**2.5. Determination of electrical conductivity**

A solution containing 20% of honey dry matter in 100 ml distilled water [7] was prepared for each sample. The 20% dry matter was determined from the moisture content reading. The amount of honey, equivalent to 20.0 g anhydrous honey, was dissolved in 70 ml distilled water, and made to a 100 ml volume. The conductivity was determined using a pH/conductivity meter (Orion Star A215 Thermo Scientific) in μS/cm) in triplicates.

#### **2.6. The determination of HMF after White**

The method according to White [8] was followed. Briefly, 5 g of honey were weighed accurately and dissolved in 25 ml of distilled water. 0.5 ml of Carrez solution I and 0.5 ml of Carrez solution II were added, and the solution made to a volume of 50 ml with water. Following filtration and dilution, the absorbance of the samples was read at 284 and 336 nm in 10 mm quartz cells within 1 hour. The HMF content in mg/kg was then obtained.

#### **2.7. The determination of diastase activity**

The Megazyme test kit (Megazyme Ireland, lot number 30602) was used for this determination. Briefly, 2 g of honey sample was dissolved in 40 ml of 100 mM sodium maleate buffer (pH 5.6) and topped to 50 ml. The Amylazyme tablet was added and following an incubation period of 10 min at 40°C, 10 ml of Trizma base (2% w/v) solution were added. The absorbance of the solution was read at 590 nm. The α-amylase activity of a sample (as Schade per gram of honey) was determined by use of the associated regression equation.

#### **2.8. The determination of proline**

The method outlined in Ref. [7] was followed. Briefly, 5 g of honey were made to the 100 ml volume with distilled water. 0.5 ml of the sample solution in one tube, 0.5 ml of water (blank test) into a second tube and 0.5 ml of proline standard solution into a third tube were pipetted. 1 ml of formic acid and 1 ml of ninhydrin solution were added to each tube. After 15 min shaking, the tubes were incubated at 70°C for 10 min. 5ml of the 2-propanol-water-solution were added to each tube and the absorbance read at 510 nm after 45 min. The proline content in mg/kg honey was calculated.

#### **2.9. Determination of sugar content**

The respective standards and honey samples were prepared as 1% solution prior to analysis. Each standard and sample (2 μl) was injected in triplicates in a Dionex Thermo Fisher Ultra High Performance Liquid Chromatography with a charged aerosol detector, equipped with an amino column from Supelco (250 × 4.6 mm, 5 μm particles). Mobile phase consisted of a water/acetonitrile mixture (volume ratio 75/25), with a flow rate of 1.5 mL/min. Detection was performed at a Data Collection Rate of 20 Hz, filtered at 5 s, peak width was 0.02 mm and oven temperature set to 35°C.

#### **2.10. Polyphenolic content**

The total phenolic content (TP) was determined using a Folin Ciocalteu test [9]. 100 μl of Folin-Ciocalteu reagent and 80 μl of sodium carbonate (1 M) were added to 10 μl of each honey stock solution (triplicates) and incubated for 20 min at room temperature. The absorbance was read at 630 nm. Tannic acid was used as a standard for the test.

#### **2.11. Total flavonoid content**

The total flavonoid (TF) content was determined by a spectrophotometric method [10, 11]. Honey samples were prepared as 50% (w/v) solutions. 25 μl of honey solutions and 100 μl of 0.15% NaNO2 solution (Fisher Scientific, UK) were allowed to mix for 6 min. 100 μl of 4% NaOH solution and 25 μl of distilled water were added for a final volume of 250 μl. After 15 min, the absorbance was read at 510 nm. Rutin (Sigma-Aldrich, USA) was used as a standard for the quantification of the total flavonoid content (0–500 mg/mL; *r*<sup>2</sup> = 0.9962) [12].

#### **2.12. Radical scavenging activity: the DPPH assay**

The DPPH assay (2,2-diphenyl-1-picrylhydrazyl, Sigma Aldrich, USA) was carried out according to Moien et al. [10]. Honey samples were prepared as 12.5% (w/v) solutions. 200 μl of a 100 μM solution of DPPH radical in methanol were added to 20 μl of honey solution, and incubated for 30 min in the dark at room temperature. The radical inhibition was measured at 490 nm against a blank containing DPPH and methanol. Ascorbic acid (BDH, UK) was used as a standard (10–100 μg/mL). The AAE-DPPH and ascorbic acid equivalence (mg AEAC/100 g honey) (*r*<sup>2</sup> = 0.9928) were calculated [12].

#### **2.13. Reducing power: the FRAP assay**

The FRAP (Ferric Reducing Antioxidant Power) activity of honey samples was determined according to Oyaizu [13]. 250 μl of honey solutions (6.25–50%) or 250 μl of distilled water (blank) were mixed with 250 μl of 0.2 M phosphate buffer (pH 6.6) and 250 μl of 1% potassium ferricyanide. Following an incubation period of 20 min at 50°C, mixtures were immediately cooled in an ice bath for 30 s. 250 μl of 10% trichloroacetic acid were added and centrifuged at 3000 rpm for 10 min. 500 μl of the upper layer were mixed with 500 μl of distilled water and 100 μl of 0.1% ferric chloride. The absorbance was read at 700 nm against a blank [14]. The EC50 was used to define the specific reducing capability (mg AEAC/100 g honey) using ascorbic acid (10–100 μg/ml; *r*<sup>2</sup> = 0.9981) as a positive control.
