本研究利用 acetic acid bacteria (AAB) type III alcohol dehydrogenase 基因 ( adh ) 設計 primer ADH III L187 / ADH III R500檢測 AAB。將 primer 與 AAB 之 reference strains 進行 PCR 反應，可產生 314 bp 的 PCR 產物。其他如 Bacillus cereus、Yersinia enterocolitica、Salmonella enteritidis D1、Lactobacillus amylovorus、Pseudomonas aeruginosa 及 E.coli 等非 AAB 之菌株，則無任何 PCR 產物生成。此外，本實驗共篩選出 79 株疑似 AAB 分離株，經 ADH III L187 / ADH III R500 增幅後，可產生 314 bp PCR 產物有 41 株，進一步將分離株進行產酸能力篩選，產酸能力強且生長快速之分離株共有 29 株。以這 29 株分離株進行 16S rDNA 序列分析。結果顯示所分析之 29 株 AAB 分離株皆為 Acetobacter spp.，由此顯示 ADH III L187 / ADH III R500 primer 對於 AAB 具有高特異性。再將這 29 株分離株進行酒精耐受性試驗，進一步馴養成具高度耐酒精能力之菌株。於29 株分離株中 AAB305 其耐酒精能力達 7％，本研究擬將 AAB305 進行醋的發酵生產製造。 In this study, primers were designed on the basis of the available acetic acid bacteria (AAB) type III alcohol dehydrogenase gene (adh ) (acc. no. AB264314) sequence . The specificity of this set of primers ADH III L187 / ADH III R500 was tested with well defined strains of AAB and non AAB strains, such as Bacillus cereus, Yersinia enterocolitica, Salmonella enteritidis D1, Lactobacillus amylovorus, Pseudomonas aeruginosa and nont6oxigenic E.coli. From the results, the specificity of this set of primers ADH III L187 / ADH III R500 was confirmed. This set of primers was further used for detection of 79 AAB isolated strains directly from different sources. The results showed that 41 isolated strains tested successfully with the same size of PCR products as reference strains of acetic acid bacteria. During the test of acidity, 29 isolated strains were selected for their ability of growth and high acetate production in YGM medium.
Identification of these 29 isolated strains were done by analysis 16S rDNA sequence and the isolated strains were clustered. All tested isolated strains were belonged to Acetobacter spp.. Further study evaluated the alcohol tolerance of AAB isolates. The results showed that AAB305 strains was able to endure 7 % alcohol in YGM medium. In the future, this strain will be used in vinegar fermentation.
