Effect of Xylitol-based Chewing Gums, Sugar-free and Sugared, on Salivary Flow and pH in Young Adults: Randomized Controlled Clinical Trial
Phase: NA
Conditions: Healthy
Sponsor: Universidad Católica de Cuenca

 
 
Form for the Submission of Protocols for Observational and Interventional Research in Humans. 
(Except Clinical Trials) 
 
Ethics Committee for Human Research at the Catholic University of Cuenca (CEISH-UCACUE) 
GENERAL INFORMATION OF THE RESEARCH 
Title: The effect of xylitol-based chewing gum, both sugar-free and sugared, on salivary flow and pH in young adults: a randomized 
controlled trial in students from the Dentistry Career at the Catholic University of Cuenca, 2023 
The study will be conducted on students from the Faculty of Dentistry at the Catholic University of Cuenca, main campus in Cuenca, 
enrolled during the period September 2023 to February 2024, aged 18 to 25, who wish to participate voluntarily. It will be executed in 
the Molecular Biology laboratory, which is equipped with a pH meter (to measure the amount of hydrogen ion in saliva) belonging to the 
Department of Research of the Dentistry program. This study will commence in December 2023 pending approval from the Ethics 
Committee. 
 
In healthy and balanced diets with reduced sugar concentration, physiological mechanisms in the mouth (such as saliva) can prevent the 
onset of tissue lesions in the teeth at the level of hydroxyapatite crystals before cavitation (dental caries) occurs, through the restoration 
of pH and remineralization of tooth tissue. Saliva plays protective roles as an adjunct to dilution and cleaning of the oral cavity, as well 
as buffering and enabling ion exchange. The activity depends on the consistency and rate of flow, with the level of salivary pH defining 
how ionic exchange occurs during enamel remineralization and demineralization processes.2 
 
Global increases in sugar consumption have prompted the development of sugar substitutes, including polyols or non-fermentable sugars, 
of which the most commonly used are nutritive sweeteners such as sorbitol and xylitol. Chewing gum stimulates salivation, neutralizes 
and raises pH levels, and serves as a means to administer therapeutic agents. Therefore, there are chewing gums that contain sugar 
substitutes like sorbitol and xylitol4. Vantipalli et al.5 indicate that chewing sugar-free gum significantly reduces caries compared to the 
use of rinses, tablets, and candies. These authors report an increase in salivary pH within 10 minutes of starting consumption. German6 
researchers claim that the consumption of this product reduces health insurance company costs by 29.13% and improves oral health 
status by 5.38%. 
 
The study possesses a degree of originality, as there are studies conducted in Ecuador that use reactive strips as instruments
to evaluate salivary pH. Therefore, appropriate and high-quality equipment is needed to determine salivary pH. The use of 
chewing gum containing polyols could play a role in preventing dental caries compared to not using gum or using sugared 
gum, most likely due to the increased salivary flow and pH, improving enamel lesion remineralization. However, xylitol has 
anticariogenic properties influencing the reduction of dental plaque. Therefore, the objective of the study is to evaluate the
effect of xylitol-based chewing gum, both sugar-free and sugared, on salivary flow and pH in young adults through a 
randomized controlled trial. 
 
 
TYPE OF RESEARCH 
Observational Studies 
Intervention Studies 
 
Cross-sectional Studies 
☐ 
 
Quasi-Experimental Studies 
☐ 
 
Ecological Studies 
☐ 
 
Field Trial 
☐ 
 
Case Reports 
☐ 
Randomized Controlled Trials without the 
use of drugs and/or medical devices 
☒ 
 
 
Case Series 
☐ 
 
Case-Control Studies 
☐ 
 
Cohort Studies 
☐ 
Others 
 
Specify 
☐ 
 
PROJECT EXECUTION TIME 
 
The study will last for 6 months, from December 12, 2023, to July 31, 2024. 
 

 
RESEARCH FUNDING 
Total amount of research funding. 
$4.054,00 
Sources of funding. 
 
