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研究生:洪淑鋒
研究生(外文):Shu-Feng Hong
論文名稱:乳酸菌抑菌活性及其plantaricin基因組之研究
論文名稱(外文):Investigation of antibacterial activity and plantaricin gene cluster of lactic acid bacteria
指導教授:葉東柏葉東柏引用關係王淑珍王淑珍引用關係
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:2010
畢業學年度:98
語文別:中文
論文頁數:113
中文關鍵詞:細菌素乳酸菌抑菌活性plantaricin基因組
外文關鍵詞:antibacterial activitybacteriocinplantaricinLactobacillus plantarum
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本研究以本實驗室所分離出之11株乳酸菌以洋菜槽擴散法(agar well diffusion test)進行抑菌活性試驗。使用的指標菌株為大腸桿菌(Escherichia coli)、沙門氏菌(Salmonella spp.)、金黃色葡萄球菌(Staphylococcus aureus)及蛀牙菌(Streptococcus mutans)。抑菌試驗顯示,11株乳酸菌對E. coli及Salmonella spp. 具有良好的抑制效果,而其中有6株乳酸菌B0013、B0039、B0106、B0125、B0126及B0134可以有效的抑制所有的指標菌。進一步以16S rDNA sequences進行序列鑑定,結果顯示B0013、B0055、B0105、B0106、B0115、B0125、B0126、B0134、B0157、B0158鑑定結果為 Lactobacillus plantarum,B0039為 Lactobacillus paracasei,而所有菌株之 16S rDNA 序列與 NCBI database進行比對,核酸相似性皆大於 99% 以上。利用聚合酶連鎖反應(PCR)及特異性引子擴增乳酸菌之 plantaricin 基因,發現B0055、B0106、B0115及B0126可擴增出大部份細菌素基因,包括 plnA、plnB、plnC、plnD、plnEF、plnI、plnJ、plnK 及 plnG 等。經鑑定後發現與Lactobacillus plantarum WCFS1之細菌素基因大多數皆具有高相似度(≧98%)。接著利用具有抑菌效果且帶有大部分細菌素基因的乳酸菌株 B0126 培養於含 2% dextrin之 DMB 培養基中發現菌數可達1.55 ×109 CFU/ml。進而利用其發酵液進行抑制 S. mutans 之儲存試驗,結果顯示儲存有效期限約為 3.5 個月。在B0126 誘導試驗中,發現B0126 與 BCRC12248 共培養時,抑制 E. coli BCRC 15375 的活性比 B0126單獨培養時佳。
In this study, 11 isolated strains of lactic acid bacteria were tested antibacterial activity by agar diffusion method, using Escherichia coli, Salmonella spp., Staphylococcus aureus and Streptococcus mutans as indicator strains. Antibacterial test results showed that all 11 lactic acid bacteria have higher inhibitin zone against E. coli and Salmonella spp.. Among of them, 6 strains of lactic acid bacteria; B0013, B0039, B0106, B0125, B0126 and B0134 showed inhibitory activity against all the indicator strains. B0013、B0055、B0105、B0106、B0115、B0125、B0126、B0134、B0157、B0158 were identified as Lactobacillus plantarum and B0039 was Lactobacillus paracasei by 16S rDNA identification. At revealed that the 16S rDNA nucleotide sequence of there isolated strains were of 99% similarity with that of NCBI database. The bacteriocin locus from isolates B0055、B0106、B0115 and B0126 showed remarkable similarity to the plantaricin loci described for Lactobacillus plantarum WCFS1. B0126 was cultured in modified DMB containg 2% of dextrin, the number of colonies were 1.55×109 CFU/ml, similar with the culture of MRS. Further more, in storage test, B0126 supernatant was against S. mutans, the results showed the antibacterial activity of storage time were about 3.5 months. In the bacteriocin induction was observed that B0126 can be induced more bacteriocin against E. coli by BCRC12248.
