Study Of The Mechanism And Active Substances Of Lactiplantibacillus Plantarum CCFM8724 On The Inhibition Of Dual Species Biofilm

The incidence of dental caries in preschool children,namely early childhood caries(ECC),ranks first among children’s diseases,seriously endangering children’s health.There are a large number of Candida albicans and Streptococcus mutans in the oral plaque biofilm of children with ECC,and interaction with C.albicans and S.mutans increases the difficulty of ECC prevention and treatment.While brushing,flossing and mouthwash,fillings,and fluoride are effective in removing plaque biofilm,children tend to be unwilling to brush their teeth and less clean.With the increasing popularity of probiotics,Lactobacillus has also become one of the potential means of preventing dental caries.Lactiplantibacillus plantarum CCFM8724,with anti-caries function,which was screened in our laboratory in the previous study,can significantly reduce the amount of S.mutans and C.albicans dual-species biofilm in vitro,and significantly reduced the caries score and demineralization in dental caries rat models.However,the mechanism of inhibition and bio-active compounds are still unclear.This study took L.plantarum CCFM8724 as the research object,and integrated multi-omics to analyze its effects on the metabolic and transcriptional levels during the dual species biofilm formation.The active components that inhibit biofilm formation were identified,and then the dose-response relationship and action targets of several potential material bases were analyzed.The main research contents include the following aspects:First of all,the inhibitory effect of L.plantarum CCFM8724 supernatant on the double-species biofilm formation was revealed by integrating metabolomics and transcriptomics multi-omics analysis methods.In the metabolomic analysis,the carbohydrate metabolism of C.albicans and S.mutans changed significantly.The addition of L.plantarum weakened their ability to utilize sugar and reduced the intermediary communication between them,thereby inhibiting the double-species biofilm formation.In the transcriptomic analysis,the expression of virulence genes in C.albicans was affected,such as adhesion-related genes encoding Als proteins,and carbohydrate utilization genes,reducing the adhesion capacity and the amount of biofilm.Next,in order to reveal the potential substance foundation of biofilm inhibition of L.plantarum CCFM8724,the synthesis metabolite synthesis gene clusters of L.plantarum CCFM8724 was analysed and the bio-active components was subsequently isolated and identified.Genomics analysis showed that L.plantarum CCFM8724 has unique carbohydrate utilization genes,such as more carbohydrate binding module genes,and it contains synthetic gene clusters for post-modified peptide products and cyclic lactone autoinducer synthesis gene clusters.Subsequent analysis of the characteristics of the active ingredients that inhibit the double-species biofilm produced by L.plantarum CCFM8724 showed that the active ingredients were thermally stable,non-protein and hydrogen peroxide substances,and had a narrow p H tolerance.Acceptable,it may be a class of organic acids or small molecules(< 3k Da)moderately polar substances that function under acidic conditions.Next,the supernatant was preliminarily separated by ultrafiltration,organic solvent extraction,and silica gel column chromatography,and the obtained effective fraction was identified by combining LC-MS and GC-MS,which was initially determined to be cyclo(leu-pro),cyclo(phe-pro),phenylpropionic acid and 3-phenyllactic acid,palmitic acid may have the effect of inhibiting the formation of biofilms of S.mutans and C.albicans.Finally,the inhibitory effect of several metabolites on the double-species biofilm formation was verified,and molecular docking of metabolites and pathogenous carbohydrate metabolic targets was simulated,revealing L.plantarum CCFM8724 potential substance foundation and its molecular mechanism.The experiments that single metabolites and complex metabolites affect the formation of double-species biofilms were carried out on several metabolites explored earlier.The results showed that the composition significantly reduced the biofilm formation of S.mutans and C.albicans(reduction rate: 79.4%),the production of EPS(reduction rate 63.8%),extracellular protein(reduction rate 63.8%),and the quorum sensing signal molecule AI-2(reduction rate 80.7%),while destroying the biofilm structure.Molecular docking results revealed that the composition could bind to targets such as S.mutans Ptx A protein and C.albicans Fba protein,affecting their sugar metabolism,thereby inhibiting the biofilms formation.In conclusion,this study explored the active ingredients and mechanisms of L.plantarum CCFM8724,which inhibited biofilm formation by producing active substances to regulate carbohydrate metabolism in cariogenic pathogen,and provided a theoretical basis for its product development.