201024320 ‘六、發明說明： 【發明所屬之技術領域】 本發明係關於一種樟芝(如的咖或如ciwwwi〇mea) 菌絲體之新穎培養方法。 【先前技術】 樟芝的型態 ❹ 棟芝又名牛樟莊、樟藏、樟窟内旅，台灣有稱陰陽對口兹。樟芝子實 艘屬多年生，具有強烈沖鼻的樟樹香氣，此與一般靈芝類有很大的差異， 其外型呈板狀或鐘狀。板狀型態者，面為橘紅(黃)色，整面全有菌孔板底 層有淺黃白色的木栓質’藉此木栓質附著在牛樟樹中空心材内壁上生長。 鐘狀型態者，子實層(鐘面)亦呈橘(黃)色，充滿菌孔(4〜5個菌孔/毫米)，内 有孢子味極苦，新鮮時為橘紅色，之後會成為橘褐色或揭色，鐘想則呈睹 綠褐色的皮殼。以顯微鏡觀察其擔抱子，其型態為平滑無色之透明微弯柱 ❿形° 樟芝的生物特性 野生的樟芝是生長在牛樟樹幹中空内壁上，因為這個特性造成很多 牛樟樹倒伏。文獻記載，樟芝是在牛樟樹上目前唯—發現的木材爲杉菌， 病徵為褐色腐朽，故為褐腐菌。但是樟芝的病原性並不強因此牛採樹很 少因此死亡。雖然樟芝對牛樟樹而言是病原菌，但因掉芝價格昂貴，超過 牛樟樹的經濟償值’因此是不是牛樟樹的病原_已經不重要了。 律芝的培養技術 201024320 梓芝的培養，人工栽培的技術，仍有待努力。所以目前仍是以深山 採集的方式來獲得。但是採集樟芝不是件容易的事因為首先要尋找牛掉 樹的產地》常有的困難料樟樹與冇樟，兩者極為相似，不絞辨。目前 最直接的方法已由藤田安二提出，冇樟幹油是以黃棟油㈣❶⑷與十五燒搭 為主，因而有沙士中黃樟素的味道。牛樟幹油則以松油醇(d娜inen〇i)為 主，而有触油的味道，藉此即可區料樟與冇棟。第二_難是要從大 片樹林中找到有中空洞的樹幹才行，此相當不易。空洞中若有棒芝，則可 Ο 定期採集。 由於找尋中空洞的牛樟樹幹不易，不肖商人乾脆將牛掉樹砍倒，以期 日後能長出樟芝’進而收集販售。因此，為環保及經濟上的考量，發展人 工栽培樟芝是必要進行的方向。可惜的是人工栽培樟芝技術一直無法突 破。樟芝在牛樟木肩上生長極為緩慢，甚至停滞。因此，若能改以現代生 物技術，來培養棟芝菌絲體，將是最經濟、最符合環保的人工培育法。 目前培養樟芝菌絲體之人工方法亟需開發及改良，常見如液態培養、 ❹固態培養、段木培養、野生菌木復育等培育方法。其中又以液態培養菌絲 體，產量快速，且成本低廉，可在相當短的時間培養出大量的菌絲醴產物· 然而，依然存在著以鏺酵槽培養時之常見問題，即醱酵過程常產生大量泡 沫。發泡常使报多醱酵工程發生問題，原因多是因培養基中的蛋白質，使 氣相與培養液的界面變性，形成不易破壞之薄膜❶當醱酵槽充滿泡泳便會 降低每單位體積可培養之容量，並且導致培養液自醱酵槽溢流等缺失。 為抑制泡沫的形成，一般的方法是加入消泡劑到培養基中，已知之消 泡劑例如聚氧化烯多元醇醚、聚氧化烯烷基醚、聚氧化烯脂肪酸酯、聚氧 201024320 化烯烷基醚脂肪酸酯等。然而，這些傳統鏺酵用消泡劑並不能自液體表面， 同時具有破壞發泡之效果及抑制泡沫形成之效果，以致於其等消泡效果不 夠充分°另一方面’這些消泡劑伴亦會伴隨抑制微生物生長及標的產物生 產的問題》甚至，該些傳統消泡劑係為界面活性劑，多屬化學物質，對於 人體、動物之健康仍有其風險。 由此可見’上述習用樟芝之鏺酵培養技術及消泡劑仍有諸多缺失及因 難’實非一良善之設計者，而亟待加以改良。 © 芝麻如汾《/« L.)屬於胡麻科，為一年生自花授粉的油料作 物。芝麻油占芝麻種子乾重45%〜60% ’因其特別的風味及所含芝麻木質 酚(lignan)的抗氧化特性，在東方食品中深受喜愛。芝麻酚(sesamin)是芝麻 油中已知含量最豐富的木質紛抗氧化分子，經動物實驗及人艘研究中證實 具有許多有益健康的功能，如保護肝臟、抗發炎、抗高血壓、降低膽固酵、 防老化、抗氧化、抗發炎反應、清除自由基、保護神經、抑制肝癌等作用。 因此有鑑於： 1·棟芝唯一寄生物種一牛樟樹，屬於保育類一級木樹種，且為有空心的牛 樟樹之不易取得； 2·樟芝子實體的試管内(invitro)及離牛樟樹心空洞外的培育之困難性；及 3·樟芝菌絲體亦有相似生物功能，且菌絲體的培養和擴大生產較可行； 4.樟芝菌絲體鏺酵培養時伴隨大量泡沫，嚴重影響其產量。 是以，本案發明人鑑於上述習用消泡劑及傳統樟芝菌絲體培養技術所 衍生的各項缺點及困難，乃亟思加以改良創新，並經多年苦心孤詣潛心研 究後，終於成功研發完成本件一種樟芝菌絲體之新穎培養方法。 201024320 【發明内容】 本發明之目的即在於提供一種樟芝菌絲體之新穎培養方法，藉由添加 芝麻油於樟芝培養基中，可達到消泡之功效。 本發明之次一目的係在於提供一種樟芝菌絲體之新穎培養方法，藉由 添加芝麻油於樟芝培養基中，可於較短之培養時間内產生較多之樟芝菌絲 艟及菌絲。 本發明之另一目的係在於提供一種樟芝菌絲體之新穎培養方法，藉由 β 添加芝麻油於樟之培養基中，所得者之顏色近似天然樟芝且更富含芝麻紛。 可達成上述發明目的之-種樟芝菌絲體之新穎培養方法，包括有： 棒芝(Antrodia camphorata 氣 Antrodia cinnamomea)第綠後； 梓芝菌絲體培養，包括將菌絲艘接種於平板上，進行平板培減，再 刮取菌絲接種於燒朗，進行餘培養，最後，賴瓶騎物接種於磨酵 槽内’並添加芝麻祕摄酵槽培養基中以培養樟芝，培養約ι〇天後，即得 樟芝菌絲體醱酵液，包括菌絲體與澄清液； ❹ 酬量此培養方法所得者，其產物近似天然樟芝顏色，且富含芝麻紛。 【實施方式】 本發明之實施例所用之樟芝洳峰“场 咖麵_則祕得自寄存於巾華民國台科新竹市食品工業研究所 菌種保存中心的樟芝菌絲體BCRC35398 ; 201024320 於適當溫度如15_寶’（較佳相關卿汀轉約2週後，到取菌絲接 種於燒瓶内’帛實施例巾所列培養基，在約3G<JC，pH2 8，較佳者pH4 7， 更佳者約pH 4.5，及振蘆料50·25〇卿之下振盘培養到1〇g期初期，亦 即’約5-7天；最後，將燒瓶培養物接種於_槽培養基(同燒瓶培養基)内， 在15-35°C ’（較佳者周温約30〇c)，槽磨〇 h 5公斤/平方公分，及pH約 4.5下，以0.5-1 wm通氣速率通入空氣，或空氣舆氧氣二氣化碟或氣氣的 混合物’較佳者空氣’在50-300 rpm攪拌速率下培養約8_16天。即得樟芝 菌絲體播酵液’包括菌絲體與澄清液。 該掉芝菌絲艘液想培養之培養基配方如下·· 培養基配方 含量(重量 0.01 〜5 0.01〜2 0.0001-0.05 0.01-10 0.001-2 0.005-0.015 威分 综合性碳氮源 動植物來源蛋白及其水解物 無機鹽類 糖類 酵母或麥芽抽出物(粉、膏） 消泡劑201024320 ‘Six, invention description: [Technical field to which the invention pertains] The present invention relates to a novel cultivation method of an anthrax (such as a coffee or a ciwwwi〇mea) mycelium. [Prior Art] The type of 樟 ❹ 栋 芝 又 又 又 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋 栋The scorpion scorpion is a perennial, scented eucalyptus scent that is very different from the general genus Ganoderma lucidum. Its appearance is plate-like or bell-shaped. In the plate shape, the surface is orange-red (yellow) color, and the bottom of the whole microporous plate has a pale yellow-white cork. The cork is attached to the inner wall of the hollow material in the burdock tree. The bell-shaped type, the sub-solid layer (clock face) is also orange (yellow) color, full