591110 A7591110 A7
本發明係有關一種胺基丁酸之製備方法，且特別者，以僅生 長於台灣特有物種牛樟樹臟仏|财/樹心内的兹類— 棒之XAntrodia camphorata 氣 Antrodia cinnamomea)一之菌絲體製備 7-胺基丁酸之方法。本發明亦有關一種按上述製備胺基丁酸之方 法製備成的含胺基丁酸物質。本發明更有關一種組成物，其包括 按上述製備環二傣之方法製備成的含7_胺基丁酸物質及醫葯上或食 品上可接受的載劑、稀釋劑或賦形劑。 先首技術說明 經濟部智慧財產局員工消費合作社印製 樟芝又名牛樟菇、樟菰、樟窟内菰，台灣有稱陰陽對口菇。樟芝 子實體屬多年生，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申 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 菌子L(4〜5個菌孔/毫米），内有孢子味極苦，新鮮時為橘紅色，之後會 成為橘褐色或褐色，鐘體則呈暗綠褐色的皮殼。以顯微鏡觀察其擔孢 子，其型態為平滑無色之透明微彎柱形。 樟芝目前已經可以用發酵法大量生產其菌絲體。在樟芝菌絲體之 發酵過程中發現亦有此類T-胺基丁酸類物質產生。根據此項發現， 观110 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明〇) 乃提出一種以樟芝菌絲體製備胺基丁酸之方法。 圈式之簡略說明 圖一為顯示出樟芝菌絲體發酵培養時間與7 _胺基丁酸產量的關 係之圖解。 綜上所述，本發明提出一種以樟芝菌絲體製備^_胺基丁酸之方 法’其包括以液體發酵培養法培養樟芝菌絲體，將經分離出且冷束乾 燥之樟芝菌絲體用水性及/或有機溶劑萃取而得含胺基丁酸物質。 r-胺基丁酸(GABA)是由穀胺酸經由穀胺酸脫叛酶催化脫羥基 化而成之非蛋白質構成性胺基酸。The present invention relates to a method for preparing aminobutyric acid, and in particular, it is a mycelium that grows only in Taiwan's endemic species, Antrodia camphora viscera | Civil / tree heart—Xantrodia camphorata (Antrodia cinnamomea). Preparation of 7-aminobutyric acid in vivo. The present invention also relates to an aminobutyric acid-containing substance prepared by the above method for preparing aminobutyric acid. The present invention further relates to a composition comprising a 7-aminobutyric acid-containing substance prepared according to the above-mentioned method for preparing cyclodifluorene and a pharmaceutically or food-acceptable carrier, diluent, or excipient. First technical description Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Antrodia cinnamomea, also known as Antrodia camphorata, Camphor sylvestris, camphor cave, Taiwan is known as Yinyang counterpart mushroom. Antrodia camphorata fruit body is perennial and has a strong nose aroma of camphor tree, which is very different from general ganoderma lucidum. Its appearance is plate-shaped or bell-shaped. The plate-shaped type has an orange-red (yellow) color, the entire surface is full of pores, and the bottom of the plate has a light yellow-white clot substance, thereby attaching the clot substance to the inner wall of A. camphor tree hollow material. For bell-shaped people, the fruit layer (bell face) is also orange (yellow), filled with fungus L (4 ~ 5 pores / mm), the spores are extremely bitter, and it is orange-red when fresh. It becomes orange brown or brown, and the bell body has a dark green brown leather case. Observing its spores under a microscope, its shape is a smooth, colorless, transparent, slightly curved cylinder. Antrodia cinnamomea can be mass-produced by fermentation. Such T-aminobutyric acids were also found during the fermentation of Antrodia cinnamomea mycelium. According to this finding, Guan 110 printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention 0) A method for preparing aminobutyric acid from Antrodia camphorata mycelium was proposed. Brief description of the circle type Figure 1 is a diagram showing the relationship between the fermentation culture time of Antrodia camphorata mycelium and the yield of 7-aminobutyric acid. In summary, the present invention proposes a method for preparing ^ _aminobutyric acid from Antrodia camphorata mycelium, which includes culturing Antrodia camphorata mycelia by a liquid fermentation culture method, and separating and anisotropically drying Antrodia camphorata The mycelium is extracted with an aqueous and / or organic solvent to obtain an aminobutyric acid-containing substance. r-Aminobutyric acid (GABA) is a non-protein constitutive amino acid that is catalyzed by glutamic acid via a glutamate detase enzyme.
