Effects of a Functional Food Supplemented with Probiotics on Biological Factors Related to Dental

Caries in Children: A pilot study.

ABSTRACT

Purpose: The aim of this study was to evaluate the effects of a functional food supplemented with probiotics

on biological factors related to dental caries in children aged 3–5 years.

Methods: A repeated measures pilot study was conducted with children who have consumed a commercial

milk containing two lactic acid bacteria as probiotics (WP milk) for a period of 3 months and another period of

3 months consuming a milk without probiotics (NP milk). Salivary pH, plaque index, pH variation before and

after a sugar rinse, quantification of Streptococcus mutans in saliva and demineralisation of the carious lesions

were determined at the beginning and at the end of both milk ingestion periods.

Results: Regarding WP milk, a non-significant decrease in terms of the concentration of S. mutans and pH

variation (p > 0.05), a significant decrease (i.e. acidification) in salivary pH (p < 0.01) and a remineralisation

of 39.4% of the caries were found. On the other hand, for NP milk, a non-significant increase in terms of the

concentration of S. mutans, pH variation, salivary pH (p > 0.05) and a remineralisation of 64.2% were found.

Conclusions: Lactic acid probiotics can contribute to the decrease in the number of cariogenic microorganisms.

However, the appropriate selection of the bacteria type with regard to its acidogenicity is fundamental to avoid

the generation of an effect contrary to that expected, e.g. a significant decrease in salivary pH.

Keywords: Functional food, probiotics, dental caries, child welfare

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1. INTRODUCTION

Early childhood caries (ECC) is a pandemic disease considered a global public health problem causing high

risk of new carious lesions, decrease in quality of life, high treatment costs, days off school, and a barrier to

educational ability (American Academy of Pediatric Dentistry 2016). In Colombia’s last National Oral Health

Study (ENSAB IV), caries was found to be prevalent in 43.77% of children aged 3 years and in 52.2% of

children aged 5 years, highlighting the need to implement more strategies for the prevention of this disease

(Ministry of Health 2014).

Dental caries is dependent on the biological factors associated with saliva and dental biofilm such as flow rate,

salivary pH, buffer capacity, presence of certain microbial communities, and the immune system. All these

factors together with oral hygiene practice and the diet, influence the demineralisation of teeth (Hicks et al.

2003; Cunha-Cruz et al. 2013). Therefore, ECC can be prevented through early and accurate management of

these most common factors.

Four key health interventions were identified in an expert panel’s report on public health intervention against

ECC conducted by the WHO in 2016, which included promotion of healthy behaviours, use of fluoride,

practices of oral hygiene and proper diet (WHO 2017). Regarding the proper diet/nutritional practices,

additional studies are needed to demonstrate the benefits of some foods in contributing to caries control, in

order to promote these kinds of food as a public health action in kindergartens. Functional foods might be a

good option once they are foods or ingredients of physiologically active food that provide additional benefits

beyond basic nutrition. Among the natural functional foods is milk and its products (e.g. yogurt and cheese),

which are sources of calcium, phosphate, casein and lipids (Vikneshan et al. 2016).

Functional foods can also be changed in order to become more effective by adding components such the ones

supplemented with probiotics, which have shown interesting results in several studies (Näse et al. 2001; Ahola

et al. 2002; Jindal et al. 2011). Probiotics are beneficial microorganisms that impart health benefits when

administered in sufficient quantities (Joint FAO/WHO 2002). They have been widely used for gastrointestinal

problems but information about their benefits in oral health is currently limited even though the literature

suggests that they could be beneficial for the maintenance of oral health, due to their ability to decrease the

colony forming units (CFU) counts of the oral pathogens. However, more studies are still needed to determine

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their efficacy in reducing the prevalence/incidence of oral infectious diseases (Seminario-Amez et al. 2017;

Nadelman et al. 2018).

Among the mechanisms of action exerted by probiotics is the reestablishment of microecological equilibrium

by inhibiting pathogens through the production of antimicrobial substances (bacteriocins and hydrogen

peroxides), competitive exclusion for binding sites and nutrients, stabilisation of oral conditions (pH and redox

potential) and modulation of immune responses (Coqueiro et al. 2018).

