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研究生:陳俊嘉
研究生(外文):Chun-chia Chen
論文名稱:由乳酸菌發酵液中分離細菌素及其特性之探討
論文名稱(外文):Isolation and characterization of bacteriocin from isolated lactic acid bacteria
指導教授:張竣凱張竣凱引用關係王淑珍王淑珍引用關係
指導教授(外文):Jiunn-Kae ChangShu-jen Wang
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:生物科技系暨研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:81
中文關鍵詞:細菌素乳酸菌過氧化氫
外文關鍵詞:DMBbacteriocinnisinStreptococcus spp.Lactobacillus spp. (LAB)
相關次數:
  • 被引用被引用:4
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Lactobacillus spp. (LAB) 屬是被廣泛研究的益生菌。在釵h實驗及臨床研究證實對人類的健康是有幫助的。乳酸菌的代謝物質已經發現對不同細菌種類具有潛在的抑制活性,包含Streptococcus spp.。在排除酸與過氧化氫的干擾後,乳酸菌分離株編號 B0105 生產的細菌素(bacteriocin),以平板擴散法分析抗菌活性,並與工業上常用來生產細菌素(nisin)的菌株進行比較,乳酸菌 B0105 生命的細菌素具有抑制 S. mutans 的能力,經由初步生理特性判定,再以 16S rDNA sequences 作序列鑑定後,確定為Lactobacillus plantarum。此外,我們發現以自製培養基 DMB(20% dextrin)生產的細菌素(bacteriocin)與MRS broth 生產之細菌素,具有相同的抑菌活性。使用吸附釋放法純化細菌素並經由 SDS-PAGE 分析後,分子量介於3.5 kDa ~ 17.0 kDa 之間。
Lactobacillus spp. (LAB) are widely studied probiotic bacterium. Its beneficial effects on human health are documented in many experimental and clinical studies. LAB have been shown to produce a substance with potential inhibitory activity against different bacterial species including cariogenic Streptococcus spp.. LAB isolated strain (B0105) producing bacteriocin against Streptococcus mutans under the condition of elimination of acid and hydrogen peroxide effect was identified and name as Lactobacillus plantarum based on the biochemical properties and 16S rDNA sequences. Antibacterial activity was examined by the diffusion plate method as compared with a reference strain used as industrial producing bacteriocin(nisin). The bacteriocin from LAB B0105 had the ability to inhibit S. mutans, suggesting that it could be a promising alternative biopreservative agent for S. mutans infections. Antimicrobial peptide, bacteriocin, produced by B0105 incubated in DMB (20% dextrin) medium produced by self-prepared had the same antimicrobial activity with bacteriocin produced by MRS medium. On the basis of absorption method, the partially purified bacteriocin had a molecular weight of approximately 17.0 to 3.5 kDa, analyzed by SDS-PAGE.
中文摘要........................ I
英文摘要......................... II
誌謝.......................... IV
本文目錄........................ V
表目錄......................... XI
圖目錄.......................... XII
縮寫表.......................... XIII

