臺灣博碩士論文加值系統

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

水野卓、合川正允，賴慶亮譯(1997) 菇類的化學、生化學。國立編譯館，台北。
王次男(1990)寄生澳洲胡桃之三種靈芝及其防治。台灣糖業研究所研究彙報第129號:1-10。
王伯徹(2000)具開發潛力食藥用菇介紹。食品工業32(5):1~16。
王青牡、孫姝媛(1997) 我國液體培養藥用虫草菌絲體的研究進展。食用菌，19(3)2-4。
王伯徹、陳啟楨、華傑(1998) 食藥用菇類的培養應用。食品工業研究所，新竹。
江國瑛、張欣暘、許垤棋、段國仁(1997) 利用深層醱酵法生產靈芝多醣的研究。第二屆生化工程研討會論文集139-142。
朱俊忠、林俊一(1994) 以實驗計畫探討製糖濾泥吸收二氧化硫之最適操作條件。技術學刊9(2):181-184。
沈明來(1999) 試驗設計學。九州圖書文物有限公司，台北市。
李時珍(1933) 本草綱目。第二十八卷，商務印書館。
李友錚(1993) 田口式品質工程。中華民國品質工程管制協會，台北市。
李明彥(1990) 松杉靈芝浸漬發酵的培養條件對產物的影響。台灣大學農業化學研究所碩士論文。
李泰興(1998) 氧氣傳送介質及消泡劑對模擬醱酵液中氧氣傳送之影響。技術學刊13(1):103-108。
李輝煌(2000)田口方法:品質設計的原理與實務。高立圖書有限公司，台北市。
吳全耀、吳黃素月、趙熹、鄭家昌(1994) 田口式品質工程在食品加工上的應用-文蛤軟糖的製造。中國水產月刊493:29-40。
吳定、劉常金(1999) 香菇液態深層發酵工藝研究。食品科學6:46~47。
呂金河、任眉眉、嵇允嬋、溫敏杰(1996) 實驗計畫在休閒食品品質提升的研究與應用。行政院國家科學委員會專題研究計畫成果報告NSC85-2121-M006-023。
林志彬(1992) 靈芝多醣的免疫藥理研究及其意義。北京醫科大學學報，24(4):271~274。
林志彬(1996) 靈芝的現代研究、特性、栽培、成份、藥理、應用。北京醫科大學、中國協和醫科大學聯合出版社，北京。212頁。
林聖哲(1990) 靈芝在深層培養之研究。國立中興大學碩士論文。
官振儀、陳漢銘、楊芳鏘(1998) 利用酒糟水於靈芝液態培養之研究。第三屆生化工程研討會論文集15-18。
洪哲穎(1999) 最適化實驗設計法。工業研究院化學工業研究所，新竹。
洪哲穎、 陳國誠(1992) 回應曲面實驗設計法在微生物酵素生產上之應 用。 化工， 39(2):3~18。
洪連發(1997) 以回應曲面法探討微胞強化超過濾技術純 化酪蛋白磷胜月太 之最佳條件。國立台灣大學化學工程學研究所碩士論文。
高益槐(2000) 世紀奇草話靈芝。元氣齋出版社，台北市。
徐新春(1997) 不同栽培因素對靈芝化學成份含量之影響。時珍國藥研究18(5):465~466。
徐敬衡、劉勝宇、張志強、文榮輝、陳怡倩、周柏甫(2000)探討培養溫度對巴西蘑菇多醣體生產之影響。第五屆生化工程研討會論文集529~532。
許瑞祥(1988) 靈芝的奧秘。正義出版社，台北市。
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。
張季娜、羅仕勇、宋振晶、高述崙、蔡彰文、陳世漣、莊泰旭、邱鎮宏(1989) 田口式品質工程導論。科建管理顧問公司，台北市。
張東柱(1983) 台灣數種靈芝生物學上之研究。國立台灣大學植病所碩士論文。
陳和賢、黃卓治、謝政峰、王志源(2000) 應用田口品質工程於蛋黃酥桿捲製程改進之研究。台灣農業化學與食品科學38(3):214-222。
陳順宇、馬瀰嘉、潘淅楠、黃銘欽(1996) 影響麵粉糊化特性之重要因素分析並探討其成品間關係。行政院國家科學委員會專題研究計畫成果報告NCS85-2121-M006-022。
陳怡宏、蘇女淳、葉錦桐、程竹青(1995) 仙草膠原之萃取與純化研究。食品工業研究所報告C009422。
陸文梁，林忠平，林志彬(1993) 靈芝的科學應用。科學出版社，北京。
梁志欽(1991) 松杉靈芝浸漬發酵培養生產的1,3-βglucanase對菌體外多醣的影響。國立台灣大學農業化學研究所碩士論文。