Lactobacillus and Bifidobacterium species are the most studied lactic acid probiotics in clinical trials owing to

their beneficial characteristics and safety aspects (Coqueiro et al. 2018). An evaluated probiotic in the oral

cavity is Lactobacillus rhamnosus GG (ATCC53103), and it is distinguished from other species by its ability

to inhibit a large variety of human pathogenic bacteria, including S. mutans, S. sobrinus, and Porphyromonas

gingivalis (Pham et al. 2011). The bacterium B. longum has been less studied with respect to its oral effect,

apart from a study in which the effect of this species in combination with L. rhamnosus and Saccharomyces

cerevisiae on S. mutans concentration in the saliva of 50 children was determined. The authors found a

significant reduction in the concentration following a 14-day intervention (Jindal et al. 2011). Despite these two

species (Lactobacilli and bifidobacterial) are bacteria associated with caries (Simón-Soro and Mira 2015), it is

also important to determine if the possible cariogenic effect outweighs the anti-caries potential of probiotics.

Therefore, the aim of this pilot study was to determine the effect of commercial milk supplemented with the

probiotics L. rhamnosus and B. longum on the biological factors associated with dental caries in children aged

3–5 years.

2. MATERIALS AND METHODS

2.1. Study design, subjects, and samples

A quasi-experimental pilot study using repeated measures was conducted through convenience sampling among

the kindergartens of the Instituto Colombiano de Bienestar Familiar (ICBF), Villavicencio and Pasto,

Colombia. This study was approved by the Universidad Cooperativa de Colombia’s Ethics Subcommittee

(Approval no. 16062015) and it was registered at Clinicaltrials.gov (NCT03078179).

Prior to the study, the parents of the kindergarten children aged between 3 and 5 years were invited to participate

and sign an informed consent. Once authorisation was obtained, information of the children’s clinical history

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was collected. The Knowledge and Attitudes survey validated by Martignon et al. (2008) was conducted, and a

clinical dental examination was performed.

Initially, the study participants comprised 121 children from Villavicencio (n = 71) and Pasto (n = 50). A final

sample of 63 children (43 from Villavicencio and 20 from Pasto) were selected by fully meeting the following

inclusion criteria throughout the study: 1) age between 3 and 5 years, 2) attending ICBF kindergarten, 3) have

at least one active carious lesion, and 4) have demineralised lesions of a degree greater than 6 according to

Castilho et al. (2016). In addition, children who had an intolerance toward lactose or any other components of

infant milk formula, did not prefer consuming milk, being administered antibiotics, had been attended by a

dentist during the study period, with systemic disease such as diabetes or cancer, taking immunosuppressants

or undergoing surgeries such as organ transplantation or bowel resection, and with criteria International Caries

Detection and Assessment System (ICDAS) 5 and 6, were excluded from the study.

2.2. Intervention

200 mL of the two types of milk (With Probiotics WP milk and No Probiotics NP milk) was administered from

Monday to Friday for 3 months. WP milk was prepared from a milk powder with probiotics (NanPro3®, Nestlé

México SA), containing 0.08 g sugar/mL of milk and the probiotics species L. rhamnosus GG (7.5 × 105

CFU/mL of milk) and B. longum (4.5 × 105 CFU/mL of milk). The NP milk was prepared from KLIM fortified

milk powder (Nestlé, Colombia SA), containing 0.09 g sugar/mL of milk and 30 % more calcium than WP

milk. The two types of milk were hydrated according to the respective manufacturer’s instructions using the

same water brand. The children belonging to Villavicencio first consumed NP milk, whereas those belonging

to Pasto first consumed WP probiotic milk (Fig. 1). Participants and dentists (n=4) were blind with respect to

the types of milks.

2.3. Measure of clinical and biological variables

All measurements were performed at the beginning and end of the intake of the two types of milk without

performing any interventions that could modify the oral hygiene habits of parents, caregivers, or children. The

measured variables were the: plaque index, salivary pH, pH variation before and after a sugar rinse,

quantification of Streptococcus mutans in saliva and demineralisation of the carious lesions. The plaque index

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was determined by using the modified Silness–Löe index (Mombelli et al. 1987) by qualified dentists (kappa

value ≥ 0.7). Salivary pH was measured using an electronic pH meter (Accumet AP110, Fisher Scientific,

Pittsburgh, USA) with a microelectrode (Fisherbrand TM AccumetTM Micro Glass Mercury-Free Combination

Electrode, Fisher Scientific, Pittsburgh, USA). Unstimulated saliva was previous collected using saliva dribble.

The variation of salivary pH following the sugar rinse was assessed as an indirect measure to determine the

effect of probiotics on the cariogenic bacteria present in saliva. For this, following the initial salivary pH

measurement, the children performed a rinse for 45 seconds with a 10% sugar solution. After 5 min the pH was

again measured and then the pH variation was determined.

