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研究生:謝宗成
研究生(外文):Tsung-Cheng Shie
論文名稱:不同培養條件對皿培式牛樟芝三萜類成分圖譜之影響
論文名稱(外文):The effect of triterpenoid profile of Antrodiacinnamomea on distinct airtifical agar medium
指導教授:洪堂耀
指導教授(外文):Tang-Yao Hong
口試委員:賈宜琛羅佑軒
口試委員(外文):Yi-Chen ChiaYu-Shiuan Lo
口試日期:2014-06-24
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:48
中文關鍵詞:牛樟芝皿培式三萜類樟芝酸
外文關鍵詞:Antrodia cinnamomeaartificial agar mediumtriterpenoidsAntcin
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台灣特有真菌牛樟芝(Antrodiacinnamomea),生長在老齡牛樟木的中空腐朽材心中,早期原住民用此來消炎、解酒、止痛,牛樟芝因稀少而珍貴,山老鼠為盜採野生牛樟芝或取得牛樟原木殖菌,盜伐保育類的牛樟木是造成的牛樟木日漸稀少的主因。牛樟芝子實體生長速度十分緩慢,價格高昂,不利於牛樟芝相關產品的推廣。近年來有生技業者利用培養皿培養出牛樟芝的類子實體,稱為皿培式牛樟芝,在培養基中不需含有任何牛樟木成分,即具有多種子實體才有的麥角固醇類三萜。本研究從牛樟段木上的野生牛樟芝子實體篩選了一株牛樟芝菌株,以內轉錄區間(internal transcribed spacer, ITS)序列比對確認所篩選的菌株確實為牛樟芝,暫時命名為Antrodiacinnamomea AC 12。此菌株生長的溫度為22.5℃~27.5℃,pH值為5.0。不同的培養基組成、碳氮比、通氣、改變培養溫度、物理性傷害或培養時間,對菌株的外觀會有明顯的影響,以HPLC分析其三萜類的成分圖譜,可能具有樟芝酸K、樟芝酸C、樟芝酸H、去氫硫色多孔菌酸、去氫層孔菌酸等6種指標成分,但外觀上的差異並沒有明顯影響三萜類圖譜的差異性。與其他市售的皿培式牛樟芝比較的話,三萜類成分圖譜峰值似乎較高些,而單片重量也較重,透過試量產的過程中,已開發建立完整皿培式牛樟芝生產流程,相關技術具有市場的競爭力。 關
Antrodia cinnamomea, the most unique fungus in Taiwan, grows up on the hollow heartwood rot of the aging Cinnamomum kanehirai. Since the early stageof Taiwanese history, aborigines have taken it as a medical solution for anti-inflammatory, hangover, and pain releasing. Due to its preciousness and rareness, some illegal logging thieves denude the wild type and plant bacteria for commercial benefits, in addition to that the fruiting body of A.cinnamomea grows up at very slow speed, leading to its decreasing quantities and over high prizes. Therefore, it’s difficult to overcome those problems to promote its related products. In recent years, some experts and biotechnology companies make good use of artificial agar medium to induce the fruiting body formation of A.cinnamomea. What’s more interesting, without any components of C. kanehirai, there are still some ergosterol class triterpenoids only from various fruiting body in the artificial agar medium. In this study, one of the individual strains from the Antrodia of fruiting body on wild C.kanehirai has been chosen to identify as a species Antrodia cinnamomum by means of the nucleotide sequences of the internal transcribed spacer (ITS) on NCBI web site as well as the contrast of BLAST. The most appropriate temperature for the strains’growing is between 22.5 ℃~27.5 ℃and the best pH is 5.0. The different medium composition, carbon-nitrogen ratio, aeration, variation of culture temperature, physical wounds or incubation period may have an apparent effect on strains’ morphology. The profile of triterpenoids analyzed by HPLC presents a solution with six target ingredients, such as Antcin C, Antcin H, Antcin K, DMB (1,4-dimethoxy-2,3-methylenedioxy-5-methylbenzene), dehydrosulphurenic acid, and dehydroeburicoic acid. Therefore, the differences from the appearances have no obvious influences on the profile of triterpenoids. Compared by other fruiting body from on artificial medium for sale, the quantity of triterpenoids, profile seems better and the individual weight is much heavier. In a word, through the process of trial mass production, the complete procedure of production in the fruiting body from on artificial medium has been set up with related technology of marketing competitive.
1 前言 1
2 文獻回顧 3
2.1 樟芝的簡介 3
2.2 三萜類化合物 3
2.3 牛樟芝學名演變 6
2.4 牛樟芝的功效研究 7
2.5 牛樟芝人工培養技術 9
3 研究動機 12
3.1 研究目的 13
4 材料與方法 13
4.1 實驗藥品與試劑套組 14
4.1.1 藥品 14
4.1.2 試劑套組 14
4.2 實驗設備 14
4.3 培養基 15
4.4 菌種來源 15
4.5 牛樟芝菌種鑑定 15
4.5.1 染色體DNA萃取 15
4.5.2 聚合酶連鎖反應 16
4.5.3 瓊膠電泳 16
4.5.4 聚合酶連鎖反應回收 17
4.5.5 製備勝任細胞 17
4.5.6 轉形 18
4.5.7 質體DNA抽取 18
4.5.8 瓊膠電泳確認是否有0.6~0.7 kb 插入DNA 19
4.6 牛樟芝菌種培養條件分析 19
4.6.1 最適生長溫度 19
4.6.2 最適培養pH值 19
4.6.3 最適培養基成分及培養時間 20
4.6.4 HPLC成分圖譜測定 20
5 結果與討論 21
5.1 原生牛樟菌株分離及培養 21
5.2 菌種鑑定 22
5.3 溫度與培養基pH對牛樟芝生長的影響 22
5.4 培養基組成、通氣、物理性對於牛樟芝生長的影響 23
5.5 HPLC分析牛樟芝三萜類成分圖譜 24
5.6 皿培式牛樟芝試量產探討 25
6 總結 28
7 圖表 29
8 參考文獻 41
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