of micropores (4~5 bacteria/mm), with spore taste extremely bitter, orange-red when fresh, afterwards It becomes orange-brown or uncovering, and the bell is green and brown. Observing the scorpion with a microscope, its shape is a smooth and colorless transparent micro-bend column. The biological characteristics of the 樟 野生 wild 樟 是 is grown on the hollow inner wall of the burdock trunk, because this characteristic causes many burdocks to fall. According to the literature, Antrodia is the only wood found in burdock trees. It is a brown rot, so it is brown rot. However, the pathogenicity of Antrodia is not strong, so there are very few cows and trees that die. Although Antrodia camphorata is a pathogen for Burdock, it is not important because it is expensive and exceeds the economic compensation of Burdock. The culture technique of Rhizoma Tobacco 201024320 The cultivation of Antrodia camphorata, the technique of artificial cultivation, still needs to be worked hard. Therefore, it is still obtained by means of deep mountain collection. However, collecting Zhizhi is not an easy task because it is necessary to first look for the origin of the cattle to drop the tree. The common difficulties are the eucalyptus and the eucalyptus. The two are very similar and do not lie. At present, the most direct method has been proposed by Fujita Anji. The sorghum oil is mainly composed of Huangdong oil (4) ❶ (4) and fifteen burning, thus having the taste of scutellaria in SARS. The burdock dry oil is dominated by terpineol (dna inen〇i), and it has a taste of oil, so that it can be used as a material. The second problem is that it is necessary to find a trunk with a hollow hole in a large forest. This is not easy. If there is a stick in the cavity, you can collect it regularly. Because the trunk of the burdock looking for a hollow hole is not easy, the unscrupulous businessman simply cut down the cow and fell down the tree, in the hope that it will grow in the future and collect and sell it. Therefore, for environmental protection and economic considerations, the development of artificial cultivation of Antrodia is necessary. It is a pity that the artificial cultivation of Antrodia technology has not been able to break through. Antrodia sinensis grows very slowly on the shoulder of the burdock, and even stagnates. Therefore, if you can change the modern biotechnology to cultivate the mycelium of Tobacco, it will be the most economical and environmentally friendly artificial cultivation method. At present, the artificial method for cultivating the mycelium of Antrodia camphorata needs to be developed and improved, and the cultivation methods such as liquid culture, solid state culture, stage wood culture, and wild fungus wood rearing are common. Among them, the mycelium is cultured in a liquid state, the yield is fast, and the cost is low, and a large amount of hyphae product can be cultured in a relatively short time. However, there are still common problems in the fermentation of the fermentation tank, that is, the fermentation process. Often a lot of foam is produced. Foaming often causes problems in the multi-fermentation project. The reason is mostly due to the protein in the medium, which denatures the interface between the gas phase and the culture solution, forming a film that is not easily destroyed. When the fermentation tank is full of bubbles, the volume per unit volume is reduced. The capacity can be cultured, and the culture solution is missing from the fermentation tank overflow. In order to inhibit the formation of foam, the