Biodiversity Of Lactobacilli From Traditional Fermented Milk In Tibet, Xinjiang And Yunnan Of China

The identification of Lactobacillus and its genetic diversity research is the basis of exploitation and development of Lactobacillus. 265 strains of Lactobacillus were isolated from traditional fermented products of Yunnan, Xinjiang, and Tibet. On the base of the experimental system of ERIC-PCR and AFLP technology had been optimized, the genetic diversity of the strains was assessed using these two technologies. The results provides the basic data for systematically understanding the genetic diversity of Lactobacillus in China minority regions, and gives useful information for further exploitation and utilization of lactic acid bacteria in traditional fermented milk products.1. Experimental methods about ERIC-PCR and AFLP markers which were suitable for Lactobacillus have been established.First, the system of ERIC-PCR technology was optimized, and the system was performed as follows: extracted genomic DNA (100 ng from each isolate) was amplified in a final volume of 25μL containing 0.2mM (each) dNTP, 25mM MgCl2, 10 pmol/μL of the ERIC primer and 2.0U rTaq DNA poly-merase(TaKaRa). Each amplification product was loaded onto a 1.8% (wt/vol) agarose gel. The bands are clear and easy to discriminate.64 pair of primers was used in 6 different species of Lactobacilli to choose as selective amplification primers, and two of them were selected. Using 2 pair of them (EcoR I-GT/Mse I-T and EcoR I-G/Mse I-TA) could got clearly and affluent bands. They were used in amplification all the 265 strains.2. Genetic diversity of 115 Lactobacilli isolated from fermented milk-products in Tibet was studied. The cluster analysis through ERIC-PCR tech-nique showed that at level of 0.8, tested strains were clustered into 14 groups. L. casei, L. fermentum and L. plantarum could be discriminated, and L. casei and L. helveticus could not be discriminated on the basis of ERIC-PCR. The cluster analysis through AFLP technique showed that at level of 0.8, tested strains were clustered into 4 groups. L. casei, L. fermentum, L. helveticus and L. plantarum could be discriminated on the basis of AFLP. The gene types of L. casei and L. fermentum were also analyzed by ERIC-PCR and AFLP tech-niques. Different techniques got different genetypes, but there was no positive relationship between the AFLP/ERIC-PCR gene types and the origin or iso-lated matrixs of the strains in Tibet. 3. Genetic diversity of 81 Lactobacilli isolated from Koumiss in Xinjiang was studied. The cluster analysis through ERIC-PCR technique showed that at level of 0.76, tested strains were clustered into 4 groups. Through AFLP technique, tested strains were clustered into 6 groups at level of 0.805. L. casei and L. helveticus could be discriminated through these two techniques. The genetypes of L. helveticus was also analyzed by ERIC-PCR and AFLP techniques. Different techniques got different genetypes, but there was no positive relationship between the genetypes and the origin or isolated matrixs of the strains in Xinjiang.4. Genetic diversity of 69 Lactobacilli isolated from Acid Whey for Dairy Fan in Yunnan was studied. All tested strains were clustered into 4 groups at level of 0.8 through ERIC-PCR technique. Through this technique, L.fermentum, L.kefirgranum, L.helveticus and L.plantarum could be dis-criminated, and L. helveticus and L.suntoryeus could not be discriminated. The cluster analysis through AFLP technique showed that at level of 0.89, tested strains were clustered into 5 groups. L.fermentum,L.kefirgranum,L.helveticus, L.plantarum and L.suntoryeus could be discriminated at certain simililarity level.The genetypes of L. helveticus was also analyzed by ERIC-PCR and AFLP techniques. Different techniques got different genetypes, but there was no positive relationship between the genetypes and the origin or isolated matrixs of the strains in Yunnan.5. Genetic diversity of 105 L.helveticus from Tibet, Xingjiang and Yunnan were studied. Through ERIC-PCR technique, the strains were clus-tered into 7 groups at level of 0.8. L.helveticus from Tibet clustered together, but strains from Xinjiang and Yunnan could not be discriminated between each other. Through AFLP technique, the 105 strains were clustered into 5 groups at the level of 0.8. Strains from different geographic origin could be discriminated through AFLP technique.