靈芝多醣液態發酵及萃取製程最適化研究

Optimization of submerged fermentation and extraction process of polysaccharide from Ganoderma tsugae

紐羅伯(Nukrob Narkprasom)

指導教授 ： 古源光 ； 黃卓治

國立屏東科技大學/農學院/熱帶農業暨國際合作系所/博士(2012年)

https://doi.org/10.6346/NPUST.2012.00198

摘要

松杉靈芝Ganoderma tsugae是眾所周知的藥用真菌，它具有許多的生理活性。松杉靈芝多醣已廣泛應用於各種食品和醫藥業中。松杉靈芝多醣是天然生成的藥物可用於保健醫學上，用在治療糖尿病和癌症患者，因此受到相當重視。這種由真菌中所萃取的化合物，可直接添加在食品中作為強化生理活性成分。然而，天然的松杉靈芝的生長速度是相當緩慢地。許多研究都試圖以開發大量生產松杉靈芝的培養系統。因此，本研究主要針對靈芝液態發酵及子實體萃取製程來進行最適化條件之探討。
在松杉靈芝的液態發酵中，針對振盪速率的供氧量的效果及培養基體積對菌絲和胞外多醣( Extracellular polysaccharide, EPS )的生產作探討。一般菌絲的生長需要適當震盪速率約134 rpm，將較大的菌絲球破壞成幾個小的菌絲球。此外，較大培養容積可提供充足營養物的用量，而有助於最大菌絲生長需要。胞外多醣則需高速的攪拌速率來提高較佳的質傳作用，來達到較高產品濃度的要求。研究發現比較合適的培養基體積為150.4 ml，並利用提高基質質傳作用以有助於胞外多醣的生產。由松杉靈芝的菌體乾重( Dry cell mass, DCM )對胞外多醣作圖可以建立兩個生產方程式，並且這圖示法對液態發酵產業將是一個有用的工具。
松杉靈芝培養條件最適化條件是利用Plackett Burman設計法( Plackett Burman design, PBD )來進行篩選，確定最佳序列上是利用陡升路徑法( Method of path of steepest ascent)來探討，以及利用反應曲面法( Response surface method, RSM )來進行最佳化條件尋求。其中，麥芽糖，脫脂牛奶，pH值是松杉靈芝培養的重要參數。在31.031 g/l麥芽糖，14.055 g/l 脫脂牛奶和pH在 7.12的條件下， EPS的最大產量為415 mg/l。在相同的發酵液中，初始pH在 6.46下可得到最大菌體產量為15.776 g/l。在這些最佳條件的確認試驗中，發現可提高菌體產量和促進胞外多醣的生產，分別為控制組的13倍和1.5倍。這些結果顯示這些策略有助於松杉靈芝液態發酵程序的改善。
在松杉靈芝的菌量和胞外多醣的動力學模型中有不錯的結果。研究中，依據之前的研究所得批式培養的最佳條件，針對松杉靈芝的菌量和胞外多醣的動力學模型進行分析。菌絲球和胞外多醣的產量，可由立方根方程式和Luedeking-Piret方程式分別描述。兩個方程式的參數，可由所實驗的觀察和演算法求解。松杉靈芝的菌量和胞外多醣的實驗值和預測模型之間可得到很高的相關性係數R2，分別為96.05%和99.16%。這兩個動力學模型有助於來預測松杉靈芝的菌體產量和胞外多醣的代謝生產。
松杉靈芝子實體的粗多醣體萃取程序是採用中心組合設計法( Central Composite design, CCD )來進行最佳化條件尋求。在這項研究中的參數為：萃取溫度( 82-98C )，萃取時間( 199-441 min )和水對原料的比例( 13-47 ml/g )。迴歸統計分析顯示，二次方程式的模型最符合來做生產最佳化的預測( R2=98.68% )。萃取程序的最佳條件發現為：94C萃取溫度，337分鐘的萃取時間和水對原料的比例為27.89 ml/g。在這些條件下，多醣的預測值是0.719%。

關鍵字

松杉靈芝 ； 液態發酵 ； 胞外多醣 ； Plackett Burman設計法 ； 反應面法 ； 立方根方程式 ； Luedeking-Piret方程式

並列摘要

The fungus Ganoderma tsugae, has long been a well known medicinal mushroom and it has many pharmacological properties. Polysaccharides from G. tsugae have various applications in food and pharmaceutical industries. G. tsugae polysaccharide has received considerable attention to produce the medicine from nature for health care, treat diabetes and cancer patient, this extracted compound from mushroom can applies to add in food as a biologically active ingredient. However, the growth rate of G. tsugae in nature is very slow. Many studies have attempted to develop culture systems for mass production of G. tsugae. Therefore, the optimal condition for submerged fermentation of fungus and extraction process of fruiting body were investigated.
The quantitative effects of oxygen supply in terms of shaking speed and medium volume on the production of mycelia and extracellular polysaccharide (EPS) using G. tsugae in submerged fermentation were investigated. Mycelia growth required the proper shaking speed at 134 rpm for breaking a larger pellet into several smaller pellets. Furthermore, high level of medium volume for consumption of abundant nutrient was needed for maximum mycelia growth. For EPS, a high agitation was needed to promote a good mass transfer for achieving high product concentrations recovery. The appropriate medium volume was found to be 150.4 ml for aiding the production of EPS by promoting the mass transfer of substrates. The dry cell mass (DCM)-EPS diagram of G. tsugae was created from both production equations, was an useful tool for submerged fermentation industry.
To determine the optimal conditions for G. tsugae, culture conditions were screened using Plackett Burman design (PBD), optimal ranges were determined by the path of steepest ascent method, and optimal searching was performed by the response surface method (RSM). Maltose, skim milk, and pH were significant parameters for G. tsugae cultivation. The conditions of 31.031 g/l maltose, 14.055 g/l skim milk and initial pH of 7.12 resulted in the maximum EPS content of 415 mg/l, and the same fermentation broth at an initial pH of 6.46 exhibited the most significant biomass at 15.776 g/l. Verification of these optimal conditions resulted in enhanced biomass and EPS production (13X and 1.5X of the control, respectively). These results showed that the strategies used here enabled improving the submerged fermentation processing of G. tsugae.
The kinetic models for biomass and EPS of G. tsugae were conducted as well. The kinetic models of biomass and EPS by G. tsugae were studied in a batch cultivation at the optimal conditions decribed previously. The productions of mycelium pellet and EPS were described by the cube-root equation and Luedeking-Piret equation respectively. The parameters of both equations were determined by the observed experiment and algorithm solving. The correlation between the experimental values and predicted models of biomass and EPS for G. tsugae obtained the high R2 at 96.05% and 99.16%, respectively. These two kinetic models may be useful to predict the productions of cell mass and EPS from G. tsugae.
Central Composite design (CCD) was applied to optimize the extraction process of crude polysaccharides from fruiting body of G. tsugae. The parameters in this study were the extraction temperature (82-98C), the extraction time (199-441 min) and the ratio of water to raw material (13-47 ml/g). The statistical regression analysis showed that a quadratic model was most fitted to estimate the optimal production (R2 = 98.68%). The optimal condition for extraction process was found at 94C of extraction temperature, 337 min of extraction time and 27.89 ml/g of ratio of water to raw material. Under these conditions, the predicted value of polysaccharides was 0.719%.

並列關鍵字

Ganoderma tsugae ； submerged fermentation ； extracellular polysaccharide ； Plackett Burman design ； Response Surface Method ； cube-root equation ； Luedeking-Piret equation

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