H2N_CHrCHrCHrC00H r-胺基丁酸除了具有降血壓之功效外，還具備控纖臟、腸胃 道和腎臟髓質等部份的内皮細胞激素分泌之功效。 於本發明以樟芝菌絲體製備了 _胺基丁酸之方法一具體實例中所 用的樟芝菌株為購自新竹食品工業發展研究所菌種中心的論感 圓Μ卿切CCRC 35398，以馬鈐薯葡萄糖瓊脂㈣伽d論⑽啊 (PDA))(購自美國DifC0公司)斜面培養基培養及保存。 · 此菌絲體之大量培養係利用液態深層培養方法連續培養，離心採 收並乾燥而得。培養條件可為例如溫度為3(rc，接種之菌數為培養 基含量之1·0 %，連續培養七天。培養基可A，例如，具有下面的組 卜紙張尺度劇㈣i?i^(CNS)A4驗咖x 2974 (請先閱讀背面之注意事項再填寫本頁)H2N_CHrCHrCHrC00H r-aminobutyric acid has the function of controlling blood pressure, but also has the function of controlling the secretion of endothelial cells hormones in fibrous organs, gastrointestinal tract, and kidney medulla. The Antrodia cinnamomea strain used in a specific example of the method for preparing _aminobutyric acid from Antrodia cinnamomea mycelium in the present invention was purchased from Hsinchu Food Industry Development Research Institute's Strain Center, MCC, CCRC 35398, and Glutinous Agar Glucose Agar (Gamma Polysaccharides (PDA)) (purchased from DifC0 Company, USA) was cultured and stored on slanted medium. · A large number of mycelium cultures are obtained by continuous cultivation using liquid submerged culture, centrifugation and drying. The culture conditions may be, for example, a temperature of 3 (rc, the number of inoculated bacteria is 1.0% of the content of the culture medium, and the culture is continued for seven days. The culture medium may be A, for example, having the following composition paper scale:? I? I ^ (CNS) A4 Coffee Inspection x 2974 (Please read the notes on the back before filling this page)
成者每A升去離子水巾含有贿：2q克：购孤：3克；MgS〇4: 3克’ KH2P〇4 · 3克’擰檬酸·· 〇·5克；酵母萃取物：5克；培養液之 pH值調整在5.5。 要從菌絲體萃取出所欲物質可為技藝中已知者，例如在陳彥霖，” 科子”技術第32卷，第12期，（2_)中所述者從紅麴萃取者，其中 發現只有用水萃取才有效果。 將此乾燥_期水性溶_如水，鹼性水溶液或水和極性有機 溶劑的混合物’其中該極性有機溶劑可為醇類例如甲醇，乙醇，異丙 醇，等，或用有機溶劑例如甲醇、氣仿、二氣甲烧、正己炫、乙酸乙 S曰丁醇等予以萃取’視需要在回流條件下進行，並離心收集萃取液。 將菌絲體與㈣分開之方法可採㈣知技術，例如離心、，過渡，沉著 (settling)，傾_c_i〇n) ’等。於本發明一較佳實施例中係採用 離。法’使用習用的離心機，例如歐式離心、脫水機，如可得自瑞典 ALFA LAVAL公司之Decater順18 s ;以遍样(4〇〇〇 X幻離心即 分離出菌絲體和澄清液。 將上述包括萃取和分離的諸步驟重複數次後，將合併萃取液濃縮 即得含r-胺基丁酸物質。 本發明製備r ·胺基丁酸之方法可視情況更包括將此含r _胺基 丁酸物質進—步純化，例如以層析術如離子交㈣析術或氧化石夕管柱 591110 A7 Β7 五、發明說明（G勹 層析術分離所欲成分，及以胺基酸自動分析儀鑑定及定量該化合物。 本發明亦提出一種按上述製備7 -胺基丁酸之方法製備成的含7 -胺基丁酸物質。 本發明更提出一種組成物，其包括按上述製備環二傣之方法製備 成的含r-胺基丁酸物質及醫葯上或食品上可接受的載劑、稀釋劑或 賦形劑。 本發明要以下面的實施例予以示範闡明，但本發明不受彼等實施 例所限制。 實施例1:棟芝8鎿《箱絲Λ之培養 1_生產菌株 樟芝¢4⑽Wia camp/^raia) CCRC 35398購自新竹食品工業發 展研九所函種中心，以馬铃薯勤萄棒複脂(p〇tat〇 dextrose agar (PDA)) (購自美國Difco公司）斜面培養基培養及保存。 2·菌絲體之培養 矛J用液態:?木層培養方法連續培養七天’溫度為3 〇 °c，接種之菌 數為培養基含量之1·〇 %，培養基之組成如下：每公升去離子水中含 有蔗糖·· 20克；卿)2犯4 ·· 3克；MgS04: 3克；KH2p〇4 ·· 3克；檸 檬酸：0.5克；酵母萃取物：5克；培養液之pH值調整在5.5。 