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研究生:湯朝棟
研究生(外文):Chao-tung Tang
論文名稱:研究白樟芝菌種鑑定與栽培
論文名稱(外文):Studies on the identify and culture ofAntrodia cinnamomea (white)
指導教授:李國陽李國陽引用關係
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:2010
畢業學年度:98
語文別:中文
論文頁數:132
中文關鍵詞:樟芝白樟芝多醣體HPLC
外文關鍵詞:Antrodia cinnamomeaAntrodia cinnamomea(white)polysaccharideHPLC
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樟芝(Antrodia cinnamomea)又名牛樟菇、牛樟芝,是台灣特有的生物之一,
在民間常被用來當作保肝、護肝、抗疲勞的聖品;近幾年樟芝開始被大家所認識,
相關的研究與文獻也不斷的推陳出新,無論是成份上的分析研究,或是功效上的
評估實驗,都再次證實樟芝是具有發展性與潛力的,但是這些研究的主軸都圍繞
在『樟芝』上,對於『白樟芝』的研究卻是乏人問津。
本研究首先鑑定博士群生技提供(DR0101)為樟芝的一種,之後進行平盤培
養、搖瓶培養及固態培養,從中找出目標菌株(DR0101)其生長習性與特點,並
且與食工所菌種中心的紅色樟芝(BCRC 35396)做比較。
實驗室成功馴化白色的樟芝菌株,並且利用ITS及5.8s核糖體基因序列,將序
列與PubMed基因資料庫比對後,顯示此菌株為樟芝的一種。平盤培養實驗中發現
DR0101在MY培養基上,生長直徑與菌絲乾重都比BCRC 35396高,而最佳培養溫
度也與前人對樟芝的研究相似,並且DR0101對agar的濃度較不敏感;添加適量動
物性或植物性peptone在平盤培養中會使菌絲生長直徑減少,但是會增加菌絲的乾
重;添加三種樟樹精油在平盤培養基中都會使菌絲生長加快,以及減少菌絲濃密
度(乾重),此外還會增加極性成份的含量。搖瓶培養實驗中發現,DR0101的菌
絲乾重、胞內多醣及胞外多醣都比BCRC 35396高;另外也發現樟芝菌絲在HPLC
成份分析上,明顯是高於DR0101菌絲。培養DR0101菌株 0~20天的實驗中,發現
培養至第16天有最高的菌絲乾重;胞外多醣含量最高的時間出現在第14天。固態
穀物培養結果中,生長較好的基質為脫殼薏仁、高粱及胚芽米;HPLC成份分析上
則是:黃豆、高粱、麥片、黑豆、紅薏仁、胚芽米有些微差異;另外也發現BCRC
35396與DR0101的發酵物有不同氣味,其中以高粱為基質的成份分析上,BCRC
35396與DR0101有些許不同。
Antrodia cinnamomea, also called Niu-Chang-Ku, is the special fungus species in
Taiwan, has recognized to use in protecting liver and function as a way to against
fatigue. Nowadays, it has become well known. No matter in the research of the
analysis in compositions, or the assessment test in function, plenty of studies and
research have proved that it has great potential and development in this section.
Unfortunately, most research focus on the main species in Antrodia cinnamomea(red
type), and leave out the white type.
In this study, we’ve identified DR0101 is a type of the Antrodia cinnamomea. After
that, we proceed to do dish culture, submerged fermentation and solid fermentation,
try finding out its growing feature and comparing with the BCRC35396.
After succeeding in taming DR0101, we utilize ITS gene sequence and 5.8 rRNA
gene sequence to compare with PubMed DNA Database, it shows that DR0101 is
certainly a type of Antrodia cinnamomea. Besides, we’ve found in MY dish that the
growth of diameter and the mycelia biomass in DR0101 are both higher than BCRC
35396. But there is no difference in the optimized cultural temperature; also DR0101
is less susceptible in the concentration of agar. We’ve also noticed adding a proper
amount of animal or plant peptone will increase the mycelia biomass but decrease the
growth of mycelium. Furthermore, add the essential oil from three kinds of camphor
trees will accelerate the growth in mycelium, and to raise the content in polarity of
component.
In the experiment of submerged fermentation, the mycelial biomass, intracellular
polysaccharide and exopolysaccharide in DR0101 are higher than BCRC35396; but
in HPLC that the Triterpenoids in DR0101 are less than BCRC35396. While in 20
days in culture DR0101, we’ve found it has the highest mycelial biomass in the sixteenth days; and the highest exopolysaccharide is in the fourteenth days.
The result of solid state fermentation experiments, the better cereal medium are
sorghum, shelled Job's tears and germ rice. The analysis of Triterpenoids in HPLC are
difference between soy beans, sorghum, oatmeal, black soy beans, Job’s tears, and
germ rice. More, the smell of ferment in BCRC35396 and DR0101 are different. And
in HPLC, the Triterpenoids are different in BCRC36396 and DR0101 which grow on
sorghum medium.
