| The environment is harsh for organisms at 4500 meters above sea level?asl? in Tibet. In order to adapt to this extreme environment, microbes have produced special response mechanism including new subspecies with special functions.As a product of microorganism fermentation, the unique flavor of Tibet yogurt could be attributed to the lactic acid bacteria species in it. In the thesis, we collected a batch of nature fermented milk at 4500 asl in Tibet, from which we isolated single colonies and preliminary evaluated the acidity of their cultured product by using their p H value. We then extracted whole genome DNAof the strains with high ability of producing acid, and obtained the 16 S r DNA by using PCR and sequencing. Based on the 16 Sr RNA sequences, we constructed the phylogenetic tree to determine their genus position. Proper high acid-produced strains were then selectedto ferment fresh milk separately. To better evaluate the ferment capability of each selected strain, thecommon organic acids contentof the fermented yogurt were determined by HPLC. The main results were as follows:1) Separated lactic acid bacteria from high altitude regions. We collected 11 nature yogurt samples at 4500 asl in Tibet and 1 market yogurt sample. We got 460 single colonies by monocolony isolation, and detected p H of 445 single colonies?443 from nature yogurt sample and 2 from market yogurt sample?. By comparison, we found that p H of most wild single colonies?p H=4.995±0.875? is less than market single colonies?p H=5.795±0.065?.2) Authenticated lactic acid bacteria from high altitude regions. We got 219 16 S r DNA sequence by PCR amplification and sequencing. Through analyzing, we found that there are 183 strains of lactobacillus?about 83.56%?, 17 strains of streptococcus?about 7.76%?, 5 strains of leuconostoc?about 2.28%?, 1 strain of bacillus?about 0.46%?, 13 strains of staphylococcus?about 5.94%?.3)The p H of high acid-produced of strains altered along with fermentation time. Fermented yoghurt of double strains is prepared with major materials of high acid lactobacillus and streptococcus thermophilus. All Fermented yoghurt samples were divided into three clusters: nature lactobacillus and nature streptococcus thermophilus?nature cluster?, nature lactobacillus and market streptococcus thermophilus?mixed cluster?, market lactobacillus and market streptococcus thermophilus?market cluster?. By detecting p H of different time, we found that there is a strong negative correlation between fermentation time and p H, and the power equation(y=6.051x-0.168) was good for fitting the relationship between fermentation time and p H. Market cluster has been fermented at 4h, nature cluster and mixed cluster have been fermented at 6h-10 h. At 14 h, the average p H of nature cluster and mixed cluster was 0.01 lower than market cluster. The average p H of nature cluster and mixed cluster was 0.22 lower than market cluster at 36 h when the different of p H was most. It`s reveal that the curd time of market cluster is shorter than nature cluster and mixed cluster, but fermentative capability of market cluster is less than nature cluster and mixed cluster.4) 8 organic acids in fermented milk were determinated by HPLC. On the concentration of oxalic acid, nature cluster was the best at Primary curd time and 36 h. On the concentration of tartaric acid, nature cluster was the best at Primary curd time, market cluster was the best at 36 h. On the concentration of malic acid, mixed cluster was the best at Primary curd time, nature cluster was the best at 36 h. On the concentration of lactic acid, mixed cluster was the best at Primary curd time, nature cluster was the best at 36 h. On the concentration of acetic acid, nature cluster was the best at Primary curd time and 36 h. On the concentration of citric acid, mixed cluster was the best at Primary curd time and 36 h. On the concentration of succinic acid, market cluster was the best at Primary curd time, nature cluster was the best at 36 h. On the concentration of propionic acid, market cluster was the best at Primary curd time, mixed cluster was the best at 36 h. We use the scoring method to compare 8 organic acid of three clusters, and found that nature cluster and mixed cluster are better than market cluster, and nature cluster is better than mixed cluster.This thesis separated a number of high acid-produced of strains from nature fermented milk at 4500 asl in Tibet, and evaluated acid-producing ability of these strains. These studies are conducive to understand?utilize and develop unique microbial resources in Tibet. |
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