Catholic University of Cuenca 
GENERAL RESEARCH INFORMATION 
SPONSOR INFORMATION 
Name of the individual/institution conducting the research 
 
Sponsor  
Vice-Rector of Research 
Pedro Martínez Suárez 
National ID/RUC /RUC 
PAB256196 
Institutional 
telephone 
number 
0964038407 
Extensión 
Email address 
pmartinezs@ucacue.edu.ec 
Address 
Av. Américas and Humboldt, Cuenca 
Institutional 
website 
https://www.ucacue.edu.ec/ 
Executing body 
Catholic University of Cuenca 
 
RESEARCH EXECUTION COVERAGE 
National ☐ 
 
Zona 1 (Esmeraldas, Carchi, Imbabura y Sucumbíos.) 
☐ 
 
Zona 2 (Napo, Orellana y Pichincha) 
☐ 
Zona 3 (Cotopaxi, Chimborazo, Pastaza y Tungurahua) ☐ 
Planning zones 
☐ 
Zona 4 (Manabí y Santo Domingo de los Tsáchilas) 
☐ 
Zona 5 (Los Ríos, Guayas, Santa Elena, Bolívar) 
☐ 
 
Zona 6 (Azuay, Cañar y Morona Santiago) 
☐ 
 
Zona 7 (El Oro, Loja y Zamora Chinchipe) 
☐ 
 
Zona 8 (Guayaquil, Durán y Samborondón) 
☐ 
 
Zona 9 (Distrito Metropolitano de Quito) 
☐ 
Provincial ☐ 
Please specify the provinces where your research will be 
conducted. 
☐ 
Local ☒ 
Please specify the province and cantons where your 
research will be conducted: Azuay-Cuenca 
☒ 
 
RESEARCH PERSONNEL 
 
ROLE 
Full Name 
Identificati
on Card 
Academic 
Background 
Institution 
Affiliation 
Personal and 
Institutional 
Email Address 
Cellular 
Phone 
Number 
Sponsor 
Pedro Martínez Suárez PAB256196 
Doctor of 
Philosophy 
Catholic 
University of 
Cuenca 
pmartinezs@ucacue.e
du.ec 
0964038407 
Principal 
Investigator 
Miriam Verónica Lima 
Illescas 
0103711925 
Doctora en Ciencias 
Estomalógicas 
Catholic 
University of 
Cuenca 
mlimai@ucacue.edu.
ec 
0999975073 
Investigator 1 
Andrea Karina Maurat 
Argudo 
0104981444 
Master of Bioethics Catholic 
University of 
Cuenca 
andrea.maurat@ucac
ue.edu.ec 
0995800436 
 

 
 
 
 
 
 
RESEARCH DETAILS 
 
 
STRUCTURED SUMMARY 
Chewing gum stimulates salivation, neutralizes, and raises pH levels, serving as a medium for administering therapeutic agents. 
However, global increases in sugar consumption have led to the development of chewing gums containing sugar substitutes such as
polyols or non-fermentable sugars, with the most common being nutritive sweeteners like sorbitol and xylitol. The use of polyol-
containing chewing gum could play a role in preventing dental caries compared to not using gum or using sugared gum, most likely
due to increased salivary flow and pH, thereby improving enamel lesion remineralization. However, xylitol has anticariogenic 
properties influencing the reduction of dental plaque. Objective: To evaluate the effects of xylitol-based chewing gum, both sugar-free
and sugared, on salivary flow and pH in young adults. Methodology: This is a quantitative, analytical, experimental study through a 
double-blind parallel randomized controlled trial. The population will consist of all students enrolled (1275 students in the period
October 2022-February 2023) in the Dentistry program at the Catholic University, aged 18 to 25, who voluntarily meet the inclusion 
criteria. The sample size will be 21 participants in each group, adjusted for a 15% loss of information or study dropout, thus requiring
25 participants in each group. Participants meeting the selection criteria will be assigned probabilistically using simple random 
sampling through a lottery system to four groups: the first group (G1) will consume gum with 100% xylitol, the second group (G2)
will consume sugar-free gum with sorbitol, maltitol, mannitol, and xylitol, the third group (G3) will consume sugared gum containing 
sugar, dextrose, glucose, sweeteners such as aspartame and acesulfame-K, and the fourth group (G4, control) will chew paraffin. Data 
will be entered into an Excel matrix and analyzed using SPSS v.25. Descriptive analysis of the data will be conducted. Chi-square will
be used for demographic data, Levene's test will determine data homogeneity, and ANOVA will determine mean differences, 
followed by Tukey's post-hoc comparison to find significant differences between group means, with a significance level of p 
<0.05.Expected Results: It is expected that salivary pH and flow means will differ among the different chewing gums. 
 

