Colonization And Movenment Of Lactobacillus Rhamnosus Labeled By Red Fluorescent Protein In Mouse Intestine

As people become increasingly concerned about the relationship between diet and health, research and development of health and functional food has become an important trend infood science research at present. According to the report, intake of probiotics can regulate the intestinal flora and enhance hostimmunity. The premise of probiotics play the beneficial effect is can smoothly through the digestive tractand adhesion and colonization in the intestinal mucus or epithelial cells. This requires the strain has a tolerance for the acid environment in gastric juice and bile salts in resistance to the small intestine. Lactobacillus rhamnosus was isolated from intestinal tract of healthy human, has been understood by the world and belongs to the third generation of probiotics. The advantage of the strain was in the tolerance of gastric acid and bile salt, colonization in a human body for a long time and a relatively stable exist in dairy products. In view of the important function and characteristics of Lactobacillus rhamnosus, the study of its mechanism of colonize the intestinal is critical. Oligosaccharides as prebiotics, is recognized bifidus factors, it can’t be absorbed by the host digest and used of pathogens, can only be use of probiotics, therefore oligosaccharides can promote probiotics multiply and enhance the competitive advantage of probiotics. In this experiment,use oligosaccharide prebiotics as experimental materials, Lactobacillus rhamnosus 05-28 as the tested strains, first of all with the red fluorescent protein dsred2 marker of Lactobacillus rhamnosus, the recombinant strains was gavage of mice, observe the bacteria in different parts of the intestinal colonization, and then screened the oligosaccharides has proliferation ability for recombinant Lactobacillus rhamnosus in vitro and further study on the effects of oligosaccharides on the recombinant strains in the intestine of mice.In this study, the recombinant plasmid p MG36e-dsred2 was transformed into recombination Lactobacillus rhamnosus, and then verified by PCR and extraction plasmid, the plasmid was successfully transferred the experimental strain. The visible red fluorescence of recombination Lactobacillus rhamnosus was observed by fluorescence microscope, the dsred2 gene was expresed. The tolerance of Lactobacillus rhamnosus and recombination strain to gastrointestinal fluid was tested. There were no obvious differences in the survival rate of the two strains. Recombination Lactobacillus rhamnosus can be used to carry out the investigation of the intestinal colonization of mice.The recombinant bacteria fed BALB/c mice, maked the ileum paraffin section, fluorescence microscopy used to detect that recombinant strains can stick to the intestinal villi in mice. Then the colonization law of the intestine was detected. After 3 h all parts of intestine in mice was detected by recombinant strains. In 4 days to reach the peak of colonization, seventh days left in the duodenum, jejunum, ileum, colon recombinant strains accounted for first days, 14.35%, 36.01%, 27.3%, 14.1%. 15 days the intestines in mice has not been detected recombinant strains. At 15 day each mouse intestinal recombinant strains were not detected, the law was ileum > colon > jejunum > duodenum.This experiment adopts the turbidimetric method to research the Oligofructose, Inulin, Xylo-oligosaccharides and Isomaltooligosaccharide in the influence on the growth of recombinant Lactobacillus rhamnosus. Two kinds of the enrichment of oligosaccharides with good effect were selected. When the oligofructose at dosage of 2.4%, oligo xylose dosage is 1.2%, had significant difference with the basic medium. Therefore, further study of fructose and oligosaccharide were the effects of recombinant strain in vivo.Male BALB/c mice were randomly divided into 7 groups,The control group was given recombinant bacteria and PBS, experimental group were givenrecombinant bacteria and low, high dose of fructose and oligosaccharide, consecutive 14 days. In the continuous administration of fos for mice in 7 days, giving mice compared with control group the number of recombinant bacteria were no obvious changes. For 14 days in addition to the low dose group had no obvious change, middle, high dose groups had significantly proliferation of recombinant bacteria. Continuous gavage in mice xylooligosaccharides 7 days, compared with the control group, low and middle dose group, the number of recombinant bacteria has no obvious change, the high dose group increased significantly. Continuous gavage for 14 days, in low, middle, high dose groups had significantly proliferation of recombinant bacteria, but in high dose group, no significant difference. Top gavage 16 days after ach bowel can still detect small amounts of recombinant strain. And most of the colon, ileum, duodenum at least, indicating a major role in colonoligosaccharides.On the basis of the exploration of Lactobacillus rhamnosus colonization in the intestine, explore the relationship between prebiotics and recombinant bacterial for colonization ability, these deepen the understanding of the bacteria, which laid a foundation for its further application.