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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 徐源泰 | |
dc.contributor.author | Yi-Ting Kao | en |
dc.contributor.author | 高怡婷 | zh_TW |
dc.date.accessioned | 2021-06-13T02:02:19Z | - |
dc.date.available | 2012-07-16 | |
dc.date.copyright | 2007-07-16 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-05 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30373 | - |
dc.description.abstract | 「乳酸菌 (Lactic acid bacteria, LAB)」特殊的代謝產物,使得食品及飲料易於保存,自古即被應用於食品加工,直至今日,乳酸菌仍在食品加工上扮演舉足輕重的角色。近年來因為乳酸菌的益生菌 (probiotics) 功效,市售含乳酸菌的產品也日漸增多,然而,這些產品所標示之菌種,是否確實?一旦標示不正確,產品之安全性、功能性均受到質疑。傳統上,對於乳酸菌之親緣關係或是分類是依據其表現型及生化反應之數據。隨著分子生物科技的發展,越來越多物種完整基因序列被解碼,利用基因型的鑑定方式是可以省時、省力且得到正確菌種的一種方法。
本研究的第一部分為利用adk, atpD, gyrB, recA, rpoB及zwf等housekeeping gene以及核糖體RNA基因來分析乳酸菌之親緣性,利用中性假說檢定以上6個基因,結果均為中性基因,因此相當適合做為探討乳酸菌種間之親緣關係。另外分別用這6個基因構築演化樹狀圖,這些中性的housekeeping gene,經計算後所構築之親源關係樹狀圖,與目前利用表現性、DDH以及16S rRNA序列之分類一致。housekeeping gene因為演化速率較快,可以用於分析較為相近之物種,綜合數個基因的分析及表現型之資料進行的polyphasic taxonomy可用來作為新品種分類,以及校正現有命名系統的工具,然需快速鑑定菌種,16S rDNA序列仍是最適合之分子標誌,同時也可將其利用於建立快速鑑定方法。 本研究的第二部份是要建立一快速且可靠的乳酸菌鑑定方法。首先自市售產品分離出乳酸菌,分別以醣類發酵及16S rRNA序列分析,兩種方法進行市售產品之菌種鑑定,並比較其優缺點。糖類發酵反應雖然可以得到不錯的結果,但因礙於資料庫不夠大,環境中之菌種無法正確的鑑定出,16S rRNA序列之分析為準確度最高的一種。 台灣地區市售含乳酸菌產品主要添加Lactobacillus acidophilus、L. delbrueckii、L. casei、L. paracasei及L. rhamnosus五種菌株,為建立快速鑑定方法,利用16S rRNA基因之序列設計種專一性引子進行PCR,但因L. casei、L. paracasei及L. rhamnosus之序列太過相近,因此無法區分,故利用即時定量PCR配合序列融點的分析以達到區別較相近物種之目的。 利用變性梯度凝膠電泳 (denaturing gradient gel electrophoresis,DGGE) 的技術,更可發展出更快速且正確鑑定產品中乳酸菌種的方法。包括13件優格產品、5件奶粉產品及6件膠囊粉末等共24件產品萃取出總微生物之DNA後, 放大其16S rRNA序列上之第三段變異區,利用變性梯度膠體電泳分離產品中混合之菌種,同時加以鑑定。整個實驗程序可以將時間縮短至12小時,與傳統利用培養分離菌種加以鑑定之方式比較,DGGE具有較高之敏感度,並且提供了一個較快, 可靠且方便之菌種鑑定方法。 | zh_TW |
dc.description.abstract | Lactic acid bacteria (LAB) are a group of microaerophilic, gram-positive, catalase-negative bacilli and cocci that ferment hexose sugars to produce primarily lactic acid. The seemingly simplistic metabolism of LAB has been exploited throughout history for the preservation of foods and beverages in nearly all societies dating back to the origins of agriculture. Today, LAB play a prominent role in the world food process. Due to the increasing healthy effect of probiotics had been published, more and more probiotic products appeared on the market. In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products in curcial. Traditionally, LAB have been classified on the basis of phenotypic characteristics, including morphology and biochemical reactions. Modern molecular techniques have become increasingly important and provide another method for analysis the phylogenetic and taxonomic relationships of LAB.