楊革(1997) 靈芝菌絲體深層培養及多糖提取工藝研究。食用菌19(2)8-9。
楊芳鏘(1996) 氣舉式生化反應器在靈芝液態培養之應用。行政院國家科學委員會專題研究計畫成果報告NSC85-2214-E029-004。
雍仲淳(1625)神農本草經。
游英欽(1996) 以搖瓶振盪及小型發酵槽培養，探討培養基組成及物理化學因子，對靈芝多醣生長形態變化的影響。國立交通大學生物科技研究所碩士論文。
黃麗娜(1998) 菇類菌絲體深層培養在食品工業上之應用。食藥用菇類的培養與應用:144~150，食品工業研究所出版，新竹。
鄭燕琴(1993) 田口式品質工程技術理論與實務。中華民國品質管制學會，台北。
趙繼鼎(1989) 中國靈芝新編。科學出版社，北京。
劉永銓、陳光宇(1998) 錐形瓶中氣液質傳效率之探討。第三屆生化工程研討會論文集171-174。
劉克琪(1994) 實驗設計與田口式品質工程。華泰書局，台北市。
謝建元(2000)靈芝的功效和栽培方法。生物資源 生物技術2(2):18-28.
賴敏男(2001)靈芝與冬蟲夏草，生物資源 生物技術3(1):8-12.
賴滋漢、 賴業超(1988)食品科技辭典 。精華出版社， 台中市。
黎中正(1993)穩健化設計之品質工程。台北圖書有限公司，台北市。
黎中正(1998) 實驗設與分析。高立圖書有限公司，台北市。
潘素美、葉至新(1997) 台灣食用藥用菇超氧歧化酵素含量之比較。中國農業化學會誌35(2):209~219。
韓紹英、趙傳孝、姜彥祥、龔國華(1989) 食用菌高產栽培及科學加工。中國食品出版社，北京。
蘇慶華(1991) 靈芝之分類學及生理活性物質。北醫學報20:1~16。
關洪昌、叢靜靜(1981) 靈芝多醣D6對核酸、蛋白質合成的影響及其初步分析。北京醫學院學報13(4)261~263。
Adaskaveg, J. E. and Gilbertson, R. L. (1986) Cultural studies and genetics of sexuality of Ganoderma lucidum and G. tsugae in relation to the taxonomy of the G. lucidum complex. Mycologia 78:694~705.
Adaskaveg, J. E. and Gilbertson, R. L. (1987) Infection and colonization of grapevines by G. lucidum. Plant Disease 71:251~253.
Ainsworth, G. C., Sparrow, F. K., and Sussman, A. S. (1973)The Fungi. Vol. IVA. A Taxonomic Review with keys:Ascomycetes and Fungi Imperfecti. Academic Press, Inc. New York pp. 621.
Billot P. and Pitard B. (1992) Taguchi design experiments for optimizing the gas chromatographic analysis of residual solvents in bulk pharmaceuticals. J. Chromatogr., 623:305-313.
Box, G. E. P. and Wilson, K. B.(1951) On the experimental attainment optimum conditions. J. Roy. Statist. Soc. B13:1-45.
Burch, G. J., Ferguson, C. H., Cartwright, G. and Kwong, F. Y.(1995) Application of Taguchi experimental design to the optimization of a baculovirus expression system. Biochem. Soc. Trans.23(1), 107S.
Caetano, A.(1998) Optimization using Taguchi methods and the effect of thermal cycling parameters on DNA amplification. Biotechniques, 25(3):472-6, 478-80.
Chen, C. Y. and Ling, Y. C.(1992) Optimizing electron-capture detector performance for gas chromatographic analysis of polychlorinated biphenyls. Chromatogr.,33:272-278.
Chen, W. C., Hau, D. M., Wang, C. C., Lin, I. H. and Lee, S. S.(1995) Effects of Ganoderma lucidum and Krestin on subset T-cell in spleen of irradiated mice. Am. J. Chin. Med. 23:289-298.
Chen, C. Y. and Ling, Y. C. (1994) Optimizing the gas chromatographic separation and detection of polychlorinated biphenyls by use of electronic pressure pressure programming and experimental design. J. High Resol. Chromatogr, 33:272-278.
Cobb, B. D. and Clarkson, J. M. (1994) A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acid Res., 22(18):3801-3805.
Corner, E. J. H. (1983) Ad Polyporaceas 1. Amauroderma and Ganoderma. Nova Hedwigia Beih. 75:1~182.
Derringer, G. and Suich, R.(1980) Simultaneous optimization of several response variables. J. Qual. Technol.12(4):241~219.
Dubois, M., Gilles, K. A.,Hamilton, J. K.,Rebers, P. A. and Smith, F.(1956) Colorimetric method for determination of sugars and related substances. Anal. Chem.28(3):350-356.
El-Mekkawy S., Meselhy M. R., Nakamura N.,Tezuka, Y.,Hattori, M., Kakiuchi, N.,Shimotohno, K., Kawahata, T., and Otake, T.（1998）Anti-HIV-1 and anti-HIV-1-protease substances from Ganoderma lucidum. Phytochemistry. 49:1651-1657.
Eo, S. K., Kim, Y. S., Lee, C. K., and Han, S. S.（2000）Possible mode of antiviral activity of acidic protein bound polysaccharide isolated from Ganoderma lucidum on herpes simplex viruses. J. Ethnopharmacol. 72(3):475 - 481.
Gan, K. H., Fann, Y. F., Hsu, S. H., Kuo, K. W., and Lin, C. N.
（1998）Mediation of the cytotoxicity of lanostanoids and steroids of Ganoderma tsugae through apoptosis and cell cycle. J .Nat. Prod. 61:485-487.
Giovanni, M.(1983) Response surface methodolgy and product optimization. Food Technology 11:41.
Glen, S. P. (1993) " Taugchi Methods ", Addison-Wesley Publishing Company, Massachusetts. pp.113~337.
Haak-Frendscho, M., K., Sone, T. and Jardieu, P. (1993) Lingzhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol.150(1):101-113.
Hijikata, Y. and Yamada, S.（1998）Effect of Ganoderma lucidum on postherpetic neuralgia. Am. J. Chin. Med.26:375-381.
Hikino, H., Konno, C., Mirin, Y., and Hayashi, T. (1985) Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies. Planta Medica. 4:339~340.
Hiroshi, K., Wakako, T., Kiyoe, S., Michko, F., Yuko, H., Kazuo, Y., Yasuo, K., Hideo, N., Shigemasa, I., and Masaru, U. (1985) The biologically activeconstituents of Ganoderma lucidum (FR.) Karst. Histamine release-inhibitory tritepenes. Chem. Parm. Bull. 33(4):1367~1374.
Hirohide, M., Naohito, O., Iwo, S., and Toshiro,Y. (1985) Purification, antitumor activity, and structural characterzation of β-1,3-Glucan from Peziza vesiculosa. Chem. Pharm. Bull. 33(11):5096~5099.
Hseu R. S., Wang H. H., Wang H. F. and Moncalvo J. M.(1996) Differention and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences. Appl. Environ. Microbio., 62(4):1354-1363.
Hu, M., Guan, D., Li, C. and Dai, G. (1998) Report on a first trial that cancer mice''s life was prolonged by crude extract from from mycelium of Korea Ganoderma. 98'' Nanjing International Symposium on Science & Cultivation of Mushrooms. Nanjning,China.
Janusz, M. J., Austen K. F. and Czop J. K.(1989)Isolation of a yeast heptaglucoside that inhibets monocyte phagocytosis of zymosan particles. J. Immunol. 142:959-965.
Jeney, C., Dobay, O., Lengyel, A., Ádám, É. and Nász, I. (1999) Taguchi optimisation of ELISA procedures. J. Immunol. Methods. 223(2):137-146.
Jong, S. C. and Birmingham, J. M. (1992) Medicinal benefits of the mushroom Ganoderma. Adv. Appl. Microbiol. 37:101~134.
Kabir, Y., Yamaguchi, M., and Kimura, S. (1987) Effect of shiitake(Lentinus edodes)and maitake (Grifola frondosa)mushrooms on blood pressure and plasma lipids of spontaneously hypertensive rats. J. Nutr. Sci. Vitaminol. 33:341~346.
Kabir, Y., Kimura, S., and Tamura, T (1988) Dietary effect of Ganoderma lucidum mushroom on blood pressure and lipid levels in spontanecusly hypertensive rats (SHR). J. Nutr. Sci. Vitaminol. J. Nutr. Sci. Vitaminol. 33:433~438.
Kabir, Y. and Kimura, S. (1989) Dietary mushroom reduce blood pressure in spontaneously hypertensive rats (SHR). J. Nutr. Sci. Vitaminol. 35:91~94.
Kawagishi, H., Mitsunaga, S., Yamawaki, M., Ido, M., Shimada, A.., Kinoshita, T. Murata, T., Usui, T., Kimura, A., and Chiba, S. (1997) A lectin from mycelia of the fungus Ganoderma lucidum. Phytochemistry.44:7-10.
Kim, K. C. and Kim, I. G. ( 1999 ) Ganoderma lucidum extract protects DNA from strand breakage caused by hydroxyl radical and UV irradiation. Int. J. Mol. Med. 4:273-277.
Kim, D. H., Shim, S. B., Kim, N. J., and Jang, I. S.（1999）Beta-glucuronidase-inhibitory activity and hepatoprotective effect of Ganoderma lucidum. Biol. Pharm. Bull. 22:162-164.
Kim, Y., S., Eo, S. K., Oh, K. W., Lee, C. K., and Han, S. S.（2000）Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with interferons . J. Ethnopharmacol. 72(3):451-458.
Kino, K., Yamsshita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., and Tsunoo, H.,(1989) Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8(lZ-8), from Ganoderma lucidum. J. Biol. Chem. 264,472-478.
Kino, K., K. Mizumoto, Sone, T., Yamaoka, J.,Yamashita, K., Watanabe, A. and Tsunoo, H.,(1990) An immunomodulatory protien, Ling Zhi-8, prevents insulitis in non-obese diabetic mice. Diabetologia. 33, 713-720.
Kohda, H., Tokumoto, W, Sakamoto, k., Fujii, M., Hirai, Y., Yamasaki, k., Komoda, Y., Nakamura, H., Ishihara, S., and Uchida, M. (1985) The biologically active constiturents of Ganoderma lucidum (Fr.) Histamine release-inhibitory triterpenes, Chem. Pharm. Bull. 33:1367~1374.
Komoda, Y., Shimizu, M., Sonoda, Y. and Sato, Y. (1985) Ganoderic acid and its derivatives as cholesterol synthesis inhibitors. Chem. Pharm. Bull. 33:1367-1374.
Koyama, K., Imaizumi, T., Akiba, M., Kinoshita, K.,Takahashi,K., Suzuki, A..,Yano,S. Horie, S., Watanabe, K., and Naoi, Y. (1997) Antinociceptive components of Ganoderma lucidum. Planta Med. 63:224-227.
Lee, S. S., Wei, Y. H., Chen, C. F., Wang, S. Y., and Chen, K. Y. (1995) Antitumor effects of Ganoderma lucidum. J. Chin. Med. 6(1):1~12.
Liang, Z.（1995）Isolation, Purification and Structural Comparison Investigation of Water Soluble Polysaccharide GFb of Fermented Mycelia From Ganoderma tsugae. Chinese Sci. Abs. 14(1):40.
Lin, C. N., Tome, W. P. and Won, S. J. (1991) Novel cytotoxic principles of Formosan Ganoderma lucidum. J. Nat. Prod. 54(4):998~1002.
Lina, J. M., Lina, C.C., Chenb, M. F., Takashic, U., and Atsushid, T.（1995）Radical scavenger and antihepatotoxic activity of Ganoderma formosanum, Ganoderma lucidum and Ganoderma neo-japonicum. J. Ethnopharmacol. 47(1):33-41.
Litchfield, J. H. (1979) Production of single cell protein for use in food and feed. In: Microbial Technology, 2nd ed. (Peppier, H.J. and Perlman, D., eds):93~145,Academic Press, New York.
Liu, F., Ooi, V. E. C. and Chang, S. T. (1997) Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci. 60:763-771.
Luo, J., Huang, R., Chen, L., Li, H. and Z, Z. (1998) Fermentation of Ganoderma lucidum and studies on polysaccharides. 98'' Nanjing International Symposium on Science & Cultivation of Mushrooms. Nanjning, China.
Min, B. S., Nakamura, N., Miyashiro, H., Bae, K. W. and Hattori, M.（1998）Triterpenes from the spores of Ganoderma lucidum and their inhibitory activity against HIV-1 protease. Chem. Pharm. Bull. (Tokyo), 46:1607-1612.
Miyazaki, T., and Nishijima, M. (1981) Studies on fungal polysaccharides. XXVII. Structural examination of a water-soluble, antitumor polysaccharide of Ganoderma lucidum. Chem. Pharm. Bull. 29(12):3611-6.
Mizuno, T. (1995) Bioactive bioactive bimolecules of mshrooms: Food function and medicinal effect of mushroom fungi. Food Rev. Int. 11:7~21.
Morgiwa, A., Kitabatake, K., Fujimoto, Y., and Ikekawa, N. (1986) Angiotensin converting enzyme-inhibitory triterpenes from Ganoderma lucidum. Chem. Pharm. Bull. 34(7):3025~8.
Oh, K. W., Lee, C. K., Kim, Y. S., Eo, S. K., and Han, S. S.（2000）Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with acyclovir and vidarabine. J. Ethnopharmacol. 72(1-2):221 - 227.
Pan, J., Cao, X., Li, F., Zuo, Z., Sun, Q. and Wang, L. (1996) Studies on the nutritional requirement of Ganoderma lucidum in submerged culture. Acta Edulis Fungi 3(4):31-34.
Pan, S. M., Ye, J. S., and Hseu, R. S.（1997）Purification and characterization of manganese superoxide dismutase from Ganoderma microsporum. Biochem. Mol. Biol. Int.42:1035-1043.
Park, E. J.,Ko, G., Kim. J., and Sohn, D. H. (1997) Antifibrotic effects of a polysaccharide extracted from Ganoderma lucidum, glycyrrhizin, and pentoxifylline in rats with cirrhosis induced by biliary obstruction. Biol. Pharm. Bull. 20:417-420.
Ryverdan L. (1995) Can we trust morphology in Ganoderma ? Proceedings of contributed symposium 59A. B, IMC5. Vancouver, August 14-21, 1994, 19-24.
Stasinopoulos, S. J., Seviour, R. J. (1990) Stimulation of exopolysacch avide production in the fungus Acremonium persicinum with fatty acids. Biotech. and Bioeng. 36:778-782.
Stejskal, J. and Potucek, F. (1985) Oxygen transfer in liquids. Biotech. and Bioeng. 27:503-508.
Sone, Y., Okuda, R., Wada, N., Kishida, E. and Misaki, A. (1985) Structure and antitumor activites of the polysacchary isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric. Biol. Chem.49(9):2641~2653.
Su, C. H., Sun, C. S., Juan, S.W., Hu, C. H., Ke, W. T. and Sheu, M. T. (1997) Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes. Biomaterials. 18(17):1169~1174.
