近年來已有學者證實由蝦蟹外殼分離出來的幾丁質具有抗菌的活性，由於靈芝殘渣中亦含有豐富的幾丁質等成份，不僅可做為廢物利用提高產品價值，且靈芝的幾丁質在萃取過程中不需經如蝦蟹幾丁質的脫鈣製程，所以在取得上較為簡便。本實驗便在於測試靈芝殘渣中的剩餘物質是否也如甲殼類動物幾丁質般具有抑制細菌生長之活性。利用靈芝子實體廢渣中經純化後之SACCHACHITIN、SACCHACHITOSAN及SACCHAGLUCOSAMINE，並取chitin、chitosan、D-glucosamine、N-acetylglucosamine之標準品進行比對。除此之外，也將其與部分抗生素併用，以測試是否可與抗生素產生協同作用。初步發現，以D-glucosamine高壓滅菌處理後的抗菌效果最佳，對Bacillus subtilis、 Staphylococcus aureus、Escherichia coli、Pseudomonas aeruginosa及Methicillin-resistant Staphylococcus aureus (MRSA)均有強烈的抑菌效果。 N-acetylglucosamine的效果次之，而靈芝及試藥級的幾丁質(Chitin)及幾丁聚醣(Chitosan)則無顯著效果。在抗生素的協同作用測試上則發現，N-acetylglucosamine及由靈芝中萃取所得的SACCHACHITIN在不同的菌種上對不同的抗生素的確具有協同作用。將SACCHACHITIN水解後取得之SACCHAGLUCOSAMINE與市售glucosamine進行比對，TLC的結果發現， SACCHAGLUCOSAMINE中亦具有glucosamine的成分。再進行生長曲線的測定比對，也可以發現兩者皆有抑制細菌生長的效果。綜合以上之結果將為靈芝殘渣開發另一用途，並為抗生素濫用而造成抗性之問題，開拓另一解決之途徑。

In recent years , many reports described chitin isolated from outer skeleton of crustaceans possessed antimicrobial activity . In previous studies of our laboratory , fruiting bodies of Ganoderma was employed as other source of chitin which is found in the waste part , after the extraction of triterpenoids and polysaccharides . The waste residue of Ganoderma provided an ready material for chitin purification since it contains no calcium as in crab shell and can be easily fabricated into membrane for its filamentous property . The purpose of this study is aimed to study the chitin and its derivatives from Ganoderma for antibiotic activity . SACCHACHITIN (chitin from Ganoderma ) , SACCHACHITOSAN (chitosan from Ganoderma ) , SACCHAGLUCOSAMINE (glucosamine from Ganoderma ) , were used for antibacterial tests as well as the pure standards of chitin , chitosan , and glucosamine . The tests including paper disc liguid culture growth curve were employed with the addition of chitin and its derivatives . The synergistic effect combined with antibiotics was also investigated . The results indicated that the autoclaved glucosamine performed significant effect on the inhibition of bacterial growth in liguid culture in the strains of Bacillus subtitis , Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa and a methicillin-resistant Staphylococcus aureus(MRSA). N-acetylglucosamine also present antibacterial activity in a less effective degree , whereas SACCHACHITSAN , SACCHACHITIN , chitin and chitosan showed no antibacterial effect in a level from 0.01﹪to 0.5﹪in the medium . However , N-acetylglucosamine and SACCHACHITIN were found to be synergistic with several antibiotics against all the tested bacteria in a concentration ranging from 0.1﹪to 0.5﹪. The autoclaved SACCHAGLUCOSAMINE , the HCl hydrolysate of SACCHACHITOSAN showed only partial antibacterial effect to all the bacteria . The analysis of TLC , H-NMR and 13C-NMR could suggested that glucosamine or SACCHAGLUCOSAMINE transformed into oligomer through the process of autoclaving , that turned uneffective glucosamine monomer into bacterial static one . The study provided on addition use for application Ganoderma fruiting body .