For the quantification of S. mutans in saliva, 1 mL of unstimulated saliva was collected, in 10% glycerol in

accordance with the procedure reported by Rojas, 2007, immediately frozen and stored at -20 °C until the

samples were processed. Sample processing was performed by shaking each sample for 30 s using a vortex

(Heidolph) and subsequently prepared microdilutions in 0.9% sterile saline. Next, 100 μl aliquots of 10−1–10−4

dilutions were inoculated in Mitis Salivarius Agar (DifcoTM, Sparks, MD, USA) supplemented with 1%

potassium tellurite (Lab M Ltd, Lancashire, United Kingdom), 20% sucrose (Fisher Scientific, Pittsburgh,

USA), and 0.2 U/mL bacitracin (Sigma-Aldrich, St Louis, MO, USA). Then, the cultures were incubated at

37°C in 5% CO2 for 48 h. Afterwards, the cultures were macroscopically reviewed at 8–32× magnification

using a stereomicroscope (Stemi DV4, Zeiss, Jena, Germany) to identify the representative S. mutans colonies.

S. mutans was counted as colony-forming unit per ml of saliva (CFU/mL) and converted to values of logarithm

in base 10.

The demineralisation of carious lesions according to ICDAS degree 1 to 4 was quantified using the

DIAGNOdent device (Classic, KaVo, Charlotte, USA) by dentists previously calibrated (kappa value ≥ 0.7) to

this equipment and to ICDAS criteria.

2.4. Statistical analysis

Statistical analysis was performed with SPSS version 25 (IBM Corp, Armonk, NY, USA). Initially, the

statistical normality of the data was determined using Kolmogorov–Smirnov test and the homogeneity of

variances was determined using Levene’s test. To compare the results obtained before and after the intake of

both milk types of the variables with continuous data that showed normality, the Paired T-test was performed.

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While for continuous data without normality, Wilcoxon test was used. Further, to compare the effect of each

milk, such as the degree of mineralisation and demineralisation, Mann-Whitney U test was performed.

For the qualitative variables, the McNemar's test was applied to compare the before and after results for each

type of milk. Further, Chi-square test was used to compare the effect between the two types of milk. Values of

p < 0.05 were considered statistically significant for all analysis.

3. RESULTS

3.1. Participant’s characteristics

Most of the children belonged to Villavicencio (68.3%), the female gender predominated with 55.6%, the

average age was 3.67 years old, and most parents or caregivers had acceptable knowledge and attitudes (93.3%

and 62.3% respectively) (Table 1). No significant association was found between the characteristics of the

participants and any measured variables obtained after intake of milk with probiotics or milk without probiotics

(Table 2).

3.2. Effect on salivary pH

The percentage of children who experienced a pH increase were significantly higher when they ingested NP

milk (p < 0.01), while the percentage of children who experienced a decrease in pH was significantly higher

when they ingested WP milk (p < 0.01; Fig. 2a). In addition, a significant decrease in the median salivary pH

was detected after the WP milk ingestion (a pH drop from 7.53 to 7.4; p < 0.01; Table 3) due to both the

reduction in the percentage of children with high pH (from 47.6% to 39.7%; p > 0.05; Fig. 2b) and the increase

in the percentage of children with an acidic pH (from 1.6% to 7.9%; p > 0.05; Fig. 2a). On the contrary, with

the administration of NP milk occurred a non-significant increase in the median salivary pH (from 7.37 to 7.66;

p > 0.05; Table 3) due to the increase in the percentage of children with high pH (from 34.9% to 55.6%; p <

0.05; Fig. 2b).

3.3. Effect on dental plaque

A higher percentage of children did not tend to demonstrate any changes in the plaque index following the

administration of both types of milk (Fig. 2c), although a non-significant increase of children without plaque

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following the administration of NP milk and a non-significant increase of children with plaque after CP milk

intake were observed (Fig. 2d).

3.4. Variation of salivary pH following the sugar rinse

Although, a decrease in the median of salivary pH variation was detected following WP milk intake (p > 0.05;

Table 3) and an increase following NP milk intake (p > 0.05; Table 3), the difference was not statistically

significant between the two types of milk. In addition, a non-significant higher percentage of children whose

pH variation decreased following WP milk intake (52.4%; Fig 3a) than that of children following NP milk

intake was detected (44.4%; Fig 3a) (p > 0.05; Fig. 3a).

3.5. Quantification of S. mutans

In quantification of S. mutans, there was no statistically significant difference between the two types of milk.