第一章 、緒論
1-1序言......................... 1
1-2乳酸菌之介紹..................... 2
1-2-1 乳酸菌簡介.................... 2
1-2-2 乳酸菌的特性................... 3
1-2-3 乳酸菌的分類................... 4
1-2-4 乳酸菌的應用................... 4
1-2-4-1 食品工業..................... 4
1-2-4-2一般工業..................... 5
1-2-5 乳酸菌增進健康之機能............... 6
1-2-5-1 緩和乳糖不耐症.................. 6
1-2-5-2 乳酸菌對胃腸道的益處.............. 6
1-2-5-3 降低血液膽固醇................. 6
1-2-5-4 抗腫瘤..................... 6
1-2-5-5 提高免疫之幼纂B抗過敏(減敏)之弁遄D..... 7
1-2-5-6 乳酸菌抑菌作用................. 7
1-3 細菌素之介紹.................... 10
1-3-1 細菌素的發現................... 10
1-3-2 可產生細菌素之菌種及條件............. 11
1-3-3 乳酸菌細菌素一般特性............... 12
1-3-4 細菌素的分類................... 13
1-3-5 乳酸菌細菌素之純化................ 14
1-3-6 細菌素抑菌機制.................. 15
1-4 轉糖鏈球菌(Streptococcus mutans)之介紹....... 17
1-4-1、轉糖鏈球菌之型態,鑑定與分類.......... 17
1-4-2 轉糖鏈球菌與人類疾病的關係............ 19
第二章 、材料與方法
2-1 實驗材料...................... 22
2-1-1培養基...................... 22
2-1-2本試驗之菌株................... 22
2-1-3試驗藥品..................... 23
2-1-4實驗設備..................... 24
2-2實驗方法...................... 25
2-2-1 實驗架構..................... 25
2-2-2 菌株保存與活化.................. 26
2-2-3 S. mutans 生長曲線................. 27
2-2-4 抑制蛀牙菌之乳酸菌篩選.............. 27
2-2-4-1 排除有機酸干擾試驗............... 27
2-2-4-2 排除H2O2干擾試驗................ 28
2-2-4-3 Pepsin影響之試驗................. 28
2-2-5 乳酸菌菌種鑑定.................. 29
2-2-5-1 染色體DNA(chromosomal DNA)之萃取...... 29
2-2-5-2 PCR引子(Primer)之設計............. 30
2-2-5-3 PCR增幅反應.................. 30
2-2-5-4 PCR產物回收.................. 30
2-2-5-5 PCR產物進行DNA定序.............. 31
2-2-5-6 16S rDNA序列分析比對.............. 31
2-2-6 替代培養基之探討................. 31
2-2-6-1替代培養基................... 31
2-2-6-2 菌數分析.................... 32
2-2-7粗萃細菌素(crude bacteriocin)特性探討....... 32
2-2-7-1 粗萃細菌素溶液之製備.............. 32
2-2-7-2 粗萃細菌素抑菌能力之測試............ 32
2-2-7-3 pH 對粗萃細菌素之影響.............. 33
2-2-7-4 粗萃細菌素之熱安定性.............. 33
2-2-7-5 粗萃細菌素對蛋白酵素之敏感性........... 33
2-2-8 細菌素之純化................... 34
2-2-8-1 菌體吸附釋放法純化細菌素............ 34
2-2-8-2 SDS-PAGE 電泳分析............... 34
2-2-8-3 膠體蛋白帶活性之偵測.............. 35
2-2-8-4 蛋白質定量................... 35
2-2-9 純化細菌素在不同蛋白質濃度對 S. mutans 之抑制活性. 35
第三章、結果
3-1 S. mutans 生長曲線分析................ 37
3-2 抑制蛀牙菌(S. mutans)之乳酸菌篩選......... 37
3-2-1抑菌試驗及有機酸排除試驗............. 37
3-2-2 過氧化氫排除試驗.................. 38
3-2-3 Pepsin作用之結果.................. 38
3-3 乳酸菌分離株B0105菌種鑑定結果........... 39
3-4 替代培養基之探討................... 39
3-5 細菌素特性探討................... 40
3-5-1 L. plantarum 細菌素對S. mutans 抑制活性....... 40
3-5-2 pH對細菌素抑制 S. mutans 抑制之影響........ 40
3-5-3熱處理對細菌素安定性分析.............. 41
3-5-4 細菌素對不同酵素之敏感性.............. 41
3-6純化細菌素之特性探討................. 41
3-6-1 蛋白質電泳分析.................. 42
3-6-2 SDS - PAGE膠體蛋白帶活性之偵測.......... 42
3-6-3 純化細菌素蛋白質濃度............... 42
3-7 細菌素中蛋白質濃度對 Streptococcus mutans 之抑制... 43
第四章、討論
4-1抑制蛀牙菌(S. mutans)之乳酸菌篩選........... 44
4-1-1 有機酸干擾之排除................. 44
4-1-2 過氧化氫干擾之排除 ................ 44
4-1-3 Pepsin影響之試驗................. 44
4-2 乳酸菌菌種鑑定................... 45
4-3細菌素特性探討................... 45
4-3-1 pH 對細菌素活性之影響.............. 45
4-3-2 熱處理對細菌素安定性分析............. 46
4-3-3 細菌素對蛋白分解酵素之敏感性........... 46
4-4 L. plantarum 替代培養基之探討............ 47
4.5 L. plantarum 純化之細菌素及生理特性之探討...... 48
4-6 純化細菌素在不同蛋白質濃度對 S. mutans 之抑制活性.. 48
第五章 結論...................... 50
參考文獻........................ 52
附表.......................... 63
附圖.......................... 76














表目錄
表1、有機酸排除抑菌試驗................. 63
表2、排除過氧化氫之抑菌試驗............... 67
表3、Pepsin作用之抑菌試驗 ................ 68
表4、乳酸菌分離株之16S rDNA 核酸序列相似度百分比.... 69
表5、不同濃度的DMB對S. mutans 的生長抑制........ 70
表6、不同 pH 對 L. plantarum 生產的細菌素抑制 S. mutans 活性之影響........................ 71
表7、不同熱處理對 L. plantarum生產的細菌素抑制 S. mutans 活性之影響....................... 72
表8、不同酵素處理對 L. plantarum生產之細菌素抑制S. mutans 活性之影響...................... 73
表9、蛋白質濃度測定................... 74
表10、純化細菌素在不同蛋白質濃度對S. mutans之抑制活性.. 75










圖目錄
圖1、實驗設計流程圖.................. 76
圖2、S. mutans 生長曲線。培養於37℃含 5% sheep blood 的 TSB broth....................... 77
圖3、乳酸菌分離株B0105之16S rDNA 序列電泳圖..... 78
圖4、乳酸菌分離株L . plantarum(B0105)之16S rDNA序列. 79
圖5、L. plantarum純化細菌素蛋白質電泳分析圖....... 80
圖6、SDS - PAGE膠體蛋白帶活性之偵測.......... 81
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