain of Cordyceps militaris cultivated in the liquid medium into the solid medium, the solid medium having the strain of Cordyceps militaris is then subjected to a) dark portion incubation step. It is known that the incubation temperature, the relative humidity of the surrounding environment, and the pH of the medium all affect the growth of the fungus of Cordyceps militaris, especially the Cordyceps militaris prefers a low temperature and moist environment. According to one embodiment of the method of the present invention, in a) dark incubation stage, the incubation temperature employed is from about 18 ° C to about 25 ° C, preferably from about 20 ° C to about 22 ° C; relative humidity is about From 60% to about 80%, preferably from about 65% to about 75%; and the pH of the solid medium is from about 5 to about 7, preferably from about 5.5 to about 6/5. After the a) dark incubation period is completed, b) the illumination incubation phase is followed. Similarly, the incubation temperature, the relative humidity of the surrounding environment, the pH of the medium, etc., 201139663 will also affect the growth of the fungus of Cordyceps militaris in the illuminating stage. According to one embodiment of the method of the present invention, the following incubation conditions are employed in b) the light incubation stage: the incubation temperature is from about 18 ° C to about 25 ° C', preferably about 20. (: to about 22 ° C; relative humidity of about 60% to about 80%, preferably about 65% to 75%; and solid medium having a pIi value of from about 5 to about 7, preferably from about 5.5 to about 6.5. In addition to the relative humidity of the surrounding environment and the pH value of the medium, the light conditions of the salt will also affect b) the growth of Cordyceps militaris under the light cultivation stage. In particular, the Cordyceps militaris in the "summer grass" stage requires a light-filled growth environment. In the method of the present invention, "lighting is carried out using LED lamps as a light source", and the color of the light source of the LED lamp is usually selected as needed. In some embodiments of the method of the present invention, a white LED lamp, a blue LED lamp, a green LED lamp, and combinations thereof are selected, and a white LED lamp is preferably used. Wherein, the daily illumination time is from about 15 to about 20 hours, preferably from about 17 to about 19 hours; and the illumination is controlled from about 300 Lux to about 500 Lux, preferably from about 350 Lux to about 450 Lux, preferably 380 Lux to about 420 Lux is the best. Under the conditions of this light condition, a relatively satisfactory Cordyceps militaris can be produced, for example, having a thick, healthy sub-solid. In the breeding method of the present invention, a) incubation time in the dark incubation stage is generally from about (7) days to about 20 days, preferably from about 13 days to about 15 days; b) incubation in the light cultivation stage usually lasts about 50 days. It is better to about 70 days 'about 55 days to about & work and private from 〇月' J 〇 :) days. In general, the longer the incubation time, the more the dry weight of Cordyceps militaris can be increased, but the longer the cultivation time can not guarantee the relative increase of the content of active ingredients, and the cost of cultivating Cordyceps militaris can be increased. The other-square δ, if the incubation time is too short , may not provide satisfactory production of Cordyceps militaris. ('201139663 The method of the present invention replaces the conventional fluorescent lamp with the LED lamp as the light source for cultivating the northern Cordyceps sinensis, which can reduce the power consumption, generate less heat energy, have strong luminosity and long service life. Moreover, the inventor of the present invention also found that When the LED lamp is used for the cultivation of Cordyceps militaris, the yield of Cordyceps militaris can be increased and the active ingredients thereof, namely cordycepin and polyphonic, can be increased, as shown in the following