general method is to add an antifoaming agent to the medium. Known antifoaming agents such as polyoxyalkylene polyol ether, polyoxyalkylene alkyl ether, polyoxyalkylene fatty acid ester, polyoxygen 201024320 olefin Alkyl ether fatty acid esters and the like. However, these conventional defoaming agents for dehydration do not adhere to the surface of the liquid, and at the same time have the effect of destroying the foaming effect and inhibiting the formation of foam, so that the defoaming effect thereof is insufficient. On the other hand, these defoaming agents are also accompanied by It is accompanied by the problem of inhibiting the growth of microorganisms and the production of the target products. Even these conventional antifoaming agents are surfactants, which are mostly chemical substances, and still have risks to the health of humans and animals. It can be seen that the above-mentioned sputum culture techniques and antifoaming agents of A. sinensis still have many defects and difficulties, and they are not a good designer, but need to be improved. © Sesame Ruan “/« L.) belongs to the family Hemp, which is an annual self-pollinated oil preparation. Sesame oil accounts for 45%~60% of the dry weight of sesame seeds. ‘Because of its special flavor and the antioxidant properties of lignan containing sesame, it is very popular in oriental foods. Sesamin is the most abundant woody antioxidant molecule in sesame oil. It has been proven in animal experiments and human studies to have many beneficial functions such as protecting the liver, anti-inflammatory, antihypertensive, and reducing cholesterol. Fermentation, anti-aging, anti-oxidation, anti-inflammatory response, scavenging free radicals, protecting nerves, inhibiting liver cancer and so on. Therefore, in view of: 1. The only parasitic species of Toshiba, a burdock tree, belongs to the first-class wood species of conservation class, and is not easy to obtain for the hollow burdock tree; 2. The invitro and the heart of the burdock tree The difficulty of cultivation outside the cavity; and 3. The mycelium of A. angustifolia has similar biological functions, and the culture and expansion of mycelium are more feasible; 4. The mycelium of A. sinensis is accompanied by a large amount of foam, which is serious. Affect its production. Therefore, in view of the shortcomings and difficulties derived from the above-mentioned conventional defoaming agents and traditional anthrax mycelium culture techniques, the inventors of the present invention have improved and innovated, and after years of painstaking research, they finally succeeded in research and development. A novel cultivation method for mycelium of Antrodia camphorata. 201024320 SUMMARY OF THE INVENTION The object of the present invention is to provide a novel culture method for the mycelium of Antrodia camphorata, which can achieve the effect of defoaming by adding sesame oil to the culture medium of Antrodia camphorata. The second object of the present invention is to provide a novel culture method for the mycelium of Antrodia camphorata. By adding sesame oil to the anthraquinone medium, more Astragalus mycelium and hyphae can be produced in a shorter culture time. . Another object of the present invention is to provide a novel culture method for the mycelium of Antrodia camphorata, by adding sesame oil to the medium of sputum by β, and the color of the obtained person is similar to that of natural yam and more rich in sesame. A novel culture method for the mycelium of Antrodia camphorata, which can achieve the