2.3.8. Sugars

For the determination of fructose, glucose and sucrose, a High-Performance Liquid Chromatography with Refractive Index Detector (HPLC-RID) technique was used. The preparation of the samples was achieved according to the literature [38], while for the separation and detection, the following parameters were used: column: Zorbax Carbohydrate Analysis (4.6 mm ID × 150 mm × 5 μm), temperature 35 ◦C, mobile phase: acetonitrile/water (75/25, *v*/*v*), flow rate: 1.8 mL min−1. For the quantification, a five-point calibration curve was created and evaluated for each sugar.

### *2.4. Sensory Evaluation*

For the sensory evaluation of samples, a two-round procedure was applied. At the first one the trained testers checked the botanical origin of the sample answering with Yes/No to the question: is the sample pine honey? At the second round, applied only for pine honeys, evaluating the visual, taste and aromatic characteristics, samples were classified in three levels: 3. very good, 2. medium, 1. good evaluated pine honey [39].

### *2.5. Bacterial Strains and Growth Conditions*

All tested bacterial strains were isolated, identified and characterized by standard laboratory methods (kindly provided by Professor Spyros Pournaras, School of Medicine, University of Athens, Athens, Greece). Antibacterial activity of pine honeys was tested against *Acinetobacter baumannii*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, *Salmonella* ser. Typhimurium and *Staphylococcus aureus*. The bacteria were routinely grown in Mueller Hinton Broth or Mueller–Hinton Agar (Lab M, Bury, UK).

### *2.6. Determination of Minimum Inhibitory Concentration (MIC)*

The determination of minimum inhibitory concentration (MIC) of tested honeys was carried out in sterile 96-well polystyrene microtiter plates (Kisker Biotech GmbH & Co. KG, Steinfurt, Germany) using a spectrophotometric bioassay as described by Tsavea and Mossialos [40]. Approximately 5 × 10<sup>4</sup> CFUs in 10 μL Mueller–Hinton broth was added to 190 μL of twofold diluted test honey (including manuka honey) at different concentrations which ranged from 25 to 0.78% (*v*/*v*). As control, Mueller–Hinton broth inoculated with bacteria was used. Optical density (OD) was determined at 630 nm using an ELx808 absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT, USA), at t = 0 (prior to incubation) and after 24 h of incubation (t = 24) at 37 ◦C. The OD for each replicate well at t = 0 was subtracted from the OD of the same replicate well at t = 24. The following formula was used to determine the growth inhibition at each honey dilution: % inhibition = 1 − (OD test well/OD of corresponding control well) ×100. The MIC was determined as the lowest honey concentration which results in 100% growth inhibition [40]. MICs were determined in triplicates in at least two independent experiments.

### *2.7. Determination of Minimum Bactericidal Concentration (MBC)*

Minimum bactericidal concentration (MBC) is the lowest concentration of any antibacterial agen<sup>t</sup> that could kill tested bacteria. In order to determine the MBC, a small quantity of sample contained in each replicate well of the microtiter plates was

transferred to Mueller–Hinton agar plates by using a microplate replicator (Boekel Scientific, Feasterville, PA, USA). The plates were incubated at 37 ◦C for 24 h. The MBC was determined as the lowest honey concentration at which no grown colonies were observed [41].

### *2.8. Determination of H2O2 Accumulation in Honey Samples*

The ability to generate H2O2 in the diluted honey samples was determined using a Megazyme GOX assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland) with some modification [27], which is based on the release of H2O2 after GOX catalysis of the oxidation of β-D-glucose to D-glucono-δ-lactone. As a standard, 9.8–312.5 μM diluted H2O2 was used. A total of 40% (*w*/*w*) of the honey solutions in 0.1 M potassium phosphate buffer (pH 7.0) were prepared and incubated for 24 h at 37 ◦C. Each honey sample and H2O2 standard were tested in duplicate in a 96-well microplate. The resultant H2O2 reacts with p-hydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase to form a quinoneimine dye complex, which has a strong absorbance at 510 nm. The absorbance of reaction was then measured at 510 nm using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA).

### *2.9. Total Protein Content*

Total protein content was measured using Quick StartTM Bradford reagen<sup>t</sup> (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.

### *2.10. 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 [Acrylamide/Bis solution, 37.5:1 (40% *w*/*v*), 2.6% C] 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).

#### *2.11. Determination of the Antibacterial Activity Due to H2O2 and Proteinaceous Compounds*

The MIC of honey treated with bovine catalase or proteinase K was determined and compared with that of untreated honey [40]. Briefly, 50% (*v*/*v*) honey in Muller–Hinton broth containing 100 μg/mL proteinase K (Blirt, Gdansk, Poland) or 600 U/mL bovine catalase (Serva, Heidelberg, Germany) was incubated for 16 h at 37 ◦C and then tested after being diluted twofold.

### *2.12. Total Phenolic Content (TPC) and Antioxidant Activity*
