臺灣博碩士論文加值系統

樟芝已廣泛應用在台灣傳統醫藥，主要有效成份來自於大分子的多醣體、小分子的三萜類化合物、半萜內酯化合物及固醇類，樟芝的生理活性成分及功能仍是目前研究重點。除多醣體外，近年來最受重視的生理活性物質就是三萜類。本研究目的為評估樟芝深層培養添加柑橘類果皮精油及樟芝平板式固態培養添加柑橘類果皮粉末提高生理活性代謝產物的可行性。
兩階段溶氧培養(在第21天靜置)對樟芝菌絲體三萜類代謝有良好的促進功效，在培養28天後三萜類含量達22.30 mg/g DW，為控制組(10.48 mg/g DW)的2.13倍。橘子果皮精油添加對樟芝菌絲體形成生理活性代謝產物促進效果最佳，在第7天添加橘子果皮精油4% (v/v)，三萜類含量和產量達到最高值，為117.17 mg/g DW和1417.77 mg/L，分別為控制組的11倍及14倍。檸檬烯為柑橘類果皮精油促進樟芝菌絲體生成三萜類的重要成分之ㄧ，檸檬烯進入細胞的質體內，抑制MCC路徑的單萜類生成，改由MVA路徑合成三萜類。
蕎麥平板式固態培養，菌絲體只能在基質表面上成長，因此取樣時可以將菌絲體與基質分離，完整分析菌絲體生理活性。葡萄柚果皮粉末添加能有效提升生理活性代謝物產量，當添加4 g/plate葡萄柚果皮粉末時，第30天三萜類的含量與產量分別為47.10 mg/g DW和166.68 mg/plate，為控制組(9.66 mg/g DW和32.26 mg /plate)的4.88倍與5.17倍。此外由HPLC圖譜中，發現經添加葡萄柚果皮粉末平板式固態培養的樟芝，含有代表子實體形成有關的5種麥角甾烷三萜類(ergostanes: antcins C and K, and zhankuic acids A, B, and C)，因此可以證明，此種培養在短時間(30天)所得到的三萜類化合物與野生樟芝子實體成份大致相同，換句話說，此培養得到的應該為樟芝子實體。

Basidiomes of Antrodia cinnamomea are used as a traditional medicine in Taiwan. Bioactive compounds found in A. cinnamomea include: polysaccharide, triterpenoids, sesquiterpene lactone, and steroids. The bioactivity and efficacy of A. cinnamomea are still the main focus in many researches. In addition to polysaccharide, triterpenoids have recently been considered as the most biologically active components. The aim of this study was to evaluate the feasibility of adding citrus peel extract in the submerged culture or adding citrus peel powder on the plate solid state culture of A. cinnamomea to enhance the formation of bioactive metabolites.
A two-stage fermentation process was proposed to enhance triterpenoids content by combining the conventional shake-flask fermentation (on the 21th day) with static culture (on the 7th day). The content of triterpenoids rose from 10.48 mg/g DW of the control to 22.30 mg/g DW on the 28th day. Adding tangerine peel extract was the most effective way to enhance bioactive metabolite production. With an addition of 4% (v/v) on the seventh day, the content and production of triterpenoids were 117.17 mg/g DW and 1417.77 mg/L, which were 10-fold and 13-fold higher than the control, respectively, on the 28th day. Limonene was an important ingredient of citrus peel extract to enhance the formation of triterpenoids in the submerged culture of A. cinnamomea. When limonene entered the mitochondria, MCC pathway was inhibited which synthesizes monoterpenes in the mitochondria. Thus MVA pathway was then enhanced to synthesize triterpenes in the cytoplasm.
Mycelia of the plate solid state culture only grew on the substrate surface so that it was easy to separate mycelia from the substrate surface. Grapefruit was the most effective element in enhancing bioactive metabolite production. With an addition of 4 g powder/plate, the content and production of triterpenoids rose from 9.66 mg/g DW and 32.26 mg/plate of the control to 47.10 mg/g DW and 166.68 mg/plate, respectively, on the 30th day. Moreover, this study also demonstrates that the plate solid state culture with an additional grapefruit powder of A. cinnamomea could contain the 5 ergostanes (antcins C and K, and zhankuic acids A, B, and C). The production of ergostanes is related to basidiomatal formation of A. cinnamomea. This result indicates that mycelia of this culture could be basidiomes of A. cinnamomea.

