**3. Results**

### *3.1. Antibacterial Activity of Commercial Honey Samples*

Antibacterial activity was determined in commercial (*n* = 36), local beekeeper (*n* = 3) and medical-grade (*n* = 3) honey samples as well as in artificial honey. The activity of the commercial honey samples against bacteria was expressed as an MIC value (Figure 1). Commercial honey samples from local food shops (Nos. 20–36) exhibited a greater antibacterial e ffect compared to samples from supermarkets (Nos. 1–19). The MIC values of about 50% of supermarket honey samples against *S. aureus* were identical to those of artificial honey, compared to 25% of honey samples from local food shops. The average MIC value of supermarket and local food shop honey samples against *S. aureus* was 28.6% and 19.2%, respectively. The highest antibacterial activity was exhibited by honey sample No. 29 with an MIC value of 4% against *S. aureus*.

Artificial honey at a concentration of 25% was able to inhibit the bacterial growth of *P. aeruginosa*. Due to the high susceptibility of *P. aeruginosa* to the sugar content in honey samples, this bacterium is not a suitable model for testing the antibacterial activity of honey.

**Figure 1.** Antibacterial activity of the commercial honey samples (*n* = 36) purchased in Slovakia. The antibacterial activity was evaluated against two bacterial pathogens by a minimum inhibitory concentration (MIC) assay. (**A**) Honey samples purchased from supermarket (*n* = 19) (**B**) Honey samples purchased from local food shop (*n* = 17). The data are expressed as the mean values with the standard deviation (SD). AH—artificial honey.

### *3.2. H2O2 Content of Commercial Honey Samples*

H2O2, a major antibacterial compound in honey, was determined in 36 commercial honey samples (Figure 2). The average H2O2 content in supermarket and local food shop honey samples was 256 and 565 μM, respectively. Honey samples purchased in local food shops were more potent in H2O2 production (Figure 2B). Three samples from supermarkets (sample Nos. 10, 11 and 12) accumulated very low levels of H2O2, at concentration below 50 μM (Figure 2A).

**Figure 2.** Hydrogen peroxide (H2O2) content in commercial honey samples (*n* = 36). (**A**) supermarket honey samples (*n* = 19), (**B**) local food shop honey samples (*n* = 17). Determination of the H2O2 content was carried out in diluted honey samples (40% (*w*/*w*) after 24 h of incubation at 37 ◦C. The data are expressed as the mean values with the standard deviation (SD). AH—artificial honey.

The correlation analysis revealed a significant correlation between the H2O2 values and the antibacterial activity of both supermarket and local shop honey samples against *S. aureus* (*r* = −0.582, *p* < 0.01; *r* = −0.648, *p* < 0.01) as well as *P. aeruginosa* (*r* = −0.584, *p* < 0.001; *r* = −0.682, *p* < 0.01) (Figure 3).

Despite a significant correlation between the content of H2O2 and the antibacterial activity of all commercial honeys was revealed, some of tested honey samples (for example Nos. 19 and 33) did not show any correlation among MICs and H2O2 content.

**Figure 3.** The relationship between the content of H2O2 and the antibacterial activity of commercial honeys: (**A**) supermarket honey samples (*n* = 19), (**B**) local food shop honey samples (*n* = 17) against two bacterial isolates. A Pearson correlation test was used for the correlation analysis.

#### *3.3. Antibacterial Activity and H2O2 Content of Local and Medical-Grade Honey Samples*

Samples of three different types of honey (honeydew, linden and acacia) from local beekeepers and three medical-grade honeys were used for the determination of antibacterial activity. The antibacterial activity of these samples is shown in Figure 4A. All honeys from local beekeepers exhibited high antibacterial activity, with average MIC values ranging from 7% to 12% depending on the bacterium. The antibacterial effect of local honey samples was even higher than that of medical-grade honeys where MIC values were in the range from 9% to 22.5%. Among the medical-grade honeys tested, the lowest antibacterial activity was documented for Revamil®. The local honeys, honeydew and acacia honey samples in particular, produced a high level of H2O2 (Figure 4B). Interestingly, the level was significantly higher than in medical-grade honeys. Activon Tube®, a manuka honey based-wound care product, did not accumulate H2O2 due to inactivated glucose oxidase enzyme. Revamil®, a blossom honey-based product, produced a low level of H2O2 and thus showed moderate antibacterial activity.

**Figure 4.** Antibacterial activity and the content of H2O2 in the honey samples from local beekeepers (*n* = 3) and medical-grade honey (*n* = 3). (**A**) The antibacterial activity was evaluated against two bacterial pathogens by a minimum inhibitory concentration (MIC) assay. (**B**) Determination of the H2O2 content was carried out in diluted honey samples (40% (*w*/*w*) after 24 h of incubation at 37 ◦C. The data are expressed as mean values with the standard deviation (SD).
