The Analysis Of Lactic Acid Bacteria Biodiversity And The Identification Of The Isolates In Chinese Hand-made ’suan-cai’ And Yogurt

The number of species with traditional fermented foods in our country is the highest in the world, such as various kinds of wine, fermented meat products,fermented dairy products and fermented vegetables. There are many kinds of microorganisms in food fermentation process, among which lactic acid bacteria are the most important one. Lactic acid bacteria(LABs) are considered as the most functionally important microorganisms during the fermentation process of suan-cai and fermented milk, of which the metabolic activities largely contributed to flavors,aromas, and textures of fermented foods. For yogurt products in Northwest China and suan-cai products in Southwest China, the family hand-made products have its unique flavor. The composition of microbial community in fermentation process is determines the different flavors of fermented food. Therefore, it is no doubt that isolation of these microbes in the fermented foods can enrich our microbial collection,diversify microbial starters, and drive development of new industry and market.This paper aims to explore the lactic acid bacteria diversity distribution in traditional Chinese fermented foods. Then we build a Culture Collection of lactic acid bacteria to promote the rapid development of fermentation industry in our country.(1)We using molecular biology method to analysis the biodiversity in our handmade suan-cai samples. At the same time, we combined with traditional culture method to select the suitable culture medium of lactic acid bacteria to isolate and identify the LAB in suan-cai samples;(2) We choose the suitable culture medium to isolate and identify the LAB in fermented dairy products, especially of Streptococcus thermophilus. We established a new method of to classify the S.thermophilus.The results of this study were summarized as follows:1. The molecular biology method which was high throughput sequencing was used to analysis the lactic acid bacteria biodiversity of 28 suan-cai samples in Sichuan,Guangi and Guizhou Provinces. The results show that lactic acid bacteria(LAB) were abundant in the suan-cai samples with 90% distributed in the Lactobacillus,Pediococcus, Lactococcus, Leuconostoc and Weissela. The content of Lactobacillus had no significant difference in three areas; Pediococcus was mainly distributed in Guizhou(52.60%) and Sichuan(43.26%) Provinces; Leuconostoc only distribution in Sichuan and Guizhou Provinces, Weissela and Lactococcus lactis mainly distributed in Sichuan suan-cai samples.2. On the other hand, we used MRS medium to isolate LAB from 28 suan-cai samples. 1255 LAB isolates were distributed in Lactobacillus and Pediococcus.However, the most predominant specie which was Lactobacillus acetotolerans was not isolated from suan-cai samples. At the same time, we also according to the physiological characteristics of Lactobacillus acetotolerans designed the improved MRS medium. We got the Lactobacillus acetotolerans isolates from suan-cai sanples for the first time with the improved MRS medium. Morever we isolated Lactobacillus paracollinoides which failed to isolated using MRS medium. A total of 2370 strains of LAB were isolated with two cultures. Compared with the molecular methods,almost all the dominant LAB in suan-cai samples could be isolated.3. We establish a novel typing method for Streptococcus thermophilus based on spacers of CRISPR 1 locus. S. thermophiles isolated from three home-made yogurts samples were apparently grouped into three types according to their sequence and arrangement of spacers of CRISPR1 locus: no sequence identical spacer was shared by different types of strains, while strains belong to the same type have almost identical arrangement of spacers with identical sequence. In addition, the result of this typing method was consistence with that obtained by a usual typing method(ERIC-PCR fingerprinting). The advantages of the sequence typing method of CRISPR1 locus typing is that the results are stable and easy to type, and the classification results are easy to be stored in the database to facilitate the comparison of different strains.4. We using selective medium of M17 and FSDA obtained 1289 S. thermophilus in 30 yogurt samples which collected from Qinghai, Xinjiang and Inner Mongolia three areas. The isolates of S. thermophiles were classified for 27 types used the typing methods in chapter three. We make a sequence alignment with the known sequence of the CRISPR 1 CRISPR locus in the database, a total of 14 species were new types of S. thermophiles.5. In order to the analysis the distribution of LAB in Chinese yogurt samples. We got 876 strains LAB from yogurt samples with MRS medium, which could indicatethe distribution of LAB in yoghurt samples. In various regions, Lactobacillus delbrueckii(100% of the isolates) was dominant LAB in Qinghai yogurt samples;Lactobacillus helveticus(34% of the isolates) and Lactobacillus kefiranofaciens(26%of the isolates) were the dominant LAB in Inner Mongolia yogurt samples;Lactobacillus helveticus(82% of the isolates) was dominant LAB in Xinjiang yogurt samples. The diversity of LAB in the three regions is from high to low turned for Inner Mongolia, Xinjiang and Qinghai.