臺灣博碩士論文加值系統

白僵蠶(Bombyx batryticatus)為蠶蛾科昆蟲(Bombyx mori L.)的幼蟲在未吐絲前感染白僵菌(Beauveria bassiana (Bals.) Vuill)後致死的乾燥蟲體，為傳统的中藥資源，已有千年以上歷史。白僵蠶的藥用目前仍十分廣泛，具有解毒、降膽固醇、抗驚厥、鎮静、祛痰等作用。白僵菌(Beauveria bassiana)為昆蟲寄生真菌，既是家蠶常見的病害，也被廣泛應用於害蟲生物防治。亦是當前世界上研究和應用最多的一種蟲生真菌，致病性和適應性都很強。本研究從不同店家所購買的白僵蠶中分離白僵菌菌株，再利用核糖体(rDNA)内轉錄間隔區(ITS)序列分析鑑定白僵菌，結果顯示市面上的白僵蠶，其品質不一，並非皆可分離到白僵菌，僅由其中一個樣品中，分離出白僵菌，菌數可達3.9x106 cfu/g，經ITS1、ITS2鑑定確定為白僵菌(Beauveria bassiana)。並探討培養基條件、營養物質以及不同碳氮比(C/N)對白僵菌生長、代謝及活性的影響。結果發現白僵菌僅對Aspergillus niger、Penicillium chrysogenum和Pseudomonas aeruginosa具有抑菌能力。而白僵菌甲醇萃取物具有較好的抗氧化能力，其清除DPPH自由基、亞鐵離子螯合能力、還原力以及總抗氧化能力呈現劑量依賴性(dose dependence)關係。以半定量之API ZYM系統分析白僵菌產生酵素的種類，結果顯示不管培養於任一培養基中，皆產生高acid phosphatase、phosphoamidase、β-glucosidase及β-glucosaminidase活性。以最適發酵生產之條件放大至200L攪拌式發酵槽中，其發酵產物亦具有良好抗氧化能力。在藥理活性成分分析方面，經高效液相層析串聯式質譜儀(LC/MS/MS)檢測出含有具特定療效之兩種產物imipramine和pergolide。另經急毒性試驗測試，分別口服投予 Sprague-Dawley (SD)大鼠單一劑量2 g/kg之試驗物質後，發現試驗期間皆未有不良反應。

Bombyx batryticatus, an oriental crude drug, has been frequently used in China for thousands of years. The silkworm larva, Bombyx mori L. is mummified often being in infected by Beauveria bassiana during the larval growth stages. In traditional Chinese medicine, B. batryticatus is widely used clinically in treating detoxification, cholesterol reducing, anticonvulsion, composure, and phlegm eliminating. Several studies indicated that the pharmacological effects of B. batryticatus are closely related with its active ingredients. Beauveria species also have been widely used as biological control agents against agricultural and forestry pests. In this study, B. bassiana strain isolated from one of three samples of B. batryticatus, and identified with nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS-5.8S-ITS2) sequences analysis. The results showed that some white B. batryticatus chinese medicine without life B. bassiana, so the isolation and identification technology can be used as quality reference of the herbal medicines B. batryticatus. The C/N ratio of cultural medium effecting on B. bassiana growth, antibacterial ability antioxidant activity extracellular and intracellular enzymes synthesis and metabolites pattern was explored. Results showed that B. bassiana could inhibit Aspergillus niger, Penicillium chrysogenum and Pseudomonas aeruginosa growth. Methanol extract of B. bassiana has good antioxidant capacity including the DPPH radical scavenging, ferrous ion chelating ability, reducing power and total antioxidant capacity. The semi-quantitative API ZYM system analysis showed that B. bassiana could produce strong activities of acid phosphatase, phosphoamidase, β-glucosidase and β-glucosaminidase in all medium. In B. bassiana fermentation products, there were two pharmacologically active metabolites, imipramine and pergolide, have been detected by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS). The cultured whole broth from 200L fermentor also has been proved with good antioxidant capacity and with active compounds imipramine and pergolide. Acute toxicity test showed no adverse reactions after oral administration of Sprague-Dawley (SD) rats at single dose of 2 g/kg.