目錄
中文摘要 ...........................................I
Abstract ........................................III
謝誌 ..............................................V
目錄 ............................................VII
表目錄 .........................................XIII
圖目錄 ...........................................XV
第一章 緒論 ........................................1
1-1前言 ...........................................1
1-2樟芝的介紹 ......................................2
1-2-1樟芝之命名 ....................................2
1-2-2樟芝的分類地位 ................................3
1-3樟芝生理活性成分 ................................5
1-3-1多醣體 .......................................5
1-3-2三萜類 .......................................7
1-3-3總多酚 ......................................10
第二章 樟芝兩階段深層培養 ...........................13
2-1前言 ..........................................13
2-1-1物理因素 .....................................14
2-1-2化學因素 .....................................16
2-1-3深層培養樟芝菌絲體功效 .........................18
2-2實驗材料與方法 ..................................19
2-2-1實驗藥品 .....................................20
2-2-2實驗儀器與設備 ................................21
2-2-3實驗方法 .....................................22
2-2-3-1菌種斜面試管保存 ............................22
2-2-3-2培養皿平面培養 ..............................23
2-2-3-3種菌的製備 .................................23
2-2-3-4三角瓶液態基礎培養試驗 .......................23
2-2-3-5兩階段培養溶氧變化試驗 .......................24
2-2-3-6兩階段培養溫度變化培養試驗 ....................25
2-2-3-7兩階段培養pH變化培養試驗 ......................25
2-2-4分析方法 ......................................26
2-2-4-1菌體濃度 ....................................26
2-2-4-2 pH值測定 .................................. 26
2-2-4-3 澱粉分析方法 ................................26
2-2-4-4胞內總多酚含量測定 ............................27
2-2-4-5胞內多醣濃度測定 ..............................27
2-2-4-6胞內三萜類含量測定 ............................28
2-3實驗結果與討論 ....................................29
2-3-1 樟芝液態基礎培養試驗 ............................29
2-3-2樟芝液態兩階段培養試驗 ...........................31
2-4結論 .............................................36
第三章 柑橘類果皮精油添加之樟芝深層培養 .................39
3-1前言 .............................................39
3-1-1萜類合成機制 ....................................39
3-1-2柑橘類果皮精油 ..................................44
3-1-3樟芝深層培養產三萜類 .............................46
3-2實驗材料與方法 ....................................47
3-2-2實驗儀器與設備 ..................................48
3-2-3實驗方法 .......................................48
3-2-3-1果皮精油製作及品質管控 .........................49
3-2-3-2不同時間添加各種果皮精油培養試驗 ................49
3-2-3-3不同濃度橘子果皮精油添加培養試驗 ................50
3-2-4分析方法 .......................................50
3-3實驗結果與討論 ....................................51
3-3-1不同時間添加各種果皮精油培養試驗 ..................52
3-3-2不同濃度橘子果皮精油添加培養試驗 ..................58
3-3-3果皮萃取液添加培養發酵動力參數之比較 ...............62
3-3-4樟芝菌絲體深層培養與野生樟芝子實體三萜類HPLC圖比較 ..64
3-4結論 .............................................66
第四章 單萜類添加之樟芝深層培養 ........................67
4-1前言 .............................................67
4-1-1乙醇效應 .......................................67
4-1-2柑橘類果皮單萜類簡介 ............................69
4-2實驗材料與方法 ....................................74
4-2-1實驗藥品 .......................................74
4-2-2實驗儀器與設備 ..................................74
4-2-3實驗方法 .......................................75
4-2-3-1不同時間添加乙醇液態培養 .......................75
4-2-3-2檸檬烯乙醇溶液與水溶液添加深層培養 ..............75
4-2-3-3不同單萜類添加深層培養 .........................76
4-2-3-4發酵液中檸檬烯濃度變化 .........................76
4-2-4分析方法 .......................................77
4-3實驗結果與討論 ....................................78
4-3-1檸檬烯乙醇溶液與水溶液添加深層培養 ................78
4-3-2不同單萜類添加液態培養 ...........................83
4-3-3發酵液中檸檬烯濃度變化 ...........................89
4-4結論 .............................................90
第五章 柑橘類果皮粉末添加之樟芝平板式固態培養 ............93
5-1前言 .............................................93
5-1-1固態培養之優缺點 .................................93
5-1-2固態發酵之應用 ...................................95
5-1-3柑橘類果皮性質與成份 ..............................95
5-1-4樟芝平板式固態培養 ................................96
5-2實驗材料與方法 ......................................99
5-2-1實驗藥品 .........................................99
5-2-2實驗儀器與設備 ...................................100
5-2-3實驗方法 ........................................100
5-2-3-1乾燥果皮粉末製作 ...............................101
5-2-3-2平板式固態培養基礎試驗 ..........................101
5-2-3-3添加不同果皮粉末平板式固態培養 ...................101
5-2-3-4添加不同重量葡萄柚果皮粉末平板式固態培養 ..........102
5-2-4分析方法 ........................................102
5-2-4-1菌體濃度 ......................................103
5-2-4-2 HPLC分析代謝產物三萜類含量 .....................103
5-3實驗結果與討論 .....................................104
5-3-1平板式固態培養基礎試驗 ............................105
5-3-2添加不同果皮粉末平板式固態培養 .....................107
5-3-3添加不同重量葡萄柚果皮粉末平板式固態培養 ............109
5-3-4果皮粉末添加平板式固態培養發酵動力參數 ..............112
5-3-5樟芝平板式固態培養與野生樟芝子實體三萜類HPLC圖比較 ...114
5-4結論 ..............................................115
第六章 結論與未來展望 ..................................117
6-1結論 ..............................................117
6-2未來展望 ...........................................118
參考文獻 ..............................................119
附錄 ..................................................133