above object, includes: Antrodia camphorata gas Antrodia cinnamomea after green; Anthocyanin mycelium culture, including inoculating a mycelium on a plate The plate is cultured and reduced, and then the mycelium is inoculated into the roast, and the remaining culture is carried out. Finally, the Lai bottle is inoculated into the grinding tank, and the sesame secret fermentation tank medium is added to cultivate the anthraquinone, and the cultivation is about ι. After the day, you can get the mycelium sputum lysate, including mycelium and clear liquid; ❹ Reward The result of this culture method, the product is similar to the natural color of Antrodia, and rich in sesame. [Embodiment] The 樟 洳 洳 “ 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 场 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 At a suitable temperature, such as 15_bao' (preferably related to 2 weeks after the transfer of the Qingting, to the inoculation of the mycelium into the flask), the medium listed in the example towel, at about 3G < JC, pH 2 8, preferably pH 4 7. The better is about pH 4.5, and the vibrating stalk is 50.25 〇 under the vibration plate to the initial stage of the 1 〇 g period, that is, 'about 5-7 days; finally, the flask culture is inoculated into the _ tank medium. (with the flask medium), at 15-35 ° C ' (preferred weekly temperature of about 30 〇 c), groove grinding 〇 h 5 kg / cm ^ 2, and pH about 4.5, at a ventilation rate of 0.5-1 wm Into the air, or air, 舆 oxygen, two gasification disc or gas mixture 'better air' at a stirring rate of 50-300 rpm for about 8-16 days. That is, Astragalus mycelium sowing solution 'including mycelium With the clear liquid. The medium of the culture of the mycelium of the mycelium is as follows: · The content of the medium formula (weight 0.01 ~ 5 0.01 ~ 2 0.0001-0.05 0.01-10 0.001-2 0.005-0.015 Weiwei Comprehensive carbon and nitrogen source Animal and plant origin protein and its hydrolyzate Inorganic salt Sugar Saccharomyces or malt extract (powder, paste) Defoamer
其中該無機鹽類可為硫酸錢、磷酸氩二鉀、硫酸鐵等； 其中該動植物來源蛋白及其水解物可為蛋白脒； 其中該糖類可為葡萄糖、蔗糖、果糖、麥芽糖等； 其中該综合性碳氮源可為榖類（如：麥粉類）或豆類（如：黃豆粉、 綠豆粉、大豆粉）； 於鏺酵槽培養基中可额外添加消泡劑以抑制於培養過程中大量泡沫之 生成，其中該消泡劑可為市售之習用消泡劑，如0.005-0.015%消泡錨 (Antifoaming KM-72，為含矽油、矽樹脂之水性消泡產品），或0.01〜1%芝麻 201024320 油(市售）。 本發鄉以τ面㈣補予料範㈣，但本發8林打妨施例所 限制。 實施例一樟芝麄絲艟之培養 菌絲體菌株： 為寄存於食品工業研究所的菌株BCRC : 35398。 平板培卷： 將菌絲體接種於平板上，使用馬铃薯糊精培養基(P〇tat〇 PDA)，於30°C下培養約2週。 燒瓶培表： 到取平板上的菌絲接種於燒瓶内，用下列培養基，在約 w l，pH 4.5 下於播動機上以振盡速率5〇_25〇啊振盪培養到i〇g期初期，亦 5-7天； 即’約 ^ 培養基配方 含量(重量㈤ 1 0.1 0.05 0.05 0.05 2 0.5 0.2 榖顓(如麥粉類） 蛋白脒 硫酸鎂 碟酸氣二钟 硫竣鐵 嚴糖 酵母抽出物、粉、膏 1類（如黃豆粉、綠豆粉、大豆粉等） 201024320 培養基壯’將燒瓶料物接胁8G公升轉槽料細释公升播 酵槽）’在3〇。(： ’槽磨1.0公斤/平方公分，及pH約4 5下以8〇升/分通 氣速率通入妓，在⑽卿挽拌速率下培養約天，殘糖降至娜ppm以 下收槽’即得樟芝_絲舰酵液，包姉絲顯澄清液。樟芝_液經冷 凍乾燥或儒乾燥後’進行乾重、乡㈣及芝麻时量測定。 上述摄酵槽培養基中’另外分别添加0.^1%消泡谢(如 KM-72 ’為含發油、梦樹脂之水性消泡產品），或添加〇 7%芝麻油(纯度， "•89%’豐利食品廠股份有限公司)於鏺酵槽培養基内。 控制組：上述鏺酵槽培養基中不添加消泡劑及芝麻油； 消泡劑組：上述鏺酵槽培養基中添加0.01%消泡劑； 芝麻油組：上述鏺酵槽培養基中添加〇.7%芝麻油。 實施例二樟芝菌絲《之乾重、多醣艘、Lab值及芝麻粉含董測定 將實施例一中各組所得之楝芝醱酵液經冷凍乾燥或噴霧乾燥後，進行 φ 乾重、多醣體、Lab值及芝麻酚含量測定》 多糖盤測定： 多糖髏分析以酚-硫酸法進行，請參閲Dubio等人之文獻(Dubi〇, M. eM/.