So far, the evolutionary relationships among LAB have been determined by comparing the sequences of 16S rRNA. But in some cases, 16S rRNA gene sequences are too conserved for closely related taxa. The rapid rates of evolutionary substitution in protein –coding genes are considered to be better molecular markers than the 16S rRNA. First, the testing the 6 housekeeping genes, adk, atpD, gyrB, recA, rpoB and zwf are fitted for the neutral mutation hypothesis. According the phylogenetic trees based on the 6 housekeeping sequences, the relationship of LAB were coincided with the taxonomy constructed by 16S rRNA gene sequence, DNA-DNA hybridization and phenotypic characteristics presently. Housekeeping gene could be used for compareing closeing strains. Polyphasic taxonomy, combined several genes and phenotypic data, could be used for clarify the taxonomy of new species and adjusted the taxonomy nowadays. The 16S rDNA sequence is still the most suitful molecular marker for rapid identification method. A rapid and reliable technique for identifying the strains of LAB was developed in the second part of this study. First, comparing the advantages and defects of carbohydrates fermentation and 16S rRNA sequence analysis of strains isolated from probiotic products in Taiwan. The carbohydrates fermentation test was good in limited database; however, the population of LAB is huge, and some species have approximate biochemical characteristics. The small divergence of biochemical characteristics between LAB caused the hardship in differentiating them by carbohydrate fermentation test. More and more 16S rRNA sequences are made public recently, and it enables the development of an extended application for analyzing LAB from the commercil products and other food-related habitats. Several species-specific primer pairs were designed base on the varialbilty of 16S rRNA gene sequences for differentiating 5 strains of lactobacilli which were added into probiotic products in Taiwan. It was simple to identify Lactobacillus acidophilus and L. delbrueckii by species-speific primers, but it could not be used to distinguish L. casei, L. paracasei and L. rhamnosus. Another PCR approach was developed with hybridization probes which were designed according to the difference among the 16S rRNA genes of L. casei, L. paracasei and L. rhamnosus, and melting curve analysis of the hybridization probe was used to distinish them. It was found that this approach could identify L. paracasei and L. rhamnosus correctly but not separate L. paracasei from L. casei, the result was due to both of them had the same 16S rRNA seauence. The results suggest that melting curve analysis of PCR approach in this part of study is a rapid, simple and accureate method in distinguishing the closely related strains of lactobacilli. In the final part of this study, a cultivation-independent method to detect and identify bacteria in probiotic was developed. A collection of 24 products, including 13 yogurt products, 5 milk powder and 6 capsular and powder products were extracted total microbial DNA directly, amplified the V3 region of the 16S rRNA gene, and separated the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture indenpent approach can be performed in less than 12 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T02:02:19Z (GMT). No. of bitstreams: 1 ntu-96-F91628209-1.pdf: 2775081 bytes, checksum: 468b5c96aff334983402c2213212995d (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 目 錄
