Effect of Lactobacillus Rhamnosus GG and Bifidobacterium Longum in Children Aged 3 to 5 Years of Villavicencio and Pasto
Phase: NA
Conditions: Caries, Dental
Sponsor: Cooperative University of Colombia

 
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PROTOCOL 
Effect of Lactobacillus rhamnosus GG and 
Bifidobacterium longum in children aged 3 to 5 years of 
Villavicencio and Pasto. 
ClinicalTrials.gov ID: NCT03078179 
Principal Investigator: María del Pilar Angarita 
Sponsor: Universidad Cooperativa de Colombia 
Funded by: 1. Universidad Cooperativa de Colombia 
2. Colgate 
Version number: 5 
 
17 June 2015 
Adapt: 01 August 2016 
 
 

 
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Table of contents 
1. 
Synopsis ....................................................................................................................................... 2 
2. 
Introduction ................................................................................................................................ 3 
3. 
Schema ........................................................................................................................................ 4 
4. 
Schedule of activities ................................................................................................................... 5 
5. 
Study Description ........................................................................................................................ 6 
6. 
Risk/Benefit assessment ............................................................................................................. 7 
7. 
Objectives and Endpoints ............................................................................................................ 8 
8. 
Study Design .............................................................................................................................. 10 
8 
Study Population ....................................................................................................................... 14 
9 
Study intervention ..................................................................................................................... 15 
10 
Statistical considerations ...................................................................................................... 18 
11. Regulatory, ethical, and study oversight consideration .............................................................. 20 
12. 
References ............................................................................................................................. 21 
 
 
 
 

 
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1.Synopsis 
Study Type 
Interventional, Pilot Study 
Intervention 
1. Commercial 
Cow 
Milk 
with 
Probiotic 
(Lactobacillus 
rhamnosus and Bifidobacterium 
longum) 
2. Commercial cow milk without 
probiotic 
Study Design:     
Repeated measures design. 
Allocation: 
Nonrandomized 
Endpoint Classification 
Efficacy Study 
Intervention Model: 
Crossover study 
 
Masking: 
No masking  
Sample size 
Convenience sampling 
Primary Purpose 
Treatment trial 
Subjects: 
Three to five-year-old children 
Genders: 
Both 
Sites 
Kindergarten in Villavicencio y Pasto, 
Colombia 
Study duration 
12 months 
Objective  
To determine the effect of consumption 
of commercial milk beverages enriched 
with 
Lactobacillus 
rhamnosus 
y 
Bifidobacterium 
longum 
on 
the 
incidence of caries in children aged 3 to 
5 years. 
 

 
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2.Introduction 
Several bacterial strains have been studied as probiotics, either isolated or in 
combination, as well as the doses used in studies, intervention periods, populations, 
experimental designs, and routes of administration. The results often include a 
decrease in S. mutans count in saliva or plaque, but few studies conduct dental 
clinical examinations to assess treatment. This is confirmed by a meta-analysis 
conducted by Laleman et al in 2013, claiming that the scientific evidence 
demonstrating caries control is insufficient (1). However, the results of studies 
describing the effect of probiotics in the index of carious lesions (2,3) or dental 
plaque, are promising (4,5). 
The probiotics most commonly evaluated so far in the oral cavity are isolated 
microbial strains, mainly of the digestive tract, like the ones belonging to the genus 
Lactobacillus (L. rhamnosus, L. reuteri, L. casei, L. brevis, L. paracasei, L. 
acidophilus, L. plantarum) and Bifidobacterium (B. bifidum, B. longum, B. lactis, B. 
animalis, B. infantis) (6,7). These bacteria have proven their safety for many years, 
allowing clinical studies and the development of commercial products, with the 
added value that some have been identified in healthy individuals (DMF = 0) (L. 
rhamnosus, L. plantarum, L. acidophilus, L. brevis, L. paracasei) (8), suggesting their 
role in micro-ecologic balance. 
One of the most commonly studied probiotic bacteria in the oral cavity was isolated 
by Gorbach and Godin in 1983 from digestive tract of a healthy adult (9). It was 
named Lactobacillus rhamnosus GG (ATCC53103) and is characterized by 
inhibiting, by means of bacteriocins, a wide variety of human pathogenic bacteria 
(10), including S. mutans, S. sobrinus, Aggregatibacter actinomycetemcomitans, 
Porphyromonas gingivalis and Prevotella intermedia. In addition, this bacterium 
colonizes the microcosm of saliva, and has no cariogenic effect. This bacterium was 
used to conduct one of the most complete clinical studies in children (aged 1 to 6 
years), reducing the incidence of caries in the age of 3 to 4 years (2). 