However, following the administration of WP milk, there was a decrease in the mean concentration of S. mutans

(p > 0.05), while following the administration of NP milk an increase in the mean concentration (p > 0.05; Table

3) was observed. Additionally, there was a non-significant higher percentage of children with decreased

concentration of S. mutans following WP milk intake (47.6%) than following NP milk intake (41.3%; p > 0.05;

Fig. 3b).

3.6. Effect on demineralisation/remineralisation of lesions

A decrease in the median measurement of demineralisation was detected following the administration of the

two types of milk, although only NP milk had a significant effect (p < 0.01; Table 3). Further, 39.4% and 64.2%

of the carious lesions showed remineralisation following the administration of WP milk and NP milk,

respectively, with significant differences (p < 0.01; Fig. 2e).

Regarding the range of demineralisation, it was found that 28.8% (after WP milk) and 30.3% (after NP milk)

of the lesions were considered healthy surfaces and thus the proportion of lesions that showed outer enamel

demineralisation significantly decreased (Fig. 2f). Notably, no significant differences were found in terms of

the degrees of demineralisation and remineralisation following the administration of both types of milk (Table

3).

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4. DISCUSSION

The present study aims to offer new evidences on this topic, by evaluating the effect of the ingestion of a

commercial milk sample containing the probiotics L. rhamnosus GG and B. longum on biologic variables

associated to dental caries among children. Following the consumption of WP milk, a decreasing effect was

found on the factors associated with the microbiological aspects of caries such as the concentration of S. mutans

and the variation in pH before and after the sugar rinse. Although this decrease was not statistically significant,

it was opposite to the effect of NP milk, which presents an increase of these two variables.

This may indicate an effect of the probiotics present in the milk like L. rhamnosus whose inhibitory capacity

has been demonstrated for different types of bacteria, including oral bacteria such as S. mutans and P. gingivalis

(Näse et al. 2001; Söderling et al. 2011; Angarita et al. 2017; Alanzi et al. 2018). The inhibitory capacity of this

bacterium (L. rhamnosus), is still unclear but has been associated with the production of bacteriocins (Joshi et

al. 2014) or lactic acid (De Keersmaecker et al. 2006). The effect of milk containing L. rhamnosus in children

has been a subject of interest in different studies with different outcomes. The studies of Näse et al. (2001) and

Jindal et al. (2011) showed a decrease in the concentration of S. mutans, while others showed no significant

effect (Villavicencio et al. 2018).

Not only S. mutans is associated with caries but a diversity of microorganisms that produce organic acids and

induce the decrease of the pH of saliva and dental plaque (Simón-Soro and Mira 2015). For this reason, salivary

pH variation following a sugar rinse was also evaluated, as an indirect measurement to determine the effect of

the probiotic on the control of the cariogenic bacteria present in saliva. Following WP milk intake, there was a

decrease in median salivary pH variation after the sugar rinse and also an increase in the percentage of children

that showed a decrease in this variation (50.8%) when compared with NP milk (44.4%). Lin et al. (2017) showed

the effect of probiotics on the pH of a dental biofilm following a sugar rinse among children aged between 7

and 11 years who were at a risk of caries who consumed a fermented beverage containing L. casei for 7 days.

These authors found an increase in the minimum pH of the biofilm and a reduction in the recovery time of pH.

Regarding other variables such as salivary pH, there was a favourable effect with the NP milk ingestion with a

slight increase in the median pH (from 7.37 to 7.66). On the other hand, following WP milk intake, a significant

decrease was detected in the median pH (from 7.53 to 7.4). The variation of this measure was focused on

children with pH > 7.5 for the two milk types and on the increase of children with acidic pH following WP milk

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intake. This significant decrease in salivary pH following WP milk intake could have been due to the use of

lactic acid bacteria for dental caries control, i.e. some studies have demonstrated the ability of L. rhamnosus to

produce acids that drastically change the pH of the medium in which they grow, associating this inhibition with

the acidity generated (De Keermaecker et al. 2006). Thus, this characteristic of L. rhamnosus may be an

explanation for the decrease in salivary pH in children during the consumption of probiotics, particularly

because it is found in saliva (Pham et al. 2011). Also the decrease in pH and increase in the acidogenicity of

dental plaque following the addition of probiotic lactobacilli have been reported in in vitro biofilm studies