 
 
 
RESEARCH PROBLEM 
Global increases in sugar consumption have prompted the development of sugar substitutes, including polyols or non-fermentable 
sugars, of which the most commonly used are nutritive sweeteners like sorbitol and xylitol. Chewing gum is a means to administer 
therapeutic agents and contribute to improving oral conditions. In the national market, there are sugared chewing gums (sucrose) and 
sugar-free gums that include xylitol plus sorbitol. These gums are consumed in high quantities, especially by teenagers and young 
adults, and the few studies conducted in Ecuador indicate a significant elevation in salivary pH from 6.4 to 7.14 7,8 after consuming 
sugar-free chewing gums, compared to sugared gums. However, these investigations evaluate salivary pH levels using reactive strips. 
Additionally, there are no locally available chewing gums solely based on xylitol, and there are also sugar-free chewing gums that use 
a blend of compounds as sweeteners without indicating the percentages. Therefore, high-quality randomized controlled trials are 
needed to determine the effectiveness on salivary pH. 
A study conducted in India5 compared the efficacy of two sugar-free chewing gums. The first group showed a significant increase in 
salivary pH levels up to 2 minutes after consuming sugar-free chewing gums (pH 7.98 ± 0.19), which gradually decreased to 7.60 ± 
0.12 at 30 minutes, compared to another group of sugar-free chewing gums where salivary pH levels at two minutes were higher (pH 
7.49 ± 0.16) than at 30 minutes (pH 6.82 ± 0.28), showing statistically significant differences within each group. Regarding salivary 
flow, there was a higher average at two minutes (1.89 ± 0.42), which gradually decreased to 1.09 ± 0.31 at 30 minutes after 
consuming sugar-free chewing gums. Similar results were observed in the second group of sugar-free chewing gums at two minutes 
(1.89 ± 0.45), which decreased to 1.04 ± 0.31 at 30 minutes, showing statistically significant differences. 
Therefore, chewing gums stimulate an increase in salivary flow, which contributes to diluting and cleaning the oral cavity, as well as 
buffering and allowing ionic exchange. Additionally, they promote a neutral or alkaline salivary pH level, which may contribute to 
enamel remineralization and, therefore, prevent dental caries.2 Furthermore, they could serve as a complement to oral hygiene in 
situations where tooth brushing is not possible. 
Based on the above, the following question is raised: Does the consumption of xylitol-based chewing gums positively influence 
salivary flow and pH compared to sugared or sugar-free gums? 
 
JUSTIFICATION 
This research is focused on determining the effects of xylitol-based chewing gums (100% xylitol, Xylichew brand), sugar-
free gums (sorbitol, maltitol, mannitol, and xylitol, Trident brand), and sugared gums (sugar, dextrose, glucose, 
sweeteners such as aspartame and acesulfame-K, Agogó brand) on salivary flow and pH in young adults: a randomized 
controlled trial in students of the Dentistry Career at the Catholic University of Cuenca, 2023. In our context, the 
distribution of sugar-free gums includes xylitol and sorbitol among its ingredients, without indicating the exact 
proportions of each. Additionally, there are no chewing gums in the Ecuadorian market that contain xylitol as the sole 
ingredient, hence the need to compare with a foreign chewing gum, which indicates the presence of only xylitol. 
Vantipalli et al.5 showed statistically significant differences between two sugar-free chewing gums in terms of unstimulated salivary 
pH and salivary pH stimulated at 2 minutes, showing an increase at this time compared to 30 minutes after gum consumption. Similar 
results were reported for salivary flow, showing a higher flow at two minutes compared to 30 minutes after gum consumption. This 
study demonstrates significant changes within each group and between subgroups for salivary pH levels, while salivary flow showed 
significant changes only within each gum group evaluated. 
This study has a degree of originality, as there are studies conducted in Ecuador that found significant changes with an increase in 
salivary pH levels in chewing gums containing sorbitol and maltitol. However, they did not evaluate chewing gum containing xylitol 
as the sole ingredient, and they used reactive strips as equipment to evaluate salivary pH. Therefore, high-quality and appropriate 
equipment is needed to determine salivary pH, and the Molecular Biology Laboratory has a pH meter, providing a precise tool for 
assessment. 
The research topic is of interest to researchers and institutions as it is part of the Medical and Health Sciences research line, within the 
field of health education and promotion. The results will be published through an article in an indexed journal, benefiting clinicians 
such as dentists, physicians, undergraduate and postgraduate students in health-related fields, and the general population who will 
learn about the benefits of consuming xylitol-based and sugar-free chewing gums compared to sugared gums. In situations where 
differences are found, it can represent an oral hygiene tool in conditions where oral hygiene with brushing technique is not feasible, 
and the use of chewing gum would improve salivary pH, preventing enamel demineralization and thus dental caries. 
The pH meter from BOECO brand (model BT-675) is a precision tool that measures pH, mV, and temperature. It has a 
built-in microprocessor that stores, calculates, and compensates all parameters related to pH determinations, including 
electrode temperature characteristics, electrode slope deviations, compensated solutions, and buffers. 
 