表目錄
表1-1 樟芝子實體中所含三萜類Ergostane型化合物 .............9
表1-2 樟芝子實體中所含三萜類Lanostane型化合物 ............10
表2-1 靈芝液態培養產靈芝酸文獻整理 .......................16
表2-2 實驗藥品清單 .....................................20
表2-3 實驗儀器清單 .....................................21
表3-1 常見幾種柑橘皮精油之含量及主要成分...................45
表3-2 樟芝液態培養產三萜類文獻整理 .......................47
表3-3 新增實驗藥品清單 ..................................48
表3-4 新增實驗儀器清單 ..................................48
表3-5 不同果皮精油添加之樟芝液態培養動力學參數 .............63
表3-6 不同濃度橘子果皮精油添加之樟芝液態培養動力學參數 ......63
表3-7 樟芝深層培養三萜類主要滯留時間的HPLC圖譜面積比較 ......66
表4-1 常見幾種柑橘皮精油之含量及主要成分 ...................73
表4-2 新增實驗藥品清單 ...................................74
表4-3 新增實驗儀器清單 ...................................74
表5-1 柑橘類果皮物理性質 .................................96
表5-2 烘乾後果皮成份比例 .................................96
表5-3 新增實驗藥品清單 ..................................100
表5-4 新增實驗儀器清單 ..................................100
表5-5 不同果皮粉末添加之樟芝平板式固態培養動力學參數 ........113
表5-6 不同重量葡萄柚果皮粉末添加之樟芝平板式固態培養動力學參數 113

圖目錄
圖1-1 樟芝子實體 ..........................................3
圖1-2 樟芝平板培養 ........................................4
圖1-3 樟芝液態培養之菌絲球 .................................4
圖1-4 樟芝固態培養 ........................................4
圖2-1 樟芝菌絲體基礎培養生長曲線及生理活性產物 ...............30
圖2-2 不同天數靜置對樟芝菌絲體生長曲線及生理活性物質代謝影響 ...33
圖2-3 第21天改變溫度對菌絲體合成總多酚的影響 .................35
圖2-4 第21天改變溫度對菌絲體合成三萜類的影響 .................35
圖2-5 第21天改變pH值對菌絲體合成總多酚影響 .................. 37
圖2-6 第21天改變pH值對菌絲體合成三萜類影響 ...................37
圖3-1 萜類合成路徑 .........................................43
圖3-2 第7天添加2% (v/v)橘子精油樟芝菌絲體生長曲線及生理活性產物 .54
圖3-3 不同時間添加2% (v/v)橘子果皮精油對樟芝菌絲體代謝影響 .....55
圖3-4 各種果皮精油(2% (v/v)添加對樟芝菌絲體總多酚之比較 ........56
圖3-5 各種果皮精油(2% (v/v))添加對樟芝菌絲體三萜類之比較 .......57
圖3-6 橘子果皮精油添加量對樟芝菌絲體總多酚影響 .................60
圖3-7 橘子果皮精油添加量對樟芝菌絲體三萜類影響 .................61
圖3-8 樟芝深層培養三萜類化合物HPLC圖譜 .......................65
圖4-1 柑橘類果皮精油中常見單萜類的結構式 ......................70
圖4-2 微生物轉化檸檬烯途徑 ..................................72
圖4-3 不同時間添加乙醇對樟芝菌絲體代謝影響 ....................80
圖4-4 檸檬烯溶液添加對樟芝菌絲體合成總多酚影響 .................81
圖4-5 檸檬烯溶液添加對樟芝菌絲體合成三萜類影響 .................82
圖4-6 不同單萜類乙醇溶液添加對樟芝菌體濃度 .....................85
圖4-7 不同單萜類乙醇溶液添加對樟芝合成總多酚之影響 ..............86
圖4-8 不同單萜類乙醇溶液添加對樟芝合成三萜類之影響 ..............87
圖4-9 第7天添加單萜類乙醇溶液後發酵液之GC圖譜 ..................88
圖4-10 檸檬烯乙醇溶液添加之樟芝生長曲線、生理活性產物及檸檬烯含量 ..92
圖5-1 人工栽培子實體 ........................................98
圖5-2 平板式固態培養 .......................................104
圖5-3 樟芝平板式固態培養菌絲體生長曲線及生理活性物質代謝 ........106
圖5-4 不同果皮粉末(2 g)添加對樟芝平板式固態培養之影響 ..........108
圖5-5 不同葡萄柚果皮粉末添加量對樟芝菌絲體代謝影響 .............111
圖5-6 三萜類化合物HPLC圖譜 ..................................116

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