examples. The following specific embodiments are used to further illustrate The invention is described by way of example only, and is not intended to limit the scope of the invention. Example φ Example 1: Incubation of Cordyceps militaris with LED lamps 12 g of potato powdered sugar, 2 g of yeast extract 1 g of magnesium sulfate and 2 g of potassium dihydrogen phosphate, dissolved in 1 liter of deionized water, sterilized in an autoclave at 121 ° C for 20 minutes, and then cooled to obtain a liquid medium. Take 20 g of Taiwan stalk 9 White rice and 30 ml of the prepared liquid culture solution were sterilized by autoclaving at 121 ° C for 20 minutes and then cooled to obtain a solid Medium: Take 12 grams of horse yam powder, 2 grams of yeast extract, 20 grams of agar, 1 gram of magnesium sulphate and 2 grams of potassium dihydrogen phosphate, dissolved in 1 liter of deionized water, autoclaved at 121 ° (: After the kettle was sterilized for 20 minutes, it was dispensed into a glass test tube, and placed obliquely until it was cooled and solidified to obtain a solid slant culture medium. Then, the Cordyceps militaris strain (registered number: BCRC930126) was cultivated on the slant medium to obtain a slant Slant culture. The strain of Cordyceps militaris is taken from the stalk strain and inoculated into the prepared liquid medium, incubated at 25 ° C and 150 rpm for 7 days, and then inoculated to The prepared solid medium is incubated in a dark room at a temperature of 20 ° C to 22 ° C and a relative humidity of 60% to 80% for 14 days; then under the same conditions, according to the 201139663 brightness of 400 Lux LED Lights (white LED lights, green LED lights and blue LED lights, respectively, of which the LED light illuminating device is shown in the figure) are used as the light source. Each illuminating light for 18 hours 'continuous illumination is cultivated for about 6 days. Northern Cordyceps The grass system is shown in Fig. 2. Comparative Example 1: Incubation of Cordyceps militaris with a neon lamp The procedure of Example 1 was repeated, except that the fluorescent lamp was used as a light source for cultivation in the illuminating stage. The cultivated Cordyceps sinensis was as shown in Fig. 2 The results are as follows: (1) Yield: The fruiting bodies of the cultivated Cordyceps militaris are directly weighed and the weight of the fruiting body per unit of culture area is calculated, as shown in Table 1. (2) Cordycepin content: The fruiting body of the cultivated Cordyceps militaris is dried and ground into powder at 4 (TC), finely weighed 10 g of the powder and added with 100 ml of water, and then extracted with 60 〇c water for 丨 hours, repeated extraction until the extract is colorless, combined with extraction The solution was concentrated in vacuo to dryness and the extract was taken quantitatively. Then 'Using a high performance liquid chromatography (HPLC) (Hitachi system, including Pump: Hitachi L-7100, Detector: Hitachi L-7420, Recorder: Hitachi D_7000, Autosampler: Hitachi L-7200, and Hitachi D-7000 HSM software) analyzes the content of cordycepin contained in the fruit powder of each gram of Cordyceps militaris, as shown in Table 1. 201139663 Table 1 Light source output a) (g/cm2) Cordycein content b) (mg/g) Xenon lamp 0.37 3.584 Soil 0.463 white LED lamp 0.41 26.390 ± 1.376 Green LED lamp 0.46 24.949 ± 0.738 Blue LED lamp 0.50 21.238 ± 0.180 a) The value is the average value of 40 groups of culture flasks b> The average value of the three groups of samples is shown in Table 1. The production of Cordyceps militaris cultivated by blue, green and white LED lamps is about to be cultivated by fluorescent lamps. The amount of Cordyceps from Cordyceps sinensis is about 5.92 to 7.36 times. Based on the above results, it can be proved that the use of blue, green, and white LED lamps to cultivate Cordyceps militaris, the yield and cordycepin content are obviously increased by 0 (3) polysaccharide content A. The glucose standard curve is drawn 0.25, 0.50, 〇 · 75, The κοο and 125 ml glucose standard solutions were placed in U) milliliters of graduated test