The Antibacterial Content of Australian Stingless Bee Honey

Honey is a common sweetener in teas and other foods; yet few are aware of its medicinal uses. Honey contains antimicrobial agents and has a history of being used topically to treat burns and wounds, both anciently as well as more recently by modern-day homeopaths.¹ According to Massaro et al., honey has a low pH and contains hydrogen peroxide, and methylglyoxal (MGO), which contribute to its antibacterial activity.

Interestingly, MGO is also found in a type of New Zealand honey variety called ma̅nuka that is derived from Leptospermum scoparium (Myrtaceae) nectars. Massaro et al., sought to determine if there were similarities between ma̅nuka and other Leptospermum derived honeys by analyzing the honey produced from a local species of Australian stingless bees (Tetragonula carbonaria).

The team harvested honey from beehives in three sites of South East Australia during Leptospermum nectar flow. In two of the sites (coastal area of Tyagarah, NSW and heathland of East Beerwah, QLD) the team identified a high density of L. polygalifolium and L. liversidgii plants. The third and control site in heathland of West Beerwah, QLD was not near a source of Leptospermum spp).

After collecting the honey, the researchers pooled the samples from each site and performed a liquid-liquid phenolic extraction followed by gas chromatography. For the phenolic characterization, the team reconstituted the honey samples and injected them into an ultra high performance liquid chromatography (UHPLC) column connected to a Dionex Ultimate 3000 UHPLC system. They pumped eluent from the column into the photodiode array detector (PDA) and then to the linked Q Exactive LC-MS (Thermo Scientific) containing a heated electrospray ionization module. For this analysis, the team set the resolving power to 140,000 FWHM, and performed separate runs for positive and negative ionization modes. They also performed HPLC with photodiode array detector to quantify individual compounds.

After making use of Xcalibur software (Thermo Scientific) to analyze the MS data, they determined the three phenolic extracts contained a flavonoid glycosides including 3-phenyllactic acid, lumichrome, diglycosylflavonoids, norisoprenoids. Surprisingly, the team found that the T. carbonaria honeys did not contain MGO, dihydroxyacetone or phenolics characteristic of honeys derived from Leptospermum nectars. From an independent protocol, the team determined that the hydrogen peroxide concentrations reached up 155.8 ± 10.0 and 91.5 ± 11.3 μM for samples diluted 44.4%.

To put the antibacterial properties to the test, the team performed antibacterial assays using prepared agar plates or broth in 96 well plates inoculated with Staphylococcus aureus and Klebsiella pneumoniae. In both cases, they used absolute ethanol and a phenol standard solution as a control. The phenolic extracts (100 μg) inhibited only S. aureus growth with diameters of inhibition ranging from 10.18 to 19.87 mm with phenol equivalents of 1.0−5.1% (w/v). The team found raw honeys (100 μL) were active against both bacterial strains. Overall, the honey reached a minimum bactericidal concentration of 1.2−1.8 mg/mL.

The authors attribute the microbial effects of T. carbonaria honey mainly to hydrogen peroxide. Since there was no MGO detected in the honeys, they posit that any non-peroxide antimicrobial activity must be caused by another factor. Future experiments will include investigating Australian stingless bee honeys to determine if there may be other phytochemicals adding to the antimicrobial activity.

References

1. Blair, S.; Carter, D. (2005) “The potential for honey in the management of wounds and infection.”Healthcare Infection, 10, (pp. 24−31)

Massaro, C. F. , Shelley, D. Heard, T.A., & Brooks, P. (2014) “In Vitro Antibacterial Phenolic Extracts from ‘Sugarbag’ Pot-Honeys of Australian Stingless Bees (Tetragonula carbonaria).”, Journal of Agricultural and Food Chemistry, 62 (50), (pp 12,209–12,217) DOI: 10.1021/jf5051848