## Materials and Methods
### Physicochemical Parameter Analyses
#### 2.4.7 Colour-Metrics  
Determination of CIELAB Colour Parameters  
CIELAB colour parameters (\(L^{*}\), a\({}^{*}\), \(b^{*}\)), were determined according to CIE (Commission Internationale de l' Eclairage) recommendations. The CIELAB system uses three parameters to evaluate colour in foodstuffs: (i) colour parameter \(L^{*}\) corresponds to the degree of brightness; (ii) colour parameter \(a^{*}\) (positive values) corresponds to the degree of redness and when \(a^{*}\) shows negative values to the degree of greenness; and (iii) parameter \(b^{*}\) corresponds to yellowness of colour (when positive) and to blueness of colour (when negative). What is remarkable is that the units within the \(L^{*}\), \(a^{*}\), \(b^{*}\) system may provide equal perception of the colour difference to a human observer [21]. The aforementioned chromaticity coordinates of honey samples were measured using a Hunter Lab model DP-9000 optical sensor colorimeter (Hunter Associates Laboratory, Reston, VA, USA). The sample consisting of 10 g of honey in 75 mL distilled water was introduced in a glass petri dish and measurements were carried out by manual rotation of the sample in 45\({}^{\circ}\) of viewing aperture. Total difference of colour was calculated based on the following formula:  
\[\Delta E^{*}=((\Delta L^{*})2+(\Delta a^{*})2+(\Delta b^{*})2)1/2 \tag{1}\]  
where \(\Delta L^{*}\), \(\Delta a^{*}\) and \(\Delta b^{*}\) are the differences between the colour parameters of honey samples and the colour parameters of the white standard (YCIE = \(L^{*}\) = 83.87, XCIE = \(a^{*}\) = 81.82 and ZCIE = \(b^{*}\) = 99.59) [25]. Reported values are the average \(\pm\) standard deviation of five measurements (\(n\) = 5). Determination of Colour Intensity: ABS\({}_{450}-\)ABS\({}_{720}\)  
The colour intensity, defined as the net absorbance Abs450\(-\)Abs720, was an alternative methodology to evaluate colour of Portuguese honey samples. Honeys were diluted to a 50% proportion (\(w/v\)) with warm water (fixed temperature 45 \({}^{\circ}\)C) (AREX Heating Magnetic Stirrer, VTF Digital Thermogelator, VELP Scientifica, Via Stazione, 16, 20.865 Usmate Velate Monza e Brianza, Italy) sonicated (Elma, Elmasonic model S 10H, Singen, Germany) for 5 min and then filtered using [PERSON] filters (Maidstone, UK) with a pore size of 0.45 \(\upmu\)m, to remove any solid particles [26]. The spectrophotometer used was SHIMADJU, UV-1280 (Kyoto, Japan). Reported results are the average \(\pm\) standard deviation values of three replicates (\(n\) = 3) and were expressed as mAU. Determination of Colour According to Pfund Scale  
Honey samples (50% aqueous honey solution, \(w/v\)) were heated to 50 \({}^{\circ}\)C to dissolve sugar crystals and the colour was determined spectrophotometrically at \(\lambda\) = 635 nm. The honeys were classified according to the Pfund scale after conversion of the absorbance values [27]:  
\[\text{Pfund (mm)}=38.70+371.39\times\text{Abs} \tag{2}\]In Vitro Determination of Antioxidant Activity of Portuguese Honeys  
Preparation of DPPH Free Radical Standard Solution. The standard solution of [DPPH\({}^{\bullet}\)] was prepared by dissolving 0.0044 g of the radical DPPH in 100 mL methanol. In that sense, the molarity of the obtained solution was 1.12 \(\times\) 10\({}^{-4}\) mol/L (M). The volumetric flask was wrapped in foil and stirred in a vortex apparatus. The solution obtained had a neutral pH (pH = 7.02), a deep purple colour and was left in the refrigerator for 2 h in order to stabilize [28]. Preparation of DPPH Free Radical Calibration Curve. A calibration curve of concentration versus absorbance of DPPH was prepared as follows: The 1.12 \(\times\) 10\({}^{-4}\) M (mol/L) solution of DPPH was diluted with the addition of methanol to cover the concentration range of 0-44 mg/L. The resulting solutions were vortexed, left in the dark (until measurements were made) and the absorbance was measured in the aforementioned UV/VIS Spectrometer at \(\lambda\)max of 517 nm.