tubes, respectively, and water was added to Μml, and another scale test containing = 1.5 ml of water was used as a blank control group. Each graduated test tube is added with 2 ml of 5 weight/_ benzene secret solution and 6 5 ml of % by weight concentrated in a water bath for 2G minutes. Then take it out and let it stand at room temperature. After shaking evenly, at 490 奈The absorbance is measured at the meter and the equation is calculated. I, the sugar standard curve back to 201139663 Β·North Cordyceps sinensis polysaccharide preparation preparation take 25 grams of comparative example 丨 北 北 北 北 北 北 北 北 北 250 250 250 250 250 250 250 250 250 250 250 250 250 250 子 子 hi hi hi hi hi hi hi hi hi hi hi hi hi In extract〇r), degreasing was carried out for 1 hour under reflux, and the ether solvent in the residue after degreasing was volatilized and removed, and then 250 ml of 80 wt% was added. Soak in ethanol overnight, reflux for 2 hours every other day to remove defatted 'repeated 2 times, rhyme, then get - ship A and residue A. Add _ ml of water to (4) A. In the 8th generation, extract and extract 3 times for 2 hours each time. After the transition, a filtrate B and a residue B are obtained, and the filtrate A and the filtrate B are combined.
著’將所合併之據液真空濃縮至'約100 $升，並以約1〇〇 升之三氣曱烧與異戊醇之混合溶液（三氣甲院與異戊醇之體積 為1)去除蛋白質’再添加4倍量之％重量%乙醇，靜置過孩 隔天’進仃離w ’將離心後之賴物分別依相無水乙 酮及乙喊洗蘇多次，將、、生收> iit 將洗滁後之樣品冷凍至·8〇〇c後， 冷凍乾燥器（Kingmechrn 罝於氣 〇. LTD.，FD-6-6P-D)内進行乾烨，嗖 乾燥條件之溫^_6Gt n成 後取出並進行枰重，所得Λ為約G至1GGmmHg，待完全乾:'Concentrate the combined liquids to a vacuum of about 100 liters, and mix the mixture with isoamyl alcohol in a gas mixture of about 1 liter (the volume of the gas and the isoamyl alcohol is 1) Remove the protein' and then add 4 times the amount of % by weight of ethanol, and let it stand for the next day, 'into the sputum, w', and the lysate after centrifugation, respectively, according to the phase of anhydrous ethyl ketone and B, shampooing several times, will, After the sample is frozen to 8 〇〇c, the lyophilizer (Kingmechrn 罝 〇 〇. LTD., FD-6-6P-D) is dried and dried. ^_6Gt n is taken out and weighted, and the obtained enthalpy is about G to 1GGmmHg, to be completely dried:
p為北冬蟲夏草之多醣純品。 c.換算因子之測定 ⑽咖Hg)將程序3所/燥溫度為俄，星力範圍為約60 其置於毫升之之該多酿純品完全乾燥錄量後，為 純品溶液。取1毫升之=，加水溶解稀釋並混勻，得到一多 線之緣製之操作步驟，^_品溶液，依照上述葡萄糖標準 用上述葡⑯標準曲線回^多醣純品溶社吸純。之後， 糖漠度後，再依多料品錢中之β 12 201139663 f = W / (C x D) 其中，w為多醣純品溶液中所含之多醣重量（毫克），c為多醣 純’品溶液中之葡萄糖濃度（毫克/毫升），D為多醣純品溶液之稀 釋倍數。 D. 北冬蟲夏草之多醣樣品溶液之製備 取5公克實施例1中利用白光LED燈所培育之北冬蟲夏草之子 實體粉末置於一圓底燒瓶中，並添加50毫升水，置於一水浴器中 煮沸1小時提取，過濾後取濾液，並重複上述煮沸提取過濾之步 φ 驟3次，再將所得之濾液合併並減壓濃縮至約100毫升，加入3 倍量之乙醇進行沉澱，靜置過夜。 隔天，進行離心，將離心後之沉澱物分別依序以無水乙醇、丙 酮及乙醚洗滌多次，再置於該減壓冷凍乾燥器内進行乾燥，設定 乾燥條件之溫度為-60°C，壓力範圍為約60至lOOmmHg，待完全 乾燥後取出秤重，所得即為一多醣樣品（即水溶性粗多醣）。取2 毫克之該多醣樣品，置於一 25毫升之量瓶中，添加水溶解至刻度， 混合均勻後製得一多醣樣品溶液X。 ® 取5公克之比較實施例1所培育之北冬蟲夏草之子實體粉末， 重複上述步驟，另製得一多醣樣品溶液Y。 E. 多醣樣品溶液之多醣含量測定 分別取1毫升之程序D所得之多醣樣品溶液X及Y，各自置於 10毫升之刻度試管中，依照上述葡萄糖標準曲線之繪製之操作步 驟，測定多醣樣品溶液X及Y之吸光值。之後，利用上述葡萄糖 標準曲線回歸方程式分別求出多醣樣品溶液X及Y之葡萄糖濃 度，再依下式分別計算出多醣樣品溶液X及Y中之多醣含量： 13 201139663 多醣含量（％)=(C’ x D，χ f / w，）χ 100 其中’C’為多醣樣品溶液中之葡萄糖濃度（毫克/毫升）,為多 醣樣品溶液之稀釋倍數’ f為換算因子，w，為㈣樣品溶液中所 含之多釀重量（毫克），結果如表2所示。 表2 光源 多醣含量（°/〇) 曰光燈 (多醣樣品溶液Y) 7.962 白光LED燈 (多醣樣品溶液X) 9.874 夕表2結果顯示，以白光燈為光源所培育之北冬蟲夏草其 多酿含量約為以曰光燈為光源所培育之北冬蟲夏草的⑶倍。 由上可知’本發明以LED燈取代日光燈作為光源進行培育北冬 蟲夏草，可提升北冬蟲夏草巾之蟲草素及/或多_之含量；另一方 面旧㈣耗電量為日光燈的1/3以下、壽命為1(>倍更可提升 培育方法的整體經濟效益。 上述實施例僅係用以例示說明本發明，而非用於限制本發明。 任何熟於此項技藝之人士均可在不違背本發明之技術原理及精神 的情況下’對上述實施例進行修改及變化。因此，本發明之權利 保護範圍應如後述之申請專利範圍所列者。 【圖式簡單說明】 第1圖係本案實施例申所用之LED燈照射裝置。 第2圖係本案實施例丨與比較實施例〗所培育之北冬蟲夏草。 14 201139663 【主要元件符號說明】p is a pure polysaccharide of Cordyceps militaris. c. Determination of conversion factor (10) Coffee Hg) The procedure 3 is dry/slow, and the star force range is about 60. It is placed in ML and the purely pure product is completely dry and recorded as a pure solution. Take 1 ml of =, add water to dissolve and mix, and obtain a multi-line edge of the operation steps, ^_ product solution, according to the above-mentioned glucose standard, using the above-mentioned Portuguese 16 standard curve back to the polysaccharide pure soluble solution. After the sugar inequality, and then according to the multi-product money, β 12 201139663 f = W / (C x D) where w is the weight of the polysaccharide contained in the pure polysaccharide solution (mg), c is pure polysaccharide ' The concentration of glucose in the solution (mg/ml), and D is the dilution factor of the pure polysaccharide solution. D. Preparation of Polysaccharide Sample Solution of Cordyceps militaris 5 g of the fruit body powder of Cordyceps militaris grown in white light LED lamp of Example 1 was placed in a round bottom flask, and 50 ml of water was added and placed in a water bath to boil 1 After hourly extraction, the filtrate was taken after filtration, and the above-mentioned boiling extraction filtration step φ was repeated three times, and the obtained filtrate was combined and concentrated under reduced pressure to about 100 ml, and precipitated by adding 3 times of ethanol, and allowed to stand overnight. The next day, centrifugation was carried out, and the precipitates after centrifugation were sequentially washed several times with absolute ethanol, acetone and diethyl ether, and then placed in the reduced-pressure freeze dryer to be dried, and the temperature of the drying conditions was set to -60 ° C. The pressure ranges from about 60 to 100 mmHg, and after being completely dried, the weight is taken out, and the obtained sample is a polysaccharide sample (i.e., water-soluble crude polysaccharide). Take 2 mg of the polysaccharide sample, place it in a 25 ml volumetric flask, add water to the mark, and mix well to prepare a polysaccharide sample solution X. ® 5 g of the fruiting body powder of Cordyceps militaris grown in Comparative Example 1 was repeated, and the above procedure was repeated to prepare a polysaccharide sample solution Y. E. Determination of the polysaccharide content of the polysaccharide sample solution Take 1 ml of the polysaccharide sample solution X and Y obtained in the procedure D, respectively, in a 10 ml graduated test tube, and determine the polysaccharide sample solution according to the operation procedure of the above glucose standard curve. The absorbance of X and Y. Then, using the above glucose standard curve regression equation to determine the glucose concentration of the polysaccharide sample solution X and Y, respectively, and then calculate the polysaccharide content in the polysaccharide sample solution X and Y according to the following formula: 13 201139663 Polysaccharide content (%) = (C ' x D,χ f / w,)χ 100 where 'C' is the glucose concentration in the polysaccharide sample solution (mg/ml), which is the dilution factor of the polysaccharide sample solution 'f is the conversion factor, w, is (4) in the sample solution The weight of the brew (mg) is shown in Table 2. Table 2 Light source polysaccharide content (°/〇) Xenon lamp (polysaccharide sample solution Y) 7.962 White LED lamp (polysaccharide sample solution X) 9.874 The result of the evening table 2 shows that the content of B. sinensis grown by white light as the light source It is about (3) times that of Cordyceps militaris cultivated with a neon light as a light source. It can be seen from the above that the present invention uses the LED lamp instead of the fluorescent lamp as a light source to cultivate the Cordyceps militaris, which can increase the content of Cordyceps and/or more of the Cordyceps militaris; on the other hand, the old (four) power consumption is less than 1/3 of the fluorescent lamp, The life expectancy is 1 (> times the overall economic benefit of the cultivation method. The above embodiments are merely illustrative of the invention and are not intended to limit the invention. Anyone skilled in the art can do without Modifications and variations of the above-described embodiments are made in the context of the technical principles and spirit of the present invention. Therefore, the scope of protection of the present invention should be as set forth in the scope of the claims described below. The LED lamp irradiation device used in the embodiment is shown in Fig. 2 is a Cordyceps militaris cultivated in the present embodiment and the comparative example. 14 201139663 [Description of main component symbols]