Re-Examining the Role of Hydrogen Peroxide in Bacteriostatic and Bactericidal Activities of Honey

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.

Glucose oxidase activity and hydrogen peroxide accumulation in Croatian honeys

“…Black locust (n = 7), chestnut (n = 3), lime (n = 5), honeydew (n = 12) and mint (n = 3) honeys were diluted by deionized water at five different mass to volume ratios (1:1; 1:2; 1:4; 1:8; 1:16) and after 60 minutes of incubation at 37 °C, the content of the produced hydrogen peroxide was measured by the MQuant peroxide test strips. However, since hydrogen peroxide formation in honey depends on the glucose oxidase (GOX) action (Brudzynski 2006;Brudzynski et al, 2011;Kerkvliet, 1996), it was necessary to determine the activity of this enzyme in honey samples before measuring the hydrogen peroxide content in honey solutions. Determination of GOX in honey samples by the standard horseradish peroxidase/o-dianisidin method revealed that all examined samples possessed the glucose oxidase activity, and can thus be analysed for hydrogen peroxide accumulation.…”

“…Such slight changes in pH upon honey dilution were not surprising, since it is well known that honey acts as a buffer (Buba et al, 2013). (Brudzynski, 2006;Brudzynski et al, 2011;Brudzynski et al, 2017), by measurement of oxygen concentration (decomposition of H 2 O 2 by exogenously added catalase) (Bang et al, 2004), while only Cooke et al (2015) measured hydrogen peroxide content in honeys using MQuant test strips, but examined special types of honey. According to our knowledge, there is only one other report on the H 2 O 2 content in honeys where MQuant test strips were used (Kerkvliet, 1996).…”

Prolonged heating of honey increases its antioxidant potential but decreases its antimicrobial activity

“…In several studies, it has been shown that hydrogen peroxide was the main compound responsible for the antibacterial action of honey (Brudzynski, 2006;Weston, 2000;White Jr, 1963) and was produced mainly during glucose oxidation, which is catalyzed by the bee enzyme glucose oxidase (White Jr, 1963). If a honey contains a high content of this oxidizing compound, bacteria cannot respond normally to proliferative signals, and their growth remains arrested even when the honey is used in diluted forms (Brudzynski, 2011). According to Kretavičius et al (2010), when honey was liquefied, glucose oxidase was activated and produces its metabolic products.…”

Colour Intensity, Polyphenol Content and Antibacterial Capacity of Unheated and Heat-Treated Sahara Honey

“…The presence of hydrogen peroxide generated by enzymatic activity of glucose oxidase in diluted honey is considered as the major antibacterial factor [11]. Furthermore, heating honey inactivates the glucose and it oxidates phenolic compounds as important factors for the non-peroxide antibacterial activity of honey [10]. The non-peroxide antibacterial activity is insensitive to heat and light [12].…”

“…Several bioactive compounds have been identified in honey which contributed to its antibacterial action. In many studies, the presences of peroxide and non-peroxide antibacterial capacity in honey have been reported [9,10]. The presence of hydrogen peroxide generated by enzymatic activity of glucose oxidase in diluted honey is considered as the major antibacterial factor [11].…”