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研究生:張明堯
研究生(外文):Chang Ming You
論文名稱:液態培養生產靈芝菌絲體與靈芝多醣最適化之研究
論文名稱(外文):Optimization of cultured condition for the production of mycelial and polysaccharide from Ganoderma lucidum
指導教授:蔡國珍
指導教授(外文):Tsai Guo Jane
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系碩士在職專班
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:111
中文關鍵詞:多醣最適化靈芝液態培養田口方法反應曲面法陡升路徑法
外文關鍵詞:polysaccharideoptimizationGanoderma lucidumsubmerged cultureTaguchi methodResponse surface methodologyPath of steepest ascent
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靈芝屬(Ganoderma sp.)是中國人自古以來珍貴的稀有藥用真菌,靈芝多醣可經口服或腹腔注射而提升免疫機能,達成間接破壞癌細胞之作用。靈芝液態培養(submerged culture)可於短時間、有限空間下生產大量靈芝菌絲體、靈芝多醣及其他生理活性物質,有其商業經濟發展潛力。
本論文以搖瓶振盪培養靈芝(Ganoderma lucidum) CCRC36124,經由實驗設計法逐步調整培養條件,期望提高靈芝菌絲體和靈芝多醣產量。由一次一因子法探討不同碳源種類、氮源種類、油脂種類、無機鹽種類、起始pH值、轉速、培養時間對搖瓶振盪培養靈芝之影響,並運用田口方法(Taguchi method),選擇直交表L18(21x37),以靈芝菌絲體濃度及靈芝多醣生成量為應變數(response),探討八項因子對其影響程度。利用期望函數(Desirability function)法折衷靈芝菌絲體濃度及靈芝多醣生成量二應變數值,作多目標函數最佳化,以利進行陡升路徑(Path of steepest ascent)法實驗,再運用直交表L9(34)及反應曲面法(Response surface methodology )進行四因子三階次及三因子三階次探討,以逼近最適培養條件。
以靈芝(Ganoderma lucidum) CCRC36124 進行研究,顯示其最適培養條件為: brown sugar 7.14 %, malt extract 1.21 %, skim milk 1.84 %, safflower seed oil 0.344 %, olive oil 0.396 %, CaCO3 0.188 %, K2HPO4 0.05 %, KH2PO4 0.05 %, MgSO4·7H2O 0.05 %,起始pH 6.5,於250 mL三角瓶中裝100 mL 培養液,於34 ℃,160 rpm振盪培養,在第十二天時,菌絲濃度2.15 g/100mL,比未最佳化培養條件前之一次一因子法(0.79 g/100mL)增加170%菌絲濃度;最佳化培養條件下,第十二天之多醣生成量0.87 mg/mL,比未最佳化培養條件之一次一因子法(0.678 mg/mL)增加28%多醣生成量。本研究結果證實運用田口方法、期望函數法、陡升路徑法及反應曲面法探求靈芝液態培養時最適醱酵條件,可提高靈芝菌絲體濃度和靈芝多醣之產量。
E-mail:netskys@yahoo.com
Ganoderma species are fungi used in traditional Chinese medicine. According to many ancient descriptions of Chinese herbs, Ganoderma sp. possesse important pharmacological activities. Recent studies on these fungi have shown that Ganderma sp. can modulate immune responses and inhibit tumor growth.
The aim of this research is to increase the productivity of the mycelium and polysaccharide during cultivation of Ganderma lucidium using different experimental designs. The strategies including one-factor-at-a-time method, Taguchi method, desirability function and response surface methodology are used to find out the optimal cultured condition in flasks for Ganoderma lucidum CCRC36124. The medium compositions including carbon and nitrogen sources, salts, vitamins and oils, and the cultural conditions of pH, temperature and rotation speed for the cultivation of Ganoderma lucidum were evaluated first by one-factor-at-a-time method. Accordingly, the main 8 affectors on the production of fungal mycelium and extracellular polysaccharides were chosen, which were further evaluated to determine their optimal concentrations by Taguchi method. The determined conditions were further optimized by the desirability function and the response surface methodology (RSM).
