Dental caries is a disease with a well-defined etiology that can be prevented and controlled [1]. Caries development depends on the stagnation of an organized biofilm on the dental surface, and its metabolic activity in the presence of sugar fuels disease progression [2, 3]. The predominance of bacteria with aciduric and acidogenic characteristics, which use sucrose as a substrate for the synthesis of exopolysaccharides, leads to the development of virulent biofilms on susceptible tooth surfaces [4, 5]. Oral streptococci are often associated with infections in various human sites and, along with other primary colonizers of human plaque, like Streptococcus sanguis and Actinomyces viscosus [6], Streptococcus mutans (SM) is considered the major pathogen of human dental caries and is often associated with the virulence of biofilms [7]. This bacterium is usually isolated from carious lesions [8, 9], and its levels are generally linked to both past caries experience and future caries activity [10, 11].

Antimicrobial treatments are generally employed to reduce the number of cariogenic bacteria in the oral cavity [12, 13]. Dental varnishes with antimicrobials as active ingredients incorporated into their formulations have been designed with the intention of adhering to the tooth surface and maintaining its activity for a longer period [14, 15]. The most common therapeutic agents are fluoride and chlorhexidine; however, in some countries, only fluoride varnishes are commercialized. There is little evidence that sufficient and bioavailable levels of fluoride in dental biofilms can significantly change the number or proportions of species found in this dynamic microbiome, not effectively addressing the infectious character of the disease [13, 16, 17].

Natural products have been tested as potential sources of new and active therapeutic agents [18]. Propolis, a bee product, has drawn interest for its safety and biological activity due to its polyphenolic compounds that exhibit proven antimicrobial activity, with potential antibiofilm and anticaries activities [17, 19–21]. Although this product has shown promising results in in vitro studies [22], only a few studies have been performed with the objective of evaluating its possible use in antibiofilm and anticaries chemotherapy via a clinical treatment protocol in vivo [7].

In the present study, we used propolis type 12, classified by number in a previous study, which corresponds to “green propolis”, which is found in southeastern Brazil [23, 24]. A previous study revealed that green propolis varnish was more effective in preventing early smooth-surface lesions under high cariogenic challenge than was the gold-standard fluoride varnish [25].

Owing to the paucity of studies in the dental literature, this study aimed to investigate the efficacy of an experimental varnish containing 15% propolis type 12 ethanolic extract on the reduction of SM in the saliva and biofilms of children over a 30-day period.

No side effects were described by the children following the topical application of propolis varnish. Propolis varnish was adhered to until toothbrushing, which was performed after 12 h of varnish application, as recommended by the researcher. Patients complained about the product’s taste, characterizing it as “very bitter”, and a burning sensation immediately after application. However, no damage to the intraoral tissues was observed. No other specific complaints were reported.

Among the 11 participants in the study, 7 had a greater than 90% reduction in salivary SM relative to baseline on the 2nd day (T1). However, the CFU/mL counts started increasing on the 3rd day after varnish application in all patients.

The results of the means and standard deviations, as well as the minimum and maximum values of CFU/mL counts at all collection times, are shown in Table 1. There was a significant difference in CFU/mL counts only between T0/T1 (baseline/2nd day) and T1/T7 (2nd day/30th day). No differences were detected from the 3rd day to the 15th day.

To confirm these results, we analysed SM colonization before and after propolis varnish treatment via a molecular and more accurate technique. In the first step, we analysed whether propolis varnish can interfere with the total bacterial load in biofilms. Our results revealed that after 2 days of propolis treatment, there was a significant reduction in the total number of bacteria colonizing the biofilm (Figure 1). Moreover, we conducted a second analysis regarding the prevalence of SM in biofilms. The results showed that in the initial days after propolis treatment, there was a great reduction in the presence of SM in biofilms from patients. SM DNA quantification revealed decreases of 44% and 82% on days 2 and 3 after propolis treatment, respectively. Moreover, this great reduction in the first days after propolis treatment was followed by a recovery of SM viability and colonization on day 4 (Figure 2).

