臺灣博碩士論文加值系統

中文摘要

背景：牛樟芝（Antrodia cinnamomea）為台灣特有且珍貴的藥用真菌，屬於多孔菌科（Polyporaceae）薄孔菌屬（Antrodia），原生長於牛樟木（Cinnamomum kanehirai Hay）樹幹腐朽心材的內壁。牛樟芝有超過78種的化合物，其最具有生物活性的化合物為多醣體和三萜類，具有抗氧化、抗腫瘤和免疫調節活性。在野外天然牛樟芝的生長速率非常緩慢，而牛樟木的數量稀少，加上濫墾濫伐和隨意盜採，導致野外越來越難找到牛樟芝的子實體。目前使用液態培養獲得菌絲體有效物質，供應保健食品和功能性食品使用；相關研究指出可加入牛樟木的樹皮萃取物或木屑有利於牛樟芝菌絲的生長。丹參（Salvia miltiorrhiza Bunge）對於抗腫瘤、抗菌、冠狀動脈阻塞性心臟病（冠心病）、特別是心血管方面如心絞痛和心肌梗塞有良好的治療功用，具有有效的抗氧化能力，對心血管疾病之預防有很大的益處，可作為醫食同源的成分之一。本實驗目的分成兩部分，其一是探討添加牛樟木屑和不同培養條件對牛樟芝菌株間的生長影響及菌絲體中化合物成分之變化；其二為添加牛樟木和丹參根萃取物對牛樟芝的生長影響及化合物成分之變化。
第一部分：添加牛樟木屑和不同培養條件對牛樟芝菌株間的生長影響及菌絲體中化合物成分之變化
材料與方法：試驗菌株為三株標準菌株BCRC37848、BCRC37849和BCRC37850及兩株野生株Chu3PP和Chu1030615。以固態培養的方式，在溫度22℃和28℃、1%和3%牛樟木屑、MEA和PDA培養基中培養，分析生長半徑、菌絲體乾重、含水率、顏色變化和菌絲體中的化合物的變化。結果：(1) 牛樟芝培養的最適合的溫度為28℃，最培養基為MEA，添加1%和3%比例的牛樟木屑皆可以促進牛樟芝的菌絲體生長，以3%牛樟木屑最能有效促進生長。菌株間的生長速度以Chu1030615最快，其次依序為BCRC37849、Chu3PP、BCRC37850和BCRC37848。(2) 在HPLC分析中，不同成分的培養基和添加牛樟木屑會影響菌絲體中化合物的生成，五株牛樟芝皆可生成6種已知化合物，以菌株Chu1030615培養在MEA培養基中生成的種類與含量較高；菌株BCRC37848、BCRC37849、BCRC37850、Chu3PP和Chu1030615培養在MEA培養基的菌絲體，分別可促進產生4、8、1、5種和無產生未知化合物；培養在PDA培養基的菌絲體，分別可促進產生4、2、6、2和2種未知化合物。結論：培養基成分是影響牛樟芝生長的因素之一，添加牛樟木屑可促進牛樟芝生長，以菌株Chu1030615生長最快；培養基的成分和添加牛樟木屑可以促進菌株BCRC37849產生較多的化合物。
第二部分：添加牛樟和丹參萃取物對牛樟芝的生長影響及化合物成分之變化
材料與方法：試驗菌株為標準菌株BCRC37849、兩株野生株Chu3PP和Chu1030615。以液態培養的方式，分別添加牛樟和丹參萃取物培養，分析菌絲體乾重、pH值和菌絲體中的化合物成分的變化。結果：(1) 牛樟芝添加牛樟和丹參萃取物培養中，以添加30 ppm牛樟萃取物有效地促進菌絲體的生長，添加丹參萃取物會抑制菌絲體的生長，以添加牛樟萃取物為最適合促進牛樟芝菌絲體生長的添加物。生長速度以菌株Chu3PP為最快，依序為菌株BCRC37849和Chu1030615。(2) 在HPLC分析中，菌株BCRC37849、Chu3PP和Chu1030615添加牛樟萃取物分別促進7、3和7種未知化合物的產生；添加丹參萃取物分別促進8、5和6種未知化合物的產生。菌株BCRC37849和Chu3PP添加牛樟萃取物培養，菌絲體會分別分泌3種未知化合物至培養液；菌株BCRC37849添加丹參萃取物培養菌絲體會分泌6種為未知化合物至培養液。結論：由於菌株不同代謝途徑也不同，添加牛樟萃取物可以促進菌絲體生長，但會促進或抑制化合物的產生，也會調控化合物存在於菌絲體或是培養液中，因此牛樟芝可以作為生物轉換的工具之一，添加物質誘導產生新的化合物。

