Mechanism Of Limosilactobacillus Fermentum B44 Inhibiting The Formation Of Streptococcus Mutans Biofilm

Dental caries is one of the most common microbial-mediated oral diseases in human beings.At present,the accepted etiology of dental caries is based on the theory of four factors including oral microorganisms,oral environment,host and time.Excessive exposure to dietary carbohydrates can lead to the accumulation of acids and acid-resistant microorganisms in the oral cavity.Dental caries is caused by an imbalance in the attachment of biofilm to the enamel surface.Effective prevention methods include inhibition of cariogenic microorganisms,treatment with an anti-biofilm agents and control of sugar intake,the aim is to reduce the total amount of biofilms or the level of specific pathogens.Today,Probiotics could be recommended for preventing dental caries,since they may possess fewer side effects in comparison with synthetic antimicrobials.In this experiment,4 strains of Lactobacillus isolated from these different sources were used as experimental subjects.Their hydrophobicity,electrostatic action,aggregation ability,lysozyme resistance,biofilm formation ability and ability to eliminate oral biofilm produced by oral pathogenic bacteria Streptococcus mutans were evaluated.According to the relevant indicators of the tested strains,PCA principal component analysis was used to select candidate strains with more potential to prevent dental caries,and the effect on biofilm structure and the mechanism of inhibiting the growth of Streptococcus mutans biofilm were further analyzed.The results are as follows:(1)Four strains belonging to different Lactobacillus species were evaluated in vitro for their potential as oral probiotics candidates.Cells of all the 4 tested strains of Lactobacillus species demonstrated high hydrophobicity(?77.24%)and electrostatic charge(?15.86%),tolerance to lysozyme and lower biofilm formation ability.The auto-aggregation ability of the the 4 tested latobacillus spp.i.e.L.rhamnosus B6,L.fermentum B44,L.casei LC2 W and L.plantarum ST-III were 37.67% ? 60.89%,while their co-aggregatiion ability with Streptococcus mutans,the predominant pathogen causing dental caries,could reach 63.47%,46.42%,60.5% and 61.51%,respectively.When coincubated with S.mutans in BHI containing 0.3%(w/v)sucrose,the cell free supernatant of the comsumed MRS broth of the 4 Lactobacillus species could significantly inhibit the growth of S.mutans which might be attributed to the synergetic effect of organic acids as well as other antagonistic substances screted by the tested lactobacilli,Besides,the cell free supernatant of the comsumed MRS broth of the 4Lactobacillus species could the reduced the formed S.mutans biofilm by31.04%?54.28%.And using PCA principal component analysis,a strain of Lactobacillus fermentans B44,which not only has good probiotic characteristics in the oral cavity,but also can inhibit the growth of oral pathogenic bacteria Streptococcus mutans and the formation of biofilm,was selected as the candidate strain,and the mechanism of inhibiting the growth of biofilm of Streptococcus mutans was further analyzed.(2)First using scanning electron microscope and laser confocal microscope for the B44 before and after the intervention S.mutans,then observe the changes of biological membrane structure.The results showed that the number and thickness of biofilm decreased obviously after the treatment of Lactobacillus fermentans B44,and the compact island structure became sparse;(3)Secondly,according to the 16 sRNA,the internal reference genes were designed,and the target genes related to the synthesis of exopolysaccharides,acid tolerance and population effect of Streptococcus mutans were introduced to calculate and analyze the changes in the expression levels of related genes,the results showed that the expression of glycosyltransferase,which are related to exopolysaccharide formation,was down regulated,and that of Com CDE,which is related to population effect,was up regulated,this is because Lactobacillus produces organic acids and peroxide during the intervention,allowing Streptococcus mutans to respond to environmental stress at the gene level before and after the intervention of Lactobacillus fermentum B44,In addition,the expression of atp D and agu D decreased,which indicated that the survival ability of the pathogen was weakened in the environment,causes of apparent changes in biofilm formation and growth of Streptococcus mutans;(4)Finally,protein components in fermentation broth were extracted before and after Lactobacillus fermentum intervention,analyzed by LC-MS mass spectrometry,searched by Proteome Discoverer(PD)and built-in SEHT Quest search engine,the differentially expressed proteins were screened by statistical method,and the reasons of inhibition of Streptococcus mutans growth and biofilm formation were analyzed from proteomics.Eight kinds of proteins(hydrolase,cytokinin,cell surface protein,collagen binding protein,Penicillin binding proteins,bacteriocin,signal peptide domain protein,carbohydrate binding protein)related to the formation and growth of Streptococcus mutans biofilm were screened,the changes in the expression of these proteins reduced the adhesion,self-division,exopolysaccharide formation,collagen binding and nutritional competitiveness of the pathogenic bacteria,Therefore,one of the ways of Lactobacillus fermentum inhibiting the formation and growth of biofilm is explained.Therefore,in this study,Lactobacillus fermentans B44 was selected as the strain to be tested through in vitro indicators,and characterization analysis confirmed that B44 could inhibit the formation of Streptococcus mutans biofilm and bacterial growth,and the preliminary mechanism of its inhibition was explored from two aspects of genomics and proteomics analysis.