Despite advances in oral health care, cavities remain highly prevalent worldwide, especially among children. Conventional treatment methods focus on removing damaged tooth tissue but fail to completely eliminate or control pathogenic bacteria in the oral cavity, leading to a high risk of caries recurrence in the same or adjacent teeth. Probiotics are a promising biological therapy that can help restore balance in the oral microbiome.

Among probiotics, Bacillus species are unique because they form spores that make them stable under harsh conditions, such as heat and storage. They can also produce enzymes and natural compounds with antibacterial, anti-inflammatory, and protective effects. These features make Bacillus probiotics strong candidates for oral health applications.

This study is designed to evaluate the safety and efficacy of an oral-spray probiotic containing Bacillus subtilis ANA48 and Bacillus clausii ANA39 (LiveSpo® Smile CARE) in preventing and supporting the treatment of tooth decay in children. We hypothesize that this probiotic spray, when used regularly, will be safe and well-tolerated, while effectively reducing harmful bacteria in the mouth, preventing the progression of cavities, and improving overall oral health.

Study Design:

• Sample Size: 130 participants
• Study Location: a primary school in Hanoi, Vietnam Intervention Description: A total of 130 eligible participants will be randomly assigned to two groups (n = 65 per group).

Placebo Group will receive a 0.9% NaCl physiological saline, spray twice daily (morning and afternoon). Each application consists of two sprays at three sites (both dental arches and the front teeth), for a total of six sprays per use, continued for three consecutive months.

Smile Care Group will receive an oral-spray probiotics containing ≥ 1 billion CFU/mL × 20 mL of Bacillus subtilis and Bacillus clausii (LiveSpo Smile CARE). The dosage, frequency, and duration of use will be the same as in the placebo group.

All participants will receive the same standard dental care throughout the study, including the use of identical toothpaste, supervised weekly fluoride rinsing with 0.2% sodium fluoride (NaF), and scheduled follow-up visits. The probiotic and placebo sprays will be packaged in blinded bottles and administered according to the study protocol to maintain objectivity.

Study Duration: 12-18 months

Tooth decay is one of the most common oral health problems in children. It not only causes pain and discomfort but also interferes with eating, daily activities, and overall quality of life. In severe cases, untreated cavities may progress to pulp infection requiring more complex treatment. One of the major causes of dental caries is an imbalance of the oral microbiome, with Streptococcus mutans being the main pathogen. Improving the oral microbiome provides a safe, long-term strategy for managing cavities, particularly in children, while avoiding the risks of antibiotic resistance.

Bacillus probiotics have been extensively studied for their benefits on gastrointestinal and respiratory health but remain underexplored in the context of oral health. This trial is among the first to evaluate the safety and potential synergistic effects of Bacillus subtilis and Bacillus clausii in improving clinical symptoms, reducing pathogenic bacterial load, and preventing the progression of dental caries after 1, 2, and 3 months of intervention.

This is a randomized, double-blind, placebo-controlled clinical trial conducted over 3 months. A total of 130 primary school children aged 7-10 years (grades 2-4) with diagnosed dental caries will be enrolled. Children will be stratified by age group (7-<8, 8-<9, 9-10 years) and number of decayed teeth (≤2 or >2), then randomized in a 1:1 ratio to receive either: Placebo group and Smile Care group.

Both sprays will be administered twice daily (morning and afternoon), two sprays per site (both dental arches and front teeth), totaling six sprays per use, for three consecutive months.

- Primary Outcome: Changes in clinical symptoms, including Decayed Teeth index using ICDAS classification, Progressive caries rate, Early caries rate and Early caries rate code 1) at months 1 and 3 vs. day 0.

- Secondary Outcomes:

Changes in cytokine levels (ELISA) and semi-quantification of bacterial pathogens (real-time PCR) at months 1 and 3 compared with day 0.

- Exploratory Outcomes: Detection of B. subtilis ANA48 and B. clausii ANA39 in soft dentin and saliva samples (months 1, 2, and 3) of the Smile Care group, but not in the Placebo group.

Comparative analysis of oral microbiota diversity and bacterial density at month 1 between the placebo and Smile Care groups, using a reference group of at least 10 healthy children without dental caries.

Statistical Analysis:

Categorical variables will be analyzed using chi-square or Fisher's exact tests, while continuous variables will be assessed using t-tests, Mann-Whitney U, or Wilcoxon signed-rank tests depending on distribution.

Inclusion Criteria:

• Children (male or female) aged 7-10 years, currently enrolled in primary school (grades 2-4).
• At least one decayed tooth (any stage) at the time of screening.
• Commitment to comply with study requirements, including refraining from using any other probiotic products for the ear, nose, and throat that could affect study outcomes.
• Written informed consent obtained from a parent or legal guardian.

Exclusion Criteria:

Participant-level exclusion:

• Presence of severe chronic disease, allergy, or use of antibiotics within 1 month prior to study initiation.
• Use of oral or throat probiotic sprays/lozenges within the past 30 days (excluding gastrointestinal probiotics).
• Participation in another clinical study within the past 30 days.
• Acute oral conditions (e.g., abscess, severe gingivitis) interfering with examination or intervention.
• Currently receiving dental treatment at specialized dental/oral health facilities.
• History of congenital immunodeficiency or infectious disease.
• Psychiatric disorders or sleep disturbances.
• Known sensitivity or allergy to components of the investigational product.

Tooth-level exclusion:

• Hard tissue lesions not caused by dental caries.
• Supernumerary teeth or teeth not part of the functional dental arch.
• Carious teeth with pain lasting >10 seconds during examination.
• Carious teeth with visible pulp hyperemia (high risk of pulp complications).
• Teeth showing signs of pulp disease, pulp necrosis, or periapical pathology.
• Teeth previously treated with endodontic therapy or permanently restored with crowns.
• Primary teeth with mobility grade ≥2 (due to disease or natural exfoliation).

Data or samples share that will be coded, with no PHI include. Approval of the request and execution of all applicable agreements (i.e. a material transfer agreement) are prerequisites to the sharing of data with the requesting party.

Data requests can be submitted starting 9 months after article publication and the data will be made accessible for up to 24 months. Extensions will be considered on a case-by-case basis.

Access to trial IPD can be requested by qualified researchers engaging in independent scientific research and will be provided following review and approval of a study protocol, informed consent form (ICF), and clinical study reports (CSR). For more information or to submit a request, please contact anabio.rd2021@gmail.com

LiveSpo Smile CARE and placebo are indistinguishable regarding taste and smell. The color and turbidity of LiveSpo Smile CARE suspension are unrecognizable to investigators, trainers, caregivers, and paticipants due to opaque plastic container. Only the independent statistician is aware of the group codes.