| In recent years, There are much report about quorum sensing (QS) system mediating pathogenic microorganism's pathogenicity, but the research about probiotics, especially for Lactobacillus physiological characteristics of inhibiting pathogenic microorganisms, adhesiveness, acid resisting, animal viability in the digestive tractand is barely, and AI-2/LuxS QS system mediating Lactobacillus bile salt tolerance, maintaining intestinal micro-ecological balance and enhancing the immunity of body is none reported. Based on the current research status, We research QS system mediating two strains of Lactobacillus plantarum's growth characteristics, AI-2/LuxS QS system, antibacterial activity, acid and bile salts resistant, QS luxS gene knock out vector construction and knock out progress, which isolate from Yunnan province traditional fermented food (dou chi) and Shilin fresh goat's milk.Lactic acid bacteria(LAB) is a class of bacteria which can use carbohydrate fermentation into lactic acid, it is not the name of the microbial taxonomy, just a idiomatic term. LAB has a very wide range of distribution, such as in living organisms and the natural environment, if so:human and animal digestive system, urogenital system, waste; also in fermentation of animal and plant food and beverage, plants, soil, available for distribution of lactic acid bacteria in sludge and sewage.Lactobacillus is one of the largest genera of LAB, named because of its shape rods or globular and can ferment carbohydrates (mainly glucose) and generate a large amount of lactic acid. As a main kind of Lactobacillus, Lactobacillus plantarum has many probiotic properties, Such as has a good inhibitory effect to the pathogen, the acid and bile salt tolerance, good adhesion on the intestinal mucosa cells, etc.Quorum sensing (QS) is a form of bacterial cell-cell communication through production and perception of chemical signal molecules. The informational molecules can be divided into three categories:gram-positive bacteria:oligopeptides; gram-negative bacteria:acyl-homoserine lactone (AHL) and diffusible signal factor (DSF); interspecific:autoinducers-2 (AI-2). Among them, AI-2 and its synthase LuxS constitute the AI-2/LuxS QS system, which mediates the intraspecies and interspecies information communication among gram-positive bacteria (G+) and gram-negative bacteria (G-)-This research two strains of Lactobacillus plantarum which isolate from Yunnan province traditional fermented food (dou chi) and Shilin fresh goat's milk. To do the following research and the corresponding results are obtained.1, Determination of the two strains of Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 OD600, pH and cfu growth curve, OD600 and cfu growth curve shows the microbial growth characteristics of typical (demurrage, exponential period, stable period, and decline period), pH growth curve has completely opposite characteristics to the growth curve of OD600 and cfu.2, Using the method of oxford cup double layer tablets antibacterial research two strains of Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3's different growth stages (early exponential (a,4.5 h), middle exponential (b,12 h), middle late exponential (c,18 h) and stability (d,27h)) the supernatant fluid and bacteria break fluid's bacteriostasis to Enterohemorrhagic Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus. Found two strains with similar bacteriostatic. Namely that of supernatant fluids have antibacterial effect, and has the increase with the increase of growth time is characteristic of the change. In addition, the bacteria break fluid in a, b, c and d four points to three kinds of pathogenic bacteria had no antimicrobial effect, illustrate antimicrobial metabolites may be largely secrete substances or enzymes.3,Extract two strains of Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 total RNA at four point of point a(4.5 h):develop early; point b(12 h):mid exponential; point c, (18 h):late exponential and point d (culture 27 h):stable of middle, and reverse transcription reaction generated the template chain cDNA. By relative semi-quantitative determination luxS gene expression, found that two strains of Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 luxS gene expression has the similarity, namely Lactobacillus plantarum AY01 increasing trend with the increase of the growth time, exponential of early no expression, expression appear at exponential of mid, late exponential expression quantity increased obviously, express maximum stability; Lactobacillus plantarum YM4-3 exponential of early appear expression, expression maxmium appear at exponential of mid, late exponential expression quantity reduced obviously, express very little stability.4, Research of two strains Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 at the grow point of point a:early exponential (4.5 h); brmiddle exponential (12 h); c:late exponential (18 h) and d:middle stability (27h), research of acid and bile salt tolerance, study found that two strains of Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 has strong tolerance to acid and bile salt, at pH around 3 is in a state of balance, in about 0.2% mass fraction of bile salts in a state of balance.5, Application of pUC19, pFED760 plasmid to building a successful two strains Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 luxS knockout plasmid pKML01 and pKML02 (Lactobacillus plantarum YM4-3 luxS knockout plasmid) and pKML05 (Lactobacillus plantarum AY01 luxS knockout plasmid).And the two strains Lactobacillus plantarum AY01 and Lactobacillus plantarum YM4-3 luxS gene knockout has made certain progress.This paper not only has the important theory significance, but also have important potential application value. |
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