Assessing the antibacterial potency of royal jelly: Minimum inhibitory concentration and minimum bactericidal concentration evaluation against Streptococcus mutans and Staphylococcus aureus

Abstract

Introduction:

Royal jelly (RJ) has been widely regarded as nature’s elixir, owing to its numerous biological properties, including its antimicrobial efficacy. Royal Jelly, a natural secretion produced by honeybees, is rich in Major Royal Jelly Proteins (MRJPs) and various bioactive compounds, notably 10-hydroxy-2-decenoic acid (10-HDA). These constituents are primarily responsible for its bacteriostatic and bactericidal properties. With the increasing resistance of pathogens such as Streptococcus mutans and Staphylococcus aureus to conventional antibiotics, there is a need to explore alternative substances for controlling these bacteria.

Materials and Methods:

Standard strains of S. mutans (ATCC strain) and S. aureus (ATCC 25923) were cultivated under aerobic conditions. RJ was acquired and stored at 4°C. It was tested in various concentrations (ranging from 1.25 mg/mL to 20 mg/mL). The minimum inhibitory concentration (MIC) was determined as the lowest concentration of RJ that inhibited visible bacterial growth after incubation. The minimum bactericidal concentration (MBC) was determined by identifying the lowest concentration of RJ capable of killing 99.9% of the initial bacterial population.

Results:

The MIC for RJ against S. mutans was determined to be 10 mg/mL, and against S. aureus, it was 5 mg/mL. The MBC for RJ against S. mutans was 20 mg/mL, while for S. aureus, it was 5 mg/mL. RJ demonstrated stronger antimicrobial effects against S. aureus compared to S. mutans.

Conclusion:

The study demonstrates that RJ has effective antimicrobial properties against S. mutans and S. aureus. These findings suggest that RJ could be incorporated into anti-cariogenic treatments. However, additional research is required to explore the practical application of RJ in clinical practice.

INTRODUCTION

Periodontal infections and dental caries are two of the most prevalent oral health issues in the globe, primarily caused by bacterial pathogens such as Streptococcus mutans and Staphylococcus aureus.[1,2] S. mutans is a key contributor to dental plaque formation and tooth decay due to its ability to produce acids from carbohydrates, leading to enamel demineralization. In addition to producing a lot of acids and fermenting different kinds of carbohydrates, S. mutans also secretes enzymes that break down tooth enamel, create extracellular polysaccharides, and cause plaque or biofilm formation.[3,4] On the other hand, S. aureus, though not primarily associated with dental caries, is often found in various oral infections, including abscesses, and can complicate periodontal diseases.[5,6]

Traditionally, calcium hydroxide was used as a pulp-capping agent. However, because of its alkaline pH, calcium hydroxide has natural antibacterial capabilities that can aid in the eradication of microbial penetration and consequent pulpal tissue unpleasant irritation. Calcium hydroxide has intrinsic limitations such as dissolving in oral fluids and the occurrence of tunnel defects in the development of dentin bridges.[7,8,9]

The antibacterial activity of royal jelly (RJ) has been well documented against various microorganisms, including Escherichia coli, Candida albicans, and Pseudomonas aeruginosa. However, the specific interactions between RJ and S. mutans and S. aureus are less explored. Considering the significant role of S. mutans in cariogenesis and the potential pathogenicity of S. aureus, understanding how RJ affects these bacteria is critical for developing alternative antimicrobial treatments in conservative dentistry. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are key metrics in evaluating the antimicrobial efficacy of a substance. MIC refers to the lowest concentration of an antimicrobial agent that inhibits visible bacterial growth, while MBC represents the lowest concentration that results in bacterial death. Determining the MIC and MBC of RJ against S. mutans and S. aureus could provide valuable insights into its potential use as a natural anticariogenic agent and for managing oral infections.[10]

The growing concern of antibiotic resistance has driven the search for alternative antimicrobial agents that are both effective and safe. Natural materials have drawn attention for their broad-spectrum antibacterial properties, especially those produced from honeybee products such as RJ. Worker bee hypopharyngeal gland secretions, or RJ, are well-known for their complex composition, which includes proteins, fatty acids, and bioactive substances such major RJ proteins and 10-hydroxy-2-decenoic acid (10-HDA).[11] These elements support the antioxidant, antibacterial, and anti-inflammatory characteristics of RJ, which makes it a possible therapeutic agent in dentistry. The aim was to evaluate the MIC and MBC of RJ against S. mutans and S. aureus under laboratory conditions. The results could contribute to the growing body of evidence supporting the use of natural products in dentistry, paving the way for safer, alternative therapeutic approaches. Propolis is a honey bee (Apis mellifera L.), where pro means “at the entrance” and polis means “community.” It is also known as hive trash or bee glue.[12] Tree species such as willow, birch, poplar, pine, alder, and so on are the sources of it. It has 50% resin, 30% wax, pollen, chemical components, and essential oil.