， 1956. Colorimetric method for determination of sugars and related substances.The inorganic salt may be sulfuric acid money, dipotassium arsenate phosphate, iron sulfate or the like; wherein the animal-derived protein and its hydrolyzate may be peptone; wherein the sugar may be glucose, sucrose, fructose, maltose, etc.; The carbon and nitrogen sources may be steroids (eg, wheat flour) or beans (eg, soy flour, mung bean powder, soy flour); an additional defoaming agent may be added to the fermentation tank medium to inhibit a large amount of foam during the cultivation process. The antifoaming agent can be a commercially available conventional antifoaming agent, such as 0.005-0.015% antifoaming anchor (Antifoaming KM-72, which is an aqueous defoaming product containing eucalyptus oil or bismuth resin), or 0.01 to 1%. Sesame 201024320 oil (commercially available). This issue of the township to the τ face (four) to fill the material (four), but this is 8 restrictions. Example 1 Culture of Astragalus membranaceus Mycelium strain: A strain BCRC: 35398 deposited in the Food Industry Research Institute. Flat plate: The mycelium was inoculated on a plate and cultured at 30 ° C for about 2 weeks using potato dextrin medium (P〇tat〇 PDA). Flask culture table: The hyphae on the plate were inoculated into the flask, and the culture medium was shaken at a speed of 5 〇 _25 于 on the seeding machine at about wl, pH 4.5 to the beginning of the i〇g period at about wl, pH 4.5. Also 5-7 days; that is, 'about ^ medium formula content (weight (five) 1 0.1 0.05 0.05 0.05 2 0.5 0.2 榖颛 (such as wheat flour) protein 脒 magnesium sulfate dish acid gas two bell sulphate iron sugar yeast extract, Powder, paste 1 (such as soy flour, mung bean powder, soy flour, etc.) 201024320 medium strong 'the flask material is threatened with 8G liters of trough material to release the liters of sowing fermenter) 'at 3 〇. (: 'The groove grinding is 1.0 kg/cm 2 , and the pH is about 4 5 at a rate of 8 liters per minute. The medium is incubated at a rate of (10) and the residual sugar is reduced to below the ppm. That is to get the 樟 _ _ silk broth, 姊 姊 显 显 樟 樟 樟 樟 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Add 0.^1% defoaming Xie (such as KM-72 'for water-based, dream resin water-based defoaming products), or add 〇 sesame oil (purity, "•89%' Fengli Food Factory limited stock The company is in the fermentation tank medium. Control group: no defoaming agent and sesame oil are added to the above fermentation tank medium; defoaming agent group: 0.01% antifoaming agent is added to the above fermentation tank medium; sesame oil group: the above fermentation 〇.7% sesame oil was added to the trough medium. Example 2 Anthocyanin hyphae "dry weight, polysaccharides, Lab value, and sesame powder containing Dong determination The lycopene fermentation broth obtained in each group of Example 1 was freeze-dried. Or spray drying, determination of φ dry weight, polysaccharide, Lab value and sesame phenol content Analysis of polysaccharide skull phenol - sulfuric acid method, see Dubio et al's document (Dubi〇, M. eM /, 1956. Colorimetric method for determination of sugars and related substances..
Anal Chem. 28,350.) 〇 1·樣品處理》 取1克均質之粉末，加入99倍體積之蒸餾水’於121°C下加熱萃取30 分鐘，上清液以1號濾紙過濾於濃縮瓶中，重複萃取步驟兩次，收集濾液 加以濃缩，定容至l〇ml。再以透析膜透析，以去除小分子多醣。透析後所 201024320 留下之溶液’再定容至10ml，作為分析之用》 2.分析方法： 取樣品及葡萄糖標準品1〇〇 μΐ，分別加入0 5 ml的5%酚溶液，再加入 2.5 ml的濃硫酸，混合均勻後，靜置使其回到室溫，另取1〇〇 μι的去離子 水，進行空白試驗，最後以490 nm波長測定其吸光值，比較樣品及標準品 之吸光值，以算出多醣含量。Anal Chem. 28,350.) 〇1·Sample treatment 》 Take 1 gram of homogeneous powder, add 99 times the volume of distilled water 'heat extraction at 121 ° C for 30 minutes, the supernatant is filtered in a concentrated bottle with No. 1 filter paper, repeat The extraction step was carried out twice, and the filtrate was collected and concentrated to a volume of 10 ml. The dialysis membrane is then dialyzed to remove small molecule polysaccharides. After the dialysis, the solution left in 201024320 is re-constant to 10ml for analysis. 2. Analytical method: Take sample and glucose standard 1〇〇μΐ, add 0 5 ml of 5% phenol solution, then add 2.5. 1 ml of concentrated sulfuric acid, mix well, let stand to return to room temperature, take 1 〇〇μιη deionized water, carry out blank test, and finally measure the absorbance at 490 nm wavelength, compare the absorbance of samples and standards Value to calculate the polysaccharide content.
