Effect Of Bacteriocin-Producing Pediococcus Acidilactici On Intestinal Function Of Mice

Intestine is the largest digestion organ and immune organ of human,so it is closely related to human's health.The stability of the intestinal microecology,the balance of the intestinal flora,the completion of the intestinal barrier,and the expression of intestinal immune cells are the keys to maintaining the normal functioning of the intestine,responding to external food-borne stimuli,and protecting human health.A large number of studies have shown that the disorder of intestinal function can cause a variety of diseases,and the occurrence of a variety of diseases can also cause the disorder of intestinal function,and the two are causally related to each other.Lactobacillus bacteriocins can inhibit pathogenic bacteria from invading the human body and competitively inhibit strains of similar homology in the intestine,but a definite reaserch of oral bacteriocin-producing lactic acid bacteria regulate the intestinal flora structure and immune response in human body and how to recover the intestinal dysfunction caused by the disease model has not yet been clearly studied.Therefore,this study aims to screen and obtain bacteriocin-producing lactic acid bacteria,and explore the effects of bacteriocin-producing lactic acid bacteria on intestinal function under different disease models.The main research contents are as follows:1.Two strains of bacteriocin-producing Pediococcus acidilactici CCFM28 and CCFM18 were screened in vitro,through MALDI-TOF and gene draft sequencing,the two strains were analyzed for their antibacterial properties and differences in gene levels.Eliminating the antibacterial effect of hydrogen peroxide and organic acid produced in the fermentation process,and the protease sensitivity test proved that the two strains of Pediococcus acidilactici have a bacteriostatic effect by producing protein substances through metabolism.It can be preliminarily concluded that the two strains of Pediococcus acidilactici can synthesize and secrete bacteriocins.Since other Lactobacillus may become the most prominent competitor of Pediococcus acidilactici in the ecological niche,Pediococcus acidilactici CCFM28 and CCFM18 strains were compared to 4 indicator strains:Lactobacillus thermophiles,Lactobacillus delbrueckii,Lactobacillus helveticus and Enterococcus faecalis.Through the oxidative stress test,we found that CCFM28 and CCFM18 significantly increased the levels of ROS and MDA in the cells of the indicator strains,and enhanced cell membrane water permeability.MALDI-TOF imaging mass spectrometry(MSI)analysis was used to explore the temporal and spatial distribution of bacteriocins when the antagonism of Pediococcus acidilactici CCFM18 and CCFM28 strains occurred.It is found that CCFM18 not only produces the same pediocin PA-1 as CCFM28 when interacting with the indicator bacteria,but also specifically produces penocin A and coagulin when interacting with Lactobacillus thermophilus,and inhibits the secretion of acidocin 1B from Lactobacillus thermophilus.Analyzeing the gene draft sequencing,it was found that only the biosynthetic gene encoding pediocin PA-1 was present on the CCFM28 chromosome,while the gene clusters encoding pediocin PA-1,penicillin penocin A and coagulin exist in the genome of Pediococcus acidilactici CCFM18.2.The bacteriocin-producing Pediococcus acidilactici adjusted the immune factors in the serum of normal mice,increased the diversity of mice intestinal microbes,and up-regulated the relative abundance of some beneficial bacteria in the intestinal flora.Two bacteriocin-producing strains Pediococcus acidilactici CCFM28,CCFM18 and one nonbacteriocin-producing strain NT17-3 were administered to normal mice daily for 14 days.It was found that the bacteriocin-producing and nonproducing Pediococcus acidilactici had no significant effect on the body weight and gastroinestinal transit rate of mice.Detecting the expression of immune factors by ELISA kit,the bacteriocinproducing Pediococcus acidilactici has different degrees of regulation on the expression of immune factors in the serum of normal mice.The expression of pro-inflammatory factors such as IL-1? and TNF-? was down-regulated,and the expression of anti-inflammatory factors such as IL-10 and IFN-? was down-regulated.Through sequencing and analysis of the mice intestinal flora genome,it was found that the bacteriocin-producing and non-producing Pediococcus acidilactici increased the alpha diversity of the gut microbes in normal mice.At the phylum level of intestinal flora,it reduces the relative abundance of Firmicutes and Bacteroides,and at the genus level,CCFM28 increased the relative abundance of probiotics such as Lactobacillus and Akkermansia,and CCFM18 increased the relative abundance of Bifidobacterium.3.The bacteriocin-producing and nonbacteriocin-producting Pediococcus acidilactici had different degrees of relief effects on mice with constipation induced by loperamide hydrochloride.Loperamide hydrochloride was given to mice in each treatment group every day to induce constipation.In the treatment group,200 ?L of bacteriocin-producing and nonbacteriocin-producting Pediococcus acidilactici was given daily for 14 days.It was found that the bacteriocin-producing Pediococcus acidilactici CCFM28 and CCFM18 had a certain degree of relieving the symptoms of constipation in mice,and the gastroinestinal transit rate and the first case of black stool time of constipated mice have been improved.Enzyme-linked immunosorbent assay was used to determine the expression of gastrointestinal regulatory peptides in the blood of mice.It was found that the bacteriocin-producing Pediococcus acidilactici can increase the expression of excitatory neurotransmitters(Substance P,Motilin MTL,Gas)and decrease the expression of inhibitory neurotransmitters(Somatostatin SS,Vasoactive Peptide VIP,Endothelin ET).Through highthroughput Mi Seq sequencing,it was found that the bacteriocin-producing Pediococcus acidilactici improved the structure of the intestinal flora and increased the relative abundance of Bifidobacterium and Lactobacillus in constipated mice.4.The bacteriocin-producing and nonbacteriocin-producting Pediococcus acidilactici were used to antagonize the invasion of Listeria monocytogenes in vivo.By measuring the body weight and the weight of each organ of mice,and selectively counting Listeria monocytogenes in the organs,we found that the bacteriocin-producing Pediococcus acidilactici prevention group can alleviate weight loss and hepatosplenomegaly caused by the invasion of Listeria monocytogenes,and reduce the detection rate of Listeria monocytogenes in various organs.Through the intestinal tight junction protein and intestinal permeability test,we found that the bacteriocin-producing Pediococcus acidilactici can effectively alleviate the decrease in claudin-1 tight junction protein expression caused by the invasion of Listeria monocytogenes.At the same time,bacteriocin-producing strains can also alleviate the increase in intestinal permeability caused by Listeria monocytogenes through the FITC detection test in the blood.The detection of immune factors in serum revealed that the bacteriocinproducing Pediococcus acidilactici had a positive regulatory effect.The bacteriocin-producing Pediococcus acidilactici also played a role in restoring the structural disorder of the intestinal flora caused by the Listeria monocytogenes,they reduced the increase in the relative abundance of Bacteroides caused by Listeria monocytogenes invasion,and increased the relative abundance of Lactococcus and Lactobacillus.