Heat-Inactivated Lactobacillus Salivarius HM-6 in Preschool Children With ECC

The study was a prospective, randomized, controlled trial with two parallel groups. It was conducted at the Department of Developmental Dentistry, Institute of Dentistry, Jagiellonian University Medical College in Krakow. The study protocol was implemented in accordance with the principles of the Declaration of Helsinki and the CONSORT recommendations. Approval for the study was obtained from the Bioethics Committee of the Jagiellonian University in Krakow, No. 1072.6120.31.2018.

Eighty generally healthy children aged 3-6 years, with or without clinical signs of early childhood caries (ECC), were enrolled in the screening study. Eight children were excluded due to failure to meet the inclusion criteria. Ultimately, 72 participants were enrolled in the study and randomly assigned in a 1:1 ratio to the intervention group receiving the study product (n=36)(n=36)(n=36) or the control group (n=36)(n=36)(n=36). The process of qualification, randomization, and follow-up is presented in a flowchart compliant with the CONSORT standard.

Inclusion criteria included:

• age 3-6 years;
• generally good health;
• no use of antibiotics or probiotics in the three months preceding study entry;
• ability and willingness of the child to chew tablets and participate in study pro-cedures;
• obtaining written informed consent from a parent or legal guardian. Exclusion criteria included severe infections, systemic diseases, immune disorders, congenital defects, food allergies, active inflammatory diseases of the oral cavity, peri-odontal disease, and the presence of fixed orthodontic appliances. Children who had taken antibiotics, anti-inflammatory medications, glucocorticosteroids, probiotics, vitamin supplements, or other products that could affect the parameters assessed within three months of the study were also excluded. Partial or complete refusal of a dental exami-nation by the child or their guardian was also an exclusion criterion.

2.2. Randomization and Blinding A 1:1 randomization sequence was generated by an investigator not involved in the further stages of the study using IBM SPSS Statistics, version 27.0, and the Microsoft Excel randomization tool. Each participant was assigned a unique allocation code corresponding to the control or intervention group.

To maintain allocation concealment, randomization codes were placed in sealed, opaque envelopes and were not revealed to the clinical assessors and biopsy sample collectors. Individuals responsible for participant care, sample collection, and clinical procedures remained unaware of the group allocation throughout the study.

Because the control group did not receive a placebo, the study was not fully dou-ble-blind. Blinding primarily affected the clinical and laboratory assessors and data an-alyzers.

2.3. Standardization of Oral Hygiene Before the study began, parents and guardians received standardized instructions regarding toothbrushing and the amount of toothpaste containing 1000 ppm fluoride to use. Children were advised to brush their teeth twice daily, morning and evening, throughout the observation period. This practice was a fundamental element of standard caries prevention in both groups. Caregivers recorded their oral hygiene practices in a dedicated diary. Both groups also received identical dietary recommendations, including maintaining appropriate intervals between meals.

2.4. Intervention Children in the intervention group received commercially available chewable tablets containing 10 mg of heat-inactivated Lactobacillus salivarius HM-6 Paradens. The preparation also contained isomalt, sucralose, natural strawberry flavor, magnesium stearate, and xylitol at 200 mg per tablet (Acidolac Dentifix Kids, Polpharma, Poland).

The preparation was administered for 14 consecutive days at a dose of two tablets per day: one after breakfast and one at bedtime, each time after toothbrushing. After taking the tablet, participants were advised to refrain from eating and drinking for two hours.

Adherence was assessed based on the number of unused tablets returned to the study team. Missing no more than two tablets per week was considered acceptable adherence to the protocol, while more frequent missed doses were classified as questionable compli-ance. Caregivers were also required to report adverse events, illnesses, and other cir-cumstances that might affect the study. Oral hygiene and dietary recommendations were the same in both groups.

2.5. Clinical Assessment of Dental Condition A dental examination was performed before the intervention. The presence of early childhood caries was classified as ECC- or ECC+, and the severity of caries was assessed using the International Caries Detection and Assessment System II (ICDAS II).

The ICDAS II score was recorded at baseline and at subsequent follow-up points according to the study protocol. The ICDAS II score obtained before the intervention was used for analyses of the effect of baseline caries severity on changes in salivary parameters.

2.6. Saliva Sample Collection and Storage Saliva samples were collected before the intervention and after 14 days of follow-up, between 8:00 AM and 10:00 AM. Parents and guardians were instructed to ensure that children did not consume any food or drink, except water, for at least 60 minutes prior to collection, and did not engage in oral hygiene. Unstimulated whole saliva was collected in sterile polypropylene tubes. Immediately after collection, samples were refrigerated and then centrifuged for 10 minutes at 4°C to remove cells, food debris, and other con-taminants. The resulting supernatant was divided into working aliquots and stored at -80°C until assayed. Samples underwent one freeze-thaw cycle.

For stimulated saliva collection, a Salivette kit was used according to the manufacturer's instructions (Sarstedt, Nümbrecht, Germany). The child chewed a sterile insert for approximately 2-3 minutes, after which it was placed in a dedicated tube using sterile forceps. Samples were centrifuged at 1000 × g for 5 minutes at 4°C. The resulting saliva was divided into aliquots and stored at -80°C.

2.7. Salivary pH Measurement Salivary pH was measured at baseline and after 14 days of intervention. Meas-urements were performed under comparable preanalytical conditions for all participants. Results were presented as a dimensionless pH value.

2.8. Determination of Antioxidant Potential by FRAP Saliva antioxidant potential was determined by FRAP according to the procedure described by Benzi and Strain. The method is based on the reduction of the iron complex Fe³⁺-TPTZ to the intensely blue Fe²⁺-TPTZ complex in a low pH environment. The increase in absorbance at 593 nm is pro-portional to the total reducing capacity of the antioxidants present in the sample. Con-centrations were calculated based on a calibration curve prepared from solutions of iron(II) sulfate heptahydrate FeSO4×7H2O at known concentrations. Results were expressed in mmol/L. The FRAP assay provided a collective measure of saliva reduction potential and was not interpreted as a measurement of the activity of a single antioxidant [34-36].

2.9. Secretory IgA and hBD-2 Assay Secretory immunoglobulin A concentration in saliva was determined by a quanti-tative sandwich ELISA using the Human IgA ELISA Kit (Abcam, Cambridge, UK; catalog no. ab196263), according to the manufacturer's instructions. The analytical sensitivity of the assay was 0.25 ng/mL, and absorbance was measured at 450 nm.

Human β-defensin 2 concentration was determined using the quantitative sandwich ELISA Human Beta Defensin 2 ELISA Kit (Abcam; catalog no. ab213770). Before assays, reagents and samples were equilibrated to room temperature. Standards, controls, and saliva samples were added to wells coated with the appropriate antibody. After further incubation and washing steps, a detection antibody conjugated to horseradish peroxidase was added, followed by the tetramethylbenzidine substrate. The reaction was stopped with stop solution, and absorbance was read at 450 nm. sIgA and hBD-2 concentrations were calculated based on calibration curves fitted with a four-parameter logistic regression model. All samples were assayed in duplicate, and the mean of two measurements was used for statistical analysis. Quality control procedures were performed according to the kit manufacturers' recommendations.