THEORETICAL FRAMEWORK 

 
 
One of the most prevalent oral health conditions is dental caries, affecting 35% of the global population. 1 Since 1960, it has been 
considered an infectious and transmissible disease, related to the presence of microorganisms, susceptible teeth, and fermentable 
substrates. The ecological plaque hypothesis is one of the most accepted, stating that oral environments with a low pH level disrupt 
the resident microflora, leading to an enrichment of pathogenic bacteria. 9 
 
En dietas saludables y equilibradas con reducida concentración de azúcar, los mecanismos fisiológicos de la boca (como la saliva) 
detiene la aparición de lesiones en los tejidos del diente a nivel de los cristales de la hidroxiapatita antes de la cavitación (caries 
dental) al restaurar el pH y remineralizar.1 One of the most prevalent conditions in oral health is dental caries, affecting 35% of the 
world's population.1 Since the 1960s, it has been considered an infectious and transmissible disease, related to the presence of 
microorganisms, susceptible teeth, and fermentable substrates. The ecological plaque hypothesis is one of the most accepted, stating 
that oral environments with a low pH level create an imbalance in the resident microflora, leading to bacterial pathogenic enrichment. 
9 In healthy and balanced diets with reduced sugar concentration, the physiological mechanisms of the mouth (such as saliva) prevent 
the onset of lesions in tooth tissues at the hydroxyapatite crystal level before cavitation (dental caries) by restoring pH and 
remineralizing.1 
Excessive consumption of sugary foods creates an imbalance in oral microbiota (dysbiosis), which favors bacterial competitiveness in 
dental biofilm (accumulation of bacteria and food deposits). Fructans and glucans are polymers produced from sucrose that stimulate 
microbial cohesion and adhesion. 10,11 
Studies link the consumption of fermentable carbohydrates containing sucrose to the risk of dental caries because microorganisms 
depend on them for their survival and on the diet provided by the metabolic activity of carbohydrates in oral microbiota. Additionally, 
bacteria require fermentable carbohydrates for the synthesis of insoluble glucans and fructans, which allow adhesion to the tooth 
surface, thereby promoting the formation of bacterial plaque. 12 
 
