Effect Of LPE On The Growth Of Five Cariogenic Bacteria And A Variety Of Cariogenic Enzymes Activity Of Streptococcus.Sobrinus

Objectives Dental biofilms is the basis of dental caries, S.mutans、S.sobrinus etc are the major cariogenic bacteria. The bacterium is initially adsorbed by an acquired pellicle formed on the enamel surface via salivary proteins through hydrophobic action, after which it then synthesizes adhesive glucans from sucrose by the action of glucosyltransferases (GTF), which mediate the firm adherence of bacterial cells to tooth surfaces. Adherent glucan synthesized by MS also contributes to the formation of dental plaque,in which the accumulation of acids leads to localized decalcification of the enamel surface, and dental caries begins to proceed.Our study group for the first time carried out a series of studies of the lemon extracts against dental caries. Lemon peel extract (LPE)is a yellow liquid which is extracted from the rutaceae citrus plant lemon and the main ingredient is the limonene (79%). This paper studied the effect of LPE on the growth of L.acidophilus、A.naeslundii、S.sangius、S.mutans and S.sobrinus, a variety of cariogenic enzymes activity and metabolic products of S.sobrinus to detect the effect of Lemon peel extract on growth, the anti-adhesion and acid-suppression mechanism including analyzing the role of metabolic enzyme.Methods1. LPE extraction and composition analysis Volatile oil was extracted by steam distillation. Under the conditions of GC-MS, Lemon Essential Oil Components was measured by GC-MS total ion chromato graphy(TIC). According to the retrieval NIST2008library, the relative content of each component was quantitatively obtained by peak area normalization method.2. The effect of LPE on the growth of the five cariogenic bacteria The minimal inhibitory concentration(MIC) of LPE on the growth of the five cariogenic bacteria was tested by dilution method. Reference to MIC test results, diluting LPE with TPY liquid medium. Taking bacterial suspension and TPY liquid medium with the ratio of1:10(v/v) into LPE diluted solution and TPY liquid medium without LPE, and inoculated at anaerobic incubator37℃. Bacterial suspension was extracted which was cultured respectively0、0.5、1、2、4、6、1、24h, then used CFU counting method to calculate the number of viable cells. At last, we can draw the grown curve of five experimental groups with the number of colony forming units of log (CFU/ml) for the longitudinal coordinates and time as the abscissa.3. Effect of LPE on GTF and water-insoluble glucan(WIG) of S.sobrinus Diluting LPE with TPY liquid medium by double dilution method to get four different concentrations, that is lowerd than4.5mg/ml (2.250mg/ml,1.125mg/ml,0.562mg/ml,0.281mg/ml). TPY liquid medium without LPE is a negative control.Use CFU counting method to detect the effect of LPE with different concentrations which are lower than MIC on the growth of S.sobrinus. Neson-Somogyi method were used to measure the content of reducing sugar at6、18、24、48hours, and the specific activity of enzyme was alculated. Measure the content of WIG by anthrone method, and do correlative analysis between the activity of WIG and enzyme activity.4Effect of LPE on Lactate Dehydrogenase (LDH) activity and acid generation. LDH activity was assayed by the pyruvate-dependent oxidation of NADH. Before and after treatment was measured with a pH meter of△pH medium to detect the anti-acid capability of LPE on S.sobrinus, and do correlative analysis between the activity of LDH and the capability of acid generation.Results1. From the the lemon GC-MS (TIC), were detected in more than100peaks were identified, of which40peaks accounted for93.954percent of the total volatile oil content. The main ingredient is the limonene, beta-pinene, et al.2. The MIC value of LPE against S.mutans、S.sangius、S.sobrinus is4.5mg/ml, and the L.acidophilus、A.naeslundii is2.25mg/ml. From0.5h to24h, with the concentration of LPE solution increasing, the ability inhibiting bacterial growth was more obvious. Selecting0,0.5,1,2hour points, the experimental group and blank control group were analysed by ANOVA of Repeated Measures. There are significant difference between the control groups and the experimental groups(P<0.05).3. The comparisons between the the experimental group and blank control group,which are lower than MIC has obvious ability of inhibiting the growth of S.sobrinus at the Oh、0.5h...48h point. At the same time point, the GTF and WIG of S.sobrinus decreased gradually with the increase of each concentration of LPE. From the group of0.563mg/ml to0.281mg/ml, there were highly significant differences between reducing sugar and enzyme activity (P<0.01), between GTF activity or WIG content of each experimental group and control group as well (P<0.01); at the same concentration excepting48h,(the concentration group-the control group) enzyme activity and the WIG content of the overall mean were statistically significant(P<0.01). The overall mean of the WIG control group at four time points were no statistically significant(P>0.05). There was positive correlation between GTF activity and WIG content(r=0.865-0.94).4. At the same time point, the LDH activity and△pH of S.sobrinus decreased gradually with the increase of each concentration of LPE. From the group of0.563mg/ml to0.281mg/ml groups, there were significant differences(P<0.07). There were significant differences of LDH activity between each experimental group and control group (P<0.01). At the sanme concentration, the overall mean of (the concentration of group-the control group) LDH activity and△pH at four time points were no statistically significant(P>0.05). There was positive correlation between LDH activity and ApH (r=0.926-0.982).Conclusion1LPE extracted by steam distillation, vinyl compounds are the main component account for79%of the total content.2The growth of cariogenic bacteria can be inhibited by the lemon peel extracts.3. LPE could inhibit the GTF and WIG of S.sobrinus; with the increase of concentration from0.281mg/ml to2.25mg/ml, the inhibiting effect increased.4. LPE could inhibit the LDH activity and acid generation of S.sobrinus; with the increase of concentration(0.281-2.25mg/ml), the inhibiting effect increased.5. The inhibitory effects of LPE on the GTF and WIG of S.sobrinus in18h-24h phase is the best phases.6. LPE could inhibit the GTF and LDH activity, reducing the WIG synthesis and acid production, but do not affect the plaque flora imbalance, with increase of concentration from0.281mg/ml to2.25mg/ml.