| The proteins can be enzymatic hydrolysis into small molecules of nutrients which are easy absorbed by human body by lactic acid bacteria in yoghurt. While there is a problem that is serious post-acidification has been considered not only seriously affects the flavor, also greatly shortens the shelf life of yogurt. Previous studies indicated that controlling the growth of Lactobacillus delbrueckii subsp. bulgaricus in the late fermentation stage is the key to solve this problem. Before lactic acid bacteria arrive at small intestine playing a role of beneficial function, they need to overcome the acidic environment of fermentation medium of the process of production, storage, transportation, but also overcome the acidic environment (pH2.5-3.5) from human gastrointestinal tract. The ability of acid-resistant of Lactobacillus delbrueckii subsp. bulgaricus plays an important role in the process of fermentation yogurt, post-acidification stage and in gastrointestinal tract.Lactobacillus delbrueckii subsp. bulgaricus is one of the economically most important representatives of homofermentation lactic acid bacteria, because of its worldwide application in yogurt, cheese and other fermented dairy production. Adolfsson O.’s and Shihata A.’s study showed that Lactobacillus delbrueckii subsp. bulgaricus have probiotic function of maintaining the ecological balance, inhibiting the growth of intestinal pathogenic bacteria and improving the immunity of the organism. While there are a lot of work on its fermentation performance and application, the research of its metabolic mechanism has been studied rarely.In this study, the basic biological characteristics and produce acid characteristics of model strain Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 cultivating in RSM are detected. The total RNA extraction time is selected according to the detection results, and then semi-quantitative RT-PCR experiments are conducted with 16S rDNA gene as internal gene. The target genes participated in the acid resistant mechanism are detected in 4 different growth stages. According to the results of semi-quantitative RT-PCR, differentially expressed gene which is closely related to post-acidification is chose out of all of the detection genes. The differentially expressed gene is further in-depth studied, in order to explore the mechanism related to yogurt post-acidification.The results are as follow:(1) the fermentation termination of model strain Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 cultivating in MRS is 11 h, when its pH is 4.8 and the number of living bacterium is 2.3 × 10 CFU/mL. After fermentation termination the culture is stored at 4 ℃ immediately, its pH is 4.6 and the the number of living bacterium is 1.5 × 108 CFU/mL when the culture are stored at 4 ℃ for 7 d. (2) 4 different fermentation stages of RNA extraction:late logarithmic phase (pH 5.3), fermentation termination (pH 4.8), stored at 4 ℃ for 2 h and stored at 4℃ for 7 d(pH 4.6). The total RNA of 4 phases is detected by agarose gel electrophoresis, UV spectrophotometry and RT-PCR. The gene expression of tpiA is different in 4 different fermentation periods, and its gene expression level significantly higher than the other 3 fermentation periods. (3) Construct recombinant vector V1 andV2. Recombinant vector V1 consists of forward tpiA gene and plasmid pMG36e, in order to over-expression of tpiA gene. Recombinant vector V2 consists of reverse tpiA gene and plasmid pMG36e, in order to silence gene tpiA. It turned out that the over-expression of tpiA gene makes no big effect on the production of acid of Lactobacillus delbrueckii subsp. bulgaricus.This study provides more valuable information for the further study of tolerance mechanisms regulating network of Lactobacillus delbrueckii subsp. bulgaricus, and provides the theoretical basis for yoghurt starter cultures which possess the independent intellectual property rights and resisting yogurt post-acidification. |
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