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研究生:楊欣儀
研究生(外文):Hsin-Yi Yang
論文名稱:蜂膠之抗致突變性及其對李斯特單胞菌和轉糖鏈球菌之抗菌作用
論文名稱(外文):Antimutagenicity and the Antimicrobial Activities of Propolis Against Listeria monocytogenes and Streptococcus mutans
指導教授:張正明張正明引用關係周正俊周正俊引用關係
指導教授(外文):Cheng-Ming ChangCheng-Chun Chou
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:82
中文關鍵詞:抗致突變性抗菌作用李斯特單胞菌轉糖鏈球菌
外文關鍵詞:AntimutagenicityAntimicrobial ActivitiesListeria monocytogenesStreptococcus mutans
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中文摘要
蜂膠 (propolis) 為一多源性物質,係蜜蜂採集特定植物之樹脂物質,與其大顎腺分泌物作用後,並混合花粉、蜜蠟等所得之黏稠性衍生物。本研究乃將蜂膠以 80% 酒精萃取後,測試蜂膠酒精萃出液 (EEP) 之抗致突變性及其對Listeria monocytogenes BCRC 14930、 Streptococcus mutans BCRC 15256 之抗菌活性。
結果顯示,EEP在所試之濃度 (7.5~60 μg/plate) 下對Salmonella typhimurium TA 100 皆未呈現毒性及致突變性。惟對4NQO 誘發 S. typhimurium TA 100之致突變性具有抑制作用。EEP 在60 μg/plate 之劑量下,其抗致突變能力可高達88.8%。
EEP 對 L. monocytogenes BCRC 14930及S. mutans BCRC 15256 皆具有抗菌活性。S. mutans BCRC 15256 較L. monocytogenes BCRC 14930 對蜂膠敏感。大致而言,EEP 在 37℃ 下對 L. monocytogenes BCRC 14930 及 S. mutans BCRC 15256 所表現之抗菌效果明顯優於4℃ 及 25℃ 者。在酸性環境下,EEP 之抗菌效果則比在鹼性環境下強,隨著pH 值之上升,其抗菌作用則愈差。不同菌齡會影響 L. monocytogenes BCRC 14930 及 S. mutans BCRC 15256 對 EEP 之敏感性。其敏感性依序皆為對數期中期、對數期末期及定常期之菌體。加熱處理不會降低 EEP 對所測菌株之抗菌活性。在 50、80、100℃ 加熱1小時後,這些 EEP 所呈現之抗菌活性並未有顯著性之差異 (p>0.05),具有良好之熱穩定性。此外,EEP 會破壞菌體細胞膜之穿透性,而導致胞內260 nm吸光物質之洩漏。
Propolis is a heterogeneous material composed of really complex substances, produced by honeybees mixing with the gum of various plants, pollen, bee wax and enzymes secreted by bees. The present study used 80% ethanolic extract of propolis to examination antimutagenic and antibacterial activities against Listeria monocytogenes BCRC 14930 and Streptococcus mutans BCRC 15256.
Results showed that different concentrations of ethanolic extract of propolis (EEP) (7.5~60 μg/plate) did not present toxicity and mutagenicity against Salmonella typhimurium TA 100. Antimutagenic activity of EEP (60 μg/plate) against 4-nitroquinoline-N-oxide (4NQO) was 88.8%.
The Data showed EEP have antimicrobial activity against L. monocytogenes BCRC 14930 and S. mutans BCRC 15256. Besides, S. mutans BCRC 15256 showed more susceptibility than L. monocytogenes BCRC 14930. Temperature, pH and cell age were found to affect the susceptibility of L. monocytogenes BCRC 14930 and S. mutans BCRC 15256 to EEP. At 37℃ EEP showed stronger antimicrobial activity of L. monocytogenes BCRC 14930 and S. mutans BCRC 15256 than at 4℃ or 25℃. Antimicrobial activity of EEP in acid environment was better than in base environment. Cells in the mid-exponential phase were most sensitive, followed by the late exponential phase cells and stationary phase cells. In addition, EEP possessed good thermal stability, after heating at 50, 80 or 100℃ for 1 h, its antibacterial activity remained unchanged. It was also noted that EEP caused the leakage of 260-nm-absorbing materials (nucleotide, DNA, RNA) from L. monocytogenes BCRC 14930 and S. mutans BCRC 15256 cells.
