*2.2. Microorganisms*

The tested bacterial isolates *Pseudomonas aeruginosa* CCM1960 and *Staphylococcus aureus* CCM4223 were acquired from the Department of Medical Microbiology at the Slovak Medical University in Bratislava, Slovakia.

### *2.3. Determining the Antibacterial Activity*

The honey samples were subjected to an antibacterial minimum inhibitory concentration (MIC) assay to determine the antibacterial activity against *P. aeruginosa* and *S. aureus* following the method of Bucekova et al. [27]. Bacteria were cultured in Mueller-Hinton broth (MHB) at 37 ◦C overnight. Bacterial culture was suspended in phosphate-buffered saline (PBS), with a pH of 7.2, and the turbidity of the suspension was adjusted to 10<sup>8</sup> colony-forming units (CFU)/mL and diluted with MHB medium (pH 7.3 ± 0.1) to a final concentration of 10<sup>6</sup> CFU/mL. The final volume in each well of sterile 96-well polystyrene U-shaped plates (Sarstedt, Germany) was 100 μL, consisting of 90 μL of sterile MHB medium or diluted honey sample and 10 μL of bacterial suspension. After 18 h incubation at 37 ◦C and 1250 rpm, the inhibition of bacterial growth was determined by monitoring the optical density at 490 nm using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA). The final MIC values correspond to the lowest concentrations of honey that completely inhibited bacterial growth. All the tests were performed in triplicate and repeated three times.

Each honey sample dilution was prepared from a 50% honey solution ( *w*/*w* in MHB medium) by further dilution with the MHB medium, resulting in final concentrations of 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6% and 4%.

### *2.4. Determining the H2O2 Content*

The H2O2 content in the honey samples was determined using a Megazyme GOX assay kit (Megazyme International Ireland Ltd., Bray, Ireland) based on the release of H2O2 after glucose oxidase catalysis of the oxidation of β-<sup>d</sup>-glucose to d-glucono-δ-lactone. As a standard, 9.8–312.5 μM diluted H2O2 was used. Honey solutions (40% *w*/*w* in 0.1 M potassium phosphate bu ffer, pH 7.0) were prepared and immediately measured. Each honey sample and H2O2 standard was tested in duplicate in a 96-well microplate. The absorbance of the reaction was measured at 510 nm using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA).

### *2.5. Determining the Protein Profile of Honey Samples*

For protein determination, 15 μL aliquots of diluted honey samples (50% *w*/*w* in distilled water) were loaded on 12% SDS-PAGE gels and separated using a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA). Protein content was assessed after gel staining with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, Darmstadt, Germany) or Serva Blue (Serva, Heidelberg, Germany).