 
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3.Schema 
 
1. Parents meeting
2. Begin:  Diagnodent 
measurement, 
microbiological analysis, 
salivary pH, dental plaque 
index
3. Intake of milk without 
probiotic during 3 months.
4. Ending: Clinical diagnostic, 
Diagnodent measurement, 
microbiological analysis, salivary 
pH, dental plaque index. Stop 
drinking milk for a week.
Intake of milk with 
probiotic during 3 months.
Ending: Clinical 
diagnostic, Diagnodent 
measurement, 
microbiological analysis, 
salivary pH, dental plaque 
index. 
Inclusion criteria
Exclusion criteria
Informed Consent
100 children from 
Villavicencio y Pasto
Clinic diagnostic about 
caries (ICDAS 1-4), 
medical history

 
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4.Schedule of activities 
Activities 
Months 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Parents 
meeting 
 
 
 
 
 
 
 
 
 
 
 
 
Informed 
consent 
 
 
 
 
 
 
 
 
 
 
 
 
Personal 
interview 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical 
diagnostis 
 
 
 
 
 
 
 
 
 
 
 
 
Selection 
criteria 
 
 
 
 
 
 
 
 
 
 
 
 
Begin: 
diagnostic 
tests 
 
 
 
 
 
 
 
 
 
 
 
 
Intake milk 
without 
probiotic 
for 3 
months 
 
 
 
 
 
 
 
 
 
 
 
 
Ending: 
diagnostic 
tests 
 
 
 
 
 
 
 
 
 
 
 
 
Intake milk 
with 
probiotic 
for 3 
months 
 
 
 
 
 
 
 
 
 
 
 
 
Ending: 
diagnostic 
tests 
 
 
 
 
 
 
 
 
 
 
 
 
Analysis of 
information 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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5.Study Description 
Brief Summary: The objective of this study is to determine if the presence of bacteria 
with probiotic characteristics (Lactobacillus rhamnosus GG and Bifidobacterium 
longum) in a commercial milk, control some measures that indicate cariogenic 
processes. Detailed Description: Dental caries is a disease caused by a shift in pH 
balance, resulting in the formation of a biofilm composed mainly of cariogenic 
microflora. The interaction between this microflora and host factors conditions the 
deceleration or acceleration of the disease. Some biotechnology tools have been 
tried based on all this research, including probiotics, intended for a selective control 
of the etiological agents of caries and the maintenance of oral homeostasis. 
Methodology: In this study, preschool children belonging to public kindergarten in 
Villavicencio and Pasto, Colombia will be invited to participate. After obtaining 
parent´s informed consent, the children will start commercial milk intake without 
probiotic for 3 months (Klim fortificada Nestlé). One week after, they will start 
commercial milk with probiotic during others 3 months (nanPro3 Nestlé). Clinical and 
microbiological testing of saliva at baseline, 3 m, and 6m will be performed. 
Differences in salivary pH (before and after a sugar solution), remineralization and 
demineralization in early Stage Decay and Established decay tooth (according to 
ICDAS) and quantification of Streptococci of the mutans group. Expected results: 
Statistically significant decrease in pH drop, demineralization in decay tooth and 
quantification of Streptococci of the mutans group. 
 
