實驗設計方法探討批次式靈芝菌絲最佳化液態培養

Experimental design method for optimal batch fermention of Ganoderma Lucidum

張哲緒(Che-Hsu Chang)

指導教授 ： 張炎林

中原大學/工學院/醫學工程研究所/碩士(2002年)

https://doi.org/10.6840/cycu200200448

摘要

靈芝，這一健康食品在療效上備受肯定其原因不外乎它有著悠久的藥典見證、多年來研究文獻和臨床上的佐證。在一系列靈芝研究熱潮中，大多數是探討它的療效、菌株分類、培養方式改良與培養條件的控制。本研究先以參考文獻為基礎，實作探討靈芝液態培養的環境影響因素包括培養的溫度、培養時增加溶氧的培養箱迴轉速率、pH值的起始值與培養基本身各項可改變因素等對產量的影響，並尋找最佳操作條件。
Two-Level 因素實驗設計部分，主要是探討培養基中因素之pH值起始值、橄欖油的添加量及靈芝代謝物添加量三因素之個別效應與三因素之間交互效應為初期實驗目標。初期結果顯示橄欖油添加的個別效應最大（E2值＝708.3±53.6）遠大於pH起始值（E1=-58.8±53.6）與代謝物添加（E3=88.8±53.6），顯示在培養基添加橄欖油後能顯著增進菌絲生長。在交互效應中值得注意的是同時添加橄欖油與代謝物（E23 =-81.3±53.6），是呈現負面影響而抑制菌絲產量。
第二階段部分因素實驗設計的五項因素，是前三項培養基之控制因素再加入溫度及培養箱轉速率兩環境因素。利用部分因素實驗設計觀點，可以減少實驗次數，即可了解各因素之個別效應（Ei）；此外亦針對橄欖油添加量因素，探討與其他因素間交互作用（E12、E23、E24與E25）。
最後由因素設計實驗藉由回歸分析設計推演，找到同時考慮三項培養基控制因素（pH值起始值、橄欖油的添加量與靈芝代謝物添加量）最佳化培養模式為：pH值起始值4.42、橄欖油的添加量2.95 ml、靈芝代謝物添加量1.16 ml、培養溫度30℃與培養箱迴轉速率100 rpm下，可得最佳化產量1614 mg。

關鍵字

實驗設計 ； 部分因素設計 ； 菌絲體培養 ； 靈芝 ； 回歸分析

並列摘要

Ganoderma lucidum, has enjoyed a critically acclaimed reputation for its healing properties as a form of health food lies in none other than an extended encyclopedic testimonies, backed by documented evidence derived from recent research publications and clinical findings. Amongst a deluge of sensational Ganoderma lucidum studies of late, a majority of them have focused largely on examining its treatment properties, mycelium colony typing, improvement of means in culturing, and manipulation of culturing criteria. The study takes to developing a premise built upon reference archives with which to examine factors that may come to affect the liquid culture of Ganoderma lucidum, which entail validating the the cultivating temperature, rate of rotating the cultivation box for increasing soluble oxygen, the commencing pH rating, and how some other variables in the culturing process may come to affect to output, and in search of establishing an optimized operating criteria.

The deign of the two-level factorial experiment lies in examining variables behind the culture solution, particularly of the individual yield and compound yield among the three critical factors of the commencing pH rating, metering of olive oil, and fungal metabolic additive with which to define the preliminary experimental objectives. Preliminary findings conclude that olive oil metering offers the highest individual yield, where E2 is measured at 708.3 ± 53.6, to far exceed the commencing pH rating, where E1 equals to 58.8 ± 53.6, or the fungal metabolic additive, where E3 equals to 88.8 ± 53.6, indicating that the presence of olive oil during the culturing process can indeed onerously excel the rate of mycelium culture. Noteworthy in the are of compound yield is how the simultaneous adaptation of olive oil and the metabolic addition, where E23 equals to 81.3 ± 53.6, would negatively impacted the results to lead to a decline in mycelium growth.

Phase two of the experiment has the experiment’s factorial design extended to include five factors, which entail continuing with the previous three factors but adding two more factorial elements of temperature and rate of rotating the cultivation box. And the perspective of a fractional elemental experiment design has proven effective to conclude the individual yield, E1, of all elements with a reduction to the number of experiments required, and to examine the compound effect the element of olive oil metering comes to response to other elements, such as E12, E23, E24 and E25.

Lastly, the experiment of factorial design takes to a regression analysis as the basis for design extrapolation and led to the conclusion an optimal model for the three critical cultivation control elements, namely the commencing pH rating, the metering of olive oil and Ganoderma lucidum metabolic additive, is at a commencing pH rating of 4.42, olive oil metering of 2.95 ml, Ganoderma lucidum metabolic additive at 1.16ml, cultivation temperature at 30℃. Celsius and rate of rotating the cultivation box at 100 rpm to conclude an optimized output of 1,614 mg.

並列關鍵字

fractional factorial design ； Granoderma lucidum ； experiment design ； regression analysis ； mycelium culture

參考文獻

5.丁自勉.2001.靈芝.中國中醫葯出版社.

23.Yeh, Zeng-Yung and Zuei-Ching Chen. 1990. Preliminary investigatns of Ganoderma australe（Subgen ,Elfvingia）in Taiwan.Taiwania 35:127-141.

6.Ya-Jie Tang, Jian-Jiang Zhong. Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganoderic acid. Enzyme and Microbial Technology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, China 2002;31:20-28.

8.Qing-Hua Fang,Jian-Jiang Zhong, Effect of PH on Production of ganoderic acid and polysaccharide by submerged fermentation of Ganoderma lucidum, Process Biochemistry, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, China 2002;37:769-774.

16.許瑞祥.1990.靈芝屬菌株鑑定系統之研究. 國立台灣大學農業化學研究所博士論文.