以4-acetylantroquinonol B為指標改善樟芝液態培養之方法

Abstract

樟芝(Antrodia cinnamomea)是台灣特有真菌，子實體生長在牛樟樹（Cinnamomum kanehirai Hay）上，具有抗癌、護肝、抗氧化、抗B型肝炎病毒、抗發炎等活性。由於樟芝子實體生長非常緩慢，目前多藉由液態培養樟芝方式以獲得活性成分。本實驗室於樟芝菌絲體中發現了能有效抑制肝癌細胞生長之物質4-acetylantroquinonol B，其抑制HepG2細胞之IC50為0.1 μg/mL。因此，本研究以4-acetylantroquinonol B為指標，以兩種策略提升其產量。由於樟芝發酵液抑制肝癌細胞能力與其香氣強度呈相關性，且從化學結構上推測其主要揮發性物質2, 4, 5-trimethoxybenzaldehyde (TMBA)及nerolidol可能為4-acetylantroquinonol B前驅物，因此，本研究之策略一乃於液態培養樟芝時添加TMBA或nerolidol作為前驅物，以期提升4-acetylantroquinonol B含量。實驗結果證實，添加0.2% TMBA或0.25% nerolidol，皆可提升4-acetylantroquinonol B含量，鑒於添加過量nerolidol可能會產生substrate inhibition效應，且4-acetylantroquinonol B含量以添加0.2% TMBA組別在第六週最高(0.75 mg/g DW)，故以TMBA作為前驅物，可能是提升樟芝發酵液之4-acetylantroquinonol B產量較好的策略。策略二乃以不同稀釋率進行連續式培養樟芝，使樟芝持續生產4-acetylantroquinonol B。由於4-acetylantroquinonol B為二次代謝產物，本實驗分為兩階段發酵，第一階段採批式發酵，使樟芝生長期達穩定期後，第二階段採連續式發酵，以不同稀釋率(dilution rate，D=0.01、0.005、0.0025、0.0015及0.001 h-1)進行實驗，以得到培養樟芝之最適稀釋率。結果發現，在高稀釋率(D=0.01、0.005及0.0025 h-1)培養下，菌絲體及4-acetylantroquinonol B含量均下降，而於低稀釋率(D=0.0015及0.001 h-1)狀態下培養樟芝，可有效維持樟芝持續穩定生長，且持續產生4-acetylantroquinonol B，其中以D=0.0015 h-1狀態下培養樟芝31天，其4-acetylantroquinonol B產量與第一階段批式發酵39天所得者相當，可有效節省重新發酵一槽之時間。

Antrodia cinnamomea is a unique fungus that grows on the inner cavity of Cinnamomum kanehirai Hay in Taiwan. This fungus has many biological activities, such as anti-cancer, hepatoprotective, antioxidant, anti-viral hepatitis B and anti-inflammatory etc. Due to the fact that cultivation of fruiting body of A. cinnamomea is a time consuming process, the production of mycelium by submerged culture has attracted much attention for the production of bioactive compounds. Our lab has conducted submerged fermentation of A. cinnamomea for many years, and we have found that the bioactive compound, 4-acetylantroquinonol B, isolated from the mycelium of A. cinnamomea possesses very high inhibitory activity to the hepatocarcinoma cells. The IC50 value of 4-acetylantroquinonol B for inhibiting HepG2 cells is 0.1 μg/mL. In this research, we used two strategies to enhance the anti-hepatoma activity of A. cinnamomea by using 4-acetylantroquinonol B as marker. The anti-proliferation activity of the ethanol extract of A. cinnamomea mycelium on hepatocellular cancer cells was found to be associated with aroma intensity of the broth during fermentation. We inferred that 2, 4, 5-trimethoxybenzaldehyde (TMBA) and nerolidol are the precursors of 4-acetylantroquinonol B based on their chemical structures. The first strategy for enhancing the bioactivity was to use TMBA or nerolidol as precursors during submerged fermentation to increase the production of 4-acetylantroquinonol B. It was found that 0.2% TMBA or 0.25% nerolidol as supplements could increase the production of 4-acetylantroquinonol B. Further investigation found that the existence of too much of nerolidol in the system would result in substrate inhibition effect, and the 0.2% TMBA supplementation could yield the highest amount of 4-acetylantroquinonol B at the 6th week (0.75 mg/g DW) during fermentation. Therefore, supplementation of TMBA as precursor appeared to be a better strategy for increasing the production of 4-acetylantroquinonol B during fermentation of A. cinnamomea. The second strategy was to use continuous culture techniques for cultivating A. cinnamomea in airlift bioreactor and tried to maintain microbial metabolism at steady state. Due to 4-acetylantroquinonol B is a secondary metabolite so we used two-stage cultivation. The first stage was batch culture for A. cinnamomea to reach stationary phase, the then second stage was to use continuous culture with proper dilution rates to to continuously produce 4-acetylantroquinonol B. The results showed that the production of mycelium and 4-acetylantroquinonol B decreased at high dilution rate (0.01, 0.005 and 0.0025 h-1), but both of mycelium and 4-acetylantroquinonol B could be maintained at low dilution rate (0.0015 and 0.001 h-1). Accumulated amount of 4-acetylantroquinonol B removed from the fermenter by using continuous culture at dilution rate 0.0015 h-1 for 31 days was the same as the amount of 4-acetylantroquinonol B obtained by using bath culture for 39 days. Thus, continuous culture can be a time-saving strategy because it can save the time for restarting the fermentation in the bioreactor.