Detection And Identification Of LAB By PCR-DGGE

Lactic acid bacteria (LAB) commonly present in the intestinal trace of human and animal or the surface of fresh plant. Most of them were used as starter cultures in the manufacturing processes of various fermented foods, beverages, and feed products. For their long history of safe use, LAB were commonly referred to as the "generally recognized as safe (GRAS)" strains. Analysis of the species of LAB and changes in microbial communities during the fermentation process was traditionally relied on the bacteriological culture techniques and microscopy. Bacteriological culture techniques have been showed the limitations in microbial ecological studies, particularly during to the presence of the cells that are not detectable by culture methods. The application of recently developed culture-independent molecular techniques permits more detailed investigations of the microflora. Denaturing gradient gel electrophoresis (DGGE) has been demonstrated to be a suitable tool for the analysis of microbial community. In this thesis, PCR-DGGE technique combined with classically plate culture method and sequencing of partial 16S ribosomal RNA (rRNA) genes were applied to analyze the microbial diversity, especially lactic acid bacteria diversity in the fermentation products and special ecosystem.DGGE analysis was widely applied in the detection of LAB in food samples and environments. DGGE conditions were firstly optimized for direct identification LAB populations such as gradient of denaturant and length of time of electrophoresis. The perpendicular DGGE revealed that 30%～55% was a suitable denaturant range of gradient for partially melting DNA fragments. To determine the length of time of electrophoresis, 16S rDNA fragments were applied to perform a time-travel DGGE onto a parallel gel with a linearly increasing gradient of from 30% to 55% denaturant, and the results indicated that about 220min was a proper length to obtain good separation in given gel. The different amplification of efficiency during PCR by using...