口試委員審定書 i 謝誌 ii 中文摘要 iii Abstract v 第一章 序論 1 第一節 乳酸菌 1 第二節 益生菌 (probiotics) 1 第三節 乳酸菌的分類 2 一、乳酸桿菌屬 (Lactobacillus) 3 二、雙叉乳酸桿菌屬 (Bifidobacterium) 7 三、嗜熱鏈球菌 (S. thermophilus) 9 第四節 乳酸菌的鑑定 9 一、表現型鑑定系統 9 (一)糖類發酵鑑定系統: 9 (二)脂肪酸鑑定系統 (fatty acid methyl ester, FAME) 10 二、分子分型鑑定系統 10 (一)核糖體DNA之研究 10 (二)分子標誌方法之應用 11 (三)遺傳變異分析 15 (四)親緣分析 (phylogenetic analysis) 17 第五節 研究目的 18 第二章 乳酸菌親緣關係之研究 22 第一節 前言 22 第二節 材料與方法 24 一、實驗材料 24 二、方法 24 第三節 結果與討論 26 一、乳酸菌基因體的比較 26 二、中性假說檢定 26 三、親緣關係 27 第三章 糖類發酵與16S rRNA鑑定方法之比較 52 第一節 前言 52 第二節 材料與方法 53 一、材料 53 二、方法 57 第三節 結果與討論 60 一、市售產品分離菌株與標示是否相符 60 二、生化鑑定及16S序列鑑定之比較 61 三、市售菌株之16S rRNA 序列親緣分析 63 第四章 基因型快速鑑定方法之建立 70 第一節 前言 70 第二節 材料與方法 70 一、材料 70 二、方法 71 第三節 結果與討論 79 一、以16S rRNA序列設計之種專一性引子鑑別乳酸菌株 79 二、以ITS rRNA序列設計引子鑑別L. casei及L. rhamnosus 79 三、雜合探針之融化曲線分析 80 第五章 不需培養的乳酸菌鑑定方法 90 第一節 前言 90 第二節 材料與方法 92 一、材料 92 二、方法 96 第三節 結果與討論 98 一、片段的選取 98 二、DGGE條件的取得 98 三、以參考菌株進行鑑定 98 四、市售產品之檢測 99 第六章 總結 105 參考文獻 106 附錄 121 表目錄 表1-1 乳酸菌過去與現行分類比較表 4 表1-2 乳酸桿菌依發酵型式分類 5 表1-3 乳酸桿菌中主要菌株依據表現型分類 8 表2-1 本實驗所使用之基因體一般特性 25 表2-2 中性檢定統計值 26 表2-3 乳酸菌依不同基因分支 28 表3-1 本實驗所使用之參考菌株 54 表3-2 本實驗所使用之產品、標示含有之菌株以及分離出之菌株編號 55 表3-3 本實驗使用之參考菌株16S rRNA序列及醣類發酵反應檢驗結果 64 表3-4 本實驗分離自產品之乳酸菌株16S rRNA序列及糖類發酵反應檢驗結果 65 表4-1 PCR反應使用引子 77 表4-2 14株乳酸桿菌進行聚合酶鏈鎖反應之結果 81 表4-3 24株乳酸桿菌進行聚合酶鏈鎖反應之結果 88 表4-4 9株參考菌株及3株產品中分離之乳酸菌Tm值 89 表5-1 本實驗所使用之樣品及其含有之菌株 93 圖目錄 圖1-1 Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Kuthia, Clostridium, Propionobacterium, Bifidobacterium以及Bacillus之16S rRNA序列親緣關係樹狀圖。 6 圖1-2 原核生物核醣體RNA基因的結構 12 圖1-3 Hybridization probe作用原理 20 圖1-4 不同細菌,不同核酸片段之Tm值之示意圖 21 圖2-1乳酸菌之adk基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 31 圖2-2乳酸菌之adk基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 32 圖2-3乳酸菌之adk基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 33 圖2-4乳酸菌之atpD基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 34 圖2-5乳酸菌之atpD基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 35 圖2-6乳酸菌之atpD基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 36 圖2-7乳酸菌之gyrB基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 37 圖2-8乳酸菌之gyrB基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 38 圖2-9乳酸菌之gyrB基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 39 圖2-10乳酸菌之recA基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 40 圖2-11乳酸菌之recA基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 41 圖2-12乳酸菌之recA基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 42 圖2-13乳酸菌之rpoB基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 43 圖2-14乳酸菌之rpoB基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 44 圖2-15乳酸菌之rpoB基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 45 圖2-16乳酸菌之zwf基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 46 圖2-17乳酸菌之zwf基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 47 圖2-18乳酸菌之zwf基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 48 圖2-19乳酸菌之16S rRNA基因序列neighbor joining所計算之親源關係圖(bootstrap=1000) 49 圖2-20乳酸菌之16S rRNA 基因序列maximum parsimony所計算之親源關係圖(bootstrap=1000) 50 圖2-21乳酸菌之16S rRNA 基因序列MrBayes所計算之親源關係圖 (every 100th sample out of 106 generations) 51 圖3-1 核糖體 DNA 上 16S 區域universal引子位置。 