(Pham et al. 2009; Schwendicke et al. 2014). Therefore, several authors recommend the use of non-acidogenic

probiotics of oral origin, which can provide beneficial effects (e.g., antimicrobial and/or production of alkali

substances) without the negative contribution of acid production (Mira 2018; Marsh 2018). However, it is also

important to mention that there are in vitro studies that report insignificant changes in the pH of a medium

following the exposure of L. rhamnosus GG to several carbohydrates (fructose, glucose, lactose, sorbitol, and

sucrose), which should not affect the dental tissue (Pham et al. 2011; Jiang et al. 2018). In contrast, Haukioja

et al. (2008) found significantly lower pH due to the production of acids from glucose metabolised by L.

rhamnosus GG, similarly to that observed for S. mutans. These results were similar to those of the B. longum

strain studied, indicating that although glucose levels are relatively low in saliva, glucose formed by sugar in

saliva and the buccal biofilm are available to bacteria within the dental biofilm (Haukioja et al. 2008).

Concerning the measurement of demineralisation/remineralisation of carious lesions, a positive effect was

detected with both types of milk. However, the effect of NP milk was significantly higher as 64.2% of the

lesions were remineralised in comparison to the effect of WP milk where only 39.4% of the studied lesions

remineralised. This could be due to the salivary pH detected following the administration of the two types of

milk and to the concentration of calcium being 30% higher in NP milk than in WP milk. The physicochemical

processes of demineralisation and remineralisation of dental hydroxyapatite are continuously being influenced

by salivary pH, buffer capacity, exchange of ions in the enamel, and presence of ions that facilitate (calcium

and phosphate) or not (hydrogen ions) the remineralisation process (Cunha-Cruz et al. 2013).

Respect to the plaque index, no significant effects were detected following the administration of the two types

of milk with the majority of children (73% with WP milk and 71.4% with NP milk) showing no change for this

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variable. Similarly, in the study by Villavicencio et al. (2018), where subjects consumed milk with probiotics

of the same brand used in this study, also showed no significant effects in this variable.

Despite the exclusion of some children due to noncompliance with the selection criteria during the study, the

effect of the commercial milk supplemented with probiotics could be verified in a single group of children with

active caries. Then, these results could be a guideline for the design of future studies based on selection of

probiotics, which essentially promote a balance of oral microflora. One limitation of this work was that the milk

used with probiotics present a higher concentration of calcium than the milk without probiotics, and this could

have influenced the results obtained in remineralisation of the lesions. However, this highlight the argument for

the important role milk and calcium play in the oral health of children with caries.

Therefore, it is recommended that clinical studies should continue to determine the effect of functional foods

supplemented only with probiotics that have low capacity for acid production, and subsequently define ideal

functional foods that promote oral health among children.

5. CONCLUSIONS

In this pilot study, the effects of a commercial milk supplemented with the probiotics L. rhamnosus GG and B.

longum was evaluated. A significantly unfavourable effect on salivary pH was identified, and non-significant

decrease effect was observed for variables such as concentration of S. mutans and pH variation after a sugar

rinse. Overall, the results emphasize the importance of selecting an appropriate probiotic not only for its

beneficial characteristics (e.g., the antimicrobials shown in this study) but also for its lack of cariogenic

properties such as acid production. In this sense, the selection of other strains of less acidogenic lactobacilli or

bifidobacteria or the use of novel oral probiotics with antimicrobial and pH buffering capacity could be a more

feasible alternative.

ACKNOWLEDGMENTS

This project was funded by the Comité Nacional para el Desarrollo de la Investigación (CONADI),

Universidad Cooperativa de Colombia (INV1532) and by the Colgate-ACFO Award 2015.

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The authors thank Dr. Alex Mira of FISABIO, Valencia, Spain, for reviewing and recommending the

manuscript and Dr. Lorena Duran from Universidad Cooperativa de Colombia, Campus of Villavicencio, for

her support in the project execution.

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Fig. 1 Phases of the study conducted to determine the effect of a functional food supplemented with probiotics.