實施例2 :用水萃取 ΐ紙張斤適用家標準（LNS)A4規格⑵〇 χ视公6 (·請先·閱讀背面之注意事項 本頁) 經 濟 部 智 慧 財 產 局 員 工 消 費 合 作 社 印 製 591110 A7Adults per 1 liter of deionized water towels contain bribes: 2q grams: purchase orphan: 3 grams; MgS〇4: 3 grams of 'KH2P〇4 · 3 grams of citric acid · · 0.5 grams; yeast extract: 5 G; the pH of the culture was adjusted to 5.5. To extract the desired substance from the mycelium can be known in the art, such as those described in Chen Yanlin, "Kezi" Technology Volume 32, Issue 12, (2_). Extraction with water is effective. This dry-phase aqueous solution such as water, an alkaline aqueous solution or a mixture of water and a polar organic solvent, wherein the polar organic solvent may be an alcohol such as methanol, ethanol, isopropanol, etc., or an organic solvent such as methanol, gas, etc. Extraction is carried out with imitation, digas, hexamethylene chloride, ethyl acetate, butanol, etc., if necessary, under reflux conditions, and the extract is collected by centrifugation. The method of separating the mycelium from the tadpole can adopt known techniques, such as centrifugation, transition, settling, tilting, etc .. In a preferred embodiment of the present invention, separation is used. The method uses a conventional centrifuge, such as a European-style centrifuge and a dehydrator, such as Decater, available from ALFA LAVAL, Sweden, for 18 s; the mycelia and clarified solution are separated by centrifugation in a sample (400X). After repeating the above steps including extraction and separation several times, the combined extracts are concentrated to obtain an r-aminobutyric acid-containing substance. The method for preparing r · aminobutyric acid according to the present invention may further include this r- Aminobutyric acid material is further purified, for example, by chromatography such as ion chromatography or stone column 591110 A7 B7 5. Description of the invention (G 勹 chromatography to separate the desired components, and amino acids An automatic analyzer identifies and quantifies the compound. The present invention also provides a 7-aminobutyric acid-containing substance prepared according to the method for preparing 7-aminobutyric acid. The present invention further provides a composition including the preparation according to the above. The r-aminobutyric acid-containing material prepared by the method of cyclodifluorene and a pharmaceutically or food-acceptable carrier, diluent, or excipient. The present invention is illustrated by the following examples, but the present invention Not limited by their embodiments. Example 1: Dongzhi 8 鎿 "Cultivation of Box Silk Λ_Production Strain Antrodia cinnamomi ¢ 4⑽Wia camp / ^ raia) CCRC 35398 was purchased from the Hakoza Center of Nine Institute of Hsinchu Food Industry Development Research Institute. Fat (potatodextrose agar (PDA)) (purchased from Difco, USA) was cultured and stored on slanted medium. 