Abstract

Background: Antrodia cinnamomea (synonyms A. camphorata, Taiwanofungus camphoratus) is a medicinal fungus, only native to Taiwan and belongs to Polyporaceae. A. cinnamomea is found exclusively on the inner wall of the endemic species Cinnamonum kanehirai Hay. Over 78 compounds found in this species, include terpenoids, benzenoids, lignans, benzoquinone derivatives, succinic and maleic derivatives with biological activity. The triterpenoids in A. cinnamomea are considered to be potential anticancer agents due to their activity against growing tumors. C. kanehirai Hay is becoming scarce leading to difficulty in finding the basidiomes of A. cinnamomea in the field. The addition of bark extract or wood chips of C. kanehirai Hay is favorable for the growth of A. cinnamomea in mycelial cultures. Salvia miltiorrhiza Bunge contained a large number of lipophilic diterpenoids (such as various tanshinone analogues), hydrophilic phenolic compounds (such as salvianolic acids), flavonoids, and triterpenoids.
Purpose: The aims of this study were to determine the effects of wood chips and different culture conditions on the growth and the synthesis of main components of A. cinnamomea in solid culture and the effects of crude extracts of C. kanehirai Hay and S. miltiorrhiza Bunge on the growth and the synthesis of other new compounds of A. cinnamomea in liquid culture.

1. The effects of wood chips and different culture conditions on the growth and the synthesis of main components of A. cinnamomea.
Materials and Methods: The test strains were three standard strains BCRC37848, BCRC37849 and BCRC37850 and two wild strains Chu3PP and Chu1030615. The culture conditions included temperature at 22℃ and 28℃, different proportions of 1% and 3% wood chips and MEA and PDA media, The growth rate, mycelium dry weight, moisture content, color change and compounds changes in solid cultures were analyzed.
Results: (1) The best culture conditions for the A. cinnamomea growth 28℃ and MEA. Addition of 1% and 3% of the wood chips could promote the growth of the mycelium of A. cinnamomea, with 3% wood chips as the most effective growth. The strain Chu1030615 showed the best growth rate, followed by BCRC37849, Chu3PP, BCRC37850 and BCRC37848. (2) In the HPLC analysis, the addition of wood chips to the medium could change the compound synthesis. Five A. cinnamomea strains synthesized six known compounds, and strains of Chu1030615 produced the more types and higher contents on MEA medium. The addition of wood chips to BCRC37848, BCRC37849, BCRC37850、Chu3PP and Chu1030615 were cultured in the mycelium of MEA medium, which increased the production of 4, 8, 1, 5 and 0 compounds, respectively; the mycelium cultured in the PDA medium increased the production of 4, 2, 6, 2 and 2 compounds, respectively.
Conclusions: The composition of the medium was a main factor to affect the growth of A. cinnamomea. Addition of wood chips increased the growth of A. cinnamomea, and the strain Chu1030615 appealed the best growth. The mycelium produced more compounds due to the medium components and the addition of wood chips stimulated the growth of strains BCRC37849.

2. The effects of crude extracts of C. kanehirai Hay and S. miltiorrhiza Bge on the growth and the synthesis of other new compounds of A. cinnamomea.
Materials and Methods: The test strains included strain BCRC37849 and two wild strains Chu3PP and Chu1030615. The extracts of C. kanehirai Hay and S. miltiorrhiza Bunge were added in the culture and the mycelium dry weight, moisture content, the pH value and compounds changes were analyzed in liquid cultures.
Results: (1) The addition of 30 ppm C. kanehirai Hay extracts stimulated much faster A. cinnamomea growth than the addition of S. miltiorrhiza Bunge extracts. The addition of S. miltiorrhiza Bunge extract inhibited the mycelial growth in the initial and middle growth of A. cinnamomea. Strain Chu3PP revealed the fastest growth rate after adding extract, followed by the strains BCRC37849 and Chu1030615. (2) HPLC analysis demonstrated that the addition of C. kanehirai Hay extracts to BCRC37849, Chu3PP and Chu1030615 increased the production of 7, 3, and 7 compounds, respectively. The addition of S. miltiorrhiza Bunge extracts increased the synthesis of 8, 5, and 6 compounds, respectively.
Conclusions: The strains of A. cinnamomea have different metabolic pathways. The addition of C. kanehirai Hay extract can promote the growth of mycelium, but it also promotes or inhibits the production of compounds. It also regulates the presence of compounds in mycelium or culture medium. Because the addition of substances induces the production of new compounds, A. cinnamomea can be a tools for biotransformation.