 

 
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6.Risk/Benefit assessment 
Minimal Risk: 
This study has a minimal risk because the probability and magnitude of possible 
harms implied by participation in the research is no greater than those encountered 
by participants in the aspects of their everyday life that relate to the research (TCPS 
Chapter 2B ). The intervention is safe because the study will use two commercial 
milk with Sanitary Registry of health products. During the reconstituting of powdered 
milk, the person responsible will use bottled water and good hygienic conditions. 
Moreover, during the clinical diagnostic and the clinical laboratory, this research will 
practice principles and practice about biosafety.  
 
Benefit:  
The immediate potential benefits of this research with children, is the daily intake of 
two nutritive commercial milk, for 6 months. Also, the researcher gives information 
about the oral health, the presence of caries, and the requirements of specific 
treatment. 
 

 
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7.Objectives and Endpoints 
OBJECTIVES 
 
ENDPOINTS 
Justification for 
endpoints 
To determine the effect of 
consumption of milk 
beverages enriched with 
Lactobacillus rhamnosus 
and Bifidobacterium 
longum in diagnodent 
measurement. 
Diagnodent 
measurement of 
tooth decay lesions 
(ICDAS 1-4). The 
idea is to compare 
the effect of 
probiotics in children 
during the intake of 
milk without 
probiotic versus 
during the intake of 
milk with probiotic. 
DIAGNOdent is a laser 
fluorescence device 
used for dental caries 
diagnosis in occlusal 
and smooth surfaces. 
Diagnodent 
measurement allows 
knowing if the effect of 
probiotic suppresses 
the demineralization of 
the cariogenic lesion. 
The reason is, if the 
probiotic inhibit the 
cariogenic bacteria, 
reduce the 
demineralization and 
improve the 
mineralization 
To determine the effect of 
consumption of milk 
beverages enriched with 
Lactobacillus rhamnosus 
and Bifidobacterium 
longum in the change of 
pH of saliva after a rinse 
with sugar. 
For this objective, 
the purpose is to 
compare the points 
difference between 
salivary pH before 
and after a rinse 
with sugar, in 
children during the 
intake of milk 
without probiotic 
versus during the 
intake of milk with 
probiotic 
The scientific literature 
shows when there are 
many cariogenic 
bacteria, the drop of Ph 
after a rinse with sugar 
is greater compared 
with people who have 
less cariogenic bacteria 
levels.   
To determine the effect of 
consumption of milk 
beverages enriched with 
Lactobacillus rhamnosus 
y Bifidobacterium longum 
in the concentration of 
group mutans 
streptococci in saliva. 
The comparison of 
the quantification of 
group mutans 
streptococci from 
saliva, during the 
intake of milk 
without probiotic 
versus during the 
Other studies have 
shown the inhibition of 
Lactobacillus 
rhamnosus in 
Streptococcus mutans. 

 
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intake of milk with 
probiotic   
To determine the effect of 
consumption of milk 
beverages enriched with 
Lactobacillus rhamnosus 
y Bifidobacterium longum 
in dental plaque index. 
The comparison of 
dental plaque index 
during the intake of 
milk without 
probiotic versus 
during the intake of 
milk with probiotic, 
show us if 
Lactobacillus 
rhamnosus and 
Bifidobacterium 
longum reduce the 
formation of dental 
plaque.   
Other studies, showed 
that some probiotics 
reduce the dental 
plaque. 
 
 
 

 
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8.Study Design 
7.1 Hypothesis 
1. The commercial milk beverages enriched with Lactobacillus rhamnosus 
and Bifidobacterium longum reduce the demineralization of caries lesion 
(ICDAS 1-4). 
2. The commercial milk beverages enriched with Lactobacillus rhamnosus 
and Bifidobacterium longum reduce the change of  salivary pH after a 
rinse with sugar. 
3. The commercial milk beverages enriched with Lactobacillus rhamnosus 
and Bifidobacterium longum reduce the quantification of mutans 
streptococci in saliva. 
4. The commercial milk beverages enriched with Lactobacillus rhamnosus 
and Bifidobacterium longum reduce the dental plaque.  
 
