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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 蔣丙煌(Been-Huang Chiang) | |
dc.contributor.author | Yi-Jun Chen | en |
dc.contributor.author | 陳奕均 | zh_TW |
dc.date.accessioned | 2021-06-17T08:13:08Z | - |
dc.date.available | 2022-08-18 | |
dc.date.copyright | 2019-08-18 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73897 | - |
dc.description.abstract | 4-acetylantroquinonol B (4-AAQB) 及 Antroquinonol (AQ) 為牛樟芝菌絲體中最具代表性的兩個泛醌類成分,過去之研究顯示 4-AAQB 比AQ具有較強的抗肝癌效果,故本研究之目的為提升 4-AAQB 之產率。從生合成途徑來看 4-AAQB 及 AQ 生合成的分界點為六碳環的 farnesylation,4-AAQB 側會接上帶有 γ-lactone 的 FPPB,AQ 側則是接上 FPP,而本研究提出之假說為 FPP 經由 Mevalonate pathway 形成後,再經由 lactonization 形成 FPPB,因此,若能提高 FPP 轉化為 FPPB 的能力則能提升 4-AAQB 產率,並同時降低 AQ 產率。相關研究顯示 γ-lactone 的形成需要在酸性環境下進行。因此本研究嘗試以調酸來提高 4-AAQB 產率,並且透過添加 Farnesol 作為前驅物與調酸共同作用而達到加乘效果。結果顯示,在發酵第 14 天將發酵液調酸至 pH 2.75 可以顯著提升 4-AAQB 產率並降低 AQ 產率,且對於菌絲體的生長沒有負面影響。以 LC MS/MS 分析生合成途徑中間產物,結果顯示,調酸可以促進 5-DMQ0 形成 CoQ3B 以及 5-DMQ0 形成 CoQ3 的步驟,而從 CoQ3B 形成 4-AAQB以及 CoQ3 形成 AQ 的步驟則沒有受到影響。此外,調酸對於 4-AAQB 側以及 AQ 側六碳環的 farnesylation 都有提升的現象,藉此推斷調酸會增加上游 FPPB 的形成。另外,添加 0.1% farnesol 會同時提升 4-AAQB 側以及 AQ 側 farnesylated 的中間產物,但對於 AQ 側六碳環修飾步驟卻有降低的影響,特別是 CoQ3 形成 AQ大幅降低了95%,而添加 farnesol 對 4-AAQB 側影響較小,只有 AQB 形成 4-AAQB 的步驟略為降低。因此,添加farnesol最終導致 AQ 之產率大幅降低,而 4-AAQB 之產率卻沒有受到影響。由以上結果可以推論,FPPB 極有可能是由 FPP 經 lactonization 而來,因為添加 farnesol 後 4-AAQB 側以及 AQ 側 之 farnesylated 中間產物都大量提升了,而調酸確實可以促進 FPPB 的形成,後續若適當的結合調酸以及添加 farnesol,則有機會大幅提升 4-AAQB 之產率。 | zh_TW |
dc.description.abstract | 4-acetylantroquinonol B (4-AAQB) and Antroquinonol (AQ) are two of the most iconic ubiquinones from the mycelium of Antrodia cinnamomea (AC). 4-AAQB has been proven to be more effective in inhibiting the growth of liver cancer cells than AQ. Therefore, the objective of this study was to increase the yield of 4-AAQB during cultiva-tion of AC. The dividing step in the biosynthetic pathways of 4-AAQB and AQ is farne-sylation of benzoquinone rings. The benzoquinone rings are either farnesylated with far-nesylpyrophosphate (FPP) or FPPB, the suffix B represents γ-lactone modification at the end of FPP. The hypothesis of this study was that FPPB are formed by lactonization of FPP. Hence, we supposed that if the lactonization process could be accelerated, then the yield of 4-AAQB would be increased and the yield of AQ decreased. Since acidic condi-tion favors the formation of γ-lactone, the effect of pH adjustment of the culture medium on the yields of 4-AAQB and AQ were investigated, and the effect of addition of farnesol as a precursor to FPP and FPPB was also studied. We expected that combination of these two treatment would further increase the yield of 4-AAQB. UPLC quantitative analysis has shown that adjusting the pH of the broth to 2.75 at the 14th day during fermentation could significantly increase the yield of 4-AAQB and decrease the yield of AQ. Further analyses of the biosynthetic intermediates by LC MS/MS revealed that farnesylation and O-methylation of the intermediates for the biosynthesis of both 4-AAQB and AQ were promoted by pH adjustment, but ketone group reduction and acetylation of benzoquinone rings were not affected. This phenomenon could mean that the reaction of FPP lactonized to FPPB was promoted. LC MS/MS analyses also showed that addition of 0.1% farnesol could increase farnesylation steps for the synthesis of both 4-AAQB and AQ, but it strongly inhibited the benzoquinone rings modification on AQ side of biosynthetic path-way, especially the conversion of CoQ3 to AQ. On the other hand, addition of farnesol did not affect too much on 4-AAQB side of biosynthetic pathway. Consequently, addition of farnesol during fermentation significantly reduced the yield of AQ but did not affect the yield of 4-AAQB. The results of this study suggested that pH adjustment could promote lactonization of FPP to formed FPPB. It seems promising to find a proper way to adjust pH and add farnesol at the same time to further increase the yield of 4-AAQB. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:13:08Z (GMT). No. of bitstreams: 1 ntu-108-R06641026-1.pdf: 8900875 bytes, checksum: 5d46be17a7359e4052238575d7939f9a (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 摘要 i
Abstract ii 第一章、 前言 1 第二章、 文獻整理 2 第一節、 牛樟芝 2 1.1. 牛樟芝的特性 2 1.2. 牛樟芝的生理活性 3 1.2.1. 抗氧化 3 1.2.2. 抗發炎 3 1.2.3. 免疫調節 4 1.2.4. 護肝 4 1.2.5. 抗癌 4 1.2.6. 神經保護 5 1.3. 牛樟芝的功效性成分 5 1.3.1. 多醣類 5 1.3.2. 萜類 6 1.3.3. 苯類 6 1.3.4. 木質素 7 1.3.5. 琥珀酸及蘋果酸衍生物 8 1.3.6. 泛醌類 9 第二節、 牛樟芝的培養 10 2.1. 固態發酵 10 2.2. 液態發酵 10 2.3. 牛樟芝培養的影響因子 11 2.3.1. 溫度 11 2.3.2. pH 值 12 2.3.3. 通氣量 14 2.3.4. 攪拌 14 2.3.5. 碳源及氮源 14 2.3.6. 前驅物 16 第三節、 4-Acetylantroquinonol B 及 Antroquinonol 的生合成途徑 16 3.1. Polyketide pathway 19 3.2. Shikimate pathway 20 3.3. Mevalonate pathway 22 第四節、 γ-lactone 的生合成 24 4.1. γ-lactone 在環境中的生合成 24 4.2. γ-lactone 在微生物中的生合成 24 第五節、 Mevalonate pyrophosphate decarboxylase 25 5.1. Mevalonate pyrophosphate decarboxylase 的功能 25 5.2. Mevalonate diphosphate decarboxylase 受到環境因子的影響 26 5.3. Mevalonate diphosphate decarboxylase 在牛樟芝中的變化情形 27 第六節、 Isoprenoid chain 的修飾 28 6.1. Erythrolic acid 28 第三章、 實驗構想及架構 30 第一節、 實驗構想 30 第二節、 實驗架構及設計 32 第四章、 材料與方法 34 第一節、 實驗材料 34 1.1. 牛樟芝菌株 34 1.2. 實驗藥品 34 1.3. 實驗器材 34 第二節、 實驗方法 35 2.1. 牛樟芝培養方法 35 2.1.1. 牛樟芝平板培養 35 2.1.2. 牛樟芝菌種保存 36 2.1.3. 牛樟芝菌種活化 36 2.1.4. 牛樟芝液態培養 36 2.2. 牛樟芝發酵液還原糖濃度測定 37 2.2.1. DNS 試劑配製 37 2.2.2. 還原醣標準曲線溶液配製 37 2.2.3. 還原糖測定操作步驟 38 2.3. 牛樟芝搖瓶液態發酵 pH 值調整 38 2.4. 牛樟芝發酵產物分離萃取 38 2.4.1. 牛樟芝菌絲體與濾液分離 38 2.4.2. 牛樟芝菌絲體重量測定 38 2.4.3. 牛樟芝菌絲體乙醇萃取物 39 2.5. 牛樟芝菌絲體中 4-AAQB 及 AQ 之定量 39 2.5.1. UPLC 系統及分析條件 39 2.5.2. UPLC 分析之動相及流洗條件 40 2.6. 4-AAQB 及 AQ 生合成途徑中間產物分析 40 2.6.1. UPLC-HRMS 系統及分析條件 40 2.6.2. UPLC 動向及流洗條件 40 2.6.3. 熱電灑游離器之參數 41 2.6.4. Target-Selected Ion Monitoring (t-SIM) 之參數 41 2.6.5. Parallel Reaction Monitoring (PRM) parameters 之參數 42 2.6.6. 4-AAQB 及 AQ 生合成途徑中間產物資料 43 2.7. 統計分析方法 44 第五章、 結果與討論 45 第一節、 調酸之 pH 值及調酸之時間點篩選 45 1.1. 實驗結果 45 1.1.1. 調酸對於發酵期間 pH 值之影響 45 1.1.2. 調酸對於發酵期間還原糖濃度之影響 51 1.1.3. 調酸對於菌絲體生物質量之影響 53 1.1.4. 調酸對於 4-AAQB 產率之影響 55 1.1.5. 調酸對於 AQ 產率之影響 57 1.1.6. 調酸對於 4-AAQB 及 AQ 生合成途徑之影響 59 1.2. 結果討論 61 第二節、 Farnesol 添加濃度之篩選 65 2.1. 實驗結果 65 2.1.1. 添加 Farnesol 對於發酵期間 pH 值之影響 65 2.1.2. 添加 Farnesol 對於發酵期間還原糖濃度之影響 66 2.1.3. 添加 Farnesol 對於菌絲體生物質量之影響 70 2.1.4. 添加 Farnesol 對於 4-AAQB 產率之影響 71 2.1.5. 添加 Farnesol 對於 AQ 產率之影響 72 2.1.6. 添加 Farnesol 對於 4-AAQB 及 AQ 生合成途徑之影響 73 2.2. 結果討論 74 第六章、 總結論 77 第七章、 參考文獻 78 | |
dc.language.iso | zh-TW | |
dc.title | 利用添加異戊二烯鏈之前驅物與促進內酯化反應提升牛樟芝菌絲體 4-AAQB 之產率 | zh_TW |
dc.title | Increase the yield of 4-AAQB in the mycelium of Antro-dia cinnamomea with isoprenoid chain precursor and lac-tonization promotion | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳錦樹(Chin-Shuh Chen),林育蔚(Yu-Wei Lin),周繼中(Chi-Chung Chou) | |
dc.subject.keyword | 牛樟芝,4-AAQB,pH 值,farnesol,lactonization, | zh_TW |
dc.subject.keyword | Antrodia cinnamomea,4-AAQB,pH,farnesol,lactonization, | en |
dc.relation.page | 82 | |
dc.identifier.doi | 10.6342/NTU201903751 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-15 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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