Inhibitiry Effect Of Bacillus Subtilis On Cariogenic Bacteria By Metabolomics Approaches

Objective: This research aimed to determine the possible small molecular metabolites of B.subtilis zh78 which can inhibit growth of cariogenic bacteria(S.mutans,A.viscosus and L.acidophilus)by metabolomics approaches,and to evaluate the prospect of B.subtilis zh78 in dental caries prevention and management.So as to provide a theoretical basis for the application of B.subtilis zh78 in probiotic research.Methods: 1.Antimicrobial assays.Methanol was used to extract the metabolites in the bacteria solutions from the B.subtilis zh78 growth at 0 hour,7 hours,12 hours and 5 days respectively.Then oxford cup method was utilized to verify the antibacterial activity of the extracted metabolites against S.mutans,A.viscosus and L.acidophilus.2.Untargeted metabolomics detection and analysis.After the metabolites in bacteria solutions from each sample of B.subtilis zh78 bacteria liquid(B.subtilis zh78 for the growth of 0 hour,7 hours,12 hours,and 5 days,6 cases per period)were extracted and derived,gas chromatography coupled with time of flight mass spectrometry(GC-TOF-MS)was utilized to metabonomics detection.Then,principal component analysis(PCA),cluster analysis,orthogonal partial least squares discriminant analysis(OPLS-DA)and Pearson correlation analysis were to process the data.3.Targeted metabolomics detection and analysis.The antibacterial related metabolites of B.subtilis zh78 were screened out,and then targeted gas chromatography coupled with mass spectrometry(GC-MS)approach was used for qualitative and quantitative analysis to verify whether B.subtilis zh78 can produce the metabolites relating to antibacterial action,and determine the exact content.Firstyly,standard products of target metabolites were diluted with methanol to obtain standard solution with concentration gradient(0.1 g/mL to 50 g/mL).Next,they were detected with GC-MS.Then,the standard curve was drawn.After the metabolites in bacteria solutions from each sample of B.subtilis zh78(B.subtilis zh78 for the growth of 0 hour and 5 days,6 cases per period)were extracted and derived,GC-MS was utilized to metabonomics detection.Then,in the Masshunter software,the default parameters were used to automatically identify and integrate each transition.Combined with the standard curve of target metabolites standard,the quantitative information of target metabolites in each sample was determined.Results:1.The results of Antimicrobial assays showed that,the metabolites at 7 hours and 12 hours of B.subtilis zh78 showed distinct bacteriostatic effect on L.acidophilus and A.viscosus,but not on S.mutans.The metabolites at 5 days of B.subtilis zh78 showed the most obvious inhibitory effect on A.viscosus,L.acidophilus,S.mutans(P < 0.01).2.The results of untargeted metabolomics approach showed that,certain kinds of substances,such as xylitol,amino acids and organic acids produced by B.subtilis zh78 were significantly related to its inhibitory effect on S.mutans,L.acidophilus,and A.viscosus.3.The target GC-MS metabolomics analysis showed that xylitol determined,with a concentration of 6.53 ?g/mL.Conclusion: Xylitol,the metabolite which possess the antibacterial effect and caries prevention effect,can be produced by B.subtilis zh78.Simultaneously,B.subtilis zh78 could produce certain kinds of metabolites(L-glutamic acid,and L-tyrosine).These metabolites may play a role in promoting antibacterial effects of B.subtilis zh78 on S.mutans,L.acidophilus and A.viscosus.B.subtilis zh78 is probably a strain of dental caries probiotic bacteria in oral cavity,which possess certain application prospect in the prevention of dental caries.