7.2 Study design: Nonrandomized comparison, single group assignment, controlled 
before and after study. 
7.3 Arm treatment information: 
A. Experimental: Commercial Cow Milk with Probiotic. 200 ml of commercial 
nutritive milk fortified with probiotic Lactobacillus rhamnosus and 
Bifidobacterium longum once a day. 
B. Placebo comparator: Commercial Dairy Cow Milk 200 ml of commercial 
nutritive milk without probiotic, once a day. 
7.4 Dosing and administration:  
Experimental: 200 ml of commercial nutritive milk fortified with probiotic 
Lactobacillus rhamnosus and Bifidobacterium longum (Nan Pro3, Nestlé) 
once a day. 
Placebo comparator: 200 ml of commercial nutritive milk without probiotic 
(Klim Fortificada Nestlé), once a day. 
7.5 Study intervention duration: 

 
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3 months intake the placebo comparator, and 3 months intake the 
experimental treatment. 
7.6 Sites: 
Kindergarten in Villavicencio, Colombia 
Kindergarten in Pasto, Colombia 
7.7 Phase of the trial: 
1. Invitation to participate in the trial: at the kinder garden during a meeting, 
the researchers invite parents to participate. 
2. To request the informed consent of the parents of the children. 
3. Diagnosis, selection, and collection of samples. Each child will be informed 
of the process in a simple way. 
First Day: 
3.1 First selection: the dentist checks up the oral health condition. Children 
without caries or with advanced caries will not continue in the process. 
Children who enter the criteria continue in the process.  
3.2 Second selection: Clinical assessment of demineralization with 
Diagnodent, after of dental prophylaxis. The children with measure upper to 
7, are included. 
3.3 ICDAS: dentist with calibration in ICDAS take this measure. 
Second day: 
3.4 Index dental plaque Sillness and Löe. 
3.5 Collection of saliva: Each child will accumulate saliva and then collect it 
in sterile containers (approximately 1.5-2 ml). 
3.6 1 ml of saliva with glycerol (10%) will be frozen (-20°C) in microcentrifuge 
tubes (1.5ml). This sample is for microbiological analysis of S. mutans). 
3.7 With the other 1ml is to determine the pH. The Salivary pH will be 
determined with pHmeter Fisherbrand Accumet Portable AP110. 
3.8 Rinse with 10% sugar solution. The children will make a mouthwash with 
sugar solution and they will wait 5 minutes. Finally, the pH of the saliva is will 
be determined again. 

 
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4. Microbiological Analysis of S. mutans 
The 100µl of saliva sample will be dilute with 900µl of sterile normal saline. 
Then will be performed decimal dilutions of saliva were made up to 10-3. 
100µl of every dilution will be uniformly spread in Agar Mitis Salivarius with 
tellurium and bacitracin. Finally, the plates will be incubated via anaerobically 
system with 5% CO2 for 48 hours at 37°C. The confirmation of morphology 
of the bacterial colonies will be determined under a stereo microscope. Also, 
the CFU will be count under the stereomicroscope. 
 
5. Interview with the parents to know the information about the children (state 
of health, diet, teeth brushing frequency) 
 
6. INTERVENTION 
Children were given 200 mL of milk with after the breakfast every day, for five 
days a week. First, the children will drink milk without probiotic, during the 3-
months period. And then the children drink milk with probiotic during the 3-
months period (L. rhamnosus and B. longum). The nursery school staff 
prepared the commercial milk with researcher supervision. The milk will be 
prepared with safe water (packaged water). 
6. After the intervention with each milk, the next outcome measures: 
6.1 Index dental plaque Sillness and Löe. 
6.2 Collection of saliva: Each child will accumulate saliva and then collect it 
in sterile containers (approximately 1.5-2 ml). 
6.3 1 ml of saliva with glycerol (10%) will be frozen (-20°C) in microcentrifuge 
tubes (1.5ml). This sample is for microbiological analysis of S. mutans). 
6.4 With the other 1ml is to determine the pH. The Salivary pH will be 
determined with pHmeter Fisherbrand Accumet Portable AP110.6.5 Rinse 
with 10% sugar solution. The children will make a mouthwash with sugar 

 
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solution and they will wait 5 minutes. Finally, the pH of the saliva is will be 
determined again. 
6.6 Dental prophylaxis 
6.7 ICDAS 
6.8 Clinical assessment of demineralization with Diagnodent. 
 
