Analysis Of Influencing Factors Of Co-aggregation Of Lactobacillus And Helicobacter Pylori

Infection with Helicobacter pylori is associated with gastrointestinal diseases and is classified as a class 1 carcinogen by the World Health Organization.At present,the method of eradicating H.pylori infection is mainly quadruple antibiotic therapy,supplemented with probiotics to improve the therapeutic effect.The antagonistic mechanisms of probiotics against pathogenic bacteria mainly include production of antibacterial substances,competitive adhesion and colonization sites,enhancement of mucosal barrier and immune regulation.Lactic acid and other metabolites in Lactobacillus fermentation supernatant have always been considered as the main reasons for inhibiting H.pylori growth.Different from previous reports,a German patented strain DSM 17648 that does not depend on the biological activity of probiotics has attracted wide attention.L.reuteri DSM 17648 can bind to H.pylori to form copolymers,preferentially occupying sites on H.pylori surface for colonization and adhesion.A large number of clinical studies have demonstrated that DSM 17648 can reduce H.pylori load in patients and improve the cure rate of treatment.The previous study of our research group found that DSM 17648 was not the best strain compared with Lactobacillus in the domestic strain bank.Therefore,this project is expected to develop the co-aggregation of H.pylori ability of Chinese probiotics,and find a targeted screening method of probiotics through the analysis of macro influencing factors and comparative genome research.The main conclusions are as follows:Firstly,an artificial gastric juice screening model was established to screen Lactobacillus strains from different species with similar or superior characteristics than DSM 17648 that could effectively co-aggregate with H.pylori.A total of 56 strains of Lactobacillus were evaluated for H.pylori aggregation in artificial gastric juice environment.The co-aggregation rate of Lactobacillus and H.pylori was mainly in the range of 10%～80% except for a few strains.Compared with positive reference strains(15.50%),six strains of Lactobacillus kitasatonis S7,Lactobacillus reuteri LYS2,Lactobacillus casei GL14,Lactobacillus rhamnosus SLT1 and SLT2,Lactobacillus acidophilus S5 showed significant high coaggregation rates(> 40%).Subsequently,laser confocal microscopy(LSCM)and scanning electron microscopy(SEM)were used to analyze the micromorphology of the co-aggregation process between Lactobacillus and H.pylori.It was found that the surface of one Lactobacillus could combine with multiple H.pylori and self-aggregation could occur between Lactobacillus,which was conducive to the formation of larger copolymers.Then select the representative strains with different co-aggregation effect to analyze the environmental factors and macro characteristics of Lactobacillus surface respectively.The results showed that the co-aggregation of Lactobacillus and H.pylori was a rapid and stable effect under low p H conditions,and was independent of the biological activity of Lactobacillus,suggesting that the effect may depend on surface substances rather than metabolites of Lactobacillus.Furthermore,the correlation analysis between surface characteristics of Lactobacillus and co-aggregation showed that the co-aggregation rate was significantly positive correlated with the surface hydrophobicity of Lactobacillus,but not with the surface Zeta potential.On this basis,the genomic characteristics of two groups of strains LYS1 and LYS2,SLT1 and SLT6,with significant different co-aggregation rate were analyzed by comparative genomic techniques.The total number of genes in the two groups were similar and belonged to lineal homologous genes.Comparing the core functional genes in different strains with the protein lineal homology cluster database,it was found that most of the core genes were mainly used to encode the most basic metabolism functions of the bacteria.The amino acid transport and metabolism ratio of the strains with better co-aggregation effect were higher than those with poor co-aggregation effect.COG0531 and COG0765 were the most different functional genes in L.reuteri and L.rhamnosus,respectively.The annotation analysis of carbohydrate active enzymes showed that there was no significant correlation between the co-aggregation rate of different strains and genes of five carbohydrate families.Finally,the material basis of coaggregation was investigated by stripping bacterial components and extracting surface material components.The results showed that both wall removal and protein/polysaccharide removal of Lactobacillus resulted in a decrease in the co-aggregation rate,and the most significant decrease was in the removal of surface protein.The results showed that a surface protein with molecular weight between 40 k Da and 50 k Da was the potential material base for co-aggregation of Lactobacillus.In summary,six strains of Lactobacillus with strong H.pylori co-aggregation ability were screened in this study,and the aggregation between strains was proved to be a real and tight effect.Furthermore,it was found that this aggregation could occur stably and effectively in gastric acid environment independent of Lactobacillus activity by exploring the influencing factors and proved that H.pylori binding effect of Lactobacillus was related to amino acid transport and metabolism related functional genes by comparing the genomes.Finally,the important role of surface proteins in co-aggregation was verified by the experiment of molecular separation and extraction of components from Lactobacillus.This study provided a new perspective for resource mining of antagonistic H.pylori probiotics,and also provided related gene and protein targets for directional breeding of functional strains.