The final optimal medium compositions for the cultivation of Ganoderma lucidum CCRC36124 include 7.14% brown sugar, 1.21% malt extract, 1.84% skim milk, 0.344% safflower seed oil, 0.396% olive oil, 0.188% CaCO3, 0.05% K2HPO4, 0.05% KH2PO4, and 0.05% MgSO4 · 7H2O with initial pH of 6.5. The optimal cultivation temperature and rotation speed are 34℃ and 160 rpm, respectively. After 12 days of incubation, the mycelial dry weight and the extracelluar polysaccharide concentration obtained were 21.5 mg/mL and 0.87 mg/mL , respectively, which were increased by 170 % and 28 % , respectively, compared to the results obtained from the cultural conditions of one-factor-at-a-time experimental design.
This research demonstrated that the optimal conditions for increasing the productivity of fungal mycelium and extracellular polysaccharides of Ganoderma lucidum CCRC36124 can be obtained by the experimental designs of Taguchi method, desirability function and RSM.
E-mail:netskys@yahoo.com
壹、前 言....................................................1
貳、文獻整理..................................................3
一、靈芝簡介.............................................3
1.靈芝屬的研究簡史.....................................3
2.靈芝屬的分類地位及生活史.............................4
3.靈芝屬的形態特徵及寄主...............................6
4.靈芝屬的生理活性物質及藥理研究.......................8
(1)多醣類(Polysaccharide).........................8
(2)蛋白多醣(Proteoglycan)........................11
(3)三帖類(Triterpenoids).........................12
(4)超氧歧化酵素(SOD).............................13
(5)類固醇(Steroid)...............................14
(6)其他..........................................14
5.靈芝屬的液態培養...................................15
(1)碳源..........................................16
(2)氮源..........................................17
(3)振盪速率及溶氧率..............................18
(4)碳氮比........................................18
(5)無機鹽類......................................19
(6)其他因子......................................19
二、田口方法(Taguchi method)簡介.........................20
1.實驗設計(Design of experiment)....................20
2.田口式直交表實驗法................................22
3.直交表(Orthogonal array) .........................22
4.田口方法實行步驟..................................25
5.變異數分析(Analysis of Variance,ANOVA)….........27
三、期望函數(Desirability function, D) ..................29
四、反應曲面法(Response surface methodology).............29
1.極值點區域近......................................29
2.反應曲面數學模式建立..............................30
3.數學模式適切性檢驗................................31
4.極值點決定........................................31
5.各因子影響效應分析............................... 32
參、材料方法
一、材料.................................................33
1.實驗菌株(Strain) .................................33
2.實驗藥品及培養基..................................33
3.實驗儀器..........................................34
二、方法.................................................35
1.一次一因子(One-factor-at-a-time)法................35
(1)菌種活化及保存..................................35
(2)菌體培養........................................35
(i)氮源種類探討.............................35
(ii)碳源種類探討............................35
(iii)油脂種類探討...........................36
(iv)維生素及無機鹽種類探討..................36
(v)培養溫度探討.............................36
(vi)起始pH值探討............................37
(vii)搖瓶轉速探討...........................37
(viii)培養時間探討..........................37
(3)菌體量測定.....................................37
(4)多醣測定.......................................37
(5)總醣測定.......................................38
