| Probiotics are a kind of live additives that exerts a beneficial effect on the host by improving intestinal microbial balance. In order to play their functions, probiotics should be able to tolerate the adverse environment of the gastrointestinal tract, and also colonize in the intestine. The beneficial effect to human body is strain dependent, however at present the domestic probiotics products only provide the name of the species without the name of the strains used, and the whole genome information. Therefore, the conbined method of screening test in vitro and functional genomic comparative analysis can provide a more comprehensive evaluation on the probiotic traits and safety of strains, which has an important significance in developing probiotics pruducts. In this thesis, 116 strains of lactic acid bacteria were isolated from the traditional fermented dairy products, which were used to screen out and identify potential probiotic strains with the ability to inhibit the growth of pathogen bacteria. Subsequently the correlation analysis of probiotic traits and safety of Enterococcus faecalis LD33 were carried out by a combination of genome sequencing and a drug sensitivity test. Finally, based on the results of transcriptome sequencing and metabolites analysis, high density proliferation conditions of E. faecalis LD33 were optimized.116 strains of lactic acid bacteria were isolated from traditional fermented dairy products, including 85 strains of lactobacillus and 31 strains of cocci. By in vitro testing four lactic acid bacteria strains which presented inhibition ability on common pathogenic bacteria by producing bacteriocin were screened. And they were chracterized as Lactobacillus plantarum subsp. plantarum W2, Lactobacillus helveticus H1, Enterococcus faecalis 1103 and LD33 through the API 50 CH system, 16 S r RNA gene homology analysis and phylogenetic tree construct ion based on 16 S r RNA and phe S gene sequences. Then adhesion and acid tolerance and bile salt tolerance ability of the four strains were investigated by in vitro tests. Finally E. faecalis LD33 was confirmed as potential probiotic strain. And the bacteriocin produced by E. faecalis LD33 was purified and the molecular weight of which was determined approximately as 10 k Da by Tricine SDS-PAGE electrophoresis, which tolerants to high temperature and shows a good stability under the different acid and alkali environment and high temperature.The whole genome sequence of strain E. faecalis LD33 was obtained by genome sequencing, and its genome size is 2 803 429 bp in length, with GC content of 37.58% and no plasmid. The comparative genomics analysis showed that, its chromosome contains encoding genes of ATPase, Na+/H+ antiporter protein, BSH, CFA synthase and a plurality of two-component regulatory system which is closely related to the acid and bile salt tolerance. And it also contains encoding genes of adhesion related adhesion exoprotein, fibronectin-binding protein, cell surface protein, sortase and elongation factor binding protein. In addition, there is also a bacteriocin synthesis gene cluster which is composed of two-component regulatory system, antibacterial peptide ABC transporter permease and bacteriocin encoding gene. These functional genes can be exactly associated with the function which showed in previous in vitro experimental results with the good acid tolerance, bile tolerance and adhesion ability, as well as the inhibitory effect of the pathogen of E. faecalis LD33. The comparative genomic analysis also indicated that the chromosome of E. faecalis LD33 contains no gene encoding pathogenic virulence factors and does not show any mobile genetic elements such as gene island and plasmid that is the basic conditions for drug resistance spreading. Antibiotic susceptibility test showed that it only had aminoglycoside antibiotics and rifampicin resistant, which can be used as probiotics safely.The differentially gene expression profiles of respiratory chain and sugar metabolism related genes of E. faecalis LD33 undergoing fermentation and aerobic respiration metabolism were compared by transcriptome sequencing technology. It was noted that coding genes of respiratory chain of E. faecalis LD33 were constitutively expressed, which was unrelated to whether it was undergoing aerobic respiration metabolism or not. Combined with metabolic product analysis, the results showed that when it conducted aerobic respiration, the metabolic pathways of glucose was changed by regulating the expression of genes of pyruvate metabolism pathway and butyric acid metabolism pathway. For example, the expression of gap, pdh, eut D, ack A, als S and but A gene were up-regulated, and the expression of ldh gene was down-regulated. And the major end products of carbohydrate metabolism was changed from lactic acid into lactic acid, acetic acid and acetoin. When glucose was depleted, it can be continued to proliferate through the metabolism of other carbohydrates and amino acids, then a higher biomass and a strong long-term survival rate were obtained. Based on results of the above transcriptome and metabolites analysis, the optimization results of high density culture conditions of E. faecalis LD33 indicated that when inoculum was 1%, initial p H was 6.4, rotational speed was 220 r/min and the content of heme added was 0.01 g/L, furthurmore the optimum formular of medium was as follows: whey 60 g/L, peptone 30 g/L, glucose 30 g/L and yeast powder 10 g/L, and after cultivated at 37 ℃ for 24 h the viable cell counts of fermentation broth of E. faecalis LD33 could reach 6.37 × 109 CFU/m L. Long term preservation experiment data showed that after storage at 4 ℃ for five days, the viable cell count of E. faecalis LD33 cultured under fermentation conditions had decreased to 106 CFU/m L. And after 30 days the viable cell count of was hard to detect, while after 45 days preservation, the viable cell counts of E. faecalis LD33 cultured under respiratory conditions remained at about 10~8 CFU/m L. |
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