論文摘要
幾丁質與幾丁聚醣之抑菌機轉，目前並不清楚，本研究室先前對幾丁聚醣抗菌試驗中發現，G（+）及G（-）細菌在洋菜膠培養基(nutrient agar)或是液態的培養基(nutrient broth)中，都無法有效地完全抑制細菌的生長。因此，假設一個抑菌機轉，其模式為幾丁聚醣與G（+）及G（-）細菌的胞外蛋白質水解酵素結合，且抑制酵素的活性，在蛋白質酵素活性被抑制的情形下，細菌便無法將大分子蛋白質水解為小分子蛋白質或氨基酸，作為養份的來源，進而達到抑制細菌的生長。
初步實驗時，選取的四株菌種，在只有蛋白質Gelatin的養份來源的前提之下，測試是否具有分泌水解大分子蛋白質的酵素。結果顯示：革蘭氏陽性菌Bacillus subtilis、Staphylococcus aureu與革蘭氏陰性菌E.coli、Pseudomonas aureginosas，皆具有分泌水解gelatin的酵素之能力。以mineral medium培養所選取的菌種，並投與幾丁聚醣時，證實了幾丁聚醣抑菌的機轉乃是傾向於抑制蛋白質酵素，而並非抑制氮源的途徑。本實驗室也發展出一套利用96孔盤的吸光值來偵測幾丁聚醣之抗菌活性。
實驗以zymography之測試方法得到結果：細菌的生長在有幾丁聚醣的介入下，抑制住了某種蛋白質酵素的活性。這證實了幾丁聚醣抑制細菌生長的機轉為抑制蛋白質水解酵素的活性。
所以利用流式細胞儀觀察細菌型態的變化，發現Ｇ（-）膜遭破壞的比例高於Ｇ（+），顯示Ｇ（-）因有LPS，所以遭損壞的膜比例較高如E.coli 90%、P. aureginosa 30%，死亡的比例E.coli 28%、P. aureginosa 10%，而Ｇ（+）死亡的比例B.subtilis 22 % 、S. aureus 6% 顯然幾丁聚醣對Ｇ（+）菌的抑菌作用在膜上的比例並不高，因此更證明了幾丁聚醣抑菌機轉為抑制細菌所產生的蛋白水解酵素。

Abstract
The exact mechanism of the antimicrobial action of chitin, chitosan is still unknow. However, according to our previous studies , chitosan can not completely inhibit the growth of bacterial on medium in nutrient agar or nutrient broth. So the hypothesis proposed that bacteria was unable to obtain amino acids through hydrolyzing protein in presence of chitin and chitosan and the growth would be rendered if small molecule nitrogen sourced were deprived. The binding ability of chitin and chitosan to proteases produced by gram-negative and gram-positive bacteria that resulted in growth inhibition of bacteria with protein was used as a sole nitrogen source. Gelatin medium was used to detect gelatinase production of bacteria and it was found that E. coli , Pseudomonas aureginosa and Bacillus subtilis, Staphylococcus aureus were positive in the test. Based on this mechanism spectrophotometry with a 96 well plate were developed to detect the antimicrobial activity of chitin and chitosan. Zymographic gel analysis indicated that proteases from E.coli, P. aureginosa , B. subtilis, S. sureus were inhibited by chitosan with a dose response reaction. The result confirmed that chitosan inhibited protease activity and it is a distinct the mechanism of the antimicrobial action of chitosan . Annexin Ⅴ-PI flow cytometry analysis on the bacteria indicated that chitosan or chitin caused asymmetric charge of bacteria plasma membrane to 90 % in E.coli,and 30% in P. aureginosa with a death ratio of 28 %and 10 % ,respectively. However, only slightly change was observe in B. subtilis 22 % and S. sureus 6 % . This information also in good agreement to the hypoesis that chitin or chitosan might effected more on G(-) that on G(+) bacteria , due to the binding of LPS in the outer membrane of G(-) bacteria.