1. Meeting with the parents

Inclusion Criteria,
Informed Consent Villavicencio
Clinical History, Pasto
Knowledge and Attitudes

2. Begin Villavicencio: 2. Begin Pasto: clinical

clinical examination, examination, salivary pH
salivary pH and and microbiological
microbiological analysis analysis

3. Ingestion of milk
3. Ingestion of milk with
without probiotics for 3
probiotics for 3 months
months

4. Ending: clinical 4. Ending: clinical

examination, salivary pH examination, salivary pH
and microbiological and microbiological
analysis analysis

5. One week without 5. One week without

ingesting milk ingesting milk

6. Begin: clinical 6. Begin: clinical

examination, salivary pH examination, salivary pH
and microbiological and microbiological
analysis analysis

7. Ingestion of milk
7. Ingestion of milk with
without probiotics for 3
probiotics for 3 months
months

8. Ending: clinical 8. Ending: clinical

examination, salivary pH examination, salivary pH
and microbiological and microbiological
analysis analysis

14
Fig. 2 Effect of WP milk and NP milk on salivary pH (n = 63), dental plaque (n = 63) and demineralisation of
lesions (n = 162), McNemar’s and Chi-square tests; *p < 0.05 and **p < 0.01. a. Effect of both types of milk
on salivary pH. b. Comparison of the effect of both types of milk on saliva pH ranges. The pH ranges are
according to the study by Fejerskov (2008); normal pH (range: 6–7.5), high pH (>7.5), and acidic pH (<6). c.
Comparison of the effect of both types of milk on dental plaque. d. Effect of both types of milk on dental plaque.
e. Comparison of the effect both types of milk have on the mineralisation of lesions. f. Effect of both types of
milk on demineralisation ranges. The ranges of demineralisation are according to the study by Castilho et al.
(2016) in which no demineralisation was scored 0°–5°, outer enamel demineralisation as 6°–14°, inner enamel
demineralisation as 15°–20°, and dentine demineralisation as 21°–99°. These ranges were determined by
initially evaluating the lesions > 6°.

15
Fig. 3 Effect of WP milk and NP milk on pH variation and the concentration of Streptococcus mutans (n = 63),
Chi-square test; *p < 0.05 and **p < 0.01. a. Comparison of the effect of both types of milk on variation of
salivary pH. b. Comparison of the effect of both types of milk on the quantification of S. mutans.

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Table 1. Characteristics of the study sample.
Characteristics Frequency Percentage

City

Villavicencio 43 68.3

Pasto 20 31.7

Gender

Male 28 44.4

Female 35 55.6

Age Median ± DS: 3.67 ± 0.68

3 28 44.4

4 28 44.4

5 7 11.2

ICDAS

1–2 112 69.1

3 44 27.2

4 6 3.7

Total 162 100

Knowledges

Scarce 2 3.2

Acceptable 58 93.6

Good 2 3.2

Attitudes

Scarce 2 3.3

Acceptable 38 62.3

Good 21 34.4

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Table 2. Analysis influence of participant’s characteristics on the results.
City Gender Age Knowledge Attitudes
p value
Salivary pH. After
0.12 0.75 0.22 0.062 0.06
WP
Salivary pH. After
0.65 0.40 0.15 0.35 0.49
NP
Variation of pH.
0.49 0.18 0.29 0.23 0.07
After WP
Variation of pH.
0.73 0.53 0.69 0.96 0.98
After NP
Quantification of
S. mutans. After 0.24 0.21 0.60 0.25 0.57
WP
Quantification of
S. mutans. After 0.61 0.66 0.15 0.06 0.31
NP
Dental Plaque.
0.66 0.48 0.56 0.82 0.71
After WP
Dental Plaque.
0.91 0.27 0.79 0.58 0.70
After NP
ICDAS
Grade for
demineralisation. 0.62
After WP
Grade for
demineralisation. 0.66
After NP
*p < 0.05 and **p < 0.01. Chi-squared test.

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Table 3. Effect of NP milk and WP milk on biological factors and demineralisation.
Before After WP Before NP After NP
Variables p-value p-value
WP milk milk milk milk

7.53 7.4 7.37 7.66

pH (median, IQR) 0.00**1 0.06
(6.98-7.93) (6.80-7.66) (6,80-7.67) (6.93-8.00)

Variation of pH 0.45 0.34 0.40 0.47

0.561 0.481
(median, IQR) (0.15-0.74) (0-0.80) (0-0.82) (0,14-0.78)

Quantification of S. 2.78log10 2.70log10 2.66log10 2.78log10

0.373 0.963
mutans (average, SD) ±0.95 ±0.83 ±0.92 ±0.99

Measurement of 7 8
demineralisation 8 0.861 0.00**1
(4.8-12.2) 10 (7-16) (5-13.3)
lesions (median, IQR) (5-13)

Grade for 3 4
remineralisation - (1-7) - - (2-6.3) 0.232
(median, IQR)

Grade for 3 2
demineralisation - (2-6) - - (2-5) 0.262
(median, IQR)
*p < 0.05 and **p < 0.011Wilcoxon test 2 Mann-Whitney U test 3Paired samples T-test.

19