Isolation And Identification Of Lactobacilli From Healthy Human Feces And Initial Research On Genetic Polymorphism

Lactobacilli commonly present in the intestinal trace of human and animal or the surface of fresh plant. In resent years, a great number of researches on lactobacilli have been made by scholars in morphology, physiology, taxology, genetics, ecology, function and application. As a result of their probiotics function, there is a great deal of probiotics products containing lactobacilli all over the world, and they won the favor of consumers. For promoting exploitation of these products, isolating lactobacilli from human is much more important, because lactobacilli from human is much more easy to survive in intestinal tract.Along with the rapid development of molecular biology and the application of biophysics technology, identification technology of lactobacilli has already developed from the traditional morphology analysis, the biochemistry experiment method to the application of various molecular biology technologies based on PCR. Even using molecular markers can identify lactobacilli as strains. Conventional methods may more easily misidentified microorganisms, so the molecular technology is required to combine with the identify methods of traditional physiology and biochemistry to identify lactobacilli exactly.The project is mainly divided into four following parts:1.Isolation and purification of LactobacilliFecal samples were collected from health individuals of different ages(adult and school age children).Samples were taken immediately after defecation. Lactobacilli counts of fecal samples were determined by selective plating MRS under anaerobic conditions.Through the physiology biochemistry tests of morphology, hydrogen peroxide enzyme and sugar fermentation to identify these isolates initially, then record the results, and 56 strains were primarily identified as lactobacilli.2. Using 16S rDNA sequence homology analysis to identify LactobacilliThe extensional steps of the method of 16S rDNA sequence homology analysis included: lactobacilli genomic DNAs preparation, PCR amplification, products sequencing, and homology analysis. The sequence results about these 20 test strains were compared with other 16S rDNA sequences of lactobacilli in GenBank, thus these strains could be differentiated exactly. Finally, 1 strain of Lactobacillus acidophilus, 1 strain of Lactobacillus crispatus, 1 strain of Weissella. cibaria, 1 strain of Lactobacillus salivarius subsp. salivarius, 1 strain of Lactobacillus pentosus, 2 strains of Lactobacillus mucosae, 5 strains of Lactobacillus fermentum, 8 strains of Lactobacillus plantarum were obtained.3. Using Denaturing Gradient Gel Electrophoresis (DGGE) to construct the standard finger printing of LactobacilliUsing the method of DGGE to construction the standard finger printing of these test organisms.The PCR amplification was carried out at V7-V8 variable region of 16S rDNA of genomic DNA. 35~55%was a suitable denatuant range of gradient with good results of isolation, the test organisms were divided into 7 species by observating the finger printing. The results of PCR-DGGE were different from the results of identification, this illustrated that the method of DGGE also presents some limitations.4. Using Amplified Fragment Length Polymorphic DNA (AFLP) markers to research genetic polymorphism of LactobacilliScreening 1 primer which with better polymorphism from 64 random primers, which was used in the analysis of genetic polymorphism of lactobacilli. Trial and error demonstrated that: AFLP finger printing has well repeatability and highly accuracy, the obtained polyacrylamide gel electrophoretogram could display genetic differences between lactobacilli, this illustrated that the polymorphism between differrent strains belong to the same species of lactobacilli were based on AFLP markers.In this study, to aim directly at intestinal tract flora of healthy human, combinating the methods of traditional physiology-biochemistry and molecular biology, these 20 isolates which from healthy human were identified to the level of species, onstructing the standard finger printing of Lactobacilli by DGGE, and using AFLP markers to research genetic polymorphism of different strains between Lactobacilli. In order to establish the foundation in developmenting resources of lactic acid bacteria in our country, probiotics strains, and intestinal microecology health care products.