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標題: Identification and subtyping of wine yeasts by molecular techniques
作者: 鄔美雲
Wu, Mei-Yun
關鍵字: 酵母菌;yeast;內轉錄區間;限制性片段長度多態性;微衛星PCR;PCR-DGGE;Internal transcribed spacer;Restricted fragment length polymorphism;Microsatellite PCR
出版社: 食品科學系
Wine fermentation is a complex ecological and biochemical process, in which yeasts play a central role. These fermentative species are not only for the alcoholic production, but also contribute the final taste and flavor in wine. It is laborious and time-consuming to identify and classify the yeasts in traditional methods. In this work, we applied molecular techniques to characterize the yeasts of wine origin, and denaturing gradient gel electrophoresis of polymerase chain reaction (PCR-DGGE) analysis was used to characterize the yeast flora during fermentation. Yeast isolates from grapes were firstly classified by morphological characteristics into 19 groups. Restriction patterns were generated from PCR-amplified products of ITS1-5.8S-ITS2 region of nuclear ribosomal gene complex using primers ITS1/ITS4. Sixteen different restriction profiles were obtained from the restriction analysis of PCR-ITS products with four endonuleases of HaeⅢ、CofⅠ、HinfⅠand MspⅠ. The D1/D2 region of 26S ribosomal DNA was amplified by PCR with primers NL1/NL4 and subjected to DNA sequencing analysis for the species identification of yeast isolates. Microsatellite PCR technique was further utilized for yeast isolates subtyping. Finally, PCR-DGGE amplified D1 region of 26S ribosomal DNA was used for characterization of the yeast flora during wine fermentation. With this method the microbial changes in model wine fermentation were profiled, and the results showed discriminatory profiles of individual yeast strains during wine fermentation process.

酵母菌在葡萄酒醱酵過程中,除了產生酒精外,也提供葡萄酒特殊的風味與口感。鑑於傳統鑑定方法繁複且費時,本研究應用靈敏度及再現性高之分子生物技術進行酵母菌種之快速鑑定及菌株分型,並進一步利用PCR-DGGE技術分析葡萄酒醱酵過程中酵母菌相之變化情況。首先自市售葡萄及釀酒用葡萄,共篩選出一百零二株酵母菌,經光學顯微鏡鏡檢及形態觀測後,先初分為十九群。然後使用引子對IT-1及IT-4以PCR增幅酵母分離株之rDNA內轉錄區間(Internal transcribed spacers;ITS)後,以具專一性之限制酶HaeⅢ、CofⅠ、HinfⅠ及MspⅠ作用於不同菌株之ITS-PCR產物,進行限制性片段長度多態性(Restricted fragment length polymorphism, RFLP)分析,依照反應結果之DNA片段數目與長度可將測試菌株區分為十六型。再利用PCR增幅26S rDNA中D1/D2區域作序列分析進行菌種的鑑定與分類。菌株分型方面,採用微衛星PCR (Microsatellite PCR)技術可更進一步將同種酵母菌株做種內的分型。以PCR-DGGE應用於觀測模擬葡萄酒醱酵階段酵母菌相變化情況之結果顯示PCR-DGGE技術可快速及有效區別出醱酵液中混合酵母菌相之個別菌株之變化情形。
Appears in Collections:食品暨應用生物科技學系

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