Honey is a food produced directly by the honeybees from the apicultural resources existing near apiaries. For this reason, the characteristics of this natural product are closely linked to the production area [1
]. For honey elaboration, the honeybees collect the nectar of the flowers, plant secretions or excretions whose production is mediated by sucking insects, such as aphids. Considering these sources, honey is classified as nectar or blossom honey or honeydew honey [3
]. Honeydew and nectar honey differ in terms of chemical composition, physical properties, and melissopalynological characteristics [4
]. In Spain, a few years ago, honeydew honey was an undervalued product, with a low commercial value; however, it turned out to be very valuable being one of the most demanded honey by consumers [10
]. The increase in demand is attributed to its healthy properties due to high antioxidant capacity and phenolic content [1
In Europe, honeydew honey is obtained mainly from trees. In the mountain areas, through exudates produced by scale insect, on conifers as Picea abies
, or pine species [13
]. In the Mediterranean areas, using plant secretions produced during the acorn formation in evergreen oaks and deciduous oaks (Figure 1
). In the Iberian Peninsula, Quercus ilex
(the evergreen oak) is biogeographically distributed mainly in central and southern areas, while other species of deciduous oaks, such as Quercus pyrenaica
appears mainly in the west and north mountain areas [17
The origin of honeydew, the factors influencing its production and its physicochemical characteristics are insufficiently studied. Honeydew production is not constant throughout the year and depends on the environmental conditions, the physiology and phenology of the plants, and the dynamics of vectors affecting secretions as the biological cycle of insects. In northwest Spain, honeydew honeys are obtained in the summer and their production depends on the weather [1
]. Some authors have indicated that rainy springs and warm summers favor the collection of this honey type [18
]. Hence, in Mediterranean habitats, honeydew productions are favorable. In recent decades, in the north and northwest regions of the Iberian Peninsula of Mediterranean, the change in climate is causing a significant effect on the phenology of plants and, consequently, in the beekeeping trends. Some of these changes are visible, for example, honeydew is more frequently harvested by beekeepers in the northwest region of the Iberian Peninsula.
The physicochemical and sensorial characteristics of nectar honey are better known in comparison to those of honeydew honey. In particular, unifloral honeys, such as orange blossom (Citrus
), eucalyptus (Eucalyptus
), acacia (Robinia pseudoacacia
), linden (Tilia
), lavender (Lavandula stoechas
), heather (Erica
, Calluna vulgaris
), rape (Brassica) or sunflower (Helianthus annuus
) have been extensively studied [6
]. The most studied honeydew honeys are those produced in the Mediterranean area and Central Europe. They are produced from oak (Quercus ilex
), fir (Picea abies
), or Pinus
]. Honeydew honeys are characterized by dark amber to dark color, slightly intense smell with a predominance of a wood attribute, intense flavor slightly bitter and sour in taste. However, the available information on the sensory characteristics of the honey type is still scarce [26
]. These honeys are characterized by less reducing sugar content, higher oligosaccharide content [4
], and higher electrical conductivity, as a consequence of its higher mineral and phenolic content [6
]. In addition, the palynological analysis revealed important differences between the honey sediments of honeydew and nectar honeys [4
]. Generally, honeydew honeys harbor more pollen grains from anemophilous plants, fungal hyphae, spores or green algae. At the time of honey classification, the presence or absence of fungal spores and the fungi that give rise to these fungal elements should be considered. This is because some fungal elements are not related to the presence of honeydew in honeys [32
]. However, significant relationships between the presence of certain spores of plant pathogenic fungi (e.g., Alternaria
, etc.) with the honeydew in the honey were found [5
]. On the contrary, the presence of yeasts in honey, especially, Metschnikowia reukafii
, is indicative of the nectariferous origin of honey [5
Several aspects must be considered to guarantee the safety of the food. The improvement in the procedures for the quality control of honey is the first step. The identification of the peculiar characteristics of each honey type, and the differences attributed to the geographical origin or the botanical origin are also of special interest with respect to food safety as they provide information for the traceability of the honey. This knowledge is useful to avoid counterfeit products derived from adulteration or mislabeling of food products. Therefore, the study of palynological and physicochemical characteristics of honeydew honeys produced from deciduous oaks and evergreen oaks collected in different areas of Spain provides crucial information that facilitates their differentiation.
