Analysis On Microbial Communities In Luzhou-flavor Liquor Through Culture Independent Approach

The production process of the Luzhou-flavor Liquor is a complex metabolic procedure which concerns huge microbial communities. These huge microbial communities mainly come from Jiaoni and Quyao, which are the foundation for the formation of Zaopei microbial communities. Bacteria make up an important part of the Quyao microorganisms. The formation and the changes of bacteria in Quyao play a significant role in the style of Luzhou-flavor liquor. Attributed to the dispersed research and laggard technologies, the knowledge and research are limited on Quyao.In our reasearch, Quyao were provided by the Luzhou laojiao Co. Ltd. Quyao were crushed and mixed for the analysis. Because of the complex components of the Quyao, various methods had been tried until we found the following one. Multi-beads shocker, enzymes and surfactants were combined to extract the total bacteria DNA from Quyao. Then the bacteria 16S rDNA sequences were amplified by PCR. PCR products were purified to establish the genomic library. After ligation, transformation and positive clone screening, all the 16S rDNA genes of bacteria in Quyao were sequenced. Finally, clone analysis and sequence homology analysis were used and the phylogenetic tree was established, which gave us some novel knowledge of the composition of the uncultured bacteria in Quyao. Results of the culture independent approach are as follows. Through the analysis of the bacteria in the Quyao, we found the bacterium of Luzhou-flavor was consisted of Uncultured beta proteobacterium (AJ318110.1) , Comamonas cf. acidovorans (AF181575.1) , Delftia acidovorans (AB020186.1) , Uncultured bacterium (AB176235.1) , Dysgonomonas mossii (AJ319867.2) , Uncultured Bactero idetes bacterium (AJ318110.1), Dys gonomo nas wimpennyi (AY643492.1), Delfiia tsuruhatensis (AY899912.1) , Pseudomonas sp. (AJ704794.1), Arthrobacter sp. (AJ810897.1) , Nocardiopsis sp. (AM236241.1) . And Delftia acidovorans, Dysgonomonas mossii, Dysgonomonas wimpennyi, Uncultured Bacteroidetes bacterium, Delftia tsuruhatensis, Arthrobacter sp., Nocardiopsis sp. had never been found in open literature about Quyao.According to the sequences alignment and analysis of comparability, the relationship of Uncultured beta proteobacterium, Comamonas cf acidovorans / Delftia acidovorans and Uncultured bacterium , the relationship of Dysgonomonas mossii, Uncultured Bacteroidetes bacterium and Dysgonomonas wimpennyi is near, the relationship of Delftia tsuruhatensis, Pseudomonas sp., Arthrobacter sp. and Nocardiopsis sp. is comparatively near. Each genus is on the different branches of the phylogeny tree, the relationships among them are relatively far. So it took on some kind of genetic diversity and genus diversity of the bacteria in the Quyao.Bacteria communities identified in our work shows some similarity while some diversity with which reported in former research papers.Through culture independent approach, we found that the genus Micromonospora is a dominant genus among the Quyao bacteria, which was same as the results of the traditional bacteira classification. However, there were diverse results in many aspects. For example, some researchers found common bacteria like lactobacillus, Acetobacter in Daqu by conventional methods, but they were not identified through culture independent approach. The production of Daqu follows a traditional process which includes natural growth of the bacteria and enrichment of the microorganisms in the environment. The species and quantity of the microorganisms vary with the natural environment. Our country has a vast territory, which leads to various environment and climate types. As a result, the quality of Daqu is unstable. This unstability may result from the difference of the climate, soil, Quyao produce process and experimental methods of different brewery. By means of molecular biology, researchers identified some bacteria communities which had not been qualitative by microbe taxology nor been reported through conventional identifying methods. The effects of these bacteria communities on quyao are still waiting for further research.