Isolation And Identification Of Probiotic Lactic Acid Bacteria Derived From Raw Milk And Its Protective Effects Against Dextran Sulfate Sodium-Induced Ulcerative Colitis In Mice

Ulcerative colitis(UC)is an inflammatory disease of the intestine caused by various reasons,and may even develop into cancer.Conventional drug therapy can only alleviate UC to a certain extent and cannot cure it.In recent years,LAB has been proven to regulate intestinal microbiota balance and alleviate inflammation,and is expected to become a new alternative therapy for treating UC.Raw milk is rich in lactic acid bacteria(LAB),which is an important source of probiotics in humans and animals.Therefore,this study isolated and identified 26 strains of lactic acid bacteria from raw milk samples,and evaluated the in vitro probiotic characteristics of the isolated strains,selecting Lactobacillus plantarum L19 with good probiotic potential as a probiotic candidate strain.Then,using DSS induced ulcerative colitis in mice as a model,the prevention and relief effects of Lactobacillus plantarum L19 on mouse colitis were explored from multiple perspectives such as clinical indicators,histopathology,intestinal barrier function,and short chain fatty acids,in order to develop new probiotics and provide a theoretical basis for the prevention and treatment of UC.The main research content and results of this project are as follows:Main contents(1)To obtain new LABs with high probiotic potential,we collected 22 samples of Holstein raw milk from a large-scale farm in Hongya,Sichuan.we isolated and purified 26 LAB isolates(named L1-L26)by dilution coating method.We identified them at the species level by biochemical identification and 16 S r DNA sequencing methods.Subsequently,the probiotic property and safety of 26 LAB isolated strains,including antagonistic activity,co-aggregative ability,survivability in the simulated gastrointestinal tract conditions and phenol,auto-aggregation and hydrophobicity,hemolytic activity,and antibiotic susceptibility,were evaluated in vitro.(2)We investigated the effect of Lactiplantibacillus plantarum L19 on DSS-induced ulcerative colitis in C57BL/6 mice.This experiment used C57BL/6 mice to construct a UC model in vivo by free drinking 3% DSS,and used mesalazine as a positive control.In addition,high and low concentrations of Lactobacillus plantarum L19 bacterial suspension and L19 cell-free suspension to intervene the UC mice,the clinical physiological manifestations of UC mice were recorded daily,such as loss of appetite,weight loss,fecal characteristics,diarrhea,and bloody stools.After four weeks,the mice were euthanized and feces,spleen,and colon samples were collected from each mouse.The pathological damage of colon tissue was analyzed by H&E staining,the density of goblet cell in colon tissue was analyzed by PAS staining,and the expression of tight junction protein was analyzed by IHC to explore the effect of L19 on the intestinal barrier function in DSS induced UC.Determine the content of short chain fatty acids(SCFAs)in the feces of UC mice by GC-MS.Main Results(1)26 LAB strains isolated from 22 raw milk samples.Five Lactobacillus plantarum strains(L5,L14,L17,L19,and L20)conglutinated with and inhibited all the target pathogens,endured the simulated gastric juice(92.55～99.69%),intestinal juice(76.18-83.39%),and 0.4% phenol(76.95～88.91%);In addition,five LAB isolated strains possessed considerable auto-aggregation(83.91～90.33% at 24 h)and hydrophobicity(79.32～92.70%).Meanwhile,they were non-hemolytic,sensitive to kinds of common antimicrobials;and showed acceptable growth performances.In summary,Five Lactiplantibacillus plantarum isolates(L5,L14,L17,L19,and L20)showed more promising probiotic potential than others,could be preliminarily determined as probiotic candidates.(2)High concertation of Lactobacillus plantarum L19 treatment attenuated body weight loss of DSS induced UC mice,reduced DAI,ameliorated colon tissue damage,and increased the number of goblet cell in the colon epithelium.In addition,Lactobacillus plantarum L19 promotes the upregulation of ZO-1,Occludin,and Claudin-3 expression,thus repairing intestinal barrier function.Meanwhile,compared with the model group,L19 significantly increased the levels of acetic acid,propionic acid,and butyric acid in the feces of mice treated with DSS.In summary,Lactobacillus plantarum L19 may be a novel candidate probiotic strain for UC resistance.In summary,this study isolated and screened 5 strains of LAB with high in vitro probiotic properties and safety from raw milk in Hongya,Sichuan.Among them,Lactobacillus plantarum L19 has a certain protective effect on DSS induced ulcerative colitis in mice,and is expected to become a candidate probiotic for the new treatment of UC.