目錄
中文摘要 ................................. I
英文摘要 ................................ III
目錄…………………………………………………………………….....…………..…VI
圖目錄……………………………………………………………….……………....…….X
表目錄………………………………………………………………………….…....….XII
附圖目錄……………………………………………..…………………………..…..XIV
附表目錄…………………………………………….…………………………....….XVII
第一章 文獻回顧 .............................1
第一節 牛樟芝簡介 ...........................1
1-1 牛樟芝之特性……………………………..…………………..…..1
1-2 牛樟芝命名…………………………………..……………………………….1
1-3 牛樟芝的功效和有效成分…………..…………………………….3
第二節 牛樟芝的人工培養方式………………………………………………….…5
2-1 固態培養…………………………………………..……………………….…5
2-2 液態培養………………………………………..…………………………….6
2-3 椴木培養………………………………….…….…………………………….7
2-4 培養基成份的影響……………………………..………………….…………7
第三節 牛樟樹簡介…………………………………………………………….……8
3-1 牛樟樹的型態和特性……………………….....…………………..8
第四節 丹參簡介……………………………………...……………..………………9
4-1 丹參的型態和特性………………………………………………………...…9
4-2 丹參的功效………………………………………………..…………….…..10
4-3 丹參的活性成分………………………………………………..…….……..11
第五節 研究目的與動機………………………………………………..………….11
第六節 參考資料……………………………………………………….…………..12
第二章 添加牛樟木屑和不同培養條件對牛樟芝菌株間的生長影響及菌絲體中化合物之變化……………………………………..…….………………………15
第一節 前言………………………………………………………………...………15
第二節 材料與方法 ………………………………………………………….……16
2-1 實驗流程……………………………………………..……………...………16
2-2 菌株來源……………………………………………..…………………...…16
2-3 培養基的配製………………………………………..…………………...…16
2-4 菌株活化………………………………………………………..………..….17
2-5 菌株鑑定………………………………………………………..….………..17
2-5.1 核酸萃取…………………………………………..……………...……17
2-5.2 聚合酶鏈鎖反應（PCR）…………...………………………….……..18
2-5.3 PCR產物純化……………………………………...…………………..19
2-5.4 基因定序……………………………………………....……………….19
2-6 牛樟木屑處理……………………………………..…………………..…….19
2-7 菌株生長分析…………………………………………………..……….…..19
2-8 菌絲體成分分析……………………………………………...…………..…21
2-8.1 HPLC之分析…………………………..……………………………....21
2-9 統計分析…………………………………..……………………………...…22
第三節 結果 ……………………………………...………………………...……..22
3-1 牛樟芝的菌株鑑定……………………………….…….…………..……….22
3-2 菌株生長影響因子………………………………………………………….23
3-3 菌絲體乾重影響因子……………………………………………………….29
3-4 培養條件對菌絲體顏色之影響……………………………….……………35
3-5 菌絲體之成分分析………………………………………………….………41
3-5.1 標準品………………………………………………………………….41
3-5.2 標準品檢量線……………………………………………….…………43
3-5.3 牛樟芝成分之HPLC分析…………………………….………………43
3-5.3.1 無添加牛樟木屑培養的菌絲體中已知化合物之含量比較…….43
3-5.3.2 添加牛樟木屑培養的菌絲體中已知化合物之含量比較……….45
3-5.3.3 添加牛樟木屑培養的菌絲體中未知化合物之種類比較…….…48
第四節 討論....……………………………………………………...……….……..56
第五節 結論………………………………………………………………...…..….57
第六節 參考資料………………………………………………………...….……..58
第三章 添加牛樟木與丹參萃取物對牛樟芝的生長影響及化合物成分之變化…...60
第一節 前言……………………………………………..………………………….60
第二節 材料與方法 ……………………………………………………………….61
2-1 實驗流程………………………………………………..………………...…61
2-2 菌株來源………………………………………………..…………..……….61
2-3 培養基之配製…………………………………………..………….………..61
2-4 菌株活化…………………………………………………..…………….…..61
2-5 添加物之萃取…………………………………………..…………….……..62
2-5.1 牛樟木之萃取………………………………..………………………...62
2-5.2 丹參之萃取……………………………..…………………………...…62
2-6 菌株接種………………………………………………………………...…..62
2-7 觀察與紀錄……………………………………………………….…………62
2-8 HPLC之分析………………………………………………………………..63
2-8.1 萃取物的成分分析………………………………………………...…..63
2-8.2 菌絲體的成分含量分析………………………..…………………..….63
2-9 統計分析…………………………………………………………………….63
第三節 結果 …………………………………………………………...…………..64
3-1 添加物的萃取………………………………………..………………….…..64
3-2 添加不同萃取物對菌株液態培養的生長之差異……….…….64
3-2.1 菌絲體型態……………………………………………..……………...65
3-2.2 萃取物的濃度菌絲體乾重之影響……………………..…..………….67
3-2.3 萃取物對菌絲體乾重之影響………………...…………………..……69
3-2.3.1 萃取物的種類影響………………………………………….……70
3-2.3.2 菌株間之差異…………………………………………………….74
3-3 HPLC 的分析…………………………………….…………………..……..77
3-3.1 標準品………………………………………...………………………..77
3-3.2 標準品檢量線………………………………………………………….77
3-3.3 牛樟和丹參萃取物成分之HPLC分析…………….………………....77
3-3.4 不同天數菌絲體成分之HPLC分析………………………….………78
3-3.4.1 已知化合物之含量比較………………………………………….78
3-3.4.2 未知化合物之種類比較…………………………………….……81
3-3.5 菌絲體和培養液成分之HPLC分析………………………………….86
3-3.5.1 已知化合物之含量比較…………………………………...……..86
3-3.5.2 未知化合物之種類比較………………………………………….88
第四節 討論…………………………………………….………………………….92
第五節 結論…………………………………………….………………………….94
第六節 參考資料……………………………………….………………………….95
附圖…………………………………………………………………………………97
附表……………………………………………………………………………..…133

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