2. Mycelium culture spear J. Liquid: wood layer culture method was used for seven days of continuous culture. The temperature was 3 ° C. ° C, the number of inoculated bacteria is 1.0% of the content of the culture medium, and the composition of the culture medium is as follows: 20 grams of sucrose per liter of deionized water; 4) 3 grams of 2 criminals; MgS04: 3 grams; KH2p. 4 ·· 3g; citric acid: 0.5g; yeast extract: 5g; pH value of culture broth adjusted to 5.5. Example 2: Extraction with water 斤 Paper catty (LNS) A4 specification ⑵ χ Vision 6 (· Please read the notes on the back page) Printed by the Ministry of Economic Affairs and Intellectual Property Bureau Staff Consumer Cooperative Agency 591110 A7
五、發明說明（P 用水在80C，％取1小時，然後分離出萃取液，可重複萃取數 次，將萃取液合併冷凍乾燥處理(_4〇°C，4小時；4〇<5c , 24小時）。 實施例3 : r-按基丁酸之鐘定 1·菌絲體r-胺基丁酸之鑑定 取2克經冷凍乾燥之樟芝菌絲體，加入10毫升2%之硫酸 基柳酸(5-Sulfosalicylic acid)震盪萃取，並以0.45 “ m過渡膜過滤之， 再取1毫升濾液以胺基酸自動分析儀分離並與標準品比對鑑定及定 量分析。 圖一顯示出樟芝菌絲體發酵培養時間與了 _胺基丁酸產量的關 係。可以看出r -胺基丁酸產量隨著發酵培養時間而遞增到第12天才 趨緩。 2·萃取濃縮物r-胺基丁酸含量之測定 將所分離出的諸區分物濃縮物溶於10毫升2%之5_硫酸基柳 酸中，並以0.45//m過濾膜過濾之，再取丨毫升濾液案上述胺基酸 自動分析儀分析及定量。如圖1中所示者，本發明方法所得含胺 基丁酸物質的7-胺基丁酸含量可以高達獅，，比傳统紅麵製得 物的250〜1〇〇〇 ppm (陳彥霖，上述)遠較為高。 . 本紙張尺度適用中國國家標準（CNS)A4規格⑵Q χ 297公爱了------- (請先閱讀背面之注意事項再填寫本頁) 訂·. 經濟部智慧財產局員工消費合作社印製V. Description of the invention (P is taken at 80C,% for 1 hour with water, then the extract is separated, and the extraction can be repeated several times, and the extracts are combined with freeze-drying treatment (_4 ° C, 4 hours; 4 ° < 5c, 24 Hours) Example 3: r-According to the determination of the butyric acid 1. Mycelium r-aminobutyric acid identification 2 g of freeze-dried Antrodia camphorata mycelium was added and 10 ml of 2% sulfate group was added Salicylic acid (5-Sulfosalicylic acid) was extracted by shaking and filtered through a 0.45 "m transition membrane. Another 1 ml of the filtrate was separated by an amino acid analyzer and compared with a standard for identification and quantitative analysis. Figure 1 shows Camphor The fermentation and culture time of Mycelium lucidum was related to the yield of aminobutyric acid. It can be seen that the yield of r-aminobutyric acid gradually increased with the fermentation culture time to the 12th day before slowing down. 2. Extraction concentrate r-amine Determination of butyl butyric acid content The separated concentrates were dissolved in 10 ml of 2% 5-sulfosalic acid, and filtered through a 0.45 // m filter membrane, and then the ml of the filtrate was used for the above amine. Analysis and quantification of basic acid analyzer. As shown in Figure 1, the aminobutyric acid-containing material obtained by the method of the present invention The content of 7-aminobutyric acid can be as high as lion, which is much higher than the 250 ~ 1000ppm (Chen Yanlin, above) produced by traditional red noodles.. This paper size applies the Chinese National Standard (CNS) A4 specification⑵Q χ 297 public love ------- (Please read the precautions on the back before filling this page) Order .. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs