臺灣博碩士論文加值系統

北蟲草(Cordyceps militaris )為一種昆蟲寄生真菌，主要分布在中國大陸的高山地區，因採集不易數量稀少，所以價格較為昂貴。北蟲草具有各種生物活性成分，包括多醣、腺苷、蟲草素和甘露醣醇等。且具有擴張氣管、鎮靜、抗心律失常、降血壓、抗病原微生物、抗惡性腫瘤等多種藥理活性。
本研究利用不同天數及不同濃度添加肌醇與D-果糖刺激北蟲草合成蟲草素，並分析其蟲草素含量之變化。研究結果顯示，蟲草素為二級代謝產物在第5天添加肌醇可促使北蟲草於培養後期有效利用而與腺苷進一步合成為蟲草素。在D-果糖組別中三個添加天數下皆為第10天添加組別最好，而在第10天已為培養後期可能是D-果糖容易被代謝，所以在添加後菌體可快速利用，而促使北蟲草在培養後期與腺苷合成為蟲草素。在不同濃度肌醇與D-果糖混合添加中，在培養中期此時菌體適應培養基利用營養物質進行生長而進一步進行溫度刺激使北蟲草進入二級代謝階段，而在培養後期營養物消耗殆盡的環境下，添加混合液對於菌體合成蟲草素有非常明顯的影響。

Cordyceps militaris is an insect parasitic fungus mainly distributed in the high mountains of mainland China. It is expensive because it is rare and difficult to collect from wild. Cordyceps militaris have various kinds of biologically active ingredient, include polysaccharide adenosine cordycepin mannose.It also have many Pharmacology activities, including dilated trachea, calm, antiarrhythmic, hypotensive, anti-pathogenic microorganism, anti-malignant tumor.
In this study, different days and different concentrations of inositol and D- fructose were added to stimulate cordycepin productin by Cordyceps militaris, and the changes of cordycepin content were analyzed. The results showed that cordycepin was a secondary metabolite. Adding inositol on the fifth day could promote the cordycepin synthesis with adenosine by C.militaris. In the D- fructose group, the D- fructose was easily metabolized at the later stage of culture. In the mixed addition of inositol and D-fructose at different concentrations, the growth of Cordyceps militaris was adapted to the medium at the middle stage of culture and further stimulated by temperature reduction to make Cordyceps militaris enter the secondary metabolic stage. In the environment of exhaustion of nutrients at the later stage of culture, the addition of mixed compounds was very effective for cordycepin synthesis by Cordyceps militaris.

摘要
致謝
目次………………………………………………………………………………..…..I
圖目錄………………………………………………………………………………..IV
表目錄………………………………………………………………………….….….V
第一章 緒論…………………………………………………..……………................1
1.1前言……………………………………………..…………………….…..….1
第二章 文獻探討………………………………………..…………………......…..…3
2.1 北蟲草簡介…………………………………………………………………….....3
2.1.1 北蟲草外型與分佈….....………………………...……………………..…3
2.2 北蟲草藥理分析……………………………...…………………………….....….4
2.2.1 北蟲草藥理活性………………………...………………………..……….4
2.2.2 降低血糖………………………………………………………………..…4
2.2.3 抗癌、抗腫瘤作用……………………………………………………..…5
2.2.4 抗氧化、抗衰老作用………………………………………..……………6
2.2.5 中樞神經系統作用…………………………..……………………………6
2.2.6 呼吸系統作用………………………………..……………………………7
2.2.7 毒性作用研究………………………………..……………………………7
2.2.8 北蟲草之生理活性成分……………………………..……………………7
2.3 北蟲草成分分析……………………...........………………….……………….....9
2.3.1 蟲草素………………………...……….……………………….……….....9
2.3.2 甘露醇………………………………………...….……………………....10
2.3.3 核苷類………………………………………...….……………….….…..11
2.3.4 腺苷……………………………………………...….………….……..….11
2.3.5 蟲草多醣體………………………...….……………………….……..….12
2.3.6 糖醇甾醇類………………………...….……………………….……..….13
2.4 北蟲草之應用…………………………………...…….…………..………..…...14
2.4.1 北蟲草之應用……...……………………………...………………...…...14
2.4.2 做為食品直接食用………………………………………………………14
2.4.3 研製成藥品………………............………………………................……14
2.4.4 功能性保健食品………………............…………………................……14
2.5 北蟲草液態醱酵培養………………...………………….……………………...15
2.5.1北蟲草人工培養………………...………………….……………………..15
2.5.2 北蟲草添加微量元素………………….....……………………………...17
2.5.3 接種量與培養基容量……………………...……………….………........17
2.5.4 酸鹼值與溫度…………………………….....……………….………......18
2.5.5 北蟲草液態菌絲培養基………...……………..……………...................19
2.6 肌醇………...……………..………..…........................................................22
2.7 D-果糖...……………..………………………………………………...……23
第三章 研究動機與目的…………………...……………………………………….24
第四章 實驗材料與方法…………………………………………………………....25
4.1 實驗架構…………………………..……………………………………….25
4.2 實驗材料…………………......………………………………………….....26
4.2.1真菌菌種、培養基……………………………....………………...…........26
4.2.2樣品代號……………………………………………..….…....……….......26
4.2.3實驗藥品………………...………………………………...……………....28
4.2.4實驗設備………………...………………………………...……………....29
4.3 實驗方法…………………………….......…………………………..………......30
4.3.1 北蟲草平盤培養…………………...………………………….................30
4.3.2 北蟲草液態醱酵培養…………………………………...…………….....30
4.3.3北蟲草液態菌絲體萃取………………………………….………………30
4.3.4不同濃度肌醇於不同天數下添加對北蟲草蟲草素生成之影響..……...30
4.3.5不同濃度D-果糖於不同天數下添加對北蟲草蟲草素生成之影響.…...31
4.3.6前驅物複合溫度變化對蟲草素生成之影響………..…………………...31
4.3.7 高效液相層析儀分析……………………………...…………………….32
4.3.7.1成分分析………………………..…………………………………33
第五章 結果與討論………………………………………………………………....34
5.1北蟲草液態醱酵培養之生長曲線……………….…………….………......34
5.2比較不同濃度肌醇於不同天數下添加對北蟲草液態菌絲甲醇萃取蟲草素生成之蟲草素HPLC分析………………………………………………..……36
5.3比較不同濃度D-果糖於不同天數下添加對北蟲草液態菌絲甲醇萃取蟲草素生成之蟲草素HPLC分析…………………………………………………..43
5.4前驅物複合溫度變化對北蟲草液態菌絲甲醇萃取蟲草素生成之蟲草素HPLC分析…………………………………………………...…………………49
第六章 結論…………………………………………………………………………56
第七章 未來展望………………………………………………………....................57
第七章 參考文獻………………………………………………………....................58
 
圖目錄
圖2.1北蟲草子實體……………………………………..............................................3
圖2.2蟲草素結構圖.....................................................................................................10
圖2.3甘露醇結構圖.....................................................................................................10
圖5.1北蟲草液態醱酵生長曲線…….………………………………………….…...34
圖5.2蟲草素標準品….……………………………….……………………………...38
圖5.3培養12天之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜……...38
圖5.4第0天添加不同濃度肌醇之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)1%肌醇(B)3%肌醇(C)5%肌醇….………..…………………….…...39
圖5.5第5天添加不同濃度肌醇之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)1%肌醇(B)3%肌醇(C)5%肌醇….………..…………………….…...40
圖5.6第10天添加不同濃度肌醇之北蟲草液態醱酵培養菌絲體20%甲醇萃液HPLC圖譜(A)1%肌醇(B)3%肌醇(C)5%肌醇.………..….………..……………….41
圖5.7 北蟲草液態醱酵分別於培養後不同天數添加不同濃度肌醇於25℃、150rpm下培養12天其菌絲體萃取液之蟲草素HPLC波峰面積比值………………………42
圖5.8第0天添加不同濃度D-果糖之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)1%D-果糖(B)3%D-果糖(C)5%D-果糖……………………………...45
圖5.9第5天添加不同濃度D-果糖之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)1%D-果糖(B)3%D-果糖(C)5%D-果糖……………………………...46
圖5.10第10天添加不同濃度D-果糖之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)1%D-果糖(B)3%D-果糖(C)5%D-果糖…………………………...47
圖5.11北蟲草液態醱酵分別於培養後不同天數添加不同濃度D-果糖於25℃、150rpm下培養12天其菌絲體萃取液之蟲草素HPLC波峰面積比值…………..….48
圖5.12培養12天之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜….…51
圖5.13第0天添加不同濃度肌醇與D-果糖混合液之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)M1D1(B)M1D3(C)M1D5………………………….52
圖5.14第0天添加不同濃度肌醇與D-果糖混合液之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)M3D1(B)M3D3(C)M3D5…………………………53
圖5.15第0天添加不同濃度肌醇與D-果糖混合液之北蟲草液態醱酵培養菌絲體20%甲醇萃取液HPLC圖譜(A)M5D1(B)M5D3(C)M5D5…………………………54
圖5.16北蟲草液態醱酵分別於培養後不同天數添加不同濃度肌醇與D-果糖混合液於25℃、150rpm下培養5天後進行20℃、150rpm 溫度刺激12天其菌絲體萃取液之蟲草素HPLC波峰面積比值………………………………………………...55
 
表目錄
表2.2.5.1冬蟲夏草與北蟲草生理功能的比較…………….……………...…………6
表4.2.2.1北蟲草添加不同濃度肌醇(Myo-inositol)液態培養條件代號…………..26
表4.2.2.2.北蟲草添加不同濃度D-果糖(D-Fructose)液態培養條件代號…………26
表4.2.2.3北蟲草添加肌醇與D-果糖混合液態培養條件代號……………………27

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