致謝………………………………………………………………….i
中文摘要…………………………………………………………….iii
英文摘要……………………………………………………………..v
目錄………………………………………………………………….vii
圖次目錄……………………………………………………………..viii
表次目錄……………………………………………………………..xvi
附錄目錄……………………………………………………………..xvii
前言及文獻探討………………………………………………………1
實驗材料與方法………………………………………………………12
實驗流程……………………………………………………………….15
結果與討論…………………………………………………………….21
結論…………………………………………………………………… 30
參考資料 ……………………………………………………………100
圖次目錄
Fig.1a：Escherichia coli (11758)中N-acetylglucosamine、chitin、chitosan、SACCHACHITIN、D-glucosamine的菌落生長比較……………………………………………………………33
Fig.1b：Bacillus subtilis (10058)中N-acetylglucosamine、chitin、chitosan、SACCHACHITIN、D-glucosamine的菌落生長比較…………………………………………………………….34
Fig.2a：Bacillus subtilis (10058) growth curve…………………………35
Fig.2b：Escherichia coli (11758) growth curve…………………………36
Fig.2c：MRSA growth curve……………………………………….…37
Fig.2d：Pseudomonas aeruginosa (10733) growth curve………………38
Fig.2e：Staphylococcus aureus (11863) growth curve…………………39
Fig.3a：Pseudomonas aeruginosa (10733) growth curve………………40
Fig.3b：MRSA growth curve……………………………………….…41
Fig.4 ：Staphylococcus aureus (11863)培養基成分間與D-glucosamine抑菌效果的關係，及D-glucosamine高壓溼熱處理前後，不同濃度間的比較…………………………………………………..42
Fig.5：D-glucosamine的抗菌活性……………………………………43
Fig.6：D-glucosamine經高壓溼熱處理前後之比較…………………44
Fig.7：D-glucosamine與SACCHAGLUCOSAMINE的比較……….44
Fig.8a：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ampicillin協同作用的檢測……………………………………45
Fig.8b：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Penicillin協同作用的檢測…………………………………….46
Fig.8c：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Bacitracin協同作用的檢測……………………………………47
Fig.8d：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Tetracycline協同作用的檢測…………………………………48
Fig.8e：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Erythromycin協同作用的檢測………………………………..49
Fig.8f：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Chloramphenicol協同作用的檢測…………………………….50
Fig.8g：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Streptomycin協同作用的檢測………………………………..51
Fig.8h：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Vancomycin協同作用的檢測………………………………..52
Fig.8i：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ceftriaxone協同作用的檢測………………………………..53
Fig.9a：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Ampicillin協同作用的檢測…………………………………54
Fig.9b：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Penicillin協同作用的檢測…………………………………..55
Fig.9c：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Bacitracin協同作用的檢測…………………………………56
Fig.9d：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Tetracycline協同作用的檢測……………………………….57
Fig.9e：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Erythromycin協同作用的檢測………………………………58
Fig.9f：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Chloramphenicol協同作用的檢測……………………………59
Fig.9g：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Streptomycin協同作用的檢測………………………………60
Fig.9h：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Vancomycin協同作用的檢測……………………………….61
Fig.9i：不同濃度的N-acetylglucosamine 及1﹪SACCHACHITIN對Ceftriaxone協同作用的檢測………………………………...62
Fig10a：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ampicillin協同作用的檢測…………………………………63
Fig.10b：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Bacitracin協同作用的檢測………………………………….64
Fig.10c：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Tetracycline協同作用的檢測………………………………..65
Fig.10d：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Chloramphenicol協同作用的檢測…………………………..66
Fig.10e：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ceftriaxone協同作用的檢測…………………………………67
Fig.10f：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Erythromycin協同作用的檢測………………………………..68
Fig.11a：不同濃度的N-acetylglucosamine對Ampicillin協同作用的檢測………………………………………………………………..69
Fig.11b：不同濃度的N-acetylglucosamine對Penicillin協同作用的檢測………………………………………………………………..70
Fig.11c：不同濃度的N-acetylglucosamine對Tetracycline協同作用的檢測……………………………………………………………..71
Fig.11d：不同濃度的N-acetylglucosamine對Erythromycin協同作用的檢測…………………………………………………………….72
Fig12a：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ampicillin協同作用的檢測……………………………………73
Fig.12b：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Penicillin協同作用的檢測……………………………………..74
Fig.12c：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Bacitracin協同作用的檢測…………………………………….75
Fig.12d：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Tetracycline協同作用的檢測………………………………….76
Fig.12e：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Streptomycin協同作用的檢測…………………………………77
Fig.12f：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Vancomycin協同作用的檢測…………………………………..78
Fig.13：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ampicillin協同作用的檢測……………………………………79
Fig.14a：不同濃度的N-acetylglucosamine對Ampicillin協同作用的檢測………………………………………………………………..80
Fig.14b：不同濃度的N-acetylglucosamine對Penicillin協同作用的檢測………………………………………………………………..81
Fig.14c：不同濃度的N-acetylglucosamine對Bacitracin協同作用的檢測………………………………………………………………..82
Fig.14d：不同濃度的N-acetylglucosamine對Tetracycline協同作用的檢測……………………………………………………………..83
Fig.14e：不同濃度的N-acetylglucosamine對Erythromycin協同作用的檢測…………………………………………………………….84
Fig.14f：不同濃度的N-acetylglucosamine對Chloramphenicol協同作用的檢測………………………………………………………..85
Fig.14g：不同濃度的N-acetylglucosamine對Streptomycin協同作用的檢測…………………………………………………………….86
Fig.14h：不同濃度的N-acetylglucosamine對Vancomycin協同作用的檢測…………………………………………………………….87
Fig.14i：不同濃度的N-acetylglucosamine對Ceftriaxone協同作用的檢測…………………………………………………………….88
Fig15：Chitin，chitosan，N-acetylglucosamine，SACCHACHITIN對Ceftriaxone協同作用的檢測………………………………….89
Fig.16：不同濃度的N-acetylglucosamine對Tetracycline協同作用的檢測………………………………………………………………90
Fig.17：N-acetylglucosamine及SACCHACHITIN對Ceftriaxone協同作用的檢測…………………………………………………….91
Fig.18：不同方式處理的D-glucosamine，兩者間抑菌效果的比較..92
Fig.19：Staphylococcus aureus (11863)最小抑菌濃度檢測……………93
Fig.20：SACCHAGLUCOSAMINE 的 H-NMR 圖譜………………94
Fig.21：D-glucosamine 的 NMR 圖譜……………………………….95
Fig.22：高壓溼熱處理後之 D-glucosamine 的 NMR 圖譜…………96
表次目錄
表一. D-glucosamine與培養基成分間抗菌效果的關係……………97
表二. D-glucosamine 對各抗生素協同作用的檢測…………………98
表三. 抗生素協同作用整理…………………………………………..99
附錄目錄
附錄一： D-glucosamine 與SACCHAGLUCOSAMINE製備過程之比較………………………………………………………….109
附錄二：Chitin、N-acetylglucosamine的結構…………………….110
附錄三：Chitosan、D-glucosamine的結構………………………...111

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