 

 
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8 Study Population 
8.1    General information: children from 3 to 5 years of kindergartens 
8.2    Inclusion criteria: 
•    Pre-school children from age 3 to 5. 
•    Healthy children from a general health perspective, with early stage decay and 
established decay 
8.3    Exclusion criteria 
•Children with systemic disorders who need special care and/or who have the 
intolerance to milk drinks or allergy to any of the components of the experimental 
and/or the placebo beverage. 
•Children who don’t like milk. 
•Children whit severe decay 
8.4 Lifestyle consideration: 
The children’s parent will inform when:  
-The child uses antibiotic 
-The child visits another dentist and the dentist use Fluor. 
-The child uses an antiseptic or mouthwash. 
-The child improves the brush frequency. 
 

 
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9 Study intervention 
Primary Outcome Measure: 
1.Change in remineralization of tooth decay 
DIAGNOdent peak reading in three moments,at the beginning and at the end of the 
milk without probiotic (after 3 months), and at the end of the milk with probiotic (after 
3 months) (Table 1). 
Secondary Outcome Measures: 
1. Change of salivary pH after a rinse with sugar. 
Change pH after a rinse with sugar (point of difference), in three moments, at the 
beginning and at the end of the milk without probiotic (after 3 months), and at the 
end of the milk with probiotic (after 3 months) (Table 1). 
2. Change of Dental Plaque Index 
Silness-Löe plaque index n three moments,at the beginning and at the end of the 
milk without probiotic (after 3 months), and at the end of the milk with probiotic (after 
3 months) (Table 1). 
3. Change of concentration of group mutans streptococci in saliva 
Colony Forming Units (CFUs) determination in three moments,at the beginning and 
at the end of the milk without probiotic (after 3 months), and at the end of the milk 
with probiotic (Table 1). 
 
 

 
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TABLE 1. 
Variable 
Scale 
of 
measurement 
Data 
Type of variable 
Measure Type 
Measure of dispersion 
BEFORE (Control and Intervention) 
Diagnodent 
measurement 
Quantitative 
Values 0-99 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 
Change 
of 
salivary pH after 
a 
rinse 
with 
sugar. 
Quantitative 
Point 
of 
difference 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 
Dental 
Plaque 
Index 
Qualitative 
0=absent 
1=present 
Dependent 
Frequency 
NA 
Colony Forming 
Units 
of 
the 
group 
mutans 
streptococci 
Quantitative 
UFC 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 
AFTER (Control and Intervention) 
Diagnodent 
measurement 
Quantitative 
Values 0-99 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 

 
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Change 
of 
salivary pH after 
a 
rinse 
with 
sugar. 
Quantitative 
Point 
of 
difference 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 
Dental 
Plaque 
Index 
Qualitative 
0=absent 
1=present 
Dependent 
Frequency 
NA 
Colony Forming 
Units 
of 
the 
group 
mutans 
streptococci 
Quantitative 
UFC 
Dependent 
-Mean 
-Median 
-Standard deviation 
-Inter-Quartile range 
 
 
 

 
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10 Statistical considerations 
Statistical hypothesis 
Primary Efficacy 
H01. There is no significant difference of consumption of commercial milk 
beverages enriched with Lactobacillus rhamnosus and Bifidobacterium 
longum in diagnodent measurement in comparison with commercial milk 
without probiotic. 
 
Secondary Efficacy 
H02: There is no significant difference of consumption of commercial milk 
beverages enriched with Lactobacillus rhamnosus and Bifidobacterium 
longum in change of pH of saliva before and after a rinse with sugar, in 
comparison with commercial milk without probiotic. 
 
H03: There is no significant difference of consumption of commercial milk 
beverages enriched with Lactobacillus rhamnosus and Bifidobacterium 
longum in concentration of group mutans streptococci in saliva in comparison 
with commercial milk without probiotic. 
 