2. Material and Methods
2.1. Honey Samples
Honeydew honeys were collected from different areas of Spain; 17 samples were collected from the northwest area, and 11 samples from the center-south area (the geographical origin of samples and the collection year is shown as supplementary data
). The samples were collected directly by the beekeepers and refrigerated at 4 °C and stored until further analysis. All the determinations were conducted in duplicate, following the homogenization of the samples.
2.2. Reagents and Standards
All chemical standards were HPLC-grade pure. Folin-Ciocalteu reagent, gallic acid, aluminum chloride, sodium carbonate, potassium iodide, and bisulfite were purchased from Panreac (Barcelona, Spain). Quercetin and 2,2-diphenyl-1-picrilhidrazil (DPPH) were purchased from Alfa Aesar (Massachusetts, USA) and methanol was obtained from Merck (Darmstandt, Germany). Hydrolyzed starch for diastase determination was purchased from Carlo Erba (Barcelona, Spain). The calibration of the HANNA Honey Color C221 colorimeter was with glycerin was provided by Glycerol HANNA instruments (Woonsocket, Rhode Island, USA). Standards of glucose, fructose, sucrose, maltose, trehalose, turanose and melezitose were obtained from Sigma–Aldrich (Madrid, Spain).
2.3. Melissopalynological Analysis
The botanical characteristics of samples were identified by optical microscopy based on the method established by Louveaux et al. [36
], with some modifications. Qualitative analysis was conducted to determine the proportion of pollen grains of different species present, and quantitative analysis was done to determine the amount of pollen per unit weight of honey. Ten grams of honey was dissolved in bi-distilled water to a final volume of approximately 30 mL for qualitative analysis. The samples were subjected to two centrifugations (Sigma Laborzentrifuge centrifuge model 3.0) at 4500 rpm (3383× g
). The supernatant was decanted, and the sediment was stirred to homogenize it. Subsequently, slides for microscopy were prepared using a drop (100 µL) of sediment. The pollen spectra were determined by counting and identifying a minimum of 800 pollen grains with a Nikon Optiphot II microscope (×400 and ×1000, as and when needed). The proportions of pollen types were expressed as percentages. For quantitative analysis, a volumetric method was used involving mixing 5 g of honey in bi-distilled water. Then, the honey solution was centrifuged as done previously. The sediment was re-dissolved in bi-distilled water until a known volume was obtained. Finally, two aliquots of 10 μL of sediment were used to count the number of pollen grains in them. Results were expressed as the number of pollen grains per gram of honey (pollen grains/g).
2.4. Physicochemical Analysis
The moisture of the honey was measured with a digital refractometer (ABBE URA-2WAJ-325; Auxilab S.L., Navarra, Spain). The refractive index values, at 20 °C, were converted to moisture contents using a Chataway table. The pH and electrical conductivity (EC) of a honey solution (5 g honey dissolved in 25 mL bi-distilled water) were measured directly using a pH meter (Crison micro pH 2001; Crison Instruments S.A., Barcelona, Spain) and a portable conductivity meter (Knick Portamess 913 Conductivity, Beuckestr, Berlin), respectively. Finally, the results were expressed as mS/cm.
The hydroxymethylfurfural (HMF) content and the diastase activity were determined following the methodology proposed by Bogdanov et al. [37
]. The HMF content was estimated using the White spectrophotometric method. This method considers the difference between the UV absorbance at 284 nm of a honey solution (0.2 g/mL) and the same solution after adding bisulfite. The HMF level was calculated after subtraction of the background absorbance at 336 nm (Jenway 6305 UV-Visible Spectrophotometer, Staffordshire, UK). Results were expressed in mg/100 g).
The diastase activity was determined on the basis of the hydrolysis rate of the starch solution by the α-amylase present in a honey buffer solution at 40 °C. The method used to determine the diastase activity was the Schade method described by Bogdanov et al. [37
]. The amount of starch converted was determined by measuring the absorbance of the honey solution at 660 nm using a UV-VIs spectrophotometer (Jenway 6305 UV-Visible Spectrophotometer, Staffordshire, UK) at different time points until an endpoint when the absorbance was less than 0.235. Diastase activity was calculated as diastase number (DN) or grams of starch hydrolyzed each hour per 100 g honey at 40 °C.
