The Diversity Of Lactic Acid Bacteria In Fermented Milk Products Of China

In China, some national minorities had the custom of homemade the fermented milk products (including the solid and liquid). Among these areas, the fermented milk products were the most popular food in their daily life. The local used a simple and original procedure to make the yogurt what was very abundant with the lactic acid bacteria (LAB) and good for the health. A great number of LAB were found in these fermented products. LAB is a group of Gram-positive bacteria united by a constellation of morphological, metabolic, and physiological. The general description of bacteria include in the group is Gram-positive, non-sporing, non-respiring cocci or rods, which produce lactic acid as major end-product during the fermentation of carbohydrates. According to the culture-dependent and culture-independent methods, the diversity of LAB in fermented milk products which were collected from the Tibet and Xin Jiang province to be analyzed.1. A total of 102 strains were isolated from the all twenty samples by culturing with different mediums. The medium MRS and BCP were used to culture the LAB in the samples. The BCP medium could indicate the LAB according to the color's change. After isolation, the Gram-stain were done, and all the strains were gram positive. At the same time did the CAT test, the strains were positive except thirteen strains. All the strains solidified the milk by ten percent digressed milk test.2. Extracted the DNA of the isolated strains based on the protocol of Del Sal (Del Sal et al.,1988). For PCR amplification of the V3 hyper-variable region of the 16S rDNA gene, the forward primer F341 and the reverse primer R534 were used. SSCP was performed on a Bio Rad SSCP system with the PCR products, and from the differences of the main bands of each lane we classified the strains into groups.3. The isolations were identified by the API50 and API20 test. The similarity of the test results up to 80% could be regarded as the species of the test strains. The rest were identified by 16S rRNA-based sequencing. Based on the species and isolation frequencies of the strains, the diversity analysis results showed that was 90% of them belonged to the genera Lactobacillus. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delrueckii, and Lactobacillus curvatus were the predominant species.4. PCR-DGGE, as the culture-independent method, was used to discover diversity of LAB in fermented milk products. The results showed that there were more than 8 bands in each lane. Based on the principle of the DGGE, it indirectly indicated that the diversity of LAB was higher by culture-independent than by culture-dependent method.5. Analyzed the profile of DGGE with the software Quantity One and SPSS. The Shannon index H showed that sample-18 had the highest diversity within all samples. That means it had the most kinds of lactic acid bacteria and the content of each species was uniform. Similarity analysis was performed by the UPGMA and cluster analysis. The sample 16 had the lowest similarity which was consistence with Shannon index H result. The sample 18 and sample 16 were clustered as one group and far from the other groups. These two samples were different species and content of LAB to others. Considering to the sample collected locations, these two samples were obtained from the Xin Jiang province. To less extent, the different diversity (or community) of LAB among these samples could be related to their local environmental condition.6. Comparison with the results from both methods, the culture-independent method could be better to obtain the more complete information than culture-dependent method because the culturing condition was easy to change. By using culture-independent method, at most,3 species which belonged to Lactobacillus were gotten from one sample (Sample13). Meanwhile, DGGE results got 8 bands at least from one lane. According to the principle of DGGE, each sample at least had 8 species of LAB, the diversity of LAB in samples was conspicuously higher than culture-dependent method. Hence, combination with two methods together could obtain the integrated and correct results.