By Poppy Irvine, a dental hygienist in Leeds

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©Martin Ruegner/Stone/Getty Images Plus

The aim of the experiment was to assess whether propolis (bee glue) mouthwash, a herbal mouthwash, could inhibit the growth of Streptococcus mutans in a biofilm. The study was conducted over a period of eight weeks and trials were conducted in vitro in the Biomedical sciences Laboratory of Leeds Beckett University.

Propolis itself derives from honeybees and contains approximately 55% resinous compounds and balsam, 30% beeswax, 10% ethereal and aromatic oils, and 5% bee pollen. The mouthwash being used consisted of purified water, glycerine, propolis extract, menthol, Polysorbate 80, Citrus aurantifolia oil (Lime), Thymol and Sodium benzoate. The compounds present in propolis resin have three sources: plan exudate collected by bees, secreted substances from bee metabolism, and other materials introduced during elaboration.1 Many different compounds are found within propolis including: phenolic acid, caffeic acid and flavonoids. It is believed that the structure of propolis is very dependent on the collection location, time and plant source. Propolis has had some reported side effects including contact dermatitis and skin irritation. It was also found that propolis mouthwash may not be as popular as other leading brands because it has a heavy texture, potential to stain teeth after prolonged periods of time and an acquired taste.2

Method

Pilot study

Prior to the main experiment, a pilot study was undertaken to determine the best media to be used in order for Strep. mutans to grow efficiently, the result of which was Brain Heart Infusion (BHI) agar plates.

Media preparation

The agar chosen was BHI and was supplied from stock from the Leeds Beckett University Laboratory. The sterile plates were stored in a sealed container at room temperature.

Bacterial cultures

A strain of Strep. mutans (43) NCIMB11516 was obtained from the Leeds Beckett University Culture Collections. The colony was streak-plated in duplicate onto BHI and grown aerobically for 48 hours at 37°C. The plates were re-cultured every week to ensure viability. A gram stain was performed to determine the organism was Strep. mutans.

The propolis mouthwash was supplied by the manufacturer Bee Vital UK (Nature's Laboratory Ltd, Whitby, UK).

Preparation of experiments

All experimental procedures were set up and conducted using aseptic conditions.

In order for future testing, a stick supply of BHI broth needed to be inoculated with Strep. mutans. There were two different BHI broths. One contained 5% sucrose and the other contained 2.5% sucrose. Both sets of broth were inoculated by taking a single colony of Strep. mutans from the previously scribed stock plate and mixing in within each of the broths.

Minimum Inhibitory Concentration (MIC) Test

To check the MIC of the strain of Strep. mutans supplied, tests were conducted at neat, 50% and 25%, using saline to dilute, of inoculated broths 5% and 2.5% sucrose. The test was repeated four times for each.

The broth and saline were spread-plated onto labelled BHI plates along with 100 µl of neutraliser to inhibit cell production. The plates were incubated at 37°C for 48 hours.

Biofilm formation

Biofilms were grown on Corning 24 well plates purchased from Sigma-Aldrich Ltd. (Dorset, UK).

Non-inoculated BHI was added to the first column, A1-D1, of the plate as a control. 10 µl of the inoculated broth, 5% or 2.5% sucrose respectively, neat concentration was added to A2-D6 inclusive. 100 µl of non-inoculated BHI and propolis mouthwash was added to wells A2-D6 inclusively.

Bee propolis may be a beneficial mouthwash when traditional mouthwashes are not recommended

100 µl of neutraliser was added to each well, A1-D6 inclusive, to inhibit growth. The plates were incubated aerobically at 37°C for 96 hours (four days).

After the incubation period, 25 µl of Crystal Violet and 100 µl of formalin was added to wells A1-D6 inclusively to fix the bacteria. The plate was left to air dry for 15 minutes to allow fixing to occur. The plate was then rinsed twice with 200 µl of distilled water and 1,000 µl of 99% ethanol. The plate was placed on a shaker for 15 minutes to release any dye and was read through a plate reader at a wavelength of 630 nm. The test was repeated again for each of the BHI sucrose concentrations and for a broth concentration of 50%.

Disk diffusion assay

All tests were carried out on BHI agar plates.

The plate was split into six sections, 1-5 would be for the testing oil/mouthwash and section 6 as a control (PBS buffer). 100 µl of neat, inoculated broth was spread plated onto the agar plate aseptically and 100 µl neutraliser added to stop cell reproduction.

6 mm disks, which had been previously soaked with 20 µl of the selected mouthwash and left to air dry, were added to the correct quadrant. The plates were incubated aerobically at 37°C for 48 hours. After incubation, the zones of inhibitions were recorded.

The disk diffusion assay was repeated for both 5% and 2.5% sucrose at all concentrations: neat, 50% and 25%.

Results

The results are shown in Figures 1 and 2.

Fig. 1
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Average zone of inhibition of S. mutans when grown in BHI 5% sucrose

Fig. 2
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Average zone of inhibitions of S. mutans grown in BHI 2.5% sucrose

Conclusion

Bee propolis showed promising results in inhibiting the growth of Streptococcus mutans grown in a biofilm when compared with a well-documented antiseptic mouthwash with known results. The results demonstrate that bee propolis mouthwash may be a beneficial herbal mouthwash alternative when traditional antiseptic chemical mouthwashes are not recommended, or the patient does not wish to use them. Of course, further tests on a larger scale are needed to fully understand the benefits of bee propolis for use within periodontal treatment.