樟芝产生三萜类化合物的液体培养条件的响应面法优化

Optimization of Conditions for Liquid Culture of Antrodia cinnamomea to Produce Triterpenoids with Response Surface Methodology

樟芝(Antrodia cinnamomea)是一种非褶菌目、多孔菌科的多年生蕈菌类,樟芝子实体中含有多种生理活性物质包括三萜类化合物,但野生樟芝只生长在台湾特有的牛樟树树干腐朽的心材内壁和枯死倒伏的牛樟树表面,生长极其缓慢,很难获得,价格昂贵。本研究的目的是通过液体培养樟芝的菌丝体以产生三萜类化合物,用响应面法优化了樟芝产生三萜类化合物的液体发酵条件。首先对樟芝的液体发酵的培养基成分及培养条件进行了单因素优化,研究发现樟芝液体发酵产生三萜类化合物的最佳碳源和氮源分别为葡萄糖和酵母提取物,最佳浓度分别为100 g/L和9 g/L,优化的培养基配方为(g/L):葡萄糖100.0,酵母提取物9.0,Mg SO4·7H2O 0.5,KH2PO41.0。优化的培养条件为:初始pH值6.0,温度28℃,接种量10%(v/v),摇床转速180 r/min。在此基础上,用响应面法对樟芝液体发酵产生三萜类化合物影响最大的三个因素即碳源、氮源和培养温度进行了优化,结果优化的三种因素条件为:葡萄糖104.71 g/L,酵母提取物9.93 g/L,温度28.42℃。用优化的培养条件液体培养樟芝,其三萜类化合物的产量达到391.45 mg/L,比优化前的产量223.39 mg/L提高了75.23%,为进一步研究液体培养樟芝产生三萜类化合物奠定了基础。

Antrodia cinnamomea is a kind of perennial mushroom, belonging to Polyporaceae in Aphyllophorale.The fruitbody of this mushroom contains many kinds of physiological active substances to human body including triterpenoids. However, the wild fruitbody of A. cinnamomea can grow only on the inner sides of the decaying trunk of Cinnamomum kanehirai and the wet surface of the dead laid tree. The fruitbody develops extremely slowly. It is very difficult to get the mushroom and the price of the mushroom is too expensive. In this study, the conditions for liquid cultivating the mycelium of A. cinnamomea to produce triterpenoids were optimized using surface response methodology. The cultivation conditions for producing triterpenoids in submerged culture fermentation were first optimized by the single factor experiment. As a result, the best carbon source and nitrogen source for A. cinnamomea to produce triterpenoids were glucose and yeast extract, respectively. The optimal concentration for glucose and yeast extract was 100 g/L and 9 g/L, respectively. The optimized medium composition(g/L)was: glucose 100.0, yeast extract 9.0, Mg SO4·7 H2 O 0.5, KH2 PO41.0. The optimized culture conditions were as below: initial p H6.0, temperature 28℃, amount of inoculum at 10%(v/v), shaking speed at 180 r/min. Based on the results from the single factor experiment, the effect of three most influential factors on the production of triterpenoids in submerged culture fermentation of A. cinnamomea were tested with surface response methodology.As a result, the optimal conditions for the three factors for submerged culture fermentation of A. cinnamomea were as below: 104.71 g/L of glucose, yeast extract 9.93 g/L, the temperature of 28.42℃. Under the optimized condition,the yield of triterpenoids in submerged culture fermentation of A. cinnamomea reached 391.45 mg/L, which was75.23% higher than the yield before optimization(223.39 mg/L), which provided solid basis for further industrial production of triterpenoids by A. cinnamomea.