Oral Changes With Caloric and no Caloric Sweeteners

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.

Eligibility Criteria:
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