浑浊米醋Bacillus subtilis分离鉴定及灭菌条件研究  

Isolate and identificate to the Bacillus subtilis from muddy rice vinegar and study the conditions of sterilization

作　　者：任文雅[1] 孙宝国[1] 廖永红[1] 徐瑾[1] 沈晗[1] 

机构地区：[1]北京工商大学化学与环境工程学院,北京100037

出　　处：《食品科技》2010年第12期2-7,共6页Food Science and Technology

基　　金：国家"十一五"科技支撑项目

摘　　要：利用稀释平板法从米醋沉淀中分离获得细菌,通过对其进行16S rDNA分子鉴定,表明该细菌为枯草芽孢杆菌(Bacillus subtilis)。以该菌为实验材料,研究了细菌基因组DNA的提取方法,并将ERIC-PCR法首次应用于醋液菌种,对此反应体系的主要因素进行了优化,最终建立了适合的ERIC-PCR体系。结果表明,采用改良的传统细菌基因组DNA提取方法,所提取的DNA质量较高,能够满足ERIC-PCR反应的需要;建立的反应体系为:25μL反应体积10×扩增缓冲液(含Mg2+)2.5μL,20 pmol/μL ERIC-PCR引物E1 1.0μL,20 pmol/μL ERIC-PCR引物E2 1.0μL,DNA模板2μL,2.5 mmol/L dNTPs混合液1.6μL,taq聚合酶0.9μL,双蒸水补齐;PCR反应程序为:94℃变性4 min,1个循环;94℃变性30 s,58℃退火40 s,72℃延伸1 min,30个循环;72℃延伸4 min。最后对老陈醋浑浊沉淀原因进行了探讨。Bacillus subtilis were isolated by streak platemethod.Identification of bacteria by using the 16S rDNA gene,Bacillus subtilis.In this study,Bacillus strains was used as experimental materials.The method of genomic DNA extraction was studied in Bacillus and the conditions of ERIC-PCR.Finally,ERIC-PCR reaction system suitable for our lab was established.The results showed that high-grade genomic DNA which could meet the requirements of PCR reaction was obtained by the modified methods.The established ERIC-PCR reaction system was as follows： 10×Buffer（Mg2＋） 2.5 μL,20 pmol/μL primer E1 1 μL,20 pmol/μL primer E2 1 μL,DNA template 2 μL,2.5 mmol/L dNTPs 1.6 μL,Taq polymerase 0.9 μL,dd H2O 16 μL,25 μL reaction volume.The reaction program of PCR was devised as follows： 4 min degeneration at 94 ℃（one cycle）,then 30 s degeneration at 94 ℃,40 s annealing at 58 ℃,and 1 min extension at 72 ℃（30 cycles）,and then 4 min final extension at 72 ℃.

关 键 词：枯草芽孢杆菌 米醋 分离 DNA提取 ERIC-PCR 

分 类 号：TS201.3[轻工技术与工程—食品科学]