乳酸菌快速鑑定方法之研究

Studies of Rapid Identification Methods of Lactic Acid Bacteria

高怡婷(Yi-Ting Kao)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.00221

摘要

「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。

關鍵字

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

並列摘要

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.

並列關鍵字

Lactic acid bacteria ； phlogenetics ； species-specific primer ； hybridization probe ； melting temperature ； DGGE

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