**1. Introduction**

Honey, a traditional sweetener, is considered as a functional food, and several recent clinical studies have proved its health-beneficial properties such as improving lipid profile [1], reducing postoperative pain [2] and inflammation [3] and modulating of hypertension [4]. Besides its oral consumption, honey has successfully been used topically in the treatment of a broad spectrum of surgical and chronic wounds [5,6] and mucositis [7] as well as herpes simplex labialis [8]. Whether applied topically or ingested orally, honey must fulfil all the requirements of international standards and possesses proven biological activity. In addition, honey has to be sterilised by gamma radiation when used in wound care management. In the case of medical usage, only honey of high quality and with guaranteed biological activity should be a part of honey-based medical products.

Several quality standards have been recongnised and listed as 'Current international honey standards' which are specified in the European Honey Directive (2002) [9] as well as in the Codex Alimentarius Standard for Honey (2001) [10]. Honey samples meeting all strictly defined composition criteria, including mainly moisture, sucrose and hydroxymethylfurfural (HMF) content, are recommended for human consumption and can be placed on the market.

On the other hand, the range of particular current honey standards is too wide and, in most cases, adulterated and/or heat-processed honey is still within the range for tested criteria and therefore classified as honey with proved quality. Furthermore, most importantly, none of the above-mentioned criteria are related to the biological activity of honey.

One of the most important and well-described aspects of honey's biological activity is its antibacterial activity that is mediated via multiple mechanisms of action such as osmotic pressure, low pH value and water activity, and disruption of bacterial cell membranes due to the presence of antibacterial peptide defensin-1.

Bee defensin-1, a regular but quantitatively variable antibacterial component of honey [11], is mainly e ffective against Gram-positive bacteria [12–14]. Furthermore, defensin-1 possesses antibiofilm activity against established multi-species biofilm [15] and exhibits wound healing properties [16].

After dilution, the antibacterial activity of honey is mainly mediated via enzymatically generated hydrogen peroxide (H2O2) in the diluted honey [17], excluding manuka honey where H2O2 is not accumulated [18]. This activity can be negatively a ffected by uncontrolled thermal processing or prolonged storage [19]. The major antibacterial compound in manuka honey is methylglyoxal (MGO) [20,21] that is primarily able to inhibit the growth of Gram-positive bacteria [22,23]. On the other hand, MGO in manuka honey seems to be ine ffective against *Pseudomonas aeruginosa* [24]. Similar to that of di fferent blossom honeys, the total antibacterial action of manuka honey is based on multiple mechanisms of action rather than single components. Therefore, the antibacterial potential of honey could be a suitable new additional international quality standard.

Our recent study aimed to determine the antibacterial e fficacy of 233 di fferent blossom honey samples collected in Slovakia [25]. Linden honeys showed the greatest antibacterial e fficacy followed by sunflower, multi-floral and acacia honey samples. The lowest antibacterial activity was assessed in rapeseed honey samples.

The goal of the study was to (i) evaluate the antibacterial potential of commercial honeys purchased from supermarkets (*n* = 19) and local shops (*n* = 17) in Slovakia, (ii) characterise the protein profile of honey samples and (iii) determine the overall H2O2 content in honey samples. Moreover, antibacterial activity together with the capability to generate H2O2 was assessed in three honey samples from local beekeepers and in three medical-grade honey samples in order to compare the overall antibacterial potential of di fferent honeys.

### **2. Materials and Methods**