High sugar intake and low levels of salivary pH appear to be pivotal components that disrupt oral cavity homeostasis and lead to the 
development of dental caries. A few minutes after sugar intake, the pH of dental plaque decreases, causing enamel demineralization 
when the acidity level is below the critical pH, which varies between individuals within an approximate range of 5.2 to 5.57. At this 
point, acids affect the enamel by dissolving apatite crystals and releasing calcium and phosphate ions into saliva. Although this 
process occurs naturally, over time it leads to the destruction of enamel infrastructure. 12 
Therefore, proposed strategies to prevent the disease should include inhibition of acid production, reduction in sugar consumption 
primarily between meals, and the use of non-fermentable sugars. 13 
Chewing generates salivary stimulation, neutralizes and raises pH levels, and serves as a means to administer therapeutic agents. 3 As 
a result, there is an improvement in the removal of fermentable carbohydrates in the oral cavity and it promotes mineral 
oversaturation, which promotes enamel remineralization. Vantipalli et al.5 indicate that chewing sugar-free gum significantly reduces 
caries compared to the use of rinses, tablets, and candies. These authors report an increase in salivary pH within 10 minutes of starting 
consumption. 
Chewing gum is primarily made from gum resin and certain amounts of preservatives, flavorings, and sweeteners. Nowadays, there is 
a wide variety of sweeteners that replace sugar in chewing gum. These non-cariogenic products hinder bacterial metabolism and 
prevent acid production because they are non-fermentable. They also increase their anticariogenic potential due to the provocation of a 
palatability reflex by the sweet taste, which, along with the chewing process, increases salivary flow stimulation. 12 
The labeling of "sugar-free" is allowed for gums that do not decrease the pH below 5.7 during or 30 minutes after consumption.12 The 
use of sugar-free gum can be particularly effective in conditions where daily oral hygiene and maintenance are difficult to carry out 
conventionally. 
The sweeteners most commonly used in gums are xylitol and sorbitol. 12,13 They are referred to as "sugar alcohols" because their 
chemical structure is similar to both sugar and alcohol. 12 
Xylitol is a pentahydroxy alcohol derived from D-xylose that was approved by the Food and Drug Administration (FDA) in 1960 and 
has since been used as a substitute for refined white sugar. 2,13 
It is estimated that to prevent dental caries with the use of xylitol, it is not necessary to completely replace sucrose in the diet, and 
relatively small daily doses of xylitol ranging from 4 to 10 grams per day have been shown to provide anticaries protection. It has 
been suggested that chewing gum sweetened with xylitol three to five times a day, for a minimum of five minutes each time, ideally 
after meals, is effective in achieving the desired effect on cariogenic bacteria. 4,12,14 
Xylitol is considered cariostatic due to its ability to decrease counts of Streptococcus mutans (SM) 12,14 and Lactobacillus (LB) 4, as 
well as its role in controlling dental plaque formation and, consequently, the incidence of dental caries. Therefore, it is particularly 
effective in conditions where proper oral hygiene is not possible 12, or as a complement to toothbrushing for oral hygiene. 14,15 
Additionally, it stimulates salivary secretion, an important factor in maintaining alkaline pH, and promotes mechanical cleansing of 
tooth surfaces. 13 
Its mechanism of action is to create an unfavorable environment for bacterial growth, especially for Streptococcus mutans (SM), as it 
reduces its ability to adhere to oral tissues by affecting the insoluble glucans involved in this process. This property reduces the 
possibility of dental plaque formation and, therefore, the local effect of lactic acid. Additionally, xylitol penetrates the bacterial 

 
cytoplasm, interfering with glycolysis and inhibiting the growth of microorganisms. 12 
Furthermore, its action affects other species such as Streptococcus sobrinus (SS) and Actinomyces viscosus (AV). As a result, a less 
cariogenic environment is created. 12  Tapiainen et al. 16 stated that the effect of xylitol disappears from saliva within 15 minutes. 
Sorbitol is a hexitol derived from glucose with low cariogenic potential. Like xylitol, it causes a decrease in SM, SS, AV, LB, and 
insoluble glucans, along with an increase in salivary pH, giving it anticariogenic potential. Current evidence establishes that pentitols 
like xylitol, as well as hexitols like sorbitol, have good results in reducing dental caries. However, pentitols are more effective and 
therefore more commonly used. 4,12 
RESEARCH OBJECTIVES 
General Objective 
Compare the effects of xylitol-based chewing gum (Xylichew), sugar-free (Trident), and sugared (Agogó) on salivary flow rate and 
pH in young adults 
Specific Objectives 
Determine the initial salivary flow, at 10, 20, and 30 minutes after the consumption of xylitol-based, sugar-free, and 
sugared chewing gums in young adults. 
Determine the initial salivary pH, at 10, 20, and 30 minutes after the consumption of xylitol-based, sugar-free, and 
sugared chewing gums in young adults. 
RESEARCH QUESTION OR STUDY HYPOTHESIS 
H0: The consumption of different chewing gums does not produce changes in the level and flow of initial salivary pH at 10, 20, and 
30 minutes. 
H1: The consumption of different chewing gums results in changes in the level and flow of initial salivary pH at 10, 20, and 30 
minutes. 

 
 
 
METHODOLOGY 
 
Research Design 
It is a quantitative, analytical, experimental study conducted through a double-blind, parallel randomized controlled trial. 
 Definition of the study population and, if applicable, it is necessary to detail how the sample size calculation was 
performed (formulas and development). 
The population will consist of all enrolled students (1275 students in the period September 2023-February 2024) in the Dentistry 
program at the Catholic University, aged between 18 and 25 years old, who voluntarily meet the inclusion criteria. 
 