MATERIALS AND METHODS

Royal jelly

The RJ in pure jelly form used in this study was sourced from local suppliers and stored at 4°C to preserve its bioactive properties. RJ was diluted with sterile water and tested at various concentrations (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, and 1.25 mg/mL) to evaluate its antimicrobial effect. The concentrations were prepared through serial dilution methods.

Cultivation of bacteria

Two bacterial strains were used in this study: S. mutans (ATCC: 25175), a key bacterium associated with dental caries. S. aureus (ATCC: 25923) is a common pathogen involved in various oral infections.

Both bacterial strains were obtained from the Microbiology Laboratory at Saveetha Microbiology Culture Centre and maintained on tryptic soy agar (TSA) plates for storage. For the experiment, bacterial cultures were freshly prepared by inoculating colonies from the agar plates into tryptic soy broth and incubating overnight at 37°C under aerobic conditions.

Minimum inhibitory concentration determination

Following the criteria provided by the Clinical and Laboratory Standards, the broth microdilution method was used to evaluate RJ’s MIC against S. mutans and S. aureus. Serial dilutions of RJ were prepared in 96-well microtiter plates, ranging from 20 mg/mL to 1.25 mg/mL. Each well was inoculated with 100 μL of bacterial suspension (approximately 1 × 10⁶ CFU/mL). A positive control (bacteria without RJ) and a negative control (broth without bacteria) were included. For a whole day, the microtiter plates were incubated at 37°C. The turbidity in the wells was used to determine the MIC of RJ, which was defined as the lowest concentration that prevented apparent bacterial growth.

Minimum bactericidal concentration determination

The MBC was determined by agar diffusion methods and sampling from the wells that showed no visible growth in the MIC assay. Aliquots of 100 μl were taken from these wells and plated onto TSA plates. The plates were incubated for 24 h at 37°C. A 99.9% decrease in the bacterial population was shown by the MBC, which was found to be the lowest concentration of RJ that prevented any bacterial growth on the agar plates.

Statistical analysis

All experiments were performed in triplicate. The MIC and MBC values were expressed as the mean of three independent assays. Data were analyzed using descriptive statistics, and results were compared between S. mutans and S. aureus to evaluate the differences in sensitivity to RJ. The statistical significance was set at P < 0.05.

RESULTS

The MIC and MBC of RJ against S. mutans and S. aureus were evaluated after 24 h. ). As shown in the results indicate that the antimicrobial effectiveness of RJ varies based on the concentration and bacterial strain tested.

Streptococcus mutans

Minimum inhibitory concentration

Table 1 illustrates that MIC of Royal jelly for S.mutans was 10 mg/ml. At this concentration, all sets (Set 1 to Set 5) showed no visible turbidity, indicating complete inhibition of bacterial growth. Bacterial growth was observed at the lower concentrations of 1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL, as evidenced by the presence of turbidity.

Table 1.

MIC and MBC of Royal jelly at different concentrations against S.mutans after 24 h

Dilution of RJ	1.25 mg/mL	2.5 mg/mL	5 mg/mL	10 mg/mL	20 mg/mL
MIC, turbidity for different concentrations of RJ after 24 h
Set 1	+	+	+	−	−
Set 3	+	+	+	−	−
Set 4	+	+	+	−	−
Set 5	+	+	+	−	−
MBC of RJ for different concentrations after 24 h
Set 1	+	+	+	+	−
Set 2	+	+	+	+	−
Set 3	+	+	+	+	−
Set 4	+	+	+	+	−
Set 5	+	+	+	+	−

MIC: Minimum inhibitory concentration, RJ: Royal Jelly, MBC: Minimum bactericidal concentration, +: Bacterial growth, -: Bacteria inhibited

Minimum bactericidal concentration

Table 1 illustrates that the MBC for S. mutans was determined to be 20 mg/mL. At this concentration, no bacterial growth was observed across all sets, confirming that this was the lowest concentration of RJ that resulted in bactericidal activity. Concentrations below 20 mg/mL did not produce bactericidal effects, as evidenced by bacterial growth.

