The objective of this clinical trial is to compare the effect that the intake of beverages without sweeteners, 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 43 healthy adolescents participants (sample size with alpha=0.05 and beta=0.8 ). The intervention will consist of ingesting 250 mL of natural water (pH 7.1) in the first meeting, in subsequent meetings the natural water will be added with 1) 0.1 gr. of stevioside or 2) 25 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 triple-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 to determine the changes.

• To determine the pH, saliva and dental biofilm samples will be obtained and analyzed using a HANNA potentiometer.
• To determine the bacterial proliferation, samples of dental biofilm will be obtained and the number of copies of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) will be analyzed by polymerase reaction technique (PCR) of the 16S ribosomal gene fragment.
• To analyze dental biofilm microbiome, 11 of the 43 participants will be randomized for. Genetic sequencing will be performed and the amplicon Sequence Variants (ASVs) of S. mutans and S. sobrinus will be compared.

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 considering p value ≤ 0.05. For the microbiome, the Amplicon Sequence Variants (ASVs) will be analyzed. Non-parametric multivariate analysis of variance and an analysis of similarities will be used. The p values will be calculated, identifying those that have differences in bacterial communities between the groups using the Genius (V3) software.

Inclusion Criteria:

• Habitual consumption of soft drinks
• Decayed, Missing, and Filled Teeth (DMF) index of at least 3 (considering as caries those lesions that are visible without the tooth needing to be dry)
• 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

Yes: There is a plan to make Individual Participant Data (IPD) Sharing Statement.

At the time of manuscript publication

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colors will be assigned for each sweetener