臺灣博碩士論文加值系統

本實驗的目的在初步去證實幾丁聚醣抑制細菌生長的機轉為抑制細菌胞外蛋白質酵素的活性，當蛋白質做為唯一氮源時，細菌無法利用蛋白質酵素去水解大分子蛋白質變成小分子蛋白質，作為其營養的來源，而達到抑制細菌生長的效果。以石蕊反應（litmus reaction）分析了革蘭氏陽性菌與革蘭氏陰性菌，結果Bacillus subtilis、Staphylococcus aureus以及Methicillin resistant Staphylococcus aureus（MRSA）具有水解gelatin的酵素。再將明膠（gelatin）平板培養基加入各種濃度的幾丁聚醣去了解細菌的生長情況，只有Bacillus subtilis、Staphylococcus aureus以及MRSA的控制組可生長，也可被幾丁聚醣抑制生長。其次，幾丁聚醣對Bacillus subtilis的最小抑菌濃度（MIC）是在0.2 mg/ml，對Staphylococcus aureus為0.2 mg/ml，而對MRSA為0.06 mg/ml。再者，以mineral medium培養菌種，證實了幾丁聚醣抑菌的途徑並非抑制利用氮源為營養的途徑，顯示抑菌的途徑為抑制利用小分子蛋白質為營養的途徑。根據幾丁聚醣抑制了chymotrypsin的活性，推測幾丁聚醣抑制蛋白質酵素，可能藉由相互的結合，而影響到蛋白質酵素活性。由受質凝膠電泳（zymography）也說明了此一論點。最後，以Bacillus subtilis的菌液，投與幾丁聚醣以zymography分析Bacillus subtilis胞外蛋白質酵素活性的變化。幾丁聚醣可抑制住Bacillus subtilis胞外蛋白質酵素的活性。本研究證實了幾丁聚醣抑制細菌生長的機轉為抑制蛋白質酵素的活性，亦可說明幾丁質或幾丁聚醣做為食品保存劑時，食物系統中，不得存在氨基酸或銨鹽。

The purpose of the present study was to elucidate the binding ability of chitin and chitosan to proteases produced by gram-positive bacteria that resulted in growth inhibition of bacteria with protein as a sole nitrogen source. The hypothesis proposed that bacteria would be 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. Litmus gelatin medium was used to detect gelatinase production of bacteria and it was found that Bacillus subtilis, Staphylococcus aureus and a methicillin resistant S. aureus (MRSA) were positive in the test. Minimal inhibitory concentrations (MIC) of chitosan in gelatin medium to the bacteria were 0.06, 0.2 and 0.2 to MRSA, B. subtilis and S aureus, respectively. In minimal medium, all the bacteria including gram positive and gram negative grew normally, in presence or absence of chitosan. It is evident that bacteria were able to proliferate by small inorganic nitrogen molecule (NH4+) and chitin or chitosan were not effective to inhibit the growth of bacteria. Spectrophotometry were performed to detect the binding of chitin and chitosan to proteases from various sources. The inhibition of the proteases revealed that chitin and chitosan might effect on those proteases of metallo-type. Zymographic gel analysis indicated that proteases from B. subtilis, S. sureus and MRSA were inhibited by chitosan with a dose response reaction. The result might be useful to explain the bacterial static effect on skin wound when chitin or chitosan dressing was applied. It is also meaningful that chitin and chitosan might be a good food preservative when amino acid or ammonium salts were absent in the food system.