摘要·······························································································································I
ABSTRACT··················································································································II
誌謝······························································································································IV
目次·······························································································································V
表目錄···························································································································X
圖目錄··························································································································XI
第一章 緒論··················································································································1
1.1 前言··················································································································1
1.2 研究動機和實驗目的······················································································2
第二章 文獻回顧··········································································································3
2.1 牛樟芝的介紹·································································································3
2.1.1 樟芝的起源··························································································3
2.1.2 樟芝的特徵··························································································4
2.1.3 高等真菌在生物學上分類的特徵······················································4
2.1.4 樟芝的生理活性成分··········································································7
2.2 樟芝多醣體·····································································································7
2.2.1 多醣體的研究······················································································7
2.2.2 真菌多醣的結構分類··········································································7
2.2.3 樟芝多醣結構······················································································8
2.3 三帖類·············································································································9
第三章 材料和實驗方法····························································································10
3.1 實驗材料與實驗儀器···················································································10
3.1.1 實驗材料····························································································10
3.1.2 實驗設備····························································································12
VI
3.2 實驗大綱·······································································································13
3.3 實驗方法·······································································································14
3.3.1 菌種馴化····························································································14
3.3.1.1 培養基配製···········································································14
3.3.1.2 菌種分離···············································································14
3.3.1.3 繼代培養···············································································15
3.3.1.4 菌種保存···············································································15
3.3.2 白樟芝的菌種鑑定············································································16
3.3.2.1 單孢分離法···········································································16
3.3.2.2 傳統型態鑑定·······································································17
3.3.2.3 分子生物基因鑑定·······························································19
3.3.3 不同的培養方式與條件····································································21
3.3.3.1 平盤培養···············································································21
(a)利用MY 培養基培養BCRC 35396 與DR0101 平盤
菌絲觀測其生長差異······················································21
(b)利用六種不同培養基培養DR0101 平盤菌絲觀測其
生長差異··········································································21
(c)利用三種不同培養溫度培養DR0101 平盤菌體觀測
其生長差異······································································22
(d)利用不同濃度的agar(瓊脂)培養DR0101 平盤菌
絲體觀測其生長差異······················································22
(e)添加不同濃度與來源的peptone 培養DR0101 平盤
菌體觀測其生長差異······················································22
(f)添加不同種類與濃度的樟樹精油培養DR0101 平盤
菌絲觀測其生長與成份差異··········································23
3.3.3.2 液態搖瓶培養·······································································23
(a)利用MY 液態培養基培養BCRC 35396 與DR0101
菌絲體觀測其生長與成份差異······································23
(b)利用MY 液態培養基培養DR0101 菌絲體觀測其生
長與發酵液多醣體變化··················································23
3.3.3.3 固態培養···············································································24
(a)穀物栽培一:利用10種穀物作為培養基固態發酵
DR0101並觀測生長與成份············································24
(b)穀物栽培二:利用脫殼薏仁、高粱、胚芽米作為培
養基固態發酵BCRC 35396與DR0101觀測生長與成
份上的差異······································································24
3.3.4 生長測定····························································································25
3.3.4.1 平盤培養生長測定·······························································25
3.3.4.2 平盤培養菌絲乾重測定 ·····················································25
3.3.4.3 液態搖瓶培養的菌絲乾重測定···········································25
3.3.5 成分分析····························································································26
3.3.5.1 大分子聚合物(多醣)分析····················································27
3.3.5.2 HPLC 樣品萃取液的製備·····················································29
3.3.5.3 HPLC 定性分析·····································································29
第四章 結果與討論····································································································31
4.1 菌種馴化·······································································································31
4.1.1 菌種分離····························································································31
4.1.2 單孢分離····························································································33
4.2 白樟芝的菌種鑑定·······················································································34
4.2.1 傳統型態鑑定····················································································34
4.2.2 分子生物基因鑑定············································································36
4.3 平盤培養·······································································································39
(a)利用MY 培養基培養BCRC 35396 與DR0101 平盤菌絲觀測其生
長差異······································································································39
(b)利用六種不同培養基培養DR0101 平盤菌絲觀測其生長差異···········41
(c)利用三種不同培養溫度培養DR0101 平盤菌體觀測其生長差異·······43
(d)利用不同濃度的agar(瓊脂)培養DR0101 菌絲體觀測其生長差異·45
(e)添加不同濃度與來源的peptone 培養DR0101 菌絲觀測其生長差異·47
添加動物性peptone················································································47
添加植物性peptone················································································51
綜合比較動物性peptone 與植物性peptone·········································55
(f)添加不同種類與濃度的樟樹精油培養DR0101 平盤菌絲觀測其生
長與成份差異···························································································56
添加牛樟樹精油························································································56
添加芳樟樹精油························································································61
添加本樟樹精油························································································65
綜合比較牛樟樹精油、芳樟樹精油、本樟樹精油································70
4.4 搖瓶培養·······································································································71
(a)利用MY 液態培養基培養BCRC 35396 與DR0101 菌絲體觀測其
生長與成份差異·······················································································71
(b)利用MY 液態培養基培養DR0101 菌絲體觀測其生長與發酵液多
醣體變化···································································································77
各數值的綜合比較····················································································78
4.5 固態培養·······································································································86
(a)穀物栽培一:利用10種穀物作為培養基固態發酵DR0101並觀測生
長與成份············································································86
10 種穀物的綜合比較···················································································89
(b)穀物栽培二:利用脫殼薏仁、高粱、胚芽米作為培養基固態發酵
BCRC 35396與DR0101並觀測生長與成份上的差異··104
第五章 結論··············································································································108
第六章 參考文獻······································································································111
1. 高曉薇”台灣靈芝屬新種樟芝之三帖類成分研究”台灣醫學院 天然物醫學系,
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