目錄
中文摘要…………………………………………………………………………………I
Abstract…………………………….……………………………………………………..i
第一章 文獻回顧……………………………………………………………………….1
（一）幾丁質及幾丁聚醣之起源………………………………………...1
（二）幾丁質及幾丁聚醣之結構…………………………………………1幾丁質……………………………………………2
幾丁聚醣…………………………………………4
（三）幾丁質及幾丁聚醣之應用……………………..…………………4
（1）化工方面…………………………………..………………..5
（2）食品加工方面……………………………..………………..5
（3）化妝品上的利用…………………………...……………….6
（4）農業上的應用……………………………..………………..6
（5）生物醫學材料方面的應用………………..………………..7
A.生物降解性…………………………………………………..7
B.生物相容性…………………………………………………..7
C.免疫刺激作用………………………………………………..8
（四）幾丁質與皮膚覆蓋物之關係……………………………………9
第二章 實驗假說……………………………………………………………………..13
Ⅰ幾丁質與幾丁聚醣的抑菌作用………..………………….……………….…13Ⅱ目前各方提出幾丁質抑菌之假說………….……………..………………….16
　　一.正負電荷結合說………………………………...……..….……16
二.穿透結合說..……….……………………………………….…..17
三.四級銨鹽說………………...…………………………….……..17
四.金屬離子鏊合說………...………………………………………18
　　Ⅲ假說的形成………………………………………...………………..……19
第三章 實驗設計與方法…………………………………………………………...…21
實驗一、幾丁聚醣的抑菌測試………………………………………………22
實驗二、幾丁聚醣（chitosan）的質子化之影響……………………………23
實驗三、不同乙醯化的程度、分子量以及來源的幾丁聚醣之抗菌試驗…24
實驗四、幾丁聚醣與金屬離子鏊合之試驗…………………………………25
實驗五、測定細菌型態與結構之試驗………………………………………26
實驗六、細菌蛋白質水解酶(extracellular protease)之測定…………..……27
實驗七、細菌營養應用來源測試……………………………………………27
實驗八、MTT 之檢驗方法…………………………………………….……...29
實驗九、幾丁聚醣抑制蛋白質水解酶（protease）的活性試驗.……………30
第四章 結果與討論…………………………………………………………………...35
Ⅰ 幾丁聚醣的抑菌測試…………………………………………...…………35
幾丁聚醣的質子化之影響(pH值對抗菌效果的影響)…………………35
不同乙醯化的程度、分子量以及來源的幾丁聚醣之抗菌試驗…….....41
Ⅱ.細胞外膜破壞試驗………………………………………………………...43
幾丁聚醣與金屬離子鏊合之試驗………………………………………..44
測定細菌細胞膜改變及死亡之試驗……………………………………..49
Ⅲ.細菌胞外蛋白質水解酶測定………………………………………………59
細菌氮源應用來源測試…………………….………………...…………60
MTT 之檢驗方法…………………………………………………………64
幾丁聚醣抑制蛋白質水解酶（protease）的活性試驗…….….…………66
第五章 結論…………………………………………………………………………...69
第六章 參考文獻……………………………………………………………………...72
圖表目錄
Fig. 1幾丁聚醣抑菌試驗對BP agar培養四種細菌的生長情形……...……….…….36
Fig. 2幾丁聚醣抑菌試驗pH 7.0下培養四種細菌之生長情形………………….…..37
Fig. 3幾丁聚醣抑菌試驗pH 6.5下培養四種細菌之生長情形…………………...…38
Fig. 4幾丁聚醣抑菌試驗pH 6.0下培養四種細菌之生長情形……….……….…….38
Fig. 5幾丁聚醣抑菌試驗pH 5.0下培養四種細菌之生長情形…………......….…....39
Fig. 6幾丁聚醣抑菌試驗pH 4.0下培養四種細菌之生長情形………………..….....39
Fig. 7幾丁聚醣抑菌試驗pH 3.0下培養四種細菌之生長情形…………………...…40
Fig. 8高、中、低分子量幾丁聚醣之抑菌試驗……………………………………..…41
Fig. 9不同乙醯化及來源的幾丁聚醣抑菌試驗…………..……………………...…..42
Fig. 10大腸桿菌在不同鋅離子濃度之抑菌試驗….……………………………....…45
Fig. 11綠膿桿菌在不同鋅離子濃度之抑菌試驗…………………………….……....46
Fig. 12枯草桿菌在不同鋅離子濃度之抑菌試驗…………….………………...…….47
Fig. 13金黃葡萄球菌在不同鋅離子濃度之抑菌試驗………………………...……..48
Fig. 14 E.coli 利用Annexin Ⅴ / PI 雙染色法之圖示……………..………………….50
Fig. 15 E.coli 利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖…...50
Fig. 16 P. aureginosa利用Annexin Ⅴ / PI 雙染色法之圖示…………….….……...…51
Fig. 17 P. aureginosa利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖…………………………………………………………………………….....51
Fig. 18 B. subtilis利用Annexin Ⅴ / PI 雙染色法之圖示.............................................52
Fig. 19 B. subtilis利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖.52
Fig. 20 S. aureus利用Annexin Ⅴ / PI 雙染色法之圖示…….………………………..53
Fig. 21 S. aureus利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖...53
Fig. 22 E.coli 利用Annexin Ⅴ / PI 雙染色法之圖示……………..………………….55
Fig. 23 E.coli 利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖…...55
Fig. 24 P. aureginosa利用Annexin Ⅴ / PI 雙染色法之圖示…………….….……...…56
Fig. 25 P. aureginosa利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖…………………………………………………………………………….....56
Fig. 26 B. subtilis利用Annexin Ⅴ / PI 雙染色法之圖示.............................................57
Fig. 27 B. subtilis利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖.57
Fig. 28 S. aureus利用Annexin Ⅴ / PI 雙染色法之圖示…….………………………..58
Fig. 29 S. aureus利用Annexin Ⅴ / PI 雙染色法之圖示分析四象限所得之數據圖...58
Fig. 30細菌營養應用試驗(礦物質培養基中加1%明膠)與幾丁聚醣作用狀況…….60
Fig. 31細菌營養應用試驗(礦物質培養基中加0.5%尿素)與幾丁聚醣作用狀況…...61
Fig. 32細菌營養應用試驗(礦物質培養基中加0.1%硫化銨)………………….…….62
Fig. 33 MTT 之檢驗方法…...…………………………………………………….…...64
Fig. 34 MTT 之檢驗方法經過ELISA reader 550 nm之結果………………...………65
Fig. 35液態的培養基(nutrient broth)培養四菌種所產生的酵素……………….……67
Fig. 36液態的培養基(nutrient broth)培養四菌種所產生的酵素以幾丁聚醣抑制之活性之圖…………………………………………………………………………….……67
Fig. 37幾丁聚醣產生蛋白質水解酶（protease）的活性試驗……………….………68
Fig. 38幾丁聚醣抑制蛋白質水解酶（protease）的活性試驗…………..…...………68
附錄.圖 1幾丁質的結構……………………….………………………………….…..80
附錄.圖2幾丁質及幾丁聚醣的結構………….……………………………………....80
附錄.圖3幾丁聚醣對植物與植物病原菌之作用…………………….……………....80
附表.表一幾丁質及幾丁聚醣之機能及其在食品工業上之利用.……………...……81

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