Screening And Microcapsules Preparation Technology Of Lactic Acid Bacteria With Anti-caries Properties

Dental caries is the most common bacterial infection diseases in the human oral cavity,affecting hundreds of millions of people.As one of the recognized cariogenic bacteria,Streptococcus mutans（S.mutans）plays a significant role in the progress of dental caries.Currently,mechanical methods and drug methods are the main ways to prevent and treat dental caries,but there are some problems such as poor cleanliness and imbalance of bacterial flora,a new safe and effective means should be developed urgently.At present,lactic acid bacteria show promising prospects for development,which attributes to its superior antibacterial activity on oral cariogenic bacteria.In this study,S.mutans was used as indicator bacteria,lactic acid bacteria with anti-caries properties were screened derived from multiple indexes.Then,the anti-caries mechanism of lactic acid bacteria was initially explored.Finally,lactic acid bacteria microcapsules with targeted delivery ability on caries sites were prepared,which was based on the local acid microenvironment and the surface charge properties of cariogenic biofilms.The main research conclusions are as follows:1.Screening of lactic acid bacteria with anti-caries activity and in vitro performance evaluation:34 strains with antibacterial activity on S.mutans were selected from 385 strains,and the inhibitory diameter was between 9.20 mm and15.31 mm.The inhibition rates of 34 strains on biofilm ranged from 0 to 83.20%,and the co-aggregation rates with S.mutans ranged from 54%to 95%.Combined with the above three indicators,3 strains of Lactobacillus plantarum（L.plantarum）with potential anti-caries ability were selected.Subsequently,the tolerance,adherence and safety to lysozyme of 3 strains were assessed.The results showed that NCU001774exhibited superior in vitro characteristics,with tolerance to lysozyme at a concentration of 0.8 mg/m L and excellent adhesion ability,which contributed to prolonging its residence time in the oral cavity environment.In addition,NCU001774had the most protective effect on hydroxyapatite,with 42.67%of the demineralization level being decreased during the co-culture process.Synthetically,L.plantarum NCU001774 was selected for the subsequent study.2.Study on anti-caries mechanism of lactic acid bacteria:S.mutans was used as an indicator bacteria to explore the main mechanism of L.plantarum NCU001774against dental caries in five aspects:antibacterial substance,co-coaggregation,biofilm formation,acid production and tolerance,adhesion.The results show that:the antibacterial activity of NCU001774 against S.mutans was mainly dependent on organic acids.The co-aggregation reaction was not affected by p H and sugar fermentation substrate,but depended on calcium ions.NCU001774 significantly decreased the biomass,viable bacteria and extracellular polysaccharides in the biofilm of S.mutans.NCU001774 suppressed the adhesion of S.mutans in the form of replacement,exclusion and competition.NCU001774 significantly reduced the acid production and acid resistance of S.mutans.NCU001774 could inhibit the expression of S.mutans virulence genes to exert anti-caries effect.3.Preparation of NCU001774 microcapsules with targeted delivery function to caries:using release rate and viable bacteria count as indicators,the best preparation process of microcapsules was determined as follows:compound coagulation p H 6.5,chitosan and gelatin ratio 1:5,wall material 2%and trehalose 2%.The structure and function characterization of NCU001774 microcapsules showed that most of the microcapsules were irregular spherical,with the particle size concentrated between 1and 5μm.FTIR results showed that there was strong electrostatic interaction between chitosan and gelatin molecules.Compared with empty microcapsules,the characteristic absorption peaks of NCU001774 microcapsules were slightly offset from 1700 to 1200 cm^-1,indicating that there may be intermolecular hydrogen bond formation between lactic acid bacteria and microcapsule wall material.The surface of the NCU001774 microcapsules was positively charged at p H below 6.5,and simulated saliva basically no longer affected the antibacterial activity of the NCU001774 microcapsules against S.mutans after 1 h.The targeting to S.mutans biofilm and p H-responsive release of NCU001774 microcapsules was then further validated,and it was found that the fluorescently labeled NCU001774 microcapsules specifically acted on the side with S.mutans biofilm,and their release rate was significantly faster under acidic conditions than neutral.The anti-caries properties of NCU001774 microcapsules were then investigated,and the results showed that the microcapsules significantly suppressed the growth and biofilm formation of S.mutans.Finally,the results of storage stability showed that NCU001774 microcapsules still showed strong antibacterial activity after 24 weeks of storage at 4℃and-20℃.