蔣丙煌臺灣大學：食品科技研究所劉景仁Liu, Ching-JenChing-JenLiu2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/56270樟芝(Antrodia cinnamomea)是台灣特有的食藥用真菌，因具有良好之抗腫瘤及護肝能力而聞名。藉由誘發劑(elicitor)的添加可使得真菌產生防禦反應(defense response)，所以可有效地提升樟芝發酵液中二次代謝物（三萜類）之產量。本研究針對樟芝深層培養系統，選擇幾丁聚醣、氯化鈣及組合法(幾丁聚醣＋氯化鈣)等誘發劑，探討其對三萜類生產的影響。由研究結果得知，雙網狀導管氣舉式發酵槽與第八天饋料批式發酵分別可獲得菌絲體濃度高達15.8與12.9 g/L。另外，在誘發劑實驗得知，以100 mg/L幾丁聚醣處理樟芝發酵液，不但菌體生長狀況較良好之外，尚可獲得最高總三萜類產量(1144 mg/L)。然而，因組合法嚴重影響樟芝之細胞膜通透性，使得其細胞漸失活性，故不利於長期培養，但可使樟芝在短時間即釋放出胞內三萜類。另外，結果顯示氯化鈣處理則不適合短期應用於誘發三萜類之合成。於兩階段培養中，研究證實利用氧氣限制與溫度波動策略將顯著提升三萜類產量，例如溫差靜置培養組得到的三萜類產量(1615 mg/L)顯著優於對照組。於癌細胞毒殺試驗中，經實驗結果顯示，溫差靜置培養組別所萃取的粗三萜具有良好的抑制子宮頸癌能力，其IC50約為28 μg/ml，且對於人類皮膚細胞(WS1)之生長不會有影響。Antrodia cinnamomea was a popular folk medicine that has attracted great attention due to its antitumor activity. In the last decade, the triterpenoids were isolated from A. cinnamomea and their biological activities such as anticancer and hepatoprotection have been reported. Elicitors have been effective in inducing the synthesis of secondary metabolites (e.g., triterpenoids) in submerged cultivation of A. cinnamomea. A defense response was induced in the culture when elicitors were added. Elicitors can enhance the secondary metabolites in fungal cells. The objective of this study was to investigate the effects of different elicitors (chitosan, CaCl2 and combination (chitosan+CaCl2)) on the triterpenoids production in submerged cultivation system of A. cinnamomea. It was found that the maximum biomass reached 15.8 g/L and 12.9 g/L in airlift bioreactor with dual-net draft tube and fed-batch (day 8) cultures, respectively. The elicitors study showed that treatment of chitosan (100 mg/L) not only obtained more biomass than combination treatment, but also achieved the highest total triterpenoids production. However, combination seriously affected the permeability of cell membrane and resulted in the loss of cell viability. Although the use of combination was not appropriate for long-term cultivation, it did enhance the fungus to accumulate the intracellular triterpenoids in a short-term cultivation. In addition, feeding CaCl2 had little effect on accumulation of triterpenoids for short time cultivation, so it was suitable for long-term fermentation. For two-stage culture strategies, the results proved that triterpenoids production could be highly enhanced by means of the control of oxygen limitation and temperature-shift strategy. For instance, the maximum triterpenoids production (1615 mg/L) was observed at static culture with temperature fluctuation (TF-S group), which was significantly higher than the control. In vitro anticancer test, the results indicated that the crude triterpenoids from TF-S group at concentration of 28 μg/ml inhibited 50% cell viability of HeLa cells, while exhibiting under 100 μg/ml no significant cytotoxicity to WS1 (normal cells).