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研究生:黃子寧
研究生(外文):Tzu-Ning Huang
論文名稱:樟芝發酵液揮發性成分變化與其抑制肝癌活性之關係
論文名稱(外文):Relationship between the volatile compounds profile and hepatoma inhibitory activity of the fermentation broth of Antrodia cinnamomea
指導教授:蔣丙煌
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:95
中文關鍵詞:樟芝揮發性化合物發酵肝癌細胞微固相萃取纖維
外文關鍵詞:Antrodia cinnamomeavolatile compoundfermentationHep G2 cellSolid Phase Microextraction (SPME)
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樟芝為台灣特有的菇類,以護肝及抗發炎而聞名。藉由液態發酵的方式可有效縮短發酵時間以生產具有抑制肝癌細胞的活性物質,且隨發酵天數的增加,肝癌細胞抑制率也隨之提高。發酵過程所產生的揮發性化合物隨著發酵的天數具有明顯變化。本研究之目的為了解樟芝培養過程中,發酵液揮發性化合物的變化與其抑制肝癌細胞的活性的關係。在揮發性成分分析方面,本研究發現PDMS/DVB纖維在60℃下吸附樟芝揮發性化合物30分鐘,可以有效吸附樟芝揮發性化合物,經由GC-MS之分析得知,樟芝於發酵期間產生之主要揮發性成分為1-octen-3-ol、methyl phenylacetate、linalool、nerolidol、γ-cadinene和 2,4,5-trimethoxybenzaldehyde六個化合物。於抑制肝癌細胞生長之試驗中發現,發酵液在發酵二十四天時即達到最高抑制率,而揮發性化合物2,4,5-trimethoxybenzaldehyde濃度也在同一時間急劇上升,因此,該揮發性化合物可能與發酵液之抑癌活性相關,也可以用來決定發酵收槽的時間。
Antrodia cinnamomea is a folk medicine well known for its hepatoprotective, anti-inflammation function in Taiwan. Submerged fermentation is used to produce its bioactive material under controlled conditions to overcome its time consuming cultivation in solid state. During fermentation, there is an increase in the hepatoma inhibitory effect and the volatile compounds changes during each stage.
The aim of this study is to investigate the correlation between the specific volatile compounds and bio-active components formed in due course of fermentation. In volatile compound analysis, PDMS/ DVB fiber was found to be most suitable fiber under the operating conditions of 60℃ for 30 minutes, to absorb volatile compounds of fermentation broth of A. cinnamomea. The GC-MS results indicated that the main volatile compounds were 1-octen-3-ol, linalool, methyl phenylacetate, nerolidol, γ-cadinene and 2,4,5-trimethoxybenzaldehyde. During fermentation it was observed that the fermentation broth have the highest inhibitory effect on 24th day. Also it was observed that among the various volatile compounds, there was a significant increase in concentration of 2,4,5-trimethoxybenzaldehyde in fermentation broth on the same day. Thus the volatile compound 2,4,5-trimethoxybenzaldehyde could be related to hepatoma inhibitory activity of fermentation broth, and can be used as a direct marker to decide harvesting time for the fermentation process.
目錄
中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VII
第一章 緒論 1
1.1 研究動機 1
1.2 研究目的 1
1.3 實驗架構 2
Part 1 利用GC/MS尋找纖維吸附最佳條件 2
Part 2 比較發酵過程中揮發性氣體的變化 2
第二章 文獻回顧 4
2.1 樟芝簡介 4
2.2 樟芝一般成分介紹 5
表 2-1樟芝一般成份 5
2.3 樟芝活性成分介紹 5
a. 多醣體 5
b. 三萜類 6
2.4. 樟芝生理活性探討 11
a. 抗氧化活性 & 降血脂 11
b. 抗發炎活性 11
c. 抑制癌症 12
d. 抗菌能力(抗病毒) 13
e. 降血糖 13
f. 護肝效果 13
g. 抑制血管新生(anti-angiogenic)活性 14
2.5. 固態發酵與液態發酵 14
a. 固態發酵: 14
b. 液態發酵: 14
2.6. 液態發酵影響因子 15
a. 培養基 15
b. 溫度 15
c. pH值 15
d. 通氣量 16
e. 光照 16
f. 攪拌速度 16
g. 生化反應器-攪拌式反應器 16
2.7. 菇類所產生之香氣 17
2.8. 真菌香氣介紹 18
2.8.1. 常見真菌香氣介紹 19
2.9 檢測揮發性物質的方法 21
2.8.1 溶劑萃取法 (Solvent Extraction) 21
2.9.2 蒸餾法 (Distillation Methods) 21
2.9.3 上部空氣(頂空)測定法 (Headspace Methods) 21
2.10 固相纖維介紹 22
2.10.1 纖維吸附香氣的應用 23
第三章 材料與方法 24
3.1 實驗材料 24
3.1.1 實驗菌種、細胞株 24
3.1.2 實驗藥品 24
3.1.3 實驗器材與儀器 25
3.2 發酵實驗方法 26
3.2.1 菌種保存 26
3.2.2 菌種活化 26
3.2.3 繼代培養 26
3.2.4 三角瓶培養 26
3.2.5 發酵槽接菌量 27
3.2.6 培養基組成 27
3.2.7 發酵槽分析 27
3.3 GC/MS實驗 28
3.3.1 GC/MS設定方式 28
3.3.2 GC/MS實驗條件 28
3.3.3 發酵液處理及步驟 29
3.3.4 內標 29
3.3.5 SPME 使用步驟 29
3.4 細胞實驗 30
3.4.1 細胞株培養 30
3.4.2細胞解凍 30
3.4.3 細胞冷凍 30
3.4.5細胞繼代 31
3.4.6 細胞計數 31
3.4.7 細胞存活率---MTT assay (Mosmann 1983) 31
3.5 統計分析 31
第一部份 吸附用纖維最適化條件探討 32
吸附溫度之探討 34
吸附時間之探討 40
纖維種類探討 42
第二部份 樟芝液態發酵期間揮發性化合物之變化 47
第三部份 不同培養期間發酵濾液和菌絲體乙醇萃取物活性關係 60
第五章 結論 63
第六章 參考文獻 64
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