H04: There is no significant difference of consumption of commercial milk 
beverages enriched with Lactobacillus rhamnosus and Bifidobacterium 
longum in dental plaque index in comparison with commercial milk without 
probiotic.  
 
11 Statistical analysis 
Data will be organized with Microsoft Excel. The analyses will be performed 
using the program SPPS® Statistics 25.0 version. Univariate descriptive 

 
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analyses: means, standard deviations, medians, and interquartile ranges 
according to the normality. Data comparisons will be performed with Students 
t-test for related samples. A p-value <0.05 was considered as statistically 
significant. If there are not data normally distributed, the comparison will be 
with Wilcoxon test. In the case qualitative variable (Dental plaque index), the 
comparison will be with McNermar’s test. 
 
 

 
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11. Regulatory, ethical, and study oversight 
consideration 
According to the classification in the article 11 of the Scientific, technical and 
administrative norms for the investigation in health (Resolution 008430 of 1993 of 
the Ministerio de Salud de Colombia), it is a research with minimum risk since it 
treats of a commercial milk. In addition, only a dental examination will be performed 
and plaque and saliva will be collected, without implying any harm to the child. The 
examination and taking of the dental plaque will be done by an expert in the area. 
The study will be endorsed by the university's bioethics subcommittee. The informed 
consent will be applied to the children’s parents. 
. 
The work will be carried out in the laboratory following all the norms set out in the 
Biosafety Manual in the WHO laboratory (WHO, 2005) and does not represent any 
risk to humans, since it is a controlled in vitro study. In addition, the project will not 
represent negative effects on the environment during the execution of the project, 
since the waste management of the reagents will be carried out following the waste 
disposal regulations. 
 
 
 

 
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12.References 
1. Laleman I, Detailleur V, Slot DE, Slomka V, Quirynen M, Teughels W. 
Probiotics reduce mutans streptococci counts in humans: a systematic review 
and meta-analysis. Clin Oral Investig 2014; 18(6): 1539-1552. 
2. Näse L, Hatakka K, Savilahti E, Saxelin M, Pönkä A, Poussa T et al. Effect of 
long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus 
GG, in milk on dental caries and risk in children. Caries Res 2001; 35(6): 412-
420. 
3. Hedayati-Hajikand T, Lundberg U, Eldh C, Twetman S. Effect of probiotic 
chewing tablets on early childhood caries - a randomized controlled trial. BMC 
Oral Health 2015; 15(1): 112. 
4. Burton JP, Drummond BK, Chilcott CN, Tagg JR, Thomson WM, Hale JD et 
al. Influence of the probiotic Streptococcus salivarius strain M18 on indices of 
dental health in children: A randomized double-blind, placebo-controlled trial. 
J Med Microbiol 2013; 62(Part 6): 875-884.  
5.  Toiviainen A, Jalasvuori H, Lahti E, Gursoy U, Salminen S, Fontana M et al. 
Impact of orally administered lozenges with Lactobacillus rhamnosus GG and 
Bifidobacterium animalis subsp. lactis BB-12 on the number of salivary 
mutans streptococci, amount of plaque, gingival 
6. Badet C, Thebaud NB. Ecology of lactobacilli in the oral cavity: a review of 
literature. Open Microbiol J 2008; 2: 38-48. 
7. Agrawal V, Kapoor S, Shah N. Role of “Live Microorganisms” (Probiotics) in 
Prevention of Caries: Going on the Natural Way Towards Oral Health. Indian 
J Multidiscip Dent 2012; 2(3): 491-496. 
8. Colloca ME, Ahumada MC, López ME, Nader-Macías ME. Surface properties 
of lactobacilli isolated from healthy subjects. Oral Dis 2000; 6(4): 227-233. 

 
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9. Gorbach SL, Goldin BR, inventors; New England Medical Center Inc, 
assignee. Lactobacillus strains and methods of selection. United States 
patent US 4839281(A). 1989 Jun 13. 
10. Doron S, Snydman DR, Gorbach SL. Lactobacillus GG: bacteriology and 
clinical applications. Gastroenterol Clin North Am 2005; 34(3): 483-498. 
 

 
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