利用氣舉式發酵槽生產具有抑制肝癌及子宮頸癌細胞生長之樟芝發酵液

Abstract

本研究利用不同種類5L氣舉式發酵槽培養樟芝發酵液，並找出具有最佳抑制肝癌細胞(Hep G2 cell)與子宮頸癌細胞(HeLa cell)生長之發酵條件，並擴大生長規模，以500L氣舉式發酵槽確定生產此一發酵產品之可行性。 實驗架構可分為三個階段。第一階段主要探討適合菌絲體生長的發酵槽及通氣條件，所探討之槽體種類包括：氣泡塔發酵槽、氣舉式發酵槽以及網狀內管氣舉式發酵槽，並以攪拌式發酵槽做為對照組，且分別採用三種不同通氣量，即0.1、0.5以及1 vvm。結果發現發酵7天後，以氣舉式發酵槽，0.1 vvm通氣量操作下，有最多菌絲體產量(3.13 g/L)。 第二階段為探討可以產出最多粗三萜的發酵條件，並以第一階段之最適培養樟芝的氣舉式發酵槽進行實驗，利用三種不同溫度22℃、25℃以及28℃進行六週長時間發酵，並每隔7天取樣一次，實驗結果發現，當氣舉式發酵槽以25℃發酵42天之菌絲體產量(5.02 g/L)及粗三萜含量較22℃及 28℃多。 第三階段進行500L大型發酵槽放大實驗，利用第二階段之最適培養樟芝的氣舉式發酵槽來進行實驗，結果發現在發酵達第28天時菌絲體產量已達到最大量(5.5g/L)。 第二階段與第三階段細胞實驗部分中，均以樟芝發酵濾液及菌絲體乙醇萃取物進行抑制Hep G2及HeLa細胞生長之實驗，發現半致死率IC50皆隨著發酵時間增加而降低。整體來說，以25℃抑制效果較佳，其發酵21天之菌絲體乙醇萃取物，對Hep G2及HeLa cells之IC50皆可達20 μg/ml。

The objectives of this research were to study the optimal condition for cultivating Antrodia cinnamomea in airlift reactors for inhibiting the growth of human hepatocellular carcinoma cell (Hep G2) and human cervical epithelioid carcinoma cell (HeLa cell). The experimental design divided into three parts. In the first part, various airlift reactors including bubble column reactor, airlift reactor with solid draft tube and airlift reactor with net draft tube were investigated for their suitability for cultivating A. cinnamomea. And the stirred tank reactor was used as control. The results indicated that the airlift reactor with solid draft tube operating at aeration rate 0.1 vvm could yield the highest amount of the mycelium (3.13 g/L after 7 days of fermentation). The second part of this research investigated the optimal fermentation conditions for A. cinnamomea for producing the highest amount of triterpenes. Using the airlift reactor with solid draft tube, it was found that when the reactor was operated at 0.1 vvm and 25℃ for 42 days, the mycelium concentration (5.02 g/L) and the crude triterpene content were more than that at 22 and 28℃. The operating scale was increased from 5L to 500L airlift reactor for the third part of this study. It was found that under the optimal fermentation condition, the mycelium in the fermentation broth reached 5.5 g/L in only 28 days, as compared to the 42 days of the 5L reactor. During the second and third part of this study, the inhibition effect on the growth of Hep G2 and HeLa cell by the filtrate and the ethanol extract of the mycelium of A. cinnamomea was investigated. The results indicated that the growth inhibitory effect of the filtrate and the ethanol extract of the mycelium increased with fermentation time. Moreover, the inhibitory effect of the fermentation products cultivated at 25℃ was higher than that at 22 or 28℃. The IC50 of the ethanol extract of the mycelium cultivated at 25℃ for 21 days was 20μg/ml.