Factors affecting the production and measurement of hydrogen peroxide in honey samples

Many Australian native honeys possess significant antimicrobial properties due to the production of hydrogen peroxide (H₂O₂) by glucose oxidase, an enzyme derived from the honeybee. The level of H₂O₂ produced in different honey samples is highly variable, and factors governing its production and stability are not well understood. In this study, highly active Australian honeys that had been stored for >10 years lost up to 54 % of their antibacterial activity, although almost all retained sufficient activity to be considered potentially therapeutically useful. We used a simple colourimetric assay to quantify H₂O₂ production. Although we found a significant correlation between H₂O₂ production and antibacterial activity across diverse honey samples, variation in H₂O₂ only explained 47 % of the variation observed in activity, limiting the assay as a screening tool and highlighting the complexity of the relationship between H₂O₂ and the killing power of honey. To further examine this, we tested whether H₂O₂ detection in honey was being inhibited by pigmented compounds and if H₂O₂ might be directly degraded in some honey samples. We found no correlation between H₂O₂ detection and honey colour. Some honey samples rapidly lost endogenous and spiked H₂O₂, suggesting that components in honey, such as catalase or antioxidant polyphenols, may degrade or quench H₂O₂. Despite this rapid loss of H₂O₂, these honeys had significant peroxide-based antibacterial activity, indicating a complex relationship between H₂O₂ and other honey components that may act synergistically to augment activity.