For the sample size calculation, an online calculator was used, employing the formula for comparing two means. A confidence level 
of 95% and a statistical power of 90% were set. Based on a previous study that reported a desired difference of 1 unit, with a 
precision and variance of 1 in the reference or control group, it was determined that 21 participants would be needed in each group. 
However, to account for potential information loss or dropout from the study, the sample size was adjusted by 15%, resulting in the 
requirement of 25 participants in each group. (Figure 1) 
  
Figure 1. Hypothesis testing formula for determining means.14 
Replacing the formula 
n= participants needed in each of the groups 
Zα= Z value corresponding to the desired risk 0.05 =1.96 
Zβ= Z value corresponding to the desired risk 90%= 1.282 
S2= Variance of the quantitative variable in the control group =1 
d= Minimum detectable difference desired = 1 
𝑛=
2(1.96+1.282)2∗1²
1²
=20.99 
 
 
List of establishments where observational or intervention research will be conducted on human subjects. It is 
necessary to specify whether the institutions are public or private and include their postal address and letter 
of interest (Appendix B) 
The intervention will take place among students of the Dentistry program at the University of Cuenca, main campus, in the 
Molecular Biology laboratory. Sampling and determination of salivary flow and pH level will be carried out there. (Appendix B) 
 
Inclusion Criteria 
Within the selection criteria, the inclusion criteria will be participants with at least 20 teeth, providing written informed consent, and 
being willing to comply with the study procedures. 
 
 Exclusion Criteria 
The exclusion criteria will be individuals with systemic, infectious, or inflammatory diseases or those taking medications, 
antibiotics, or fluoride in the last month; regular consumers of products and mouthwashes containing xylitol or sorbitol, 
with abnormal salivary flow (<1 ml/min), pregnant women or those on contraceptive pill treatment, or with eating disorders 
(anorexia and bulimia); subjects with periodontal disease or presence of dental caries. 
 
 
The variables described in this section should be consistent with those mentioned in (APPENDIX C) the table of 
operationalization of the variables 
The variables of interest will be chewing gums, salivary flow level, and salivary pH during 4 time periods: initial (without 
chewing gums), at 10, 20, and 30 minutes after consuming the chewing gums. 
 
Detailed and sequential description of all procedures that will be carried out to achieve each of the stated objectives. At least 
the following should be included. 
 
7.1 Procedures: Students will be invited to participate through the study's presentation in each and every classroom of the students 
enrolled in the period September 2023-February 2024. At the end of the presentation, they will be invited to sign the informed 
consent, a document in which participants will provide their cell phone numbers so that they can be contacted via WhatsApp 
messages for the preliminary examination of their oral cavity to verify compliance with the inclusion criteria, as well as for sample 

 
collection. 
 
 
7.2 Sample Selection: Participants who meet the selection criteria will be probabilistically assigned using simple random sampling 
through a lottery system and will be assigned to each group. There will be 4 groups: the first group, G1, will consume chewing 
gums with 100% xylitol; the second group, G2, will consist of individuals who consume sugar-free chewing gums; the third group, 
G3, will consist of participants who chew sugared gums; and the fourth group, G4, will serve as the control group and will chew 
paraffin (Figure 2). This procedure will be carried out by investigator 1 (Appendix D - Chewing Gum Information) 
 
The principal investigator, investigator 1 and 2, and technician 1, as well as the participants who will form the different groups 
consuming the chewing gums, will be unaware of the type of gum (double-blind). Both the chewing gums and the paraffin will 
have the same weight (1.3 g), and the blinding method will involve the presence of identical, pre-numbered containers containing 
the different chewing gums. This information will only be accessible to technician 2. Saliva samples will be collected by the 
principal investigator, and the data will be recorded on a form specifically designed for this purpose by technician 1, which will be 
executed in the Molecular Biology laboratory (Appendix E - Data Collection Form). 
 
7.3 Saliva Sample Collection: Saliva samples will be taken between 8 to 10 am, ensuring that subjects have not ingested food, 
smoked, or brushed their teeth at least 1 hour prior to sample collection. Participants will be asked to rinse their mouths with pure 
water to remove any food residue. Subsequently, they will be seated in a chair in an upright position with their head slightly tilted 
forward and eyes open, and the basal saliva sample will be taken as indicated by the established protocol for collecting unstimulated 
saliva by Tomas Seif. Before starting the test, participants will be asked to swallow saliva. They should keep their mouth slightly 
open and allow saliva to drain into the tube. The test will last five minutes, during which salivary pH will be evaluated and 
weighed, denoted as T1. For stimulated saliva, the protocol dictated by the Faculty of Dentistry at the University of Southern 
California will be used, utilizing the drainage method. Stimulated saliva will be collected in another pre-weighed test tube at three 
additional time intervals: T2 from 5 to 10 minutes, T3 from 15 to 20 minutes, and T4 from 25 to 30 minutes after the start of 
chewing gum consumption. Salivary flow and pH level measurements will be evaluated immediately using a pH meter (BT-675 
meter, Boeco-Germany), performed by the principal investigator. Sample collection will be conducted over a period of two weeks. 
 