Su, C. H., Sun, C. S., Juan, S. W., Hu, C. H., Ke, W. T., and Sheu,M. T. (1999) Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes. Biomaterials (incorporating Clinical Materials) 18(17):1169-1174.
Su, C. Y., Shiao, M. S., and Wang, C. T.（2000） Potentiation of ganodermic acid S on prostaglandin E1-induced cyclic AMP elevation in human platelets. Thromb. Res. 99(2):135-145.
Su, C. Y., Shiao, M. S., Wang, C. T.（1999）Differential effects of ganodermic acid S on the thromboxane A2-signaling pathways in human platelets. Biochem. Pharmacol. 58:587-595.
Su, C. H., Sun, C. S., Juan, S. W., Ho, H. O. Hu, C. H. and Sheu, M. T.（1999）Development of fungal mycelia as skin substitutes: Effects on wound healing and fibroblast. Biomaterials 20:61-68.
Taguchi, G. (1990) Introduction to Quality Engineering. Asian Productivity Organization, Tokyo.
Toth, J. O., Liu, B., Ourisson, G. (1983) Ganoderic acid T to Z : Cytototoxic triterpenes from Ganoderma lucidum. Tetrahedron lett. 24:1081~1084.
Tseng, T. C., Shiao, M. S., Shieh, Y. S. and Hao, Y. Y. (1984) Study on Ganoderma lucidum 1. Liquid culture and chemical composition of mycelium. Bot. Bull. Academia Sinica 25:149-157.
Tunga, R., Banerjii, R. and Bhattacharyya, B. C.(1999) Optimization of variable biological experiments by evolutionary operation-fractional design technique. J. Biosci. Bioeng.87(2):224-230.
van der Hem, L. G., van der Vliet, J. A., Bocken, C. F., Kino, K.,Hoitsma, A. J. and Tax, W. J. (1995) Ling Zhi-8: studies of a new immunomodulating agent. Transplantation. 60:438-443.
Wang, C. N., Chen, J. S., Shiao, M. S. and Wang, C. T. (1994) Activation of human platelet phospholipases C and A2 by various oxygenated triterpenes. Eur. J. Pharmacolo. 267(1):33~42.
Wang, S. Y., Hsu., M, L., Hsu, H. C., Tzeng, C. H., Lee, S. S., Shiao, M. S. and Ho, C. K. (1997) The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int. J . Cancer.70:699-705.
Won, S. J., Lin, M. T. and Wu, W. L. (1992) Ganoderma tsugae mycelium enhances splenic natural killer cell activity and serum interferon production in mice. Japan. J. Pharmacol. 59:171-176.
Yang, F. C., Ke, Y. F., and Kuo, S. S. （2000）Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures. Enzyme and Microbial Technology 27(3-5):295-301.
Yang, F. C., and Liau, C. B.（1998）The influence of
environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures. Process Biochem. 33(5):547-553.
Yen, G. C., and Wu, J. Y.（1999）Antioxidant and radical
scavenging properties of extracts from Ganoderma tsugae . Food Chem. 65(3):375-379.
Yoon, S. Y. Eo, S. K. Kim, Y. S. Lee, C. K. and Han, S. S.（1994）Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics. Arch. Pharm. Res. 17:438-442.
Zhang, K., and Howard, R. P. (1994) Influence of poly-saccharides on neutrophil function: specific antagonists suggest a model for cooperative saccharide-associated inhibition of immune complextriggered superoxide production. J. Cell. Biochem. 56:225~235.
Zhang, K., Li, P. and Huopo, X. R. (1998) Studies on the bioactive compounds produced by submerged fermentation of Ganoderma lucidum-polysaccharide. (I) 98'' Nanjng International Symposium on Science & Cultivation of Mushrooms. Nanjning, China.
Zhu, M., Chang, Q., Wong, K., Chong, F. S. and Li, R. C. (1999) Triterpene antioxidants from Ganoderma lucidum. Phytother. Res. 13:529~531.