Effects of a food enriched with probiotics on Streptococcus mutans and Lactobacillus spp. salivary counts in preschool children: a cluster randomized trial

Abstract Probiotics have provided benefits to general health, but they are still insufficient to dental health. Objective: This study aimed to evaluate milk supplemented with probiotic bacteria and standard milk, measured by levels of Streptococcus mutans (S. mutans) and Lactobacillus spp., in 3-4-year-old children after 9 months of intervention. Material and Methods: The study was a triple-blind, placebo-controlled, randomized trial. The sample was composed of 363 preschoolers attending five child development centers in Cali, Colombia. They were randomized to two groups: children in the intervention group drank 200 mL of milk with Lactobacillus rhamnosus 5x10 6 and Bifidobacteruim longum 3x10 6 , and children in the control group drank 200 mL of standard milk. Interventions occurred on weekdays and information was gathered through scheduled clinical examination. The primary result was the number of colony forming units (CFU) of S. mutans and Lactobacillus spp. in the saliva. Secondary results were dental caries, rated by the International Caries Detection and Assessment System (ICDAS), dental plaque, pH, and salivary buffer capacity. Results: The proportion of S. mutans was lower in the intervention group compared with the control group after 9 months; however, the differences did not reach statistical significance (p=0.173); on the other hand, statistically significant differences between groups were found in the CFU/mL of Lactobacillus spp. (p=0.002). There was not statistically significant difference in the prevalence of dental caries for both groups (p=0.767). Differences between groups were found in the salivary buffering capacity (p=0.000); neither salivary pH nor dental plaque were significantly different. Conclusions: Regular consumption of milk containing probiotics bacteria reduced CFU/mL of Lactobacillus spp. and increased salivary buffering capacity at 9 months of consumption.

on caries prevalence as a final goal in preschool children 9,10,17,[22][23][24] . Six of these studies reported a reduction in caries occurrence after probiotic exposure compared with control groups in children 10,17,19,22-24 ; Hasslöf, et al. 9  Using a random number generator, one of the authors (J. V.) assigned each group of the Child Development Centers either to the intervention or control group. They were allocated to a blue or black code to hide their identity. The code was only known by the lead author (J. V.) and was not revealed until all data were analyzed. Neither the researchers, clinicians, the staff of the Child Development Centers nor the relatives of the participants knew whether the children were receiving the probiotic-supplemented milk or the control. The sample type was probabilistic by clusters. The sampling frame corresponds to the groups of the five Child Development Centers, conformed by 10, which will be the primary sampling units. At random, the ten groups were selected for each site (secondary sampling unit).

Clinical Examination and Data Collection
Clinical dental evaluation was carried out at baseline and during the 9-month period of study. One dentist using an artificial light, a mouth mirror, and Collection and culturing of salivary samples A pediatric dentist was responsible for taking the sample of unstimulated saliva to each child. The deposit was made between one and two milliliters of saliva using a 50 ml conical sterile plastic tube, marked with a study entry code for each child. Each medium plate was cultured from 0.1 ml of the previously diluted sample to 10 -3 ; then, the sample was uniformly spread. Finally, the plates were sealed.
The media were incubated via anaerobically system (gas-pack jars) with a mixture of 95% N 2 and 5% CO 2 for 48 hours at 37°C 16

Outcome Measures
The primary result was to measure the CFU of S. mutans and Lactobacillus spp. from saliva. Secondary results were the presence of dental caries lesions, which was assessed using ICDAS, dental plaque, pH, and salivary buffer capacity in 3-4-year-old preschool children.

Results
The flow chart depicts the study population ( Figure   1). Table 1 shows the baseline characteristics of the probiotic and control groups. There were no statistically significant differences between the groups, nor significant demographic differences between the children who continued in the study and those who left it.
Effects on S. mutans, Lactobacillus spp., dental caries, dental plaque, pH, and salivary buffer capacity The data of this study, as collected at baseline and at the end of the clinical trial, are shown in Table 2.
The mean of the S. mutans CFU/mL was lower in the intervention group compared with the control group after 9 months, however, the differences did not reach statistically significance (p=0.173). The results showed statistically significant difference between intervention and control group in Lactobacillus spp. CFU/mL after intervention (p=0.002).
Dental caries decreased between baseline and at 9 months of intervention (dicdas2-6mft), in both the probiotic and the control group; however, these differences were not statistically significant (p=0.767).
Regarding the probiotic group, the relative risk values (95% CI) for children with initial carious lesions were 1.03 (0.87-1.23, p=0.66) at the baseline and 1.09 (0.93-1.27, p=0.28) at 9 months. The ingestion of probiotics did not show a statistical significance in RR, indicating that there was no association between the consumption of probiotics and the appearance of initial caries during intervention ( Table 2). The differences in dental plaque and pH in the probiotic and the control group were not significant after 9 months. There was statistically significant difference in the salivary buffering capacity at baseline and after 9 months of intervention between the probiotic and the control group (p=0.00) ( Table 3).

Characteristic
Probiotic

Discussion
This study determined whether the daily drinking of milk complemented with the probiotics Lactobacillus rhamnosus and Bifidobacterium longum decreased the CFU/mL of S. mutans, Lactobacillus spp., dental caries, and dental plaque and increased pH and salivary buffer capacity compared with standard milk in preschool children. The dropout rate was 11.6%. A total of 42 children abandoned the study; the participants withdrew because of the change of residence address and because they were compelled to enter primary school by Colombian law. Therefore, the age of the retired children is higher than the average of those who remained in the study. The dropout rates were 11% in the control group and 12.5% in the intervention group. This study did not record side effects associated with the bacteriotherapy with Lactobacillus rhamnosus and Bifidobacterium longum.
Cluster randomization was made for this trial. Therefore, the differences observed in the trial might be due to the probiotics instead of differences of milk brands. The probiotic strains in this study were based in Lactobacillus rhamnosus 5x10 6 CFU/g in 62.5% and the Bifidobacterium longum 3x10 6

S. mutans
Studies such as the one performed by Näse, et al. 17  It is understood that, to the extent that probiotic strains reduce bacterial counts associated with dental caries, there is a decrease in plaque, which is where the cariogenic bacteria harbor. pH Slight changes in the salivary acid reduction of preschool children were found after 9 months of milk supply with probiotics in this study; in the group that consumed standard milk, pH values were the same after 9 months of intervention. Campus, et al. 5