[[alternative]]Inhibitory activities of chitosan and its enzymatic hydrolysates against bacteria and Drosophila larval

Abstract

碩士[[abstract]]本研究係以Bacillus cereus TKU006、Serratia ureilytica TKU013、Pseudomonas sp.TKU015三株幾丁質酶/幾丁聚醣酶生產菌發酵製備出粗酵素液。以酵素法水解懸浮態幾丁質、水溶性幾丁聚醣，製備不同水解時間之酵素水解物並以其進行抗菌及抗果蠅實驗，探討酵素水解物之對E.coli、B.subtilis及果蠅幼蟲之抑制活性。 抗E.coli測試中，WSC酵素水解物依水解程度不同，對E.coli與B.subtilis在抗菌效果上有顯著的不同抑制效果，樣品WSC/013/1h有最佳抑制效果，IC50為0.1%。抗B.subtilis測試中，樣品WSC/015/12h有最佳抑制效果，IC50在0.07%以下。 在果蠅幼蟲毒殺實驗，酵素水解懸浮態幾丁質之水解物中，CC/015水解物以水解第1小時之樣品對果蠅有最佳毒殺效果，致死率達87.5%。[[abstract]]Preparation of crude enzyme solutions from three chitinase/chito- sanase-produced strains, Bacillus cereus TKU006, Serratia ureilytica TKU013, and Pseudomonas sp.TKU015 to hydrolyze prepared from colloidal chitin(CC) and water soluable chitosan(WSC). Hydrolysates were collected at several points in time. The bio- activity of these hydrolysates against bacteria, Drosophila larval were studied. The inhibitory activity has a significant difference between against E.coli and B.subtilis treating with different hydrolysates. WSC hydrolysates prepared using TKU013 enzyme at hydrolysis time of 1h which had better antibacterial activity against E.coli, IC50=0.1%. WSC/015/12h had better antibacterial activity against B.subtilis, IC50<0.07%. CC/015/0h used in anti-Drosophila activity tests shows best insecticidal effect(morality=87.5%).[[tableofcontents]]目錄 頁次 授權書 簽名頁 誌謝 中文摘要 ·································································································· I 英文摘要 ································································································· II 目錄 ········································································································ III 圖目錄 ····································································································VI 表目錄 ··································································································· VII 第一章 緒論 ........................................................................................... 1 第二章 文獻回顧 ................................................................................... 3 2.1 幾丁質與幾丁聚醣 .................................................................... 3 2.1.1 幾丁質 ............................................................................. 3 2.1.2 幾丁聚醣 ......................................................................... 3 2.2 幾丁質酶與幾丁聚醣酶 ............................................................ 4 2.2.1 幾丁質酶 ......................................................................... 4 2.2.2 幾丁聚醣酶 ..................................................................... 4 IV 2.3 水解幾丁質、幾丁聚醣之方法 ............................................... 4 2.3.1 化學法(表2.1) ................................................................ 4 2.3.2 酵素法(表2.2) ................................................................ 7 2.4 幾丁聚醣抗菌上之研究 ............................................................ 9 2.5 幾丁質、幾丁聚醣之應用 ...................................................... 11 第三章 材料與方法 ............................................................................. 12 3.1 實驗菌株、果蠅 ..................................................................... 12 3.2 實驗材料 ................................................................................ 12 3.3 實驗儀器 ................................................................................ 13 3.4 液態培養 ................................................................................ 13 3.5 幾丁質酶、幾丁聚醣酶粗酵素液之製備 .............................. 15 3.6 懸浮態幾丁質製備 ................................................................. 15 3.7 酵素活性 ................................................................................ 15 3.7.1 幾丁質酶活性測定 ...................................................... 15 3.7.2 幾丁聚醣酶活性測定 .................................................. 16 3.8 進行酵素水解之基質的製備 ................................................. 16 3.8.1 基質之製備 .................................................................. 16 3.8.2 酵素水解之基質的製備 .............................................. 17 3.9 N-乙醯幾丁寡糖製備 ........................................................... 17 V 3.10 總糖含量測定(Dubois et al., 1956) ...................................... 17 3.11 還原糖含量測定(Miller, 1959) .............................................. 