Screening Of Lactic Acid Bacteria With Potential Probiotics And Study On The Mechanism Of Anti-Salmonella Adhesion To Caco-2 Cells

Lactobacillus,a common probiotic bacteria,which belongs to dominant bacteria in the intestinal tract,has an important impact on the intestinal health of the host.Lactic acid bacteria can play their probiotic function only when they are colonized and proliferated in the intestine.Therefore,adherence and gastrointestinal tolerance are important indicators for screening lactic acid bacteria with intestinal probiotic properties in vitro.Salmonella,a common enteropathogenic bacterium,has pathogenicity to human and animal.Adhesion is also the basis for Salmonella to destroy intestinal mucosa,produce toxins and invade cells.Studying the mechanism of lactic acid bacteria inhibiting Salmonella adhesion is helpful to clarify the improvement of gastrointestinal environment and the prevention and control of pathogenic bacteria.It is of great significance to the development and utilization of lactic acid bacteria resources with intestinal probiotic characteristics.In this text,15 different lactic acid bacteria preserved in laboratory,isolated from fermented food and possessing antimicrobial activity against various spoilage bacteria,were used as materials.A probiotic Lactobacillus plantarum C174 was obtained by screening simulated gastrointestinal tolerance and adhesion to intestinal epithelial Caco-2 cells.The adhesion properties and mechanism of Lactobacillus plantarum C174 and its antagonism against Salmonella adhesion were studied.The main results are as follows:1.Preliminary screening and identification of lactic acid bacteria with intestinal probiotic characteristicsLactic acid bacteria with antimicrobial activity against Salmonella typhimurium and Salmonella enteritidis were screened by Oxford cup method.Lactic acid bacteria with strong gastrointestinal tolerance were screened by simulating gastric juice and intestinal juice,and lactic acid bacteria screened were evaluated by hydrophobicity,auto-aggregation and co-aggregation.The results showed that 15 lactic acid bacteria had strong antimicrobial activity,but the difference was not significant.Ten strains of lactic acid bacteria survived more than 106 CFU/mL in simulated gastrointestinal juice environment.The hydrophobicity,auto-aggregation and co-aggregation of 10 strains of lactic acid bacteria with good tolerance were evaluated.The most probiotic potential of 5 strains of lactic acid bacteria C37,C174,D24,D599 and D512 was obtained,and the percentage of auto-aggregation from them was more than 30%within 5 hours.Five strains of lactic acid bacteria include three Lactobacillus plantarum,one Lactobacillus fermentans,and one Lactobacillus brevis by physiological and biochemical analysis and 16S rRNA gene sequencing.2.Evaluation of lactobacillus adhesion to caco-2 cells in vitro and study on adhesion propertiesTaking Lactobacillus rhamnosus ATCC53103 as the control strain,three strains of Lactobacillus plantarum,one strain of Lactobacillus fermentum and one strain of Lactobacillus brevis were screened by Caco-2 cell model.Lactobacillus plantarum C174 with high adhesion was screened for the experiments of bacterial concentration,action time,bacterial growth stage,environmental pH value and adhesion of divalent metal ions.The experimental results are as follows:Lactobacillus plantarum C174 had the best adhesion effect.The adhesion number of Lactobacillus plantarum C174 to Caco-2 cells was 7.4 bacteria/cell,slightly higher than the Lactobacillus rhamnosus GG as control strain,and its adhesion number was 5.4 bacteria/cell.Lactobacillus plantarum C174 was used as the research object to study its adhesion properties and mechanism.It was found that Lactobacillus plantarum C174 adhered to Caco-2 cells in a dose-effect relationship between co-culture time and cell concentration.At the same time,it was found that the adhesion effect of C174 in stable stage was better than that in other growth stages.Lactobacillus plantarum C174 had stronger adhesion to Cao-2 cells in acid-biased environment,and metal Ca2+ions also promoted the adhesion of Lactobacillus plantarum C174.The S-layer proteins of strain C174 mediated the adhesion of strain C174 to Caco-2 cells by different chemical treatments.The S-layer proteins of strain C174 was extracted from 5 M LiCl and its molecular weight was 41 kDa by analysis of SDS-PAGE agarose gel electrophoresis.3.Inhibition effect of Lactobacillus plantarum C174 on Salmonella adhesion and functional properties of its S-layer proteinThe effects of concentration and mode of action of Lactobacillus plantarum C174 on inhibition of Salmonella adhesion to Caco-2 cells were studied,and the protective effect of probiotics on Caco-2 cells was analyzed by detecting the permeability of cell membrane.The results showed that the inhibitory effect of Lactobacillus plantarum C174 on the adhesion of Salmonella was dose-effect relationship.In the mode of action,C174 inhibited the adhesion of Salmonella mainly through exclusion,followed by competition,and the substitution was not obvious,indicating that the addition of probiotic C174 in advance could repel the adhesion and invasion of Salmonella.The results of cell membrane permeability showed that strain C174 can by means of competitive space-occupying effect with pathogen to protect host from damage.By removing surface proteins,the inhibition of Lactobacillus plantarum C174 on Salmonella adhesion was weakened in exclusion and competition mode,and its tolerance to gastrointestinal was also reduced.At the same time,the auto-aggregation ability of strain C174 was also significantly reduced.The results showed that surface proteins mediated the adhesion of Lactobacillus plantarum C174 was the basis of its inhibition on Salmonella adhesion.The high electric point and hydrophobic amino acid of S-layer protein also affect the aggregation of strain C174.