探討誘發劑及兩階段培養對樟芝深層發酵三萜類及抗癌作用之影響

The Influence of Elicitors and Two-Stage Culture on Triterpenoids Production and Antitumor activity in Submerged Cultivation of Antrodia cinnamomea

劉景仁(Ching-Jen Liu)

指導教授 ： 蔣丙煌

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10029

摘要

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。

關鍵字

樟芝 ； 誘發劑 ； 兩階段培養 ； 三萜類 ； 抗癌作用

並列摘要

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).

並列關鍵字

Antrodia cinnamomea ； Elicitors ； Two-stage culture ； Triterpenoids ； Antitumor activity

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