應用核醣體核酸基因序列鑑定食品酵母菌

Abstract

中文摘要 酵母菌種之鑑定，在釀酒、烘焙及其他食品工業上非常重要。傳統酵母菌之鑑定，主要是以外觀型態與生理生化特性來作區分，但既耗費時間且費力。近年來分子生物技術進展快速，科學家利用分子技術於各種微生物的檢測及鑑定之研究。本研究擬利用靈敏度及重複性高之分子生物技術來發展出一套食品酵母菌之鑑定方法。 本研究收集食品相關的酵母菌，包括市售烘焙用麵包酵母十一株、啤酒酵母七株、分離自蘋果酵母二株、分離自楊桃酵母二株、葡萄酒酵母一株、米酒酵母一株及紅酵母菌Xanthophyllomyces dendrorhous 編號為CCRC 21346等共二十五株酵母菌。經增菌培養後，接著將萃取之酵母菌染色體DNA以NL1、NL4這組核苷酸引子進行聚合酶連鎖反應，增幅出rDNA 較大次單元（Large-Subunit, LSU）中之D1/D2區域，可得長度約600 bp之DNA片段。然後將PCR產物直接回收、定序，定序的結果利用Vector NTI Suite Version 6.0 軟體進行比對分析。結果顯示果實酵母中分離自蘋果酵母及分離自楊桃酵母為Hanseniaspora uvarum，其餘的樣品包含麵包酵母粉、啤酒酵母粉、米酒酵母、葡萄酒酵母及生啤酒均為Saccharomyces cerevisiae。 為了進一步區別同種麵包酵母之不同菌株，續以RFLP（Restricted fragment length polymorphism）進行菌株分型之研究。首先以聚合酶連鎖反應增幅rDNA ITS區域，再以具專一性之限制酶HaeⅢ、EcoRI、HinfI作用於各種不同菌株之ITS-PCR產物，結果顯示所呈現之DNA片段數目、長度並無法區分菌株之差異。 因此再利用EPIC(Exon-Primed Intron-Crossing Amplification)方法，以不同核苷酸引子組之組合，針對粒線體中特定COX1 基因DNA片段的intron及exon部分序列增幅出不同大小之片段，並依所增幅片段之長度與數目來進行菌株間之區分。結果顯示可藉由一至數組不同引子組組合，而得以將11株不同來源之不同之S. cerevisiae菌株區分。 本研究結果顯示，利用酵母菌D1/D2區域之間序列之差異，可輕易及準確地區分出不同屬及不同種的酵母菌。但同種菌株之間則無法區別，而欲區分種之內的菌株，則利用不同引子對組合之EPIC方法是為簡便且效果尚稱良好之方法。

英文摘要 The importance of accurate identification of yeasts is well known to researchers working with these microorganisms. Yeast identification is traditionally based on morphological and physiological criteria. However, such methods are time consuming and are not efficient enough for the identification of strains within a single species. The fast growing molecular biology techniques in recent years have provided alternative and additional methods on the classification and identification of the yeast species and strains. By using those molecular biology techniques in this study, we hope to provide a better and faster identification of twenty-five food-related yeasts including one control strain, eleven baker''s yeasts, six food yeasts, three brewing yeasts and four fruit yeasts. In this study, yeast cells used for DNA extraction were grown for approximately 24 hr at 30℃ in 10 ml YM broth. PCR amplifications of different yeast isolates were performed with a set of primers (NL1, NL4). The amplified D1/D2 DNA region on Large-Subunit (LSU) of ribosomal RNA gene (rDNA) was approximately 600 bp in size. The PCR products were further identified by genotyping and DNA sequencing. Our results showed that the fruit yeasts are Hanseniaspora uvarum and all the other tested strains are Saccharomyces cerevisiae. In order to distinguish the different strains in the same species of S. cerevisiae, RFLP (Restriction Fragment Length Polymorphism) and EPIC (Exon-Primed Intron-Crossing Amplifications) techniques were used. The ITS regions of the eleven baker''s yeast rDNA were further amplified by PCR. The RFLP analyses with three restriction endonucleases (HinfI, EcoRI, HaeIII) were unable to differentiate all the baker’s yeast strains in our studies. For the EPIC method, several different conserved primers were designed and used to amplify the introns and exons regions of COX1 gene in the mitochondrial DNA. The EPIC method efficiently distinguished the baker’s yeast strains to five different groups. In this study, the results indicated that the differences in the nucleotide sequence of D1/D2 region could be used to differentiate species within a genus but failed to discriminate among strains.The EPIC method is an appropriate method for identification of baker’s yeast strains within the same species.