(6)葡萄糖測定.....................................38
(7)pH測量.........................................38
2.田口方法(Taguchi method).........................38
3.期望函數(Desirability function)法................39
4.陡升路徑(Path of steepest ascent)法..............39
5.反應曲面法(Response surface methodology )........39
6.統計分析.........................................40
肆、結果與討論...............................................41
一、一次一因子法.........................................41
1.氮源種類探討....................................42
2.碳源種類探討....................................42
3.油脂種類探討....................................42
4.維生素及無機鹽種類探討..........................44
5.培養溫度探討....................................45
6.起始pH值探討....................................46
7.搖瓶轉速探討....................................46
8.培養時間探討....................................47
9. 一次一因子法所得生長曲線圖.....................48
二.第一次田口方法.......................................49
1.田口方法求較高菌絲濃度培養條件之生長曲線........51
2.田口方法求較高多醣生成培養條件之生長曲線........52
三、期望函數............................................52
四、陡升路徑法..........................................53
五、反應曲面法..........................................54
六、第二次田口方法......................................55
七、最適化培養條件之生長曲線............................55
伍、結論及展望...............................................58
陸、參考文獻.................................................60
表目錄
表一、L9(34)直交表……………………………………23 表二、L8(27)之交互作用配行表………………………24 表三、一次一因子法結果…………………..........74
表四、田口氏直交表L18(21X37)實驗設計各因子
及其嘗試水準之定義…………………….…....76
表五、L18直交表實驗設計及其實驗結果…….…....77
表六、L18直交表以菌絲濃度為應變數應用SAS
方程式之變方分析結果…………………........78
表七、L18直交表以多醣為應變數應用SAS方
程式之變方分析結果……………………........79
表八、以期望函數值為應變數應用SAS 方程
式之直線迴歸變方分析結果……………80
表九、第一次陡升路徑法實驗設計及其實驗結果…….81
表十、第二次陡升路徑法實驗設計及其實驗結果…….82
表十一、三因子三階次反應曲面設計中操作
條件變數及其階次………………………........83
表十二、三因子三階次反應曲面實驗結果….........84
表十三、RSM結果以SAS方程式執行變方分析之結
果…………………………………………….…85
表十四、田口氏直交表L9(34)實驗設計各因子及
其嘗試水準之定義………………………….....86
表十五、L9(34)直交表實驗設計及其實驗結果….....87
表十六、比較不同實驗設計法所得培養條件及其菌絲
……………………………………………………88
圖目錄
圖一、靈芝生長史……………………………………….89
圖二、Ganoderma lucidum之菌絲體和扣子體
(clampconnections)…………………………….90
圖三、Ganoderma lucidum孢子…………………………90
圖四、有抗腫瘤活性的β(1→6)分支的β(1→3)-D-葡聚
糖結構及其結晶結構…………………………...91
圖五、β(1→3)-D-葡聚糖系多醣體的抗腫瘤作用顯現
的作業假說………………………………..…….92
圖六、實驗流程圖……………………………………….93
圖七、碳源種類對培養CCRC36124菌絲濃度和胞外多
醣之影響…………………………………………94
圖八、氮源種類對培養菌株CCRC36124菌絲濃度和胞
外多醣之影響………………………………….95
圖九、油脂種類對培養菌株CCRC36124菌絲濃度和胞
外多醣之影響…………………..…………….96
圖十、不同無機鹽和維生素對培養菌株CCRC36124菌
絲濃度和胞外多醣之影響…………….……..97
圖十一、培養溫度對培養菌株CCRC36124菌絲濃度和
胞外多醣之影響………….…………………..98
圖十二、起始pH對培養菌株CCRC36124菌絲濃度和胞
外多醣之影響………………………………….99
圖十三、搖瓶轉速對培養菌株CCRC36124菌絲濃度
和胞外多醣之影響………………….…....100
圖十四、由一次一 因子法求得最適培養條件(培養
條件A)下,培養Ganoderma lucidum CCRC
36124 過程中,菌絲濃度及培養基中pH值
、葡萄糖及多醣含量變…………………...101
圖十五、由田口直交表L18(21x37) 設計所得結果以
菌絲濃度為應變數時之因子反應圖……...102
圖十六、由田口直交表L18(21x37) 設計所得結果以
多醣生成量為應變數時之因子反應圖.…..103
圖十七、由田口方法求得較高菌絲濃度之培養條件
(培養條件B)下,培養Ganoderma lucidum
CCRC36124 過程中,菌絲乾重及培養基中
pH值、葡萄糖及多醣含量變化…………..104
圖十八、由田口方法求得較高多醣生成量之培養條
件(培養條件C)下,培養 Ganoderma
lucidum CCRC36124 過程中,菌絲濃度及培
養基中pH值、總醣、葡萄糖及多醣含量變
化…………………………………………….105
圖十九、由田口直交表L18(21x37) 設計所得結果以
期望函數值為應變數時之因子反應圖.....106
圖二十、陡升路徑法求培養菌株CCRC36124較適培養
條件各階段反應圖…………………..……107
圖二十一、最適培養條件(培養條件D)下, 培養
Ganoderma lucidum CCRC36124 過程中,菌絲
濃度及培養基中pH值、總醣、葡萄糖及多醣
含量變化………………………………..…108
圖二十二、G. lucidum CCRC36124 於培養條件D下,
第四天 ( a ) 、 第五天 ( b )及第七 天
( c ) 菌團之形態,放大倍率40X………109
圖二十三、由不同實驗設計法所得之培養條件培養
菌株CCRC36124過程中菌絲濃度變化….110
圖二十四、由不同實驗設計法所得之培養條件培養
菌株CCRC36124過程中多醣含量變化….111
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