PCR-DGGE分析绍兴黄酒麦曲中细菌群落方法的建立

Establishment of PCR-DGGE for analysing the bacterial community of Shaoxing rice wine wheat Qu

利用垂直电泳及时间间歇法初步确立了变性剂的梯度范围和电泳时间,首次建立了聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)法分析绍兴黄酒机制生麦曲和熟麦曲中细菌群落结构的电泳条件。机制生麦曲的变性剂梯度范围为50%～65%,电泳时间为5～5.5h;熟麦曲的变性剂梯度范围为40%～60%,电泳时间为4.5～5h。DGGE条带经切胶回收、重新扩增、T-A克隆及DNA测序后,鉴定结果显示绍兴黄酒麦曲中存在糖多孢菌、肠杆菌、棒形杆菌等多属种细菌,并且从熟麦曲中鉴定到的细菌种类都包含于机制生麦曲中的细菌群落,表明不同工艺的黄酒麦曲其细菌群落结构存在着一定的差异。研究结果丰富了对绍兴黄酒麦曲中细菌群落的认识,为进一步剖析细菌在黄酒发酵中的作用奠定一定的基础。

By using the perpendicular-electrophoresis and the Time-travel methods,the denaturing gradient range and the running time of DGGE analysis for investigation on the bacterial communities of two types of Shaoxing rice wine wheat Qu（machine-made crude wheat Qu and cooked wheat Qu） were established. The suitable denaturing gradient range and running time of machine-made crude wheat Qu were 50%-65% and 5-5.5h,respectively. And the suitable denaturing gradient range and running time of cooked wheat Qu were 40%-60% and 4.5-5h,respectively. Banded on the DGGE gel were cut,reamplified,T-A cloned,and sequenced. The results showed Saccharopolyspora sp., Enterobacter cloacae and Clavibacter michiganensis were present in wheat Qu. All the bacteria in cooked wheat Qu could be found in machine-made crude wheat Qu,this suggested that different types of wheat Qu included different bacterial communities. The information gathered from the present research enriched the present knowledge of Shaoxing rice wine-related bacteria,and provided a basis for further clarification of the role of bacterial community in wheat Qu during Shaoxing rice wine fermentation.