目錄
中文摘要…………………………………………………………………………………i
Abstract…………………………….……………………………………………………ii
第一章 文獻回顧……………………………………………………………………….1
一、靈芝廢渣中之成份組成……………………………………………………1
（一）幾丁質及幾丁聚醣之由來………………………………………...1
（二）幾丁質及幾丁聚醣之結構…………………………………………1
（三）幾丁質與幾丁聚醣的生合成………………………………………2
（四）幾丁質與幾丁聚醣的生理活性……………………………………3
（五）幾丁質及幾丁聚醣之應用…………………………………………3
（1）食品方面……………………………………………………..3
（2）化工方面……………………………………………………..3
（3）生物技術方面………………………………………………..4
（4）生理方面……………………………………………………..4
（5）臨床應用方面……………………………………………….4
（六）幾丁質與皮膚覆蓋物之關係………………………………………5
（七）幾丁聚醣的抑菌作用………………………………………………6
第二章 實驗假說……………………………………………………………………….8
第三章 實驗設計……………………………………………………………………….9
實驗一、細菌蛋白質水解酶(extracellular protease)的測定…………………9
實驗二、幾丁聚醣（chitosan）的抑菌作用…………………………………11
實驗三、幾丁聚醣的最小抑菌濃度試驗……………………………………13
實驗四、幾丁聚醣在Mineral medium中的抑菌試驗………………………15
實驗五、以黏度儀測定幾丁聚醣的相對分子量……………………………17
實驗六、幾丁聚醣抑制蛋白質水解酶（protease）的活性試驗……………...18
實驗七、受質凝膠電泳（Zymography）………………………………………20
第四章 結果與討論…………………………………………………………………...24
一、細菌胞外蛋白質水解酶測定……………………………………………24
二、幾丁聚醣抑制細菌生長…………………………………………………24
三、幾丁聚醣的最小抑菌濃度……………………………………………....25
四、幾丁聚醣抑制蛋白質水解酶的活性……………………………………26
五、幾丁聚醣相對分子量的分析……………………………………………27
六、蛋白質水解酶與幾丁聚醣的交互作用…………………………………27
第五章 結論…………………………………………………………………………...28
第六章 參考文獻……………………………………………………………………...58
圖表目錄
Fig. 1 革蘭氏陰性菌的細胞外蛋白質水解酶之測定及幾丁聚醣之抑制反應….….30
Fig. 2 革蘭氏陰性菌的細胞外蛋白質水解酶之測定及幾丁聚醣之抑制反應……..31
Fig. 3 革蘭氏陽性菌的細胞外蛋白質水解酶之測定及幾丁聚醣之抑制反應……..32
Fig. 4 Methicillin resistant Staphylococcus aureus的細胞外蛋白質水解酶之測定及幾丁聚醣之抑制反應………………………………...…………………………….33
Table 1. 革蘭氏陰性菌在明膠培養基的生長情況與石蕊反應………………..……34
Table 2. 革蘭氏陽性菌在明膠培養基的生長情況與石蕊反應……………..……....34
Fig. 5 幾丁聚醣對Bacillus subtilis的抑菌試驗………………………………….…..35
Fig. 6 幾丁聚醣對Enterobacter aerogenes的抑菌試驗…………………………...…36
Fig. 7 幾丁聚醣對Escherichia coli的抑菌試驗……………………….……………..37
Fig. 8 幾丁聚醣對Klebsiella pneumoniae的抑菌試驗……………………...….…....38
Fig. 9 幾丁聚醣對Micrococcus luteus的抑菌試驗…………………...………..….....39
Fig. 10 幾丁聚醣對Methicillin resistant Staphylococcus aureus的抑菌試驗…….…40
Fig. 11 幾丁聚醣對Proteus vulgaris的抑菌試驗……………..…………………...…41
Fig. 12 幾丁聚醣對Staphylococcus aureus的抑菌試驗……….………………...…..42
Fig. 13 幾丁聚醣對Shigella dysenteria的抑菌試驗………….…………………...…43
Table 3. 各濃度幾丁聚醣對革蘭氏陰性菌在明膠培養基的生長情況....………..…44
Table 3. 各濃度幾丁聚醣對革蘭氏陰性菌在明膠培養基的生長情況……...…...…44
Fig. 14 幾丁聚醣對革蘭氏陰性菌的最小抑菌濃度………………………………....45
Fig. 15 幾丁聚醣對革蘭氏陰性菌的最小抑菌濃度………………….………..…….46
Fig. 16 幾丁聚醣對革蘭氏陽性菌的最小抑菌濃度…………………...…………….47
Fig. 17 幾丁聚醣對Methicillin resistant Staphylococcus aureus的最小抑菌濃度….48
Table 5. 幾丁聚醣對革蘭氏陰性菌的最小抑菌濃度……………………………......49
Table 6. 幾丁聚醣對革蘭氏陽性菌的最小抑菌濃度…………………………...…...49
Fig. 18 礦物質培養基中，幾丁聚醣對革蘭氏陽性菌的抑菌圖……..………..…….50
Fig. 19 礦物質培養基中，幾丁聚醣對革蘭氏陰性菌的抑菌圖……...…………...…51
Fig. 20 礦物質培養基中，幾丁聚醣對Methicillin resistant Staphylococcus aureus的抑菌圖………………………………………………...………………………..52
Table 7. 加入幾丁聚醣對革蘭氏陰性菌在礦物質培養基的生長情況…………..…53
Table 8. 加入幾丁聚醣對革蘭氏陽性菌在礦物質培養基的生長情況………….….53
Fig. 21 幾丁聚醣對bromelain之抑制之動態測定…………………………..…….....54
Fig. 22 幾丁聚醣對Glutamyl endopeptidase之抑制之動態測定…..………………..55
Fig. 23 幾丁聚醣對Chymotrypsin之抑制之動態測定……………………………....56
Fig. 24 Bacillus subtilis的蛋白酶與幾丁聚醣交互作用之受質凝膠電泳（7.5 ％）……………………………………………………………………………...57
Fig. 25 各種濃度幾丁聚醣抑制Bacillus subtilis培養液中蛋白酶活性之量化圖 （由Fig. 24 轉換分析）…………………………..………………………...…57
附錄一：Chitin及Chitosan結構比較……………………………………………….…67
附錄二：蛋白質水解酶特性………………………………………………………...…68
附錄三：幾丁聚醣黏度原始資料………………………………………………….…..69

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