Figure 2. Random allocation of participants for the determination of salivary flow and pH level. 
7.4 Operation mechanism of the desktop pH meter BT-675 BOECO-Germany 
The to take pH measurements, the BT-675 must be calibrated before first use and will be calibrated by the company Record Dental. 
Immediately after sample collection, the pH level and salivary flow will be measured. (Appendix F - Certification of the pH 
meter BT-675 BOECO-Germany) 
Procedure for using the desktop meter: 
1. Connect the pH electrode to the BNC connector and the ATC/Temp probe to the ATC/Temp connector on the unit. The "ATC" 
icon will light up. 
2. Press the "Mode" button until the "pH" icon and the "AUTO" icon light up. 
3. Rinse the pH electrode and the ATC/temp probe with distilled water and immerse them in the sample to be measured. Remove 
any trapped air bubbles around the probe by shaking or agitating the probe. 
4. Press the "Mea. / Eff." button. The "WAIT" icon will start flashing. The unit is waiting for a stable reading. The display will 
track the pH value as detected by the pH electrode and the ATC/Temp probe. 
5. When the "WAIT" icon disappears, the reading is "LOCKED" and will not respond to further changes in the sample. The pH 
value displayed is the pH value of the sample at the sample temperature shown. 

 
 
 
7.5 Disposal of biological samples: Immediately after determining the salivary flow and pH level in the Molecular Biology 
Laboratory, disposal will be carried out using the system for managing biological waste. This will involve the disposal of 
biohazardous waste, for which the Falcon sample tubes will be capped, and the samples will be placed in red bags identified as 
biohazardous waste. Custody of the waste will be the responsibility of the principal investigator. 
 
7.6 Handling of data sheets: The physical documents will be handed over to technician 1, who will then receive them and 
immediately, the laboratory coordinator will assign technician 1 a physical space for handling the information. The information will 
not be provided to third parties, as only investigator 1 will have knowledge and access to that physical space and will develop the 
data matrix in Excel. It is worth noting that the laboratory coordinator will assign the necessary physical space, and thereafter, only 
technician 1 will have access to it. 
 
7.7 Information Management: Technician 1 will handle the physical documents until the laboratory process is completed. 
Subsequently, Technician 1 will provide the data matrix in Excel for further analysis by Investigator 1. Participants will not receive 
individual research results; instead, the final research results will be disseminated. 
 
8. Human biological samples do not require exportation as they will be disposed of immediately after the determination of 
salivary flow and pH level. 
 
9. Data Analysis Plan 
The data analysis will be conducted using SPSS v.25. Descriptive analysis of the data will be performed. Chi-square test will be 
used for demographic data, Levene's test will determine the homogeneity of the data, and ANOVA test will be used to determine 
differences in means. Post-hoc Tukey's test will be employed to find significant differences between the means of the groups, with a 
significance level set at p<0.05. All analyses will be conducted by Investigator 1. 
 
 
 
HUMAN AND MATERIAL RESOURCES 
1. Enumerate the human resources that will participate in the research, detailing the functions each one will fulfill; it should coincide 
with the section. 
 