Staphylococcus aureus

Minimum inhibitory concentration

For S. aureus, the MIC was 5 mg/ml, as shown in Table 2. At this concentration, bacterial growth was completely inhibited, as demonstrated by the absence of turbidity in the broth. Similar to the previous example, higher quantities (10 mg/mL and 20 mg/mL) did not permit observable bacterial growth, while lower concentrations (1.25 mg/mL and 2.5 mg/mL) did.

Table 2.

MIC and MBC of Royal jelly at different concentrations against S. aureus after 24 h

Dilution of RJ	1.25 mg/mL	2.5 mg/mL	5 mg/mL	10 mg/mL	20 mg/mL
MIC, turbidity for different concentrations of RJ after 24 h
Set 1	+	+	−	−	−
Set 2	+	+	−	−	−
Set 3	+	+	−	−	−
Set 4	+	+	−	−	−
Set 5	+	+	−	−	−
MBC of RJ for different concentrations after 24 h
Set 1	+	+	+	−	−
Set 2	+	+	+	−	−
Set 3	+	+	+	−	−
Set 4	+	+	+	−	−
Set 5	+	+	+	−	−

RJ: Royal Jelly, MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration, +: Bacterial growth, -: Bacteria inhibited

Minimum bactericidal concentration

The MBC of RJ for S. aureus was 10 mg/mL [Table 2]. At this concentration, no bacterial growth was observed, signifying complete bactericidal action. Lower concentrations, such as 5 mg/mL, did not achieve the bactericidal threshold, as bacterial growth was still evident.

Summary of Antimicrobial Activity

A summary of the MIC and MBC values for both bacterial strains is provided in Table 3, highlighting the concentration-dependent effectiveness of Royal Jelly. The data clearly demonstrate that RJ exhibited greater antibacterial potency against S. aureus than S. mutans, with lower MIC and MBC values observed for S. aureus.

Table 3.

Summary of results - minimum inhibitory concentration and minimum bactericidal concentration of Royal Jelly against Streptococcus mutans and Staphylococcus aureus

Bacterial strain	MIC mg/mL	MBC mg/mL
Streptococcus mutans	10	20
Staphylococcus aureus	5	10

MBC: Minimum bactericidal concentration, MIC: Minimum inhibitory concentration

DISCUSSION

The results of this study provide significant insights into the antimicrobial efficacy of RJ against S. mutans and S. aureus. The ability of RJ to inhibit the growth of both organisms, particularly at varying concentrations, underscores its potential as a natural antimicrobial agent. The antibacterial activity of RJ is attributed to its complex composition, which includes proteins, lipids, vitamins, and minerals, as well as bioactive substances such as fatty acids, phenolic acids, and flavonoids.

According to a number of studies, 10-HDA, a special fatty acid present in high proportions in RJ, is the main antibacterial component of the food.[13] This compound is thought to disrupt bacterial cell membranes, leading to the leakage of cytoplasmic contents and subsequent cell death. In addition, RJ contains a variety of peptides, such as royalisin and jelleins, which have been shown to possess antimicrobial properties.[14] The MIC and MBC results indicate that S. aureus is more sensitive to RJ than S. mutans. This difference in sensitivity may be attributed to the structural variations between the two bacteria. S. aureus, a Gram-positive bacterium, has a thick peptidoglycan layer that may be more susceptible to the bioactive compounds in RJ. In contrast, S. mutans, though also Gram-positive, possesses additional mechanisms such as biofilm formation that could confer increased resistance to RJ.

The findings of this study align with previous research indicating that RJ shows a broad spectrum of antibacterial activity, particularly against Gram-positive bacteria. Studies have demonstrated RJ’s inhibitory effects against E. coli, Listeria monocytogenes, and Bacillus subtilis, among others. The lower MIC and MBC for S. aureus compared to S. mutans suggest that RJ might be more effective against certain Gram-positive bacteria, though its exact efficacy can vary based on the species tested and their individual resistance mechanisms. These peptides can permeate bacterial membranes or interfere with bacterial replication, enhancing the overall antibacterial effect of RJ. The clinical implications of these findings are significant, particularly in the context of rising antibiotic resistance. Natural alternatives such as RJ could offer a solution to managing bacterial infections without contributing to the development of resistant strains. Literature has reported the effect of RJ in wound healing, oral infections, and even in combination with other antimicrobial agents to enhance therapeutic outcomes. In dentistry, RJ could be explored as an adjunct to traditional antimicrobial treatments for oral infections, especially for targeting cariogenic bacteria such as S. mutans, which plays a key role in dental caries formation.