中文摘要…………………………………………………………………… I
英文摘要…………………………………………………………………… III
本文目錄……………………………………………………………………… V
表目錄………………………………………………………………………… X
圖目錄…………………………………………………………………… XI
縮寫表……………………………………………………………………… XII

第一章 緒論
1.1序言………………………………………………………………………… 1
1.2乳酸菌之介紹…………………………………………………………… 2
1.2.1乳酸菌之特性………………………………………………………… 2
1.2.2 乳酸菌之分類………………………………………………………… 3
1.2.3 乳酸菌之應用………………………………………………………… 6
1.2.3.1 乳酸菌在食品工業上之應用………………………………………… 6
1.2.3.2 乳酸菌在保健上之應用……………………………………………… 7
1.2.4 乳酸菌之代謝產物…………………………………………………… 10
1.3 細菌素之介紹…………………………………………………………… 12
1.3.1細菌素起源…………………………………………………………… 12
1.3.2細菌素之分類………………………………………………………… 13
1.3.3細菌素之特性………………………………………………………… 15
1.3.4細菌素之抑菌機制…………………………………………………… 16
1.3.5 Plantaricin 基因之介紹……………………………………………… 17
1.3.5.1 Plantaricin 基因之組成…………………………………………… 17
1.3.5.1.1 L. plantarum C11 與 WCFS1 之pln 基因組………………… 17
1.3.5.1.2 L. plantarum NC8、J23及J51之pln 基因組…………………… 20
1.3.5.2 細菌素操縱子與啟動子之調控…………………………………… 22
1.3.5.3 細菌素之誘導……………………………………………………… 23
1.3.5乳酸菌細菌素在食品加工上之應用………………………………… 23
第二章 材料與方法
2.1 實驗材料………………………………………………………………… 25
2.1.1培養基………………………………………………………………… 25
2.1.2實驗藥品及試劑…………………………………………………… 26
2.1.3實驗設備…………………………………………………………… 27
2.1.4實驗用菌株………………………………………………………… 27
2.2實驗方法………………………………………………………………… 28
2.2.1實驗架構……………………………………………………………… 28
2.2.2菌株保存與活化……………………………………………………… 28
2.2.2.1 菌株保存…………………………………………………………… 28
2.2.2.2菌株活化…………………………………………………………… 29
2.2.3乳酸菌抑菌活性試驗…………………………………………………… 29
2.2.3.1乳酸菌發酵液之製備………………………………………………… 29
2.2.3.2 乳酸菌發酵液抑菌活性試驗……………………………………… 29
2.2.4乳酸菌菌種及乳酸菌plantaricin基因鑑定………………………… 30
2.2.4.1染色體DNA(chromosomal DNA)之萃取……………………… 30
2.2.4.2 16S rDNA菌種鑑定………………………………………………… 31
2.2.4.3 乳酸菌plantaricin基因擴增及鑑定……………………………… 32
2.2.4.4 PCR產物回收……………………………………………………… 32
2.2.4.5 DNA定序………………………………………………………… 33
2.2.4.6 16S rDNA及plantaricin 基因序列分析比對……………………… 33
2.2.5乳酸菌發酵液儲存試驗……………………………………………… 33
2.2.5.1乳酸菌菌數分析……………………………………………………… 33
2.2.5.2乳酸菌發酵液之製備……………………………………………… 34
2.2.5.3抑制S.mutans之乳酸菌的篩選…………………………………… 34
2.2.5.4抑制S.mutans之乳酸菌儲存試驗…………………………………… 35
2.2.6細菌素誘導試驗……………………………………………………… 35
2.2.6.1乳酸菌發酵液之製備……………………………………………… 35
2.2.6.2共培養抑菌試驗…………………………………………………… 35
第三章 結果
3.1乳酸菌抑菌活性試驗………………………………………………… 36
3.2乳酸菌菌種及乳酸菌plantaricin基因鑑定…………………………… 37
3.2.1乳酸菌菌種鑑定……………………………………………………… 37
3.2.2乳酸菌plantaricin基因擴增之結果………………………………… 38
3.2.3 乳酸菌plantaricin基因鑑定………………………………………… 39
3.3乳酸菌發酵液儲存試驗………………………………………………… 40
3.3.1乳酸菌菌數分析……………………………………………………… 40
3.3.2抑制S.mutans之乳酸菌篩選…………………………………… 40
3.3.3抑制S.mutans之乳酸菌儲存試驗……………………………… 41
3.4細菌素誘導試驗…………………………………………………… 43
第四章 討論
4.1乳酸菌抑菌活性試驗………………………………………………… 44
4.2乳酸菌菌種及乳酸菌plantaricin基因鑑定…………………………… 45
4.2.1乳酸菌菌種鑑定……………………………………………………… 45
4.2.2乳酸菌plantaricin基因擴增及鑑定………………………………… 45
4.3乳酸菌發酵液儲存試驗………………………………………………… 46
4.3.1乳酸菌菌數與培養基分析………………………………………… 46
4.3.2抑制S. mutans之乳酸菌篩選………………………………………… 47
4.3.3抑制S. mutans之乳酸菌儲存試驗…………………………………… 48
4.4細菌素誘導試驗………………………………………………………… 49
第五章 結論………………………………………………………………… 51
參考文獻…………………………………………………………………… 52
附表………………………………………………………………………… 67
附圖………………………………………………………………………… 86
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