1. Principal 
Investigator 
Miriam Lima 
Collecting the sample and measuring salivary flow and pH 
2. Investigator 1 
Karina Maurat 
Inviting participation via email and conducting statistical analysis 
3. Investigator 2 
Génesis López 
 Sending study participation information via email. 
4. Technician 1 
María Augusta Lara 
Collecting quantitative data on the data sheets. 
5. Technician 2 
Cristian Paladines 
Collecting the sample. 
2. Provide a detailed description of all the material resources used for the execution of the project, including necessary 
information        
Among the materials are nitrile gloves, masks, Falcon tubes, funnel, hand towels, xylitol chewing gums, Trident, Agogo, and 
paraffin. As for the equipment, a pH meter and a precision scale (Boeco) are needed. 
ETHICAL AND GENDER   CONSIDERATIONS 
1. Provide a detailed description of the measures to be taken to ensure the rights of individuals to minimal risk, autonomy, 
and confidentiality 
For the conduct of this research, reference will be made to the Universal Declaration on Bioethics and Human Rights, the 
Helsinki Declaration, International Ethical Guidelines for Biomedical Research Involving Human Subjects, Organic Health Law, 
and considering that research in health, particularly involving human beings or the use of biological samples of human origin, in 
this case, as it involves an intervention trial with human subjects, permission will be sought from a duly recognized entity by the 
Ecuadorian state, in this case, the Ethics Committee in Human Research at the Catholic University of Cuenca (Approval of the 
study, Declaration of Confidentiality, and Informed Consent). Subsequently, approval will be sought from the National Agency 
for Regulation, Control, and Sanitary Surveillance (ARCSA) for the registration, review, and approval of the trial.19 
 
2. Describe the process of anonymizing human biological samples, specifying if applicable, how they will be coded 
(example: First letter of the name, first letter of the surname, first digits of ID card LH1715). 
 
The process of anonymizing data involves: 
Purpose 
 
Minimizing the number of variables necessary for identifying individuals, restricting access to confidential information to the 
research team involved in the present investigation 
Coding 
Participants will be coded in an alphanumeric manner. 

 
Anonymization Process 
 
The process will be determined by a sequential number, with the appended single letter corresponding to the chewing gum: Xylitol, 
Non-sugared, Sugared, Paraffin (X, N, A, P), followed by the participant's age in years and a letter, which in this case is the letter A 
for all participants as shown in Table 1. The code consists of a length of 6 characters. Table 1. Anonymization Process 
 
Current Code 
Anonymization 
1. Miranda 
Ojeda 
Adriana 
Estefanía, Xilitol, 18 Años 
 
1.X18A 
1. Miranda Ojeda Adriana Estefanía Xilitol Femenino 18 Años 
 
3. Define the person or institution responsible for safeguarding the human biological samples. 
On the same day that the sample is taken, the salivary flow and pH level will be measured, and the samples will be disposed of in red 
biohazard bags. The custody will be under the responsibility of the principal investigator. The Molecular Biology Laboratory has a 
system for managing biological waste 
 
INFORMED CONSENT 
 
The informed consent is presented in Appendix 4, and the participants are individuals aged 18 to 25 years old, therefore, there is 
no need for assent. 

 
 
EXPECTED RESULTS 
The possible results according to the study objectives: 
Identify the benefits of chewing gum consumption on salivary flow. 
Determine significant differences in salivary pH among different types of chewing gum. In addition to finding positive results, it could
be implemented in oral health promotion programs as an alternative, especially where oral hygiene is not practiced three times a day.
Among the possible risks, allergy to chewing gum components could occur. (Annex D - Information on chewing gum) 
 
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FINAL STATEMENT 
• 
That the content, authorship, and responsibility for the study results correspond to the Sponsor and the Principal Investigator, and
that the Ministry of Public Health is exempted from any legal action arising from this cause.  
• 
That the project described in this document is an original work, whose authors are part of the research team, and therefore assume
full legal responsibility in the event that a third party claims ownership of the intellectual rights of the project; Likewise, the
Ministry of Public Health is exempted from any legal action arising from this cause.  
• 
That this project does not cause any harm to the participants in the research or to the environment, and does not violate any legal
or ethical norms. In the event that the research requires permits from other institutions outside the Ministry of Public Health prior 
to its execution, the Sponsor/Principal Investigator will provide a certified copy of these permits to the Ministry of Public Health
of Ecuador.  
• 
That they will ensure compliance with this research in the terms approved by both the Committee of Ethics of Research in 
Human Beings and by the Ministry of Public Health.  
• 
That a final report of the research with the obtained results will be sent to the Ministry of Public Health. 
 
 
Location: Cuenca, Ecuador  
Date: December 12, 2023 
                   Od. Miriam Lima Illescas 
 
Signature Principal 
Investigator 
                                                                                                                                    0103711925 
                                                 ID number Principal Investigator 
 
  
 
 
Dr. Pedro Martínez Suárez Ph.D                                     
 
         
                                                                     Signature of Vice-Rector for Research, Catholic University of Cuenca 
                                                                                PAB256196