頁碼 中文摘要………………………………………………………………I 英文摘要……………………………………………………………II 目錄………………………………………………………………III 表次………………………………………………………………VII 圖次………………………………………………………………VIII 第一章、緒論……………………………………………………1 1.1 研究動機…………………………………………………1 1.2研究目的………………………………………………2 1.3實驗架構………………………………………………2 第二章、文獻回顧……………………………………………6 2.1樟芝簡介………………………………………………6 2.1.1名稱變革……………………………………………6 2.1.2型態特徵……………………………………………7 2.1.3產地…………………………………………………9 2.2生理活性物質與機能性研究………………………………9 2.2.1生物活性成分………………………………………10 2.2.1.1三萜類……………………………………10 2.2.1.2多醣體……………………………………16 2.2.2生理機能性研究………………………………17 2.2.2.1抗氧化功能………………………………17 2.2.2.2提升免疫及抗腫瘤功能…………………18 2.2.2.3護肝功能…………………………………19 2.2.2.4預防心血管疾病及降血脂功能…………20 2.3工業液態發酵……………………………………………20 2.4真菌細胞培養高代謝物的操作策略……………………22 2.4.1饋料批式培養………………………………………23 2.4.2兩階段培養…………………………………………23 2.4.3固定化培養…………………………………………24 2.5控制菌絲體形成與形態大小變化的因子………………26 2.5.1 物理因素……………………………………22 2.5.1.1 溫度………………………………26 2.5.1.2 通氣量…………………………………26 2.5.1.3 光照……………………………………27 2.5.1.4剪切力或攪拌……………………28 2.5.1.5 接菌量…………………………………28 2.5.1.6不同形式發酵槽………………………28 2.5.2 化學因素…………………………………………29 2.5.2.1 培養基組成……………………29 2.5.2.2 pH值…………………………………29 2.5.2.3 誘發劑…………………………………30 第三章、材料與方法………………………………………33 3.1 實驗材料…………………………………………………33 3.1.1 實驗菌腫…………………………………………33 3.1.2 實驗藥品…………………………………………33 3.1.3 實驗器材與儀器………………………………34 3.2 實驗方法…………………………………………………36 3.2.1 菌株保存與活化…………………………………36 3.2.2 培養基組成………………………………………36 3.2.3 操作條件…………………………………………38 3.2.4 分析方法…………………………………………38 3.3 樟芝粗三萜之生物活性測定…………………………41 3.3.1 細胞株……………………………………………41 3.3.2 細胞株保存………………………………………41 3.3.3 細胞株解凍………………………………………41 3.3.4 細胞株繼代培養…………………………………41 3.3.5 細胞株培養液組成………………………………42 3.3.6 動物細胞實驗流程………………………………43 3.4 統計分析………………………………………………43 第四章、結果與討論………………………………………44 4.1第一部分……………………………………………44 4.1.1批式與饋料批式對菌絲體之影響………………44 4.1.2批式與饋料批式對溶氧量(DO)之影……………47 4.1.3批式與饋料批式對pH值之影響………………49 4.1.4批式與饋料批式對總多醣之影響………………50 4.1.5雙網狀與實壁導管對菌絲體之影響……………51 4.1.6雙網狀與實壁導管對溶氧量(DO)之影響………53 4.1.7雙網狀與實壁導管對pH值之影響……………55 4.1.8雙網狀與實壁導管對總多醣之影響……………56 4.2第二部分…………………………………………58 4.2.1幾丁聚醣對菌絲體之影響……………………58 4.2.2幾丁聚醣對胞外三萜類之影響………………60 4.2.3幾丁聚醣對胞內三萜類之影響………………62 4.2.4氯化鈣對菌絲體之影響……………………64 4.2.5氯化鈣對胞外三萜類之影響………………66 4.2.6氯化鈣對胞內三萜類之影響………………67 4.2.7組合使用對菌絲體之影響…………………69 4.2.8組合使用對胞外三萜類之影………………71 4.2.9組合使用對胞內三萜類之影響……………73 4.3第三部分……………………………………………77 4.3.1兩階段培養對樟芝菌絲體之影響…………77 4.3.2兩階段培養對樟芝發酵液pH之影響……………79 4.3.3兩階段培養對總多醣之影響……………………81 4.3.4兩階段培養對胞外三萜之影響…………………83 4.3.5兩階段培養對胞內三萜之影響…………………85 4.4第四部分……………………………………………92 4.4.1粗三萜之治療指數……………………………92 4.4.2胞外三萜對子宮頸癌之影響…………………94 4.4.3胞內三萜對子宮頸癌之影響………………96 4.4.4胞內外三萜類與IC50之消長關係…………98 第五章、結論與建議………………………………………100 5.1結論…………………………………………………100 5.2建議…………………………………………………101 第六章、參考文獻…………………………………………1023049305 bytesapplication/pdfen-US樟芝誘發劑兩階段培養三萜類抗癌作用Antrodia cinnamomeaElicitorsTwo-stage cultureTriterpenoidsAntitumor activity[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56270/1/ntu-95-R93641009-1.pdf