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Abstract
The aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey. Using the sensitive peroxide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quantitative relationships between the content of H2O2 and honey’s antibacterial activity was established. The results showed that: (A) the average H2O2 content in honey was over 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A supplementation of bacterial cultures with H2O2 inhibited E. coli and B. subtilis growth in a concentration-dependent manner, with minimal inhibitory concentrations (MIC90) values of 1.25 mM/107 cfu/ml and 2.5 mM/107 cfu/ml for E. coli and B. subtilis, respectively. In contrast, the MIC90 of honey against E. coli correlated with honey H2O2 content of 2.5 mM, and growth inhibition of B. subtilis by honey did not correlate with honey H2O2 levels at all. (C) A supplementation of bacterial cultures with H2O2 caused a concentration-dependent degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H2O2. DNA degradation by honey occurred at lower than ≤2.5 mM concentration of honey H2O2 suggested an enhancing effect of other honey components. (D) Honeys with low H2O2 content were unable to cleave DNA but the addition of H2O2 restored this activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H2O2 participated in the oxidative DNA damage. We concluded that the honey H2O2 was involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components.
Highlights
Hydrogen peroxide is generally thought to be the main compound responsible for the antibacterial action of honey (White et al, 1963; Weston, 2000; Brudzynski, 2006)
CONCENTRATION-DEPENDENT EFFECT OF HYDROGEN PEROXIDE ON BACTERIAL GROWTH INHIBITION Throughout this study, we used terms: endogenous hydrogen peroxide to describe H2O2 produced in honey by glucose oxidase and exogenous hydrogen peroxide, which has been added as a supplement to the bacterial cultures
These terms were introduced in order to differentiate between the effects of honey’s endogenous H2O2 whose action on bacterial cells could be modulated/obscured by other honey components as opposed to true, well-defined action of exogenous hydrogen peroxide directly added to bacterial culture
Summary
Hydrogen peroxide is generally thought to be the main compound responsible for the antibacterial action of honey (White et al, 1963; Weston, 2000; Brudzynski, 2006). Hydrogen peroxide in honey is produced mainly during glucose oxidation catalyzed by the bee enzyme, glucose oxidase (FAD-oxidoreductase, EC 1.1.3.4; White et al, 1963). Glucose oxidase is introduced to honey during nectar harvesting by bees. This enzyme is found in all honeys but its concentration may differ from honey to honey depending on the age and health status of the foraging bees (Pernal and Currie, 2000) as well as the richness and diversity of the foraged diet (Alaux et al, 2010). The total concentration of catalase depends on the amount of pollen grains in honey (Weston, 2000), and the hydrogen peroxide levels in different honeys may vary considerably (Brudzynski, 2006)
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