Lab值谢定： 以SP60分光儀（χ-rite公司，美國)測定菌絲體鏺酵液乾燥粉之Lab值 (L : +偏淺，一偏深；a : +偏紅，一偏綠；b : +偏黃，一偏藍）· .芝麻紛fSesaminjjSi丨定： 請參閱王桂芳等人之文獻(王桂芳等，1999, HPLC法侧定北細辛中芝麻 脂素和細辛脂素的含量，藥物分析雜諸，19(4),251.)。 1·芝麻盼測定樣品處理： 取樣品lg，加入l〇ml的MeOH。置於超音波震堡機，60 min萃取， 過遽後再以MeOH定容至10ml，取適量通過0.45 μιη遽膜供作檢液。 2.分析方法： a.標準品溶液配製 精確量取芝麻紛儲備溶液適量’以MeOH分別調配一系列濃度，分别 為芝麻酚：0.072、0.096、0.12、0.144、0.168 mg/ml» b·高效能液相層析法 樣品及標準品溶液，以下列條件分析之： 管柱：C18 (25〇x4.6mm，5μιη)，流速：1.〇 ml/min，管柱溫度：室溫。 201024320 移動相條件 標準品 移動相 偵測波長（UV) 滯留時間(RT) 芝麻酚Lab value Xieding: The Lab value of the mycelium lysate dry powder was determined by SP60 spectrometer (χ-rite, USA) (L: + shallow, one deep; a: + reddish, one greenish; b: + yellowish, bluedish · · sesame sesame fSesaminjjSi 丨 :: Please refer to Wang Guifang et al. (Wang Guifang et al., 1999, HPLC method for the determination of sesame and saponin in Beixinxin, Drug analysis is mixed, 19(4), 251.). 1. Sesame Hope Sample Determination: Take sample lg and add 1 ml of MeOH. It was placed in a supersonic vibrating machine and extracted for 60 min. After passing through the crucible, the volume was adjusted to 10 ml with MeOH. The appropriate amount was passed through a 0.45 μm 遽 membrane for the test solution. 2. Analytical methods: a. Prepare the standard solution to accurately measure the amount of sesame seed stock solution. Depending on the concentration of MeOH, each is a concentration of sesame: 0.072, 0.096, 0.12, 0.144, 0.168 mg/ml. The liquid chromatography sample and the standard solution were analyzed under the following conditions: Column: C18 (25 〇 x 4.6 mm, 5 μιη), flow rate: 1. 〇 ml/min, column temperature: room temperature. 201024320 Mobile phase conditions Standards Mobile phase Detection wavelength (UV) Retention time (RT) Sesame phenol
Water/MeOH=30/70 280 nm 14.4 min 比較樣品及標準溶液之分析結果，以積分面積計算可得到樣品中芝麻 酚的含量。 結果： 請參考表一所示，將實施例一中各組所得之樟芝鏺酵液經冷凍乾燥或 ❹喷霧乾燥後’進行乾重、多雜、Lab值及芝脑含制定。其中添加消泡 劑及芝麻油之培養基相較於控制組，於培養過程中消泡劑組及芝麻油組皆 無起泡現象，故，添加芝麻油可取代習用之化學類消泡劑。 另，觀察添加芝麻油之培養基(芝麻油組)可提前2天收槽天數(芝麻油 组收槽天數：9天；控制組及消泡劑組之收槽天數·· u天）。且測量乾燥後 之菌絲體重量，芝麻油組之菌絲體乾重(2.83 ± 0.15 g/100ml)明顯較高於控 制组(2.05 ± 0.21 g/100ml)及消泡劑组(2.16 ± 0.16/lOOml)所得者。更重要 φ 的，相較於控制組及消泡劑组，藉由添加芝麻油以培養楝芝所得之菌絲艎， 經測量發現其菌絲體芝麻酚含量為1.41 ± 〇.〇9 mg/g。 表一培養基中添加芝麻油對於楝芝生長之影響 培養過程收槽天數 ^ 乾重 多醣體含量芝麻酚含量 起泡情形（殘醣 <500) (g/l〇〇ml) (〇/〇) (mg/g) 0 控制組微起泡現象 11 2.05 ± 0.21 10.02 ± 0.26 消泡劑組 不起泡 11 2.16 ± 0.16 10.25 ± 0.20 0 11 201024320 — 2.83 ± 0.15 10.33 ± 0.23 1.41 ± 〇.〇9 9 芝麻油组 不起泡 另 β--—----------- 請參考表二及表三所示，表二為添加芝麻油於培養基所得之棒芝 菌絲體鏺酵液，經;東乾後觀察其顏色之變化如表二之結果，相較於控制 組及消/&劑组’芝麻油組所得之乾燥粉，其顏色有增加紅色度現象(a值： 12.〇8 ± 0.〇5)，近似天然樟芝之類色。 表二添加芝麻狄攸瞒液乾燥狀顏色變化 ❹Water/MeOH=30/70 280 nm 14.4 min Compare the analysis results of the sample and the standard solution, and calculate the content of sesame in the sample by the integrated area. Results: Referring to Table 1, the yew broth obtained from each group in Example 1 was freeze-dried or spray-dried, and the dry weight, poly-hybrid, Lab value and cirrhosis were determined. Compared with the control group, the medium in which the defoaming agent and the sesame oil were added had no blistering phenomenon in the defoaming agent group and the sesame oil group during the culture, so the sesame oil could be substituted for the conventional chemical defoaming agent. In addition, the medium in which the sesame