以回應曲面法探討樟芝高密度發酵

Abstract

「樟芝」（Antrodia cinnamomea）是一種極為昂貴的藥用真菌，又稱「牛樟芝」、「牛樟菇」、「樟菇」、「紅樟菰」等。自然界中，發現樟芝僅生長於樟科的牛樟樹（Cinnamomum kanehirai Hay），牛樟樹屬於台灣稀有保育樹種，也因此，天然生長的樟芝亦不多。所以本論文的目的是以高密度發酵的方式，大量生產樟芝菌絲體，並瞭解在培養環境改變的情況下，對相關產物、機能性的影響。 研究的試驗菌株是Antrodia cinnamomea CCRC35396，以傳統的一次一因子法配合回應曲面法（RSM），設計並分析樟芝高密度發酵的最適培養條件。先運用一階設計中的二水準因子設計法，將樟芝以搖瓶震盪實驗，探討培養液中碳源的濃度、氮源的濃度、無機鹽類、橄欖油、椰子汁、植物激素、起始pH值及轉速等因子，對樟芝菌絲體生長、發酵液終點pH值及多醣生成量的影響；再利用陡升路徑法、中心混成及二階設計，逐步定出最佳培養條件。 對Antrodia cinnamomea CCRC35396而言，在500 mL有溝槽的搖瓶培養中，裝100 mL培養液，以溫度28 ℃、轉速50 r.p.m.、接種量10 ﹪的固定條件下，先以一次一因子法固定起始pH值為5，然後再由回應曲面法可得最佳培養環境為Glucose 3.6788 ﹪、Sucrose 6.734 ﹪、Peptone 0.58 ﹪、Olive Oil 500λ、Coconut Milk 10ml、Ion 0.03 ﹪、2,4-D 0.5ppm，另由實驗可得最適化起始pH值為4.5，若分兩段式轉速培養則可提升產量(前7天100rpm，後7天50rpm)。跟原培養條件相比，菌絲體產量已經有顯著的提高，由此可知，在尋找樟芝的最適生長條件上，回應曲面法具有良好的契合度。

Antrodia cinnamomea is precious and rare fungus using for Chinese medicine. In the natural world, they are only found in Cinnamomum kanehirai Hay that protecting in Taiwan. The purpose of study is going to enhance production of Mycelium and analyze the Polysaccharide concentration in different conditions. The strategy is to find out the optimum cultured condition of Antrodia cinnamomea CCRC35396 with one-factor-at-a-time method and experimental design (response surface methodology). Screening for carbon sources and nitrogen sources are tested by one-factor-at-a-time method. Then we can determine the concentration of nutrients by response surface methodology (RSM). RSM is a systemic manner of statistic and including three steps: the two-level factorial of first order, the path of steepest ascent and the central composite design of second order. The optimum cultured conditions of Antrodia cinnamomea CCRC35396 are Glucose 3.6788 %, Sucrose 6.734 %, Peptone 0.58 %, Olive Oil 500λ,Coconut Milk 10mL,Ion 0.03 %,2,4-D 0.5ppm,when culture in 500 mL Hinton flask containing 100 mL media, 28 ℃, 50 r.p.m., initial pH 5.In our experiment we can demonstrate that the best initial pH 4.5.Our product will be increased if we use two stage submerge cultured(First seven days use 100rpm,Last seven days use 50rpm).Results of Mycelium concentrate and Polysaccharide concentrate are increased significantly.