In the present study, the MIC of RJ against S. mutans was found to be 10 mg/mL, while for S. aureus, it was 5 mg/mL. These values indicate that a higher concentration of RJ is required to inhibit the growth of S. mutans. The MBC values were 20 mg/mL and 10 mg/mL for S. mutans and S. aureus, respectively, further confirming that S. aureus is more susceptible to RJ. These findings are consistent with previous studies that reported RJ’s ability to inhibit oral pathogens and support its use in dental applications, such as in the management of dental plaque or as an additive in toothpaste.

The comparative analysis shows that S. aureus is more susceptible to RJ than S. mutans. This could be due to differences in cell wall composition and thickness between the two bacteria. The thicker peptidoglycan layer in S. mutans may hinder the penetration of RJ’s bioactive compounds, requiring a higher concentration to achieve bactericidal effects. In addition, S. mutans is known for its ability to form biofilms, which can provide an extra layer of protection against antimicrobial agents. Moreover, biofilm-forming bacteria such as S. mutans tend to be more resistant to antimicrobials due to the extracellular polymeric substances that protect the cells within the biofilm. This might explain why S. mutans required a higher concentration of RJ to achieve both inhibitory and bactericidal effects. The disruption of biofilm by RJ could be another potential mechanism, though further studies would be required to confirm this hypothesis.

Pulp-capping agents containing antibiotics prevented mutant streptococci strains from developing resistance over time.[15] According to Selvendran et al., calcium hydroxide does not dissolve the biofilm; on the contrary, it aids in its maintenance.[16] Kaspar et al. did point out that S. mutans could develop a resistance to the antibacterial effects of fluoride.[17] Thus, the need of this work was to present and contrast novel native pulp-capping materials. RJ, a bee product, was chosen as the indigenous new material. The purpose of this study was to evaluate native RJ’s antibacterial capabilities in preventing the formation of biofilms. The results of this investigation are in line with earlier studies on the antimicrobial properties of modern pulp-capping agents and aparian products such as propolis and RJ.[12,18] In addition, our results align with the well-established bactericidal properties of contemporary pulp-capping agents, as reported by Poggio et al. and Aljandan et al.[19,20] On the other hand, minerals with an alkaline pH and the ability to promote tissue repair, such as calcium hydroxide or mineral trioxide aggregate, are often found in traditional pulp-capping treatments. By creating an environment that is unsuitable for bacterial growth, these characteristics may aid in their antibacterial actions.[21,22] These findings have important therapeutic ramifications. Native RJ is a viable natural substitute due to its antibacterial properties and ability to prevent or inhibit dental cavities.[14,23] It can also be used as a substitute pulp-capping agent. These results open the door to both the development of new dental treatments and the enhancement of current pulp-capping methods. To improve oral health and treatment outcomes, its potential as a natural alternative deserves more research in clinical settings.

While this study provides valuable data on the antibacterial activity of RJ, several limitations should be acknowledged. First, the in vitro nature of the study may not fully represent in vivo conditions, where factors such as host immune response, tissue environment, and microbial interactions could influence the efficacy of RJ. In addition, the specific bioactive compounds responsible for the observed antibacterial effects were not isolated in this study. Future research should focus on identifying these compounds and exploring their mechanisms of action against a broader range of bacterial species.

Further studies are also needed to investigate the potential synergistic effects of RJ when used in combination with conventional antibiotics. Such combinations could enhance antibacterial efficacy while reducing the risk of resistance development. The exploration of RJ’s role in biofilm disruption, particularly against S. mutans, would be another promising avenue for future research.

CONCLUSION

This study demonstrates the significant antibacterial properties of RJ against S. mutans and S. aureus. The results indicate that RJ is more effective against S. aureus, with lower MIC and MBC values compared to S. mutans. These findings support RJ’s potential as a natural antimicrobial agent, particularly in the dental field, for combating oral pathogens. While promising, further research is needed to understand the mechanisms of action, explore in vivo applications, and investigate the synergistic potential of RJ with conventional antibiotics.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.