oil was added (sesame oil group) was observed in the days of 2 days in advance (the number of days of sesame oil group collection: 9 days; the number of days in the control group and the defoaming agent group). The dry weight of the mycelium was measured. The dry weight of the mycelium of the sesame oil group (2.83 ± 0.15 g/100 ml) was significantly higher than that of the control group (2.05 ± 0.21 g/100 ml) and the defoamer group (2.16 ± 0.16/). lOOml) The winner. More importantly, compared with the control group and the defoamer group, the mycelium content of the mycelium was determined to be 1.41 ± 〇.〇9 mg/g by adding sesame oil to culture the mycelium obtained from Antrodia camphorata. . Table 1 Addition of sesame oil to the growth of Antrodia camphora on the growth of Antrodia camphora. Number of days in the culture process. Dry weight Polysaccharide content Foaming of sesame phenol content (residual sugar <500) (g/l〇〇ml) (〇/〇) ( Mg/g) 0 Microbubbing in the control group 11 2.05 ± 0.21 10.02 ± 0.26 Antifoaming group No blister 11 2.16 ± 0.16 10.25 ± 0.20 0 11 201024320 — 2.83 ± 0.15 10.33 ± 0.23 1.41 ± 〇.〇9 9 Sesame oil The group can not blister another β-------------- Please refer to Table 2 and Table 3, Table 2 is the sesame oil mycelium lysate obtained by adding sesame oil to the culture medium; After the East dried, the color change was observed as shown in Table 2. Compared with the control group and the dry powder obtained from the sesame oil group, the color of the sesame oil group increased the redness (a value: 12.〇8 ± 0. 〇 5), similar to the color of natural anthraquinone. Table 2 Adding the color change of dried sesame seed ❹ ❹
*L 凍乾Lab值 *a *b 控制組 消泡劑組 芝麻油組 +54.38 ± 0.19 +61.11 ± 0.24 +59.05 ± 0.12 +10.53 ± 0.08 +10.79 ± 0.06 +12.08 ± 0.05 +26.67 ± 0.18 +24.54 ± 0.11 +31.75 ± 0.16 此外，表三為添加芝麻油於培養基所得之樟芝菌絲體醱酵液，經喷霧 乾燥後觀察其顏色之變化，如表三之結果，相較於控制組及消泡劑組，芝 φ 麻油组所得之乾燥粉，其顏色有增加紅色度現象(a值：16.54 ± 0.05)，近似 天然楝芝之顏色。故’藉由添加芝麻油以培養樟芝可得近似天然樟芝顏色 之產物。 表三添加芝麻油之樟芝鏺酵液乾燥後之顏色變化 喷霧Lab值 ♦L *a *b 控制組 +36.39 ± 0.17 +12.39 ± 0.10 +26.53 ± 0.19 消泡劑組 +38.19 + 0.22 +11.87 ± 0.05 +27.22 ± 0.15 12 201024320 芝麻油組 +38.52 ± 0.15 +16.54 ± 〇.〇5 +27.56 ±0.18 實施例三樟芝因艎培養 將木屑、黃豆粉、米糠及小麥麩皮(7:1:1:1之比例)與適量水混合均勻， 再裝進耐熱太空包袋内(每包約1公斤的固體培養基，包含木屑、黃豆粉、 米糠及小麥楚皮(7:1:1:1之比例)及適量水），套上套環，塞住棉花；另一組 為相同固體培養基配製成之太空包，並額外添加〇 7%芝麻油，再將太空包 送進滅菌釜内減g(121°C，1小時)。冷卻後於無菌操作台各接種5%#芝醱 β酵液，置於25。(：，75-85RH(相對濕度)環境下培養。3個月後取出評估菌絲 生長情形。 結果： 由菌絲生長情形評估，添加〇.7〇/0芝麻油組之菌絲顯著較未添加組好， 添加芝麻油有助於樟芝固體培養生長. 本發明所提供之一種樟芝菌絲體之新穎培養方法，與前述引證案及其 他習用技術相互比較時，更具有下列之優點： _ 本發明所提供之一種樟芝菌絲鳢之新穎培養方法，其特徵在於藉由添 加芝麻油於樟芝培養基中以取代習用化學消泡劑，亦可達到消泡現象故， 本發明提供一種新穎之天然消泡劑。 本發明提供之一種樟芝菌絲想之新穎培養方法，其十添加芝麻油於掉 芝培養基中用《培養樟芝’除可達職泡之功效外，藉此方法所得之禅芝 菌絲艘’相較於習知培養方法，添加芝麻油可使樟芝菌絲雄收槽天數缩短、 乾重增加，顏色近似天然樟芝之紅色，更重要者，藉此培養方法所得之掉 芝菌絲體富含芝麻酚，相較習用方法所得者更富營養價值。 13 201024320 本發明提供之一種樟芝菌絲艟之新穎培養方法’藉由添加芝麻油於樟 芝培養基中，可促使樟芝之菌絲生長》 上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例 並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實 施或變更，例如：習用培養基之成分、習用消泡劑種類等實施例，均應包 含於本案之專利範圍中。 综上所述，本案不但在用途上確屬創新，並能較習用物品增進上述多 β項功效，應已充分符合新穎性及進步性之法定發明專利要件，爰依法提出 申請’懇請責局核准本件發明專利申請案，以勵發明，至感德便。 【圖式簡單說明】 無 【主要元件符號說明】 ❹ 無*L freeze-dried Lab value *a *b Control group defoamer group sesame oil group +54.38 ± 0.19 +61.11 ± 0.24 +59.05 ± 0.12 +10.53 ± 0.08 +10.79 ± 0.06 +12.08 ± 0.05 +26.67 ± 0.18 +24.54 ± 0.11 +31.75 ± 0.16 In addition, Table 3 shows the change of color of Antrodia camphorata mycelium broth obtained by adding sesame oil to the culture medium. The results of Table 3 are compared with the control group and defoamer. In the group, the dry powder obtained from the Zhi φ sesame oil group has a red color increase (a value: 16.54 ± 0.05), which is similar to the color of natural yam. Therefore, by adding sesame oil to cultivate Antrodia camphora, a product similar to the color of natural Antrodia can be obtained. Table 3 Adding sesame oil to the color change of the broth after drying. Lab value ♦L *a *b Control group +36.39 ± 0.17 +12.39 ± 0.10 +26.53 ± 0.19 Defoamer group +38.19 + 0.22 +11.87 ± 0.05 +27.22 ± 0.15 12 201024320 Sesame oil group +38.52 ± 0.15 +16.54 ± 〇.