Oral Changes With Caloric and no Caloric Sweeteners

Effect of the Consumption of Beverages Added With Stevia Rebaudiana on Oral pH and Dental Biofilm in Adolescents

The objective of this clinical trial is to compare the effect that the intake of beverages without sweetwners, added with non-caloric sweeteners (stevioside) and caloric sweeteners (sucrose) on oral pH and dental biofilm microbiome in Mexican adolescents. Participants will drink on different occasions a beverage without sweetener, a beverage added with stevioside or a beverage added with sucrose. The researchers will compare the changes that each one causes in salivary pH, dental biofilm pH, dental biofilm bacterial proliferation and dental biofilm microbiome.

A randomized crossover clinical trial will be conducted including 52 healthy adolescents participants (sample size with alpha=0.05 and beta=0.8 ). The intervention will consist of ingesting 355 mL of tea lemon flavor (pH 7.1) in the first meeting, in subsequent meetings the tea will be added with 1) 2.2 grams (gr.) of stevioside or 2) 7.5 gr. of sucrose. The washout period between interventions will be 1 week. Participants will be informed of potential risks and those who sign the informed consent and complete the inclusion criteria will be randomized in a double-blind manner. The data will be collected in a special format including previous conditions, identification (age and sex) and possible adverse effects. If any possible adverse effect occurs, it will be notified to the research team in order to determine the changes. To determine the pH, saliva and dental biofilm samples will be obtained and analyzed using a HANNA potentiometer calibrated with buffer solutions. To determine bacterial proliferation, samples of dental biofilm will be obtained, cultivated in appropriate media and conditions, compared with the American Type Culture Collection (ATCC) catalog and the number of colony-forming units will be compared. To analyze the main components of the dental biofilm, genetic sequencing will be performed and the operative taxonomic units will be compared. (11 of the 52 participants will be randomized for microbiome analysis). The data will be collected at a time indicated by a stopwatch and carried out by two verifiers. The statistical analysis will be done according to the type of variable, these will be described with mean and standard deviation or frequencies and percentages. The pH will be compared by ANOVA analysis and adjusted by Bonferroni correction. Bacterial proliferation will be analyzed by Kruskal Wallis test. Statistical Package for the Social Sciences (SPSS) v. 22 program will be used and statistical significance will be considered with p ≤ 0.05. For the microbiome, the principal component analysis (PCA) of each operational taxonomic unit (OTU) will be done. Non-parametric multivariate analysis of variance (Adonis) and an analysis of similarities (ANOSIM) will be used. The p values for Adonis and for ANOSIM will be calculated, identifying those that have differences in bacterial communities between the groups using the Genius (V3) software.

355 ml of tea lemon flavor should be drunk Salivary pH will be collected at 0,5,10,15,30,45 and 60 minutes later Dental biofilm pH will be collected at 0,5,10,15,30,45 and 60 minutes later Streptococcus mutans dental biofilm formation ( Colony Forming Units) will be collected at 0 and 120 minutes later Operative Taxonomic Units of dental biofilm will be analyzed at 0 minutes and 120 minutes later

355 ml of tea lemon flavor added with 2.2 grams of stevioside should be drunk Salivary pH will be collected at 0,5,10,15,30,45 and 60 minutes later Dental biofilm pH will be collected at 0,5,10,15,30,45 and 60 minutes later Streptococcus mutans dental biofilm formation ( Colony Forming Units) will be collected at 0 and 120 minutes later Operative Taxonomic Units of dental biofilm will be analyzed at 0 minutes and 120 minutes later

355 ml de tea lemon flavor added 7.5 grams with should be drunk. Salivary pH will be collected at 0,5,10,15,30,45 and 60 minutes later Dental biofilm pH will be collected at 0,5,10,15,30,45 and 60 minutes later Streptococcus mutans dental biofilm formation ( Colony Forming Units) will be collected at 0 and 120 minutes later Operative Taxonomic Units of dental biofilm will be analyzed at 0 minutes and 120 minutes later

355 ml of tea lemon flavor added with 2.2 gr. of stevioside should be drunk Salivary pH will be collected at 0, 5, 10, 15, 30, 45 and 60 minutes later. Dental biofilm pH will be determined at 0, 5, 10, 15, 30, 45 and 60 minutes later. Streptococcus mutans dental biofilm formation ( Colony Forming Units) will be determined at 0 and 120 minutes later. Operative Taxonomic Units of dental biofilm will be analyzed at 0 and 120 minutes later.

Mean Colony Forming Units Streptococcus mutans dental biofilm with tea lemon flavor added with 2.2 gr. stevioside

Mean Colony Forming Units Streptococcus mutans dental biofilm with tea lemon flavor added with 2.2 gr. stevioside

Mean Colony Forming Units Streptococcus mutans dental biofilm with tea lemon flavor added with 7.5 gr sucrose

Mean Colony Forming Units Streptococcus mutans dental biofilm with tea lemon flavor added with 7.5 gr sucrose

Principal component analysis of dental biofilm with tea lemon flavor added with 2.2 gr. of stevioside.

Principal component analysis of dental biofilm with tea lemon flavor added with 2.2 gr. of stevioside.

Inclusion Criteria: Habitual consumption of soft drinks Decayed, Missing, and Filled Teeth (DMF) index of at least 3 Agree to participate in the study and sign informed consent Parents sign informed consent Any nutritional condition Exclusion Criteria: Orthodontic treatment Topical application of fluoride during the last 3 months Having a motor disability that interfered with tooth brushing Xerostomia Antibiotic therapy during the study period Periodontal infections

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