Characteristic Analysis Of Lactobacillus Plantarum And Its Colonization In Chicken Intestine

Lactobacillus Plantarum(LP)is a genus of Lactobacillus with genetic diversity and metabolic diversity,and it is a standard strain widely used in scientific research.LP can produce antibacterial metabolites,such as acetic acid,lactic acid,fatty acids and other organic acids,diacetyl,H2O2,bacteriocin,lactones,ethanol and oxytocin.When ingested in a certain amount,it will have a beneficial effect on the intestinal morphology,microbial population,nutrient absorption,antioxidant capacity and immune response of broilers,thus benefiting the intestinal health,intestinal immunity and production performance of broilers.The key to the selection of probiotic strains includes their tolerance to various environments in vivo and their ability to metabolize beneficial substances.The intestinal colonization ability of LP determines whether it can stably exert its probiotic function,so it is particularly important to track the whereabouts of LP in the complicated intestinal environment.In this paper,we will systematically study the biological characteristics and metabolites of LP and its colonization law in chicken intestines,in order to lay a theoretical foundation for clinical application of LP.In this experiment,the gastrointestinal environment of animals was simulated to study the tolerance of LP strain to acid,bile salt and artificial intestinal fluid.Its growth cycle was studied by fermentation,and its antagonistic ability and antibiotic sensitivity were studied by drug sensitivity test and in vitro bacteriostasis test.The study found that the optimal pH for LP growth was 6.5,and the survival rate was still 89% after pH1.0 for 3 h,indicating that LP was very well tolerated by acids.The survival rate of LP was 65% after 3 h treatment with 0.3% bile salt concentration,and it was almost unchanged after 2 h treatment with artificial intestinal fluid.The fermentation period of LP strain was 24 h,and the fermentation period was stable from 8to 12 h.50.degree.C.is that high temperature tolerance limit of LP.LP had an inhibitory effect on three pathogenic bacteria,namely avian staphylococcus aureus,salmonella and escherichia coli.The results of the above studies showed that LP had good stress resistance to the gastrointestinal environment and was not resistant to high temperature.The fermentation broth and metabolites had an inhibitory effect on some avian pathogenic bacteria,and they still had the ideal antibacterial effect after thermal killing.In this study,two novel bacteriocin genes Ⅱa and KL1Y were amplified in the LP genome by PCR,and the prokaryotic expression vector PET-32a-IIa-KL1Y was constructed.With IPTG as an inducer,the fusion protein IIa-KL1Y with high content was induced and expressed.The results showed that Ⅱa-KL1Y had high-efficiency inhibition on avian Escherichia coli,avian Salmonella and avian Staphylococcus aureus.In addition,the principle of reverse chromatography was used to verify that five organic acids such as formic acid,acetic acid,citric acid,malic acid,and lactic acid could be produced during LP metabolism.Besides,the organic acids produced by metabolism in different fermentation time periods of 4 h,8 h,12 h,16 h,20 h and 24 h were verified,and it was found that the content of organic acids produced in the early stage of fermentation was high.The results of this test demonstrate the potential of LP as a natural preservative and flavor.On the basis of the above studies,Flourescein isothiocyanate(FITC)was used to label LP strain for in vitro adhesion test and in vivo colonization rule test.The results showed that LP strain could adhere to intestinal epithelial cells with high density or scattered on the cell edge.The LP strain was colonized from 2 h in the intestinal tract,and the colonization took place in a cycle of 48 h and reached the highest value at 18 h.The LP strain can reproduce extensively in the cecum of chicken,reaching the highest colonization at 18 h and less colonization in jejunum and ileum,where almost indefinite colonization in the duodenum can be considered.Thus,the following evaluation was made for the intestinal colonization of LP strain: cecum >jejunum > duodenum.The biological characteristics and the significance of metabolites above were further interpreted,which would provide guidance for their clinical use.