*2.2. Palynological Analysis*

The pollen analysis of the honey samples was performed based on the method proposed by Louveaux, et al. [35], with some adaptations [9]. The honey samples (10 g) were diluted in distilled water and centrifuged at 4500 rpm for 10 min (first centrifugation), the obtained sediment was re-dissolved and centrifuged for 5 min (second centrifugation). The slide for the microscopical analysis was prepared by duplicate with 100 μL of the sediment. The pollen grains were identified using a Nikon Optiphot II microscope (Nikon UK Ltd., London, UK) at 400× or 1000×. The results were expressed in percentage considering the total number of pollen grains counted.

### *2.3. Determination of Quality Parameters*

The moisture was measured with a digital refractometer (ABBE URA-2WAJ-325; Auxilab S.L., Navarra, Spain) on a drop of honey sample. The refractive index values at 20 ◦C, were converted to moisture content using the Chataway table.

Then, 5 g honey dissolved in 25 mL bi-distilled water were used to determine the pH and the electrical conductivity (EC). The pH was measured directly using a pH meter (Crison micro pH 2001; Crison Instruments S.A., Barcelona, Spain). EC was measured in a honey solution considering the moisture content of sample with a portable conductivity meter (Knick Portamess 913 Conductivity, Beuckestr, Berlin). Results were expressed in μs/cm.

HMF content and the diastase activity were determined following the methodology proposed by Bogdanov et al. [36]. The HMF content (expressed in mg/100 g) was estimated using the White spectrophotometric method. This method considers the difference between the UV absorbance at 284 nm of a honey solution and the same solution after adding bisulphite. The HMF level was calculated after subtraction of the background absorbance at 336 nm using a UV-Visible Spectrophotometer (Jenway 6305 UV-Visible Spectrophotometer, Staffordshire, UK).

The diastase activity was calculated through the hydrolysis rate of the starch solution by the α-amylase present in a honey buffer solution at 40 ◦C (Schade method). The amount of starch converted was determined measuring the absorbance of the honey solution at 660 nm for different time points until an endpoint when the absorbance was less than 0.235. An UV-Visible spectrophotometer (Jenway 6305 UV-Visible Spectrophotometer, Staffordshire, UK) was used for this purpose. Diastase activity was expressed as diastase number (DN) or grams of starch hydrolyzed each hour per 100 g honey at 40 ◦C.

### *2.4. Determination of Color*

The color of samples was determined with a portable Minolta Chroma Meter CR-210 colorimeter (Konica Minolta, Tokyo, Japan). The CIE L a\*b\* coordinates were registered, where L is the luminance component (ranging from 0 to 100), while a\* and b\* are chromatic coordinates related with the gradient red/green and yellow/blue, respectively.

A HANNA Honey Color C221 colorimeter was also used to determine the color in mm Pfund. The honey must be fluid to be analyzed correctly. For crystallized or weakly fluid honeys, they were heated up to 45 ◦C in a thermostatic bath until the complete fluidification. Approximately 4 mL of sample was placed in a cuvette and sonicated to eliminate bubbles. The measure was taken after calibration with glycerin (Glycerol HANNA instruments).

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

Folin–Ciocalteu spectrophotometric method adapted to honey was used for the determination of the phenol content [37]. This method is based on the oxidation of the phenolic compounds forming a bluish complex. The samples (0.1 g/mL) were mixed with the reactive of Folin–Ciocalteu and the absorbance at 765 nm was measured using a UV–Vis spectrophotometer (Jenway 6305, UK). Gallic acid solutions (0.01–0.50 mg/mL) as a reference standard were used. Finally, the total phenolic content was expressed as gallic acid equivalents in mg/100 g honey.

The total flavonoid content was measured by spectrophotometry using the Dowd method adapted by Arvouet-Grand et al. [38]. The method uses a solution of aluminum chloride that reacts with the flavonoids present in the honey solution (0.33 g/mL). Solutions developed a yellow color for which absorbance was determined spectrophotometrically at 425 nm. Quercetin solutions (0.002–0.01 mg/mL) as reference standard were used. The results were expressed as equivalents of quercetin in mg/100 g honey.

### *2.6. Near Infrared Spectroscopy Measurement*

A portable MicroNIR Pro v2.5 equipment (MicroNIR 1700 ES, VIAVI, Santa Rosa, CA, USA) was used for acquisitions of NIR spectra data on honey samples (Figure 1). The samples properly homogenized were placed to the spectrometer housed in 4 mm borosilicate glass vials with measurements performed through the bottom of the vials. The vials were coupled directly with an adapter to the system NIR, and each sample a minimum of three times was scanned. The NIR spectrophotometer includes an instrument designed to measure diffuse reflectance in the NIR region of the electromagnetic spectrum [39]. A tungsten light bulb composes the system as the radiation source, and a linear-variable filter (LVF) connected to a linear indium gallium arsenide (InGaAs array detector) are integrated into the equipment itself. This equipment used a Spectralon® ceramic tile as a white reference (100% reflectance) of politetrafluoroetilen (~99%). Therefore, a 99% diffuse reflectance panel was used for the 100% reference value, and the 0% reference value was taken by leaving the tungsten Lampson with an empty support (known as dark Current Scan). Spectra were recorded using the instrument acquisition software MicroNIR ™ Pro v.2.2 (VIAVI, Santa Rosa, CA, USA). The measuring of reflectance in the NIR zone was recorded in a range between 900 and 1700 nm at intervals of 6 nm in the spectra. To minimize sampling errors, all the samples were analyzed in triplicate and the mean was used in statistical treatments. The diffuse reflectance signal of the NIR spectrum is referred to as reflectance (R), using log (1/R) values for performing chemometric analyses [40]. The advantage of portable MicroNIR systems is its easy handling and size. The MicroNIR dimensions are 45 mm in diameter and 42 mm in height, weighing about 60 g, and it is equipped with a 128-pixel detector array. In addition, the MicroNIR can be directly connected to a USB port of any laptop [27].

**Figure 1.** Near-Infrared (NIR) spectra of honey samples recorded by portable MicroNIR.
