Effect Of Exopolysaccharide From Enterococcus Faecium WEFA23 Against Listeria Monocytogenes Infection And Its Application In Dairy Products

Listeria monocytogenes is a foodborne pathogen.It not only causes huge economic losses,but also seriously threatens human life and health.At present,the main treatment method for L.monocytogenes is antibiotics,but the abuse of antibiotics can easily lead to the emergence of drug-resistant bacteria.Probiotic exopolysaccharide has various functions such as antagonizing pathogenic bacteria,inhibiting biofilm formation,and immune regulation,but there is relatively little research on their antagonism against L.monocytogenes.Previous studies had shown that Enterococcus faecium WEFA23 from infants could effectively inhibit L.monocytogenes,and this inhibition might be related to exopolysaccharide.Therefore,exopolysaccharide EPSA23 from E.faecium WEFA23 was isolated and purified in this study.On the one hand,effects of EPS-A23 on the adhesion and internalization of L.monocytogenes in intestinal epithelial cells and the activity of L.monocytogenes biofilm in vitro were investigated.On the other hand,antagonistic effect of different concentrations of EPSA23 on L.monocytogenes infection in vivo was evaluated.On the above basis,impact of EPS-A23 on the quality of fermented milk and its inhibitory effect on L.monocytogenes in dairy products were explored.First of all,effects of EPS-A23 from E.faecium WEFA23 on the adhesion and internalization of L.monocytogenes to Caco-2 cells and the biofilm formation of L.monocytogenes were investigated.The effect of different concentrations of EPS-A23(50,100,200,400,and 800 μg/m L on viability of Caco-2 cells was investigated by CCK8 assay,with the help of cell model,the inhibition of EPS-A23 on adhesion and internalization of Caco-2 cells by L.monocytogenes was explored by means of competition,exclusion and substitution.The results indicated that 50-800 μg/m L of exopolysaccharide is non-toxic to Caco-2 cells,exopolysaccharide significantly inhibit L.monocytogenes adhesion to Caco-2 cells through rejection and replacement,exopolysaccharide significantly inhibited adhesion of L.monocytogenes to caco-2 cells by means of competition(P < 0.0001)and rejection(P < 0.01),and the expression level of Occludin in the exopolysaccharide group is relatively high in both competition and rejection modes.Furthermore,the effect of EPS on L.monocytogenes biofilm formation was studied by crystal violet staining with microporous plates,and the effect of EPS-A23 on L.monocytogenes biofilm formation related factors was investigated by RT-q RCR method.The results showed that polysaccharide inhibited the formation of L.monocytogenes biofilm-related factors and mediated the transcription levels of L.monocytogenes adhesion related genes,thus destroying the integrity of L.monocytogenes biofilm.After that,different concentrations of exopolysaccharide were used to intervene L.monocytogenes infected mouse models to explored the role of E.faecium WEFA23 exopolysaccharide in antagonizing L.monocytogenes infection in vivo by analyzing the number of colonization of L.monocytogenes in mouse intestines and extraintestinal organs,the level of inflammatory factors in serum,the analysis of mouse colon goblet cell,mouse intestinal tissues,and mouse liver histopathology.The results showed that the intervention of exopolysaccharide EPS-A23 can alleviate the liver and spleen enlargement caused by L.monocytogenes infection in mice,alleviate liver inflammation,reduce liver AST and ALT activities,reduce the load of L.monocytogenes in various tissues such as the stomach,ileum,cecum,colon,mesenteric lymph nodes,and liver,the tight junction proteins ZO-1,Claudin-1 and Occludin in the colon of mice caused by L.monocytogenes infection were relieved to varying degrees.In addition,EPS-A23 can improve the levels of serum and colon inflammatory factors;exopolysaccharide could increase the number of goblet cells and the transcription level of MUC2 in colon tissue.These results indicate that the exopolysaccharide of E.faecium WEFA23 can enhance the intestinal barrier function of mice,regulate the immunity of mice,and antagonize L.monocytogenes infection of mice.In the end,EPS-A23 was added to fermented milk to evaluate its effect on the quality of fermented milk.L.monocytogenes contamination model was constructed using restored milk,fermented milk,soy milk and cow milk as carriers to simulate the process of L.monocytogenes contamination of dairy products,and to explore whether EPS-A23 can effectively reduce the contamination of L.monocytogenes in restored milk,soy milk,cow milk and fermented milk at 4℃ and 28℃.The results showed that EPS-A23 can effectively improve the water holding capacity of fermented milk,and EPS-A23 can effectively reduce the whey release rate of fermented milk,indicating that EPS-A23 can improve the quality of fermented milk.The experimental results of further inoculating L.monocytogenes in dairy products showed that adding 50 μg/m L of EPS-A23 to cow’s milk,soy milk,reconstituted milk,and fermented milk could all inhibit the proliferation of L.monocytogenes.The above results showed that the exopolysaccharide of E.faecium WEFA23 could improve the quality of fermented milk and effectively inhibit the proliferation of L.monocytogenes in dairy products.In conclusion,the exopolysaccharide of E.faecium WEFA23 has the effect of antagonizing the infection of L.monocytogenes in vivo and in vitro.It can reduce the adhesive internalization rate of L.monocytogenes,destroy the biological membrane activity of L.monocytogenes,maintain the intestinal barrier function,regulate the immunity of mice,and antagonize the infection of L.monocytogenes.At the same time,E.faecium WEFA23 exopolysaccharide can improve the quality of fermented milk and control the proliferation of L.monocytogenes in dairy products.The results of this study provide data support for the application of E.faecium WEFA23 exopolysaccharide in the control of L.monocytogenes.