內容 頁次
致謝…………………………………………………………………………………..……Ⅰ
目錄………………………………………………………………………………………..Ⅱ
圖目錄……………………………………………………………………………………..Ⅹ
表目錄…………………………………………………………………………..………ⅩⅣ
中文提要 …………………………………………………………………………....…ⅩⅥ
英文提要 …………………………………………………………………………..…..ⅩⅧ
壹、前言..…………………………………………………………….…………………….1
一、緒論……………………………………………………………….……………….….1
二、蟲生真菌…………………………………………………………………………..….2
三、白僵蠶……………………………………………………………………….……….3
(一)形態與特徵………………………………………………………………….……….3
(二)感染過程…………………………………………………………………….……….3
(三)古籍記載…………………………………………………………………….……….4
(四)製備及食用方式…………………………………………………………….……….5
(五)白僵菌的分類……………………………………………………………..…………6
1.白僵菌屬產孢方式……………………………………………………..……….…7
2.白僵菌之菌落特徵……………………………………………………..………….7
3.白僵菌之化學成分………………………………………………………………...7
4.白僵菌之相關應用……………………………………………………..……….…9
5.白僵菌之藥理作用…………………………………………………..…….….......11
四、DNA分析…………………………………………………………….…...…….......13
五、抗氧化性質………………………………………………………………….............16
六、生物活性……………………………………………………….……………........…18
七、白僵菌具藥理活性的代謝產物………………………………….………………....21
(一) Imipramine…………………………..………………………………….………......21
1.作用機制………………………………………………………...….......................21
2.抗憂鬱劑的使用…………………………………………………………..……...22
(二) Pergolide…………………………………………………………………….…......24
貳、材料與方法………………………………………………………………….……….28
一、白僵菌菌種培養…………………………………………………………….……....28
(一)實驗菌株…………………………………………………………………….………28
(二)平面培養基配製……………………………………………………………..……...29
(三)液態發酵(搖瓶)培養…………………………………………………….………….29
(四)篩菌……………………………………………………………………….…………29
二、白僵菌之DNA鑑定……………………………………………………….……….31
(一)實驗材料…………………………………………………………………….………31
(二)實驗方法…………………………………………………….…………………........31
1.真菌DNA之抽取……………….………………………………….…..………...31
2. ITS rDNA PCR…………………………………………………….……………..32
3. DNA 定序……………………………………………………….…..………..….32
4.基因序列之比對分析方法……………….………………………..………….…..32
三、最適化培養條件篩選………………………….…………………………………….33
(一)不同溫度對白僵菌生長的影響………………………………………….…….….. 33
(二)不同碳源對白僵菌生長的影響…………………………….……….………….…..33
(三)不同氮源對白僵菌生長的影響…………………………….…….…………….…..33
(四)不同碳氮比例(C/N)對白僵菌生長的影響…………….……………….…….…….34
(五)物理性質測定………………………………………………………….………….. 34
1.體積變化-菌絲體乾重……………………………….…………….…………….34
2. pH值測定…………………………………………….……….…………………34
四、白僵蠶培養液抑菌性之測定………………………………….…….……………..36
(一)實驗材料…………………………………………………….…………….………..36
1.抑菌材料…………………………………………………………….………........36
2.供試菌株………………………………………………………….………………36
3.試驗菌株之製備…………………………………………….………….………...36
（二）抑菌試驗……………………………………………………………….…….….36
五、抗氧化能力之分析………………………………………………………………....37
(一)實驗材料-白僵菌甲醇萃取物製備………………………………...……………....37
(二)抗氧化能力測定……………………………………………………….......……......37
1.清除DPPH (1, 1-diphenyl – 2-pichrylhydrazyl)自由基能力………...…………..37
2.還原力測定(Reducing power)………………………………………….......……..38
3.總抗氧化能力測定(Trolox equivalent antioxidant capacity, TEAC)………….....38
4.總酚類化合物測定(Total phenols)……………….………………………………39
5.亞鐵離子螯合能力(Chelating ability on fewous ions)………………….………..39
六、真菌菌株鑑定-API ZYM………………………………………………………..….40
(一)菌落挑選………………………………………………………………......…….…..40
(二) API ZYM操作……………………………………………………………..……...40
七、白僵菌之成分分析………………………………………………………..…….…..43
(一)高效能液相層析儀(HPLC)分析……………………………………...…………….43
1.不同店家白僵蠶粉之樣品……………………………………………..…………43
2.不同碳氮比例(C/N)的白僵菌培養液…………………………………..…...…...43
(二)高效液相層析串聯式質譜儀(liquid chromatography-mass spectrometry)分析…………………………………………………………...……………..…….….44
八、白僵菌於200L發酵槽培養試驗…………………..……………………………….46
(一)菌絲體乾重………………………………………….………………………..…......46
(二) pH值測定………………………………………………………………….…...…..46
(三)殘糖量……………………………………………………….……………….…...…46
(四) HPLC分析…………………………………………………….…….………...……47
九、口服急毒性試驗…………………………………………………………………..…48
參、結果與討論…………………………………………………………..…….……..….50
一、白僵菌分離與鑑定………………………………………………………..……..… 50
二、最適化培養條件…………………………………………………………..…...……57
(一)不同溫度對白僵菌生長的影響…………………………………………….....……57
(二)不同碳源物質對白僵菌生長的影響……………………………………..……...…59
(三)不同氮源物質對白僵菌生長的影響………………………….……….…………...59
(四) 不同碳氮比例(C/N)對白僵菌生長的影響……………………………….………60
三、不同碳氮比(C/N)之抑菌性試驗……………………………………….…………..75
四、不同碳氮比(C/N)之抗氧化能力………………………………….………………..82
(一)清除自由基DPPH能力測定………………………………….…………………...82
(二)總抗氧化能力測定……………………………………………….………………...82
(三)亞鐵離子螯合能力測定………………………………………….………………...83
(四)還原力測定…………………………………………………….…………………...84
五、不同碳氮比(C/N)對API ZYM系統測試結果………………………………….....90
六、白僵菌之活性成分分析結果…………………………………………….…………95
(一)不同碳氮比(C/N)培養基對所產生代謝物成分之比較…………………………....95
(二)不同店家之白僵蠶蟲體成分分析…………………………………………….……95
七、活性成分pergolide與imipramone的分析…………………………………..……99
八、200L發酵槽試量產………………………………………………………..........…104
(一)預實驗…………………………………………………………………...……....…104
(二) 200 L試量產………………………………………………………………...…….104
(二)抗氧化能力…………………………………………………...…………………....105
1.清除自由基DPPH能力測定…………………………………………………...105
2.總抗氧化能力測定……………………………………………...……………….106
3.亞鐵離子螯合能力測定……………………………………………..….……….106
4.還原力測定……………………………………………………….…..………….107
(三)代謝成分分析……………………………………………………………….……..107
(四)活性成分pergolide與imipramine的分析………………………..…..……….….108
九、口服急性毒性試驗結果……………………………………….……………………117
(一)大鼠單一極限劑量急性毒性測試結果………..………………...………………..117
(二)肉眼檢查及組織病理學檢查結果……………………………...…………..…… .117
1.腎臟……………………………………………………………………..………..117
2.肝臟…………………………………………………………………..…………..118
肆、結論…………………………………………………………………..………………125
伍、參考文獻…………………………………………………………..…………………127

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