2.3.3. Physicochemical Analysis

The quality parameters of moisture, electrical conductivity, pH, and hydroxymethylfurfural content were determined following the methodology of the International Honey Commission [23]. All determinations were made in duplicate. The results are expressed as the mean of the obtained values.

Moisture was determined with a Carl Zeiss Jena refractometer (Zeiss, Oberkochen, Germany) by measuring the refractive index at 20 ◦C. Moisture content was calculated using the Wedmore table, and results were expressed as percentages.

Electrical conductivity was measured at 20 ◦C in a 20% ( *w*/*v*) honey solution (dry-matter basis) in CO2-free deionized distilled water with an EUTECH instrument conductivity meter (Thermo Fisher Scientific, Massachusetts, USA), and results were expressed as mS/cm; pH was measured by a pH meter (WTW in Lab pH 750) in a solution containing 10 g of honey in 75 mL of distilled water.

Hydroxymethyfurfural (HMF) content was determined using the White spectrophotometric method. Briefly, 5 g of honey was dissolved in 25 mL of distilled water and transferred to a volumetric flask of 50 mL; then, 0.5 mL of Carrez Solution I and 0.5 mL of Carrez Solution II were added. The final volume of 50 mL was set with distilled water. The honey solution was filtered, and the first 10 mL of the filtrate was rejected. Lastly, aliquots of 5 mL were pipetted into 2 tubes (reference and sample solution). Then, 5 mL of sodium bisulfite solution 0.2% was added to the reference, and 5 mL of water was added to the sample solution. The absorbance of the reference against the sample solution was determined at 284 and 336 nm with a UV-vis spectrophotometer (Fisher Scientific, Leicestershire, UK).

Diastase activity was determined through the amount of starch converted by a honey solution. The absorbance of yielded blue during the reactions was spectrophotometrically determined at 660 nm with a UV-vis spectrophotometer (Jenway 6305 UV-Visible Spectrophotometer, Sta ffordshire, UK) at di fferent times until an endpoint of less than 0.235. Diastase activity was calculated as diastase index (DI) or grams of starch hydrolyzed at 40 ◦C each hour per 100 g of honey.

### *2.4. Color Determination*

Prior to color determination, all samples were decrystallized and left for 20 min in an ultrasound bath to avoid bubbles. Sample color was determined using a HANNA Honey colorimeter (HANNA Instruments, Bedfordshire, UK). This is an instrument that gives the transmittance of honey using glycerol as a reference. Samples were introduced in square optical cuvettes of 10 mm sides, and the color value was taken directly; results are expressed in mm Pfund.

### *2.5. Determination of Total Polyphenol and Flavonoid Content*

The Folin-Ciocalteu spectrophotometric method adapted to honey was used to the quantification of polyphenol content [24]. A UV-vis spectrophotometer (Jenway 6305, Sta ffordshire, UK) was used for this purpose. Absorbance at 765 nm of a honey solution (0.1 g/mL) that reacted with the Folin-Ciocalteu reactive was determined. Ethanolic solutions of gallic acid in di fferent concentrations (0.01–0.50 mg/mL) were used as a standard to construct the calibration curve. The linearity of the curve was 0.997 ( *R*2). The polyphenol content of the samples was expressed as gallic acid equivalents in mg/100 g.

Total flavonoid content was measured using a similar spectrophotometric method based on an adaptation of the Dowd method [25]. A solution of aluminum chloride reacted with the flavonoids of the samples, prepared in a concentration of 0.33 g/mL with methanol. Absorbance of yellow yielded by the reaction was measured at 425 nm. Di fferent concentrations of the quercetin flavonoid (0.002–0.01 mg/mL) were employed to construct the calibration curve; linearity was 0.998 ( *R*2). The flavonoid content of honey samples was expressed as mg equivalent of quercetin per 100 g.