目 錄
摘要 ------------------------------------------------------------------------------ I
Abstract -------------------------------------------------------------------------- II
目錄 ------------------------------------------------------------------------------ III
圖目錄 --------------------------------------------------------------------------- VI
表目錄 --------------------------------------------------------------------------- VII
壹、前言 ------------------------------------------------------------------------- 1
貳、文獻整理 ------------------------------------------------------------------- 3
一、蜂膠 --------------------------------------------------------------------- 3
1. 蜂膠之膠源植物 --------------------------------------------------- 3
2. 蜂膠之物理性質 --------------------------------------------------- 4
3. 蜂膠之化學組成 --------------------------------------------------- 6
(1) 蜂膠之組成份 ------------------------------------------------- 7
(2) 蜂膠中類黃酮成分 ------------------------------------------- 7
4. 蜂膠之抗致突變作用 --------------------------------------------- 9
5. 蜂膠之抗微生物活性 --------------------------------------------- 11
(1) 蜂膠之抗細菌作用 ------------------------------------------- 11
(2) 蜂膠之抗真菌作用 ------------------------------------------- 12
(3) 蜂膠之抗病毒活性 ------------------------------------------- 12
6. 蜂膠抑制微生物之機制 ------------------------------------------ 13
7. 蜂膠之其他生理活性 --------------------------------------------- 14
8. 蜂膠之毒性 --------------------------------------------------------- 14
二、抗致突變性檢測方法 ------------------------------------------------ 15
1. 組胺酸需求性測定 ------------------------------------------------- 16
2. raf 突變型測定 ----------------------------------------------------- 16
3. uvrB突變型測定 ---------------------------------------------------- 17
4. R-factor 測定 ------------------------------------------------------- 17
三、李斯特單胞菌 --------------------------------------------------------- 17
1. 細菌特性 ------------------------------------------------------------ 18
2. 環境分佈與傳播媒介 --------------------------------------------- 18
3. 疾病症狀 ------------------------------------------------------------ 19
四、轉糖鏈球菌 ------------------------------------------------------------ 21
1. 細菌形態及特性 --------------------------------------------------- 21
2. 疾病產生 ------------------------------------------------------------ 22
參、材料與方法 ---------------------------------------------------------------- 23
一、實驗材料 --------------------------------------------------------------- 23
1. 蜂膠原膠 ------------------------------------------------------------ 23
2. 實驗菌株 ------------------------------------------------------------ 23
3. 培養基 --------------------------------------------------------------- 23
4. 試驗藥品 ------------------------------------------------------------ 25
5. 儀器及器材 --------------------------------------------------------- 26
二、實驗方法 --------------------------------------------------------------- 27
1. 菌株之保存與更新 ------------------------------------------------ 27
2. S. typhimurium TA 100試驗菌液之製備 ------------------------ 27
3. L. monocytogenes及S. mutans之活化與接種源製備 -------- 28
4. 蜂膠酒精萃出液之製備 ------------------------------------------ 28
5. 蜂膠酒精萃出物含量之測定 ------------------------------------ 29
6. S. typhimurium TA 100 試驗菌株基因型態之確認 ----------- 29
(1) 組胺酸要求性之確認 ---------------------------------------- 29
(2) rfa突變之測試 ------------------------------------------------- 29
(3) uvrB突變之測試 ----------------------------------------------- 30
(4) R-factor之測試 ------------------------------------------------- 30
