The Prevention And Treatment Effect Of Lactobacillus Plantarum FB-T9 On Dental Caries

Dental caries is one of the chronic infectious diseases,which is caused by sugary diet,cariogenic bacteria and the susceptible host under the catalysis of time resulting in irreparable damage of body tissue of tooth.As a typical and main cariogenic bacteria,Streptococcus mutans can adhere to the tooth surface and produce acid by metabolizing sucrose to destroy the enamel,and form dental plaque biofilm with other bacteria on the tooth surface.This study screened lactic acid bacteria that could inhibit the growth and the biofilm formation of S.mutans,and investigated the specific influence of the aimed strain on the biofilm formation of S.mutans in vitro and measured the probiotic properties and safety when applied to oral cavity.Moreover,a confirmatory experiment was carried out by a caries model in rats with the purpose of providing preliminary theoretical foundation for the prevention and treatment of dental caries.The main contents of this paper are as follows:(1)The isolation and screening of lactic acid bacteria inhibiting the growth and biofilm formation of S.mutans93 LAB strains were isolated from oral samples including saliva,dental plaque and mouth mucosa of the healthy and the cariogenic,feces samples of health people and fresh vegetable leaves and so on.Their antibacterial activity to S.mutans was investigated by oxford cup agar diffusion method,then 12 LAB strains whose inhibitory zone diameters were greater than 15 mm were selected to study their influence on the biofilm formation of S.mutans when co-cultured with S.mutans for 24 h,it turned out that some of them promoted the biofilm formation of S.mutans,while others exhibited inhibition effect.(2)The determination of related properties of LAB strainsThe abilities of auto-aggregation,co-aggregation,self-biofilm formation,surface hydrophobicity and acid and alkali charge of the 12 LAB strains were inspected.And a LAB strain named FB-T9 of good comprehensive properties was selected by principal component analysis.The reduction rate of biofilm formation of S.mutans caused by FB-T9 which showed a poor self-biofilm formation ability(OD600=0.295)was 60.33%.The auto-aggregation rate of FB-T9 at 2 h and 4 h were 28.52% and 35.09%,respectively,and the rate of co-aggregation with S.mutans at 2 h and 4 h were 8.69% and 15.26%,respectively.The surface hydrophobicity and acid and alkali charge of FB-T9 were 52.98%,34.52% and 20.32%,respectively.Eventually,FB-T9 was identified as Lactobacillus plantarum by 16 S rDNA molecular identification.(3)The effect of the mediation of L.plantarum FB-T9 on the biofilm formation of S.mutans at different timesThe effect of the mediation of L.plantarum FB-T9 on the biofilm formation of S.mutans at different times was explored.The results showed that during the initial period(12 h),the middle period(24 h)and the late mature period(48 h)of biofilm formation of S.mutans,compared to control groups,the biofilm quantity of S.mutans could be reduced by 44.41%,40.43% and 23.84% by L.plantarum FB-T9,respectively.And the mediating effect of L.plantarum FB-T9 was the best in early stage of the biofilm formation of S.mutans,and during that time the early adhesion and colonization of S.mutans could be significantly suppressed.Besides,the total bacterium areas of the biofilm of S.mutans at three times were reduced by 66.83%,50.24% and 19.57%,respectively,and the activities of the biofilm of S.mutans at three times were reduced by 36.96%,53.69% and 60.96%,respectively.Therefore,the earlier L.plantarum FB-T9 mediated,the more effcetive the biofilm formation of S.mutans was inhibited.(4)The determination of oral application features of L.plantarum FB-T9To inspect the oral application features of L.plantarum FB-T9,the adhesion of L.plantarum FB-T9 to saliva-coated hydroxyapatite was determined in vitro.It indicated that the adhension rate to the tooth surface of FB-T9(32.93%)was obviously lower than that of S.mutans(69.13%),which illustrated L.plantarum FB-T9 had a low risk of acid etching to tooth surface.Results also showed that the maximum tolerated concentration of lysozyme of FB-T9 was 1.2 mg/mL,which was far higher than that in saliva of human oral(0~57 ?g/mL),manifesting that L.plantarum FB-T9 had the ability to survive under the environment of human oral.(5)The evaluation of the prevention and treatment effect of L.plantarum FB-T9 on caries model in ratsThe anti-caries properties of L.plantarum FB-T9 was verified by a caries model in rats.Chlorhexidine acetate was selected as a positive drug control and another L.plantarum 5D-3,which could promote the biofilm formation of S.mutans in the process of strain screening was selected as a negative reference strain.During the first 5 days,a dose of 0.2 mL 108 CFU/mL of S.mutans was administrated to rats by cotton swab scrubbing to establish caries model in rats.Results showed that the total bacterial population in rats cavity maintained at 2~10×105 CFU/mL,indicating that the level of total bacterial population in rats cavity was relatively stable during the animal experiment.In the treatment groups,the level of S.mutans in rats cavity could be reduced by two orders of magnitude.The results of prevention groups revealed L.plantarum FB-T9 could inhibit the later colonization of S.mutans to a level of 4×103 CFU/mL and the retention capability of L.plantarum FB-T9 in oral cavity was notably better than that of another L.plantarum 5D-3(P<0.05).The final result of caries lesion scoring demonstrated that L.plantarum FB-T9 could reduce the incidence of rats molar teeth enamel caries(E)and dentin shallow caries(Ds)and remit dentin moderate caries(Dm)to some extent.L.plantarum FB-T9 had a superior effect on prevention and treatment of dental caries than that of L.plantarum 5D-3.