A HANNA Honey Color C221 colorimeter was used to determine the color in the honey, after calibration with glycerin (Glycerol HANNA instruments). The sample (approximately 4 mL) was placed in a plastic bucket with smooth walls. The honey must be fluid to be analyzed correctly; in the case of crystallized or weakly fluid honey; it is heated up to 45 °C in a thermostatic bath (J. P. SELECTA S.A.) and allowed to stand to eliminate the bubbles. Results were expressed in mm Pfund. The color of the honey was also measured on the CIELab scale. This system defined three colorimetric coordinates (L, a*, and b*), which are dimensionless magnitudes. L coordinate defines the brightness of honey samples; a* and b* are the chromatic coordinates and they represent variation between reddish-green and yellowish-blue, respectively.
2.5. Determination of The Total Polyphenol and Flavonoid Content
For the determination of the phenol content, the Folin-Ciocalteu spectrophotometric method, adapted to honey by Singleton et al. [38
] was used. This method was based on the oxidation of the phenolic compounds of phosphomolybdic and phosphotungstic acids, forming a bluish complex, which is analyzed at 765 nm using a UV-Vis spectrophotometer (Jenway 6305, UK). Solutions of honey samples (0.1 g/mL) were prepared. A calibration curve was obtained using gallic acid solutions (0.01–0.50 mg/mL) as a reference standard. 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. [39
]. 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. The total flavonoid content was determined using a standard curve with quercetin (0.002–0.01 mg/mL) as a reference standard. Finally, the results were expressed as equivalents of quercetin in mg/100 g honey.
2.6. Radical Scavenging Activity
The antioxidant activity of honey was determined by the DPPH discoloration method. It involves the determination of the antioxidant capacity of 2,2-diphenyl-1-picrilhidrazil (0.0006 M) by spectrophotometrically [40
]. A honey solution in methanol (0.1 g/mL) was prepared. This solution (0.3 m) was mixed with 2.7 mL of a DPPH solution (6 × 10−5
M). The sample mixture and blank DPPH solution were maintained in the dark at room temperature for 30 min. Then, the absorbance was measured at 517 nm using a UV-Vis spectrophotometer (Jenway 6305, Staffordshire, UK). Discoloration of the DPPH in each sample was calculated by the percentage of RSA (radical scavenging activity): RSA = [(AbsB−AbsS)/AbsB] × 100, where AbsB is the absorbance of the DPPH solution and AbsS is the absorbance of the honey sample solution.
2.7. Sugar Composition
The sugar composition was determined using an ion Dionex ICS-3000 chromatography system (Sunnyvale, California, EEUU) [6
]. The system separated the sugars by using an analytical polyvinylidene/polyvinylbenzene CarboPac PA1 column (Dionex 3 × 250 mm) suitable for mono-, di-, tri-, and oligo-saccharides and a pulsed amperometric detector with a gradient of two mobile phases (A and B). Phase A was ultrapure water, while phase B was 200 mM NaOH (HPLC grade, Merck). The sugars in honey solutions (10 mg/L) were calculated using the calibration curves of the standard solution for each pure sugar. The Chromeleon Chromatography Management System was used for acquisition of the chromatograms. The linear ranges of identified sugars were between 10–45 mg/L (glucose and fructose), 0.5–10 mg/L (sucrose, maltose), 0.1–10 mg/L (turanose, melezitose, and trehalose). The retention time as well the LOD and LOQ are included in the Supplementary Material
. Finally, the concentrations of the sugars were expressed as g/100 g honey
2.8. Statistical Analysis
The difference between pollen characteristics, physicochemical parameters, bioactive compounds and sugars of both honey types was determined using a Student’s t-test. The significance was determined at p < 0.05. The cluster multivariate analysis or conglomerate analysis was used as the classification method of honeydew honeys. This statistical approach groups the samples together based on a set of case-variable data. The objective is to place the cases (individuals) in homogeneous groups, suggested by the essence of the data so that individuals that can be considered similar are assigned to the same cluster, while different (dissimilar) individuals are located in different clusters. The cluster analysis allowed classification of honeydew honey samples while considering all the variables (palynological and physicochemical). The statistical analyses were carried out with the SPSS Statistic 23.0 (IBM SPSS Statistics, Armonk, New York, USA) and Statgraphics Centurion 17.0 for Windows (Statgraphics Technologies, Inc., The Plains, VA, USA).