〇5 +27.56 ±0.18 Example 3 Astragalus sinensis is cultivated with wood chips, soybean powder, rice bran and wheat bran (7:1:1: 1 ratio) mixed with the right amount of water, and then packed into a heat-resistant space bag (about 1 kg of solid medium per pack, including wood chips, soy flour, rice bran and wheat Chupi (7:1:1:1 ratio) And the right amount of water), put on the collar, plug the cotton; the other group is the space bag made of the same solid medium, and add 〇 芝麻 sesame oil, and then send the space bag into the sterilizer to reduce g (121 ° C, 1 hour). After cooling, inoculate 5% #芝酦 β fermentation solution on the aseptic workstation, and place it at 25. (:, 75-85RH (relative humidity) culture. After 3 months, the hyphal growth was evaluated. Results: As assessed by mycelial growth, the hyphae added to the 〇.7〇/0 sesame oil group was significantly less than that added. The sesame oil is added to help the solid culture growth of Antrodia camphorata. The novel culture method of the mycelium of Antrodia camphorata provided by the present invention has the following advantages when compared with the above-mentioned cited cases and other conventional techniques: _ 本The invention provides a novel culture method for the mycelium of the genus Antrodia camphorata, which is characterized in that the saponin is added to the sage medium to replace the conventional chemical antifoaming agent, and the defoaming phenomenon can also be achieved. The anti-foaming agent provides a novel culture method for the mycelium of the genus Antrodia camphorata, and the sesame oil obtained by the method is the same as the effect of the cultivating 樟 ' 除Compared with the conventional culture method, the addition of sesame oil can shorten the number of days and increase the dry weight of the mycelium of the mycelium, and the color is similar to the red color of the natural anthraquinone. The mycelium obtained by the method is rich in sesame phenol, which is richer in nutritive value than the one obtained by the conventional method. 13 201024320 The novel culture method of the genus Antrodia camphorata provided by the present invention is provided by adding sesame oil to the medicinal medium The detailed description of the present invention is directed to the specific embodiment of the present invention, which is not intended to limit the scope of the invention, and is not deviated from the spirit of the invention. Equivalent implementation or modification, for example, the composition of the conventional medium, the type of conventional defoamer, etc., should be included in the scope of the patent in this case. In summary, the case is not only innovative in use, but also more customary. Articles that promote the above-mentioned multi-beta effects should fully comply with the statutory invention patents of novelty and progressiveness, and submit an application in accordance with the law's request for the approval of this invention patent application to encourage invention and to the sense of convenience. Brief description] No [Main component symbol description] ❹ No