2.4.1. Honey Freshness

HMF content was determined through the White spectrophotometric method. The absorbance of a honey solution was measured at 284 and 336 nm with a UV-visible spectrophotometer (Thermo Scientific Helios Gamma, Chorley, UK) against a blank. The determination of diastase activity was based on the quantity of starch converted by a honey solution and the absorbance of the resulting blue color. It was determined spectrophotometrically at 660 nm using a UV-visible spectrophotometer (Thermo Scientific Helios Gamma, Chorley, UK) at different times to an end point below 0.235. Diastase activity was expressed in diastase index (DI) Gothe Scale.

### 2.4.2. Other Quality Parameters

Water content was determined with a Carl-Zeiss Jena refractometer by measuring the refractive indices at 20 ◦C. The percentage of water was calculated using the CHATAWAY table. Electrical conductivity was performed at 20 ◦C in a 20% ( *w*/*v*) honey solution (dry matter basis) in CO2-free deionized distilled water using a portable conductivity meter (Knick Portamess® 913 Conductivity, Beuckestr, Berlin, Germany). The values were expressed in mS/cm. pH was measured by a pHmeter (WTW inoLab pH 750) on a solution containing 10 g of honey dissolved in 75 mL of distilled water; the same solution was titrated for free acidity with 0.1 M sodium hydroxide (NaOH) solution up to a pH of 8.3. The results were expressed in meq/kg.

### 2.4.3. Color of Honey

Color determination was carried out using a HANNA Honey colorimeter (HANNA C221 Honey Color Analyzer, Rhode Island, RI, USA), previously calibrated with glycerin (Glycerol HANNA instruments, Rhode Island, RI, USA), which gives the values in millimeters Pfund.

### 2.4.4. Polyphenol and Flavonoid Content

The total phenolic content (TPC) was determined using an adapted Folin-Ciocalteu method [15]. Briefly, a honey solution for each sample was prepared (0.1 g/mL) and mixed with 10 mL of distilled water, 1 mL of Folin–Ciocalteu reagen<sup>t</sup> and 4 mL of 7.5% sodium carbonate (Na2CO3) solution up to a final volume of 25 mL. After incubation at room temperature in dark for 1 h, the absorbance of the solution was measured by spectrophotometry at 765 nm. Gallic acid (GA) was chosen as a standard, using various concentrated solutions (0.01–0.50 mg/mL), and then a calibration curve was obtained. The results were expressed as gallic acid equivalents in mg/100 g of honey.

Total flavonoid content (TFC) was measured using an adaptation of the Dowd method [16]. The same first stock honey solution (0.1 g/mL) prepared for the determination of phenol content was used. This solution was dissolved until a concentration of 0.33 g/mL and 0.5 mL of 5% aluminum chloride (AlCl3) solution was added prior to incubation in dark for 30 min. The reaction yields a yellow color and their absorbance was determined spectrophotometrically at 425 nm after incubation in dark for 30 min. The TFC was calculated using a calibration curve with different quercetin solutions (0.002 to 0.01 mg/mL) and the results were expressed as mg equivalent of quercetin per 100 g of honey.

### 2.4.5. Mineral Composition Analysis

Mineral content of honey was quantified by inductively coupled plasma mass spectrometry (ICP-MS) and by Atomic Absorption spectrometry (AAS) using a Spectrometer

Varian SpectraAA-600 (Agilent Technologies, Santa Clara, CA, USA). First, the samples were warmed and sonicated to facilitate the homogenization of honey, then 5 mL of HNO3- H2O2 9:2 were added to 0.5 g aliquots of the homogenized honey and afterwards were digested in a microwave oven (CEM MARSX press model) [17]. ASS was used to determine Na, K, Ca, Mg and Fe and ICP-MS for quantifying Mn, Cu, Zn, Cd, Pb and P. The results were expressed as mg per 100 g of honey.