This study aimed to evaluate the efficacy of green propolis varnish in reducing the cariogenic oral microbiota. After a single application of propolis varnish in children, salivary and biofilm levels were reduced for three days, confirming that its antimicrobial activity was maintained.

Strategies for managing dental lesions, both carious and non-carious, have developed in an era that aims to conserve dental tooth tissue in order to limit removal and preserve tooth structure [12, 21]. Nonrestorative or noninvasive caries treatment (fluoride- and nonfluoride-based interventions) are being employed to manage the caries disease process at the initial lesion level and minimize the loss of sound tooth structure [12].

Varnishes can be employed in pediatric dentistry because they are easy to apply and well accepted by children [13–15]. The main advantage of polymeric materials such as varnishes over gels is that they increase the contact time of the active compound on the tooth surface through a controlled or sustained system where a natural or synthetic polymer plays a crucial role [15]. Our formulation has chitosan as a polymeric base used in a wide range of dental applications, and previous in vitro studies have shown a variety of release times for the active ingredients contained in the formulation, whether synthetic or natural. In a study with chitosan and fluoride, fluoride was released for a few hours depending on the concentration of chitosan in the polymer [15]. When propolis was added to chitosan, it could be released for at least seven days, depending on its concentration. Although in vitro tests do not exactly correspond to in vivo performance, they are important for predicting the potential of a material and its probable clinical application. Through an in vitro study of propolis varnish, we hypothesized that a controlled release system using chitosan would maintain propolis release at a low level over time [26].

Clinical studies have demonstrated that the professional application of fluoride varnish prevents dental caries, and the amount of fluoride released from a varnish typically decreases within a few hours and continues to decrease for up to 24 h [14, 15]. In this clinical analysis, propolis release in saliva was not measured; only the SM amount in biofilms and saliva was measured. The results revealed that there was a significant reduction in this bacterium until the third day after application, leading us to conclude that this occurred due to the antimicrobial activity of propolis during these days.

Chlorexidine varnish, which has antimicrobial activity and substantivity, effectively decreases the microbial load in both buccal tissues and dental biofilms but has weak effects on the prevention of dental caries. Another formulation of varnish containing red propolis with antimicrobial properties was tested in a clinical trial [14]. The results revealed that the red propolis group developed dental caries, even if not in the teeth to which the product was applied, unlike the fluoride varnish group. Our research had no comparison groups, just preliminary results of a product in the development phase. However, the results of previous tests [25] revealed that the use of antimicrobial products in the prevention of tooth decay remains insufficient for disease prevention.

Furthermore, since individual antimicrobial resistance has emerged as a major worldwide health concern, research is being developed using natural products with inherent antibacterial action, like propolis, as substitute antimicrobials. Propolis’ antimicrobial effectiveness against oral bacteria has been thoroughly evaluated, and it has been found to be generally effective, making it a very appealing option for treating or preventing a variety of oral and dental disorders [20, 21].

Although the outcome was not the prevention of dental caries, it was the primary motivation for developing this product. Future research on the efficacy of new materials in the prevention of dental caries disease must consider its multifactorial aspects and not just its biological aspects. It is a biofilm-sucrose-dependent disease but has great social influence, and variables such as access to fluoridated water and dental services must be taken into consideration. Products that combine an antimicrobial substance with fluoride and that might have a low cost, combined with collective actions, can be allies in the prevention of dental caries in low-income communities.

The main limitations of this study are the small sample size and the absence of comparison groups. Future studies should expand the sample and compare the tested product with materials already available, following the study participants for a longer period, to verify whether the product is truly effective in preventing tooth decay.

In conclusion, according to initial research on the effectiveness of green propolis, the number of cariogenic bacteria decreased until the third day following application. It is important to conduct more research to refine the original composition and determine the appropriate duration of follow-up to confirm its efficacy in preventing dental caries.