59 圖3-2 本實驗分離之34株與資料庫61株乳酸菌之16S rRNA基因序列neighbor joining所計算之親源關係圖 68 圖3-3 本實驗分離之34株與資料庫61株乳酸菌之乳酸菌之16S rRNA基因序列maximum parsimony所計算之親源關係圖 69 圖4-1 本實驗使用之11株與資料庫中之7株乳酸桿菌屬菌株之16S rDNA序列。 72 圖4-2 核糖體DNA上16S區域種專一性引子之位置。 73 圖4-3 PCR反應引子對之位置圖 74 圖4-4 SNP, 雜合引子及探針之設計 78 圖4-5 利用Acido/Lac引子放大之乳酸菌PCR產物2%洋菜膠體電泳圖,編號1, L. acidophilus BCRC 10695; 2, L. delbrueckii subsp. bulgaricus BCRC 10696; 3, L. casei BCRC 10697; 4, Lactobacillus sp. BCRC 16000; 5, 空白對照組。 82 圖4-6 以Cas1/Pr2引子對對14株乳酸桿菌進行PCR後,經由2%洋菜膠體電泳圖:(1)BCRC 10695; (2)BCRC10696; (3)BCRC 10697; (4)BCRC 11051; (5)BCRC 12193; (6)BCRC12195; (7)BCRC 16000; (8)BCRC 10069; (9)BCRC 10940; (10)BCRC 12190; (11)BCRC12248; (12)BCRC 12936; (13)BCRC 17002; (14)BCRC 17226; B, 空白對照; M, DNA大小標記。 83 圖4-7 以Cas1/Rha2引子對對14株乳酸桿菌進行PCR後,經由2%洋菜膠體電泳圖:(1)BCRC 10695; (2)BCRC10696; (3)BCRC 10697; (4)BCRC 11051; (5)BCRC 12193; (6)BCRC12195; (7)BCRC 16000; (8)BCRC 10069; (9)BCRC 10940; (10)BCRC 12190; (11)BCRC12248; (12)BCRC 12936; (13)BCRC 17002; (14)BCRC 17226; B, 空白對照; M, DNA大小標記。 84 圖4-8 以Cas1/Rha2引子對對七株Lactobacillus paracasei及三株Lactobacillus rhamnosus進行PCR後,經由2%瓊膠電泳膠片: (1)2C; (2)3C; (3)4C; (4)9C; (5)10C; (6)15C; (7)16C; (8)5D; (9)11D; (10)14D; B, 空白對照; M, DNA大小標記。 85 圖4-9 以RhamF/RhamR引子對對14株乳酸桿菌進行PCR後,經由2%洋菜膠體電泳圖:(1)BCRC 10695; (2)BCRC10696; (3)BCRC 10697; (4)BCRC 11051; (5)BCRC 12193; (6)BCRC12195; (7)BCRC 16000; (8)BCRC 10069; (9)BCRC 10940; (10)BCRC 12190; (11)BCRC12248; (12)BCRC 12936; (13)BCRC 17002; (14)BCRC 17226; B, 空白對照; M, DNA大小標記。 86 圖4-10 15株乳酸菌之Melting temperatures示意圖 87 圖5-1 L. acidophilus, L. delbrueckii, L. paracasei, O.oeni, B. longum, S. thermophilus, L. lactis, B. bifidum, B. animalis, S. bovis等10株乳酸菌之341f/518r片斷進行變性程度為0﹪-100﹪的垂直DGGE 101 圖5-2 以9種乳酸菌之341f/518r片段進行水平DGGE。1:,L. acidophilus, 2: L. paracasei, 3: O. oeni, 4: B. longum, 5: S. bovis, 6: L. bulgaricus, 7: S. thermophilus, 8: L. lactis, 9: B. lactis, Ma: 混合1~4, Mb: 混合5~9。 102 圖5-3 以10種乳酸菌之341f-gc/518r片段進行水平DGGE。1:,O. oeni, 2. L. acidophilus, 3: S. bovis, 4: S. thermophilus, 5: L. paracasei, 6: B. lactis, 7: B. longum, 8: L. lactis, 9: L. bulgaricus, 10: B. bifidum, Ma: 混合1~5, Mb: 混合6~10。 103 圖5-4 以A~X,24種產品萃取之乳酸菌341f-gc/518r片段進行水平DGGE。圖A, 1:, L. acidophilus, 2: L. bulgaricus, Ma: 混合O. oeni, L. acidophilus, L. paracasei, B. longum以及B. bifidun, Mb: 混合B. animals, L. bulgaricus, L. lactis, S. bovis以及S. thermophilus。圖B, Ma: 混合O.oeni, L. acidophilus, S.bovis, S. thermophilus, L. paracasei, Mb: 混合B. animals, L. bulgaricus, L. lactis, B. longum, B. bifidum, Mc: 混合L. acidophilus和L. bulgaricus. 104 | |
dc.language.iso | zh-TW | |
dc.title | 乳酸菌快速鑑定方法之研究 | zh_TW |
dc.title | Studies of Rapid Identification Methods of Lactic Acid Bacteria | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳陸宏,何國傑,許輔,曾文聖 | |
dc.subject.keyword | 乳酸菌,親緣關係,種專一性引子,雜合探針,融化曲線,變性梯度凝膠電泳, | zh_TW |
dc.subject.keyword | Lactic acid bacteria,phlogenetics,species-specific primer,hybridization probe,melting temperature,DGGE, | en |
dc.relation.page | 120 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-09 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
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