17 3.12 抗菌活性測試 ....................................................................... 19 3.12.1 抗大腸桿菌活性測試 ................................................ 19 3.12.2 抗枯草桿菌活性測試 ................................................ 19 3.13 毒殺果蠅幼蟲活性試驗 ....................................................... 20 3.14 質譜分析(MALDI-TOF-MASS) ........................................... 20 第四章 結果與討論 ............................................................................. 21 4.1 幾丁質酶/幾丁聚醣酶生產菌之選擇 ..................................... 21 4.2 幾丁質/幾丁聚醣酶之粗酵素液製備..................................... 21 4.3 粗酵素液活性測定 ................................................................. 21 4.4 總糖與還原糖變化量 .............................................................. 24 4.5 抗菌活性 ................................................................................. 29 4.5.1 抗大腸桿菌活性測試 ................................................... 29 4.5.2 抗枯草桿菌活性測試 ................................................... 30 4.6 抗菌之綜合討論 ...................................................................... 31 4.7 毒殺果蠅幼蟲活性測定 .......................................................... 41 第五章 結論 ......................................................................................... 51 參考文獻 ............................................................................................... 52 VI 圖目錄 圖2.1 幾丁質、幾丁聚醣、纖維素之分子結構 ................................... 5 圖4.1、三種粗酵素液水解CC 不同時間總糖、還原糖含量之變化 (A)B. cereus TKU006 (B)S. ureilytica TKU013 (C)Pseudomonas sp. TKU015) ... 27 圖4.2、三種粗酵素液水解WSC 不同時間總糖、還原糖含量之變化 (A)B. cereus TKU006 (B)S. ureilytica TKU013 (C)Pseudomonas sp. TKU015) ............................................................................................................... 28 圖4.3、WSC 及WSC/006/1h、WSC/013/1h 與WSC/015/1h 水解物濃 度對抗大腸桿菌之影響 ........................................................................ 33 圖4.4、水解時間對WSC/013 水解物抗大腸桿菌之影響(樣品濃度為 0.1%) ...................................................................................................... 34 圖4.5、水解時間對WSC/015 水解物抗大腸桿菌之影響(樣品濃度為 0.1%) ...................................................................................................... 35 圖4.6、WSC 及WSC/006/1h、WSC/013/1h 與WSC/015/1h 水解物濃 度對抗枯草桿菌之影響。 .................................................................... 36 圖4.7、水解時間對WSC/006 水解物抗枯草桿菌之影響(樣品濃度為 0.08%) .................................................................................................... 37 圖4.8、水解時間對WSC/013 水解物抗枯草桿菌之影響(樣品濃度為 0.05%)………………………………………………………………………………………………….38 圖4.9、水解時間對WSC/015 水解物抗枯草桿菌之影響(樣品濃度為 0.05%)………………………………………………………………………………………………….39 圖4.10、添加不同劑量幾丁質、幾丁聚醣、纖維素之果蠅幼蟲致死率 ............................................................................................................... 44 VII 圖4.11、CC/015不同水解時間水解物對果蠅幼蟲毒殺效果(添加劑量： 30μL) ...................................................................................................... 45 圖4.12、WSC(●)、WSC/015/1h(○) 添加不同劑量樣品對果蠅幼蟲毒 殺效果 ................................................................................................... 46 圖4.13、WSC/006 不同水解時間水解物對果蠅幼蟲毒殺效果(添加劑 量：30μL) .............................................................................................. 47 圖4.14 WSC/013 不同水解時間水解物對果蠅幼蟲毒殺效果(添加劑量： 30μL) ...................................................................................................... 48 圖4.15 CC/015/1h 水解物樣品經製備所得寡糖之MALDI-TOF-MASS 分 析圖 ....................................................................................................... 49 圖4.16 (A)WSC/006/1h 及(B)WSC/013/1h 水解物樣品經製備所得寡糖 之MALDI-TOF- MASS 分析圖 ................................................................ 50 表目錄 表2.1、化學法水解幾丁質、幾丁聚醣優劣之比較 ............................. 6 表2.2、酵素法水解幾丁質、幾丁聚醣優劣之比較 ............................. 8 表2.3、幾丁聚醣在抗菌上之討論 ...................................................... 10 表3.1、以B. cereus TKU006、S. ureilytica TKU013、Pseudomonas sp. TKU015 生產幾丁質酶及幾丁聚醣酶之發酵條件 .............................. 14 表3.2 DNS 試劑的組成 ........................................................................ 18 表4.1、B. cereus TKU006、S. ureilytica TKU013、Pseudomonas sp. TKU015 三株細菌所生產之幾丁質酶、幾丁聚醣酶粗酵素液活性 .. 23 表4.2、三組酵素水解物中分別對E.coil 及B.subtilis 抗菌效果較佳之 樣品的IC50............................................................................................ 40[[note]]學號: 698160545, 學年度: 9