7. 蜂膠之抗致突變性測試 ------------------------------------------ 30
(1) 毒性試驗 ------------------------------------------------------ 31
(2) 致突變性試驗 ------------------------------------------------- 31
(3) 抗致突變性試驗 ---------------------------------------------- 32

8. 蜂膠酒精萃出物對 L. monocytogenes及 S. mutans之抗菌活性 ------------------------------------------------------------------ 33
(1) 不同濃度蜂膠酒精萃出物之抗菌作用 ------------------- 33
(2) 不同溫度對蜂膠酒精萃出物之抗菌作用 ---------------- 34
(3) 不同 pH 環境下對蜂膠酒精萃出物之抗菌作用 ------ 34
(4) 加熱處理蜂膠酒精萃出物之抗菌作用 ------------------- 35
(5) 蜂膠酒精萃出物對不同菌齡菌體之抗菌作用 ---------- 35
(6) 經蜂膠酒精萃出物作用後菌體之穿透性試驗 ---------- 36
(7) 以掃瞄式電子顯微鏡觀察蜂膠酒精萃出物作用於菌體之情形 --------------------------------------------------------- 36
9. 菌數之測定 --------------------------------------------------------- 38
10. 統計分析 ----------------------------------------------------------- 38
肆、結果與討論 ---------------------------------------------------------------- 39
一、S. typhimurium TA 100 試驗菌株基因型態之確認 ------------- 39
1. 組胺酸要求性之確認 --------------------------------------------- 39
2. rfa突變測試 --------------------------------------------------------- 39
3. uvrB 突變測試 ----------------------------------------------------- 41
4. R-factor之測試 ------------------------------------------------------ 41
二、蜂膠酒精萃出物對S. typhimurium TA 100之毒性與致突變性------------------------------------------------------------------------- 41
三、蜂膠酒精萃出物之抗致突變性 ------------------------------------ 43
四、不同濃度蜂膠酒精萃出物之抗菌作用 --------------------------- 46
五、不同溫度對蜂膠酒精萃出物之抗菌作用 ------------------------ 49
六、不同 pH 環境下對蜂膠酒精萃出物之抗菌作用 --------------- 54
七、不同菌齡之菌體對蜂膠酒精萃出物敏感性測試 --------------- 58
八、加熱處理對蜂膠酒精萃出物抑菌效果之影響 ------------------ 61
九、蜂膠酒精萃出物對菌體細胞膜之穿透性試驗 ------------------ 64
伍、結論 ------------------------------------------------------------------------- 71
陸、參考文獻 ------------------------------------------------------------------- 73

表 目 錄
表一、蜂膠之膠源植物 ------------------------------------------------------- 5
表二、蜂膠中常見之類黃酮 ------------------------------------------------- 8
表三、S. typhimurium TA 100 試驗菌株之基因型態確認 -------------- 40
表四、蜂膠酒精萃出物對S. typhimurium TA 100 之毒性測試 ------- 42
表五、蜂膠酒精萃出物對S. typhimurium TA 100 之致突變性 ------- 44
表六、蜂膠酒精萃出物對4NQO 於S. typhimurium TA 100之抗致突變性 ------------------------------------------------------------------- 45
表七、蜂膠酒精萃出物對李斯特菌及轉糖鏈球菌之最小抑菌濃度與最小殺菌濃度 ---------------------------------------------------------- 50















圖 目 錄
圖一、南投草屯6 - 7月所生產之蜂膠原膠 ------------------------------- 24
圖二、蜂膠之酒精萃出物對李斯特菌之抗菌作用 ---------------------- 47
圖三、蜂膠之酒精萃出物對轉糖鏈球菌之抗菌作用 ------------------- 48
圖四、溫度對蜂膠酒精萃出物於李斯特菌抗菌作用之影響 ---------- 51
圖五、溫度對蜂膠酒精萃出物於轉糖鏈球菌抗菌作用之影響 ------- 52
圖六、不同pH對蜂膠酒精萃出物於李斯特菌抗菌作用之影響 ------ 55
圖七、不同pH對蜂膠酒精萃出物於轉糖鏈球菌抗菌作用之影響 --- 56
圖八、蜂膠酒精萃出物對不同菌齡李斯特菌之抗菌作用 ------------- 59
圖九、蜂膠酒精萃出物對不同菌齡轉糖鏈球菌之抗菌作用 ---------- 60
圖十、經50、80、100℃加熱處理之蜂膠酒精萃出物對李斯特菌之抗菌作用 --------------------------------------------------------------- 62
圖十一、經50、80、100℃加熱處理之蜂膠酒精萃出物對轉糖鏈球菌之抗菌作用 --------------------------------------------------------- 63
圖十二、蜂膠酒精萃出物對李斯特菌菌體內260 nm吸光物質洩漏之影響 ------------------------------------------------------------------ 65
圖十三、蜂膠酒精萃出物對轉糖鏈球菌菌體內260 nm吸光物質洩漏之影響 --------------------------------------------------------------- 66
圖十四、蜂膠酒精萃出物對李斯特菌菌體細胞膜影響之掃瞄式電子顯微鏡照片 (10,000X) ----------------------------------------------- 67
圖十五、蜂膠酒精萃出物對轉糖鏈球菌菌體細胞膜影響之掃瞄式電子顯微鏡照片 (20,000X) -------------------------------------------- 68
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