1. Introduction
In Europe, beekeeping and honey production are a common part of agriculture, also in view of the appropriate climatic, environmental, and economic conditions. According to Al-Badri [
1] the honey production in Iraq is influenced by environmental issues like little rain, desertification, using of pesticides, and economical problems. Iraq is climatically diverse, with continental, subtropical semi-arid type, and Mediterranean climate in the north and north-eastern region. Rainfall is very seasonal. Summers in Iraq are characterized by drought, hot to extremely hot, with temperatures of over 43 degrees of Celsius during July and August [
2]. The environment of Kurdistan is very diverse and consists of areas of forests and mountains herbs (mainly with oaks, almonds, walnuts, pines, pistachios, grasses, and shrubs), plains regions (with grasses, bulbous, thorny, and spiny plants), and areas of riverbanks (with willows, shrubs, grasses, liquorice, and thorny plants). This diverse flora provides pasture for the bees. In 2008, 3 258 beekeepers were recorded in Dohuk Governorate of Iraqi Kurdistan, which is the second highest number after Sulaimania Governorate. Total annual production of honey in Iraqi Kurdistan ranges from 600 to 800 tons [
3].
To date, no works related to the analysis of honey from the Dohuk Governorate of Iraqi Kurdistan have been published and a narrow range of work has been published about the analysis of honeys originating from Kurdistan province of Iran [
4,
5,
6]. Some works are focused specifically on the biological activity of Iraqi honey and little data have been published on the physicochemical properties. Ali et al. [
7] identified by GC-MS method in Iraqi honeys wide range of volatile compounds like alcohols, phenols, ketones, organic acids, and esters with antibacterial, antifungal, and anticancer activity. Hamed et al. [
8] focused to determination of water-soluble vitamins of B-complex group in Iraqi honeys by HPLC method. Al-Hasani et al. [
9] detected antibacterial effect of Iraqi honey samples against gram-positive and gram-negative bacteria at concentration of 100 % and the inhibition effect of honey decreased as honey diluted. Abu-Almaaly [
10] focused on identification of some heavy metals in samples of Iraqi honeys by atomic absorption technique. The same author detected cadmium, lead, nickel and iron in honey samples and concentrations of these heavy metals varied according to the areas of honey collection.
The chemical composition of honey is variable depending on the floral source and geographical origin. Honey is a mixture of different components such as carbohydrates, water, proteins, amino acids, enzymes, vitamins, phenolic acids, minerals, volatile compounds, pigments, and other substances [
11].
The moisture content in honey usually ranges from 15% to 21 %. The moisture content of honey is important for honey stability, and the low moisture content avoids the honey fermentation [
12]. The hydroxymethylfurfural (HMF) content and diastase activity reflect the freshness and heat treatment of the honey. Shortly after extraction, the honey has a low HMF content and relatively high diastase activity, depending on the origin of the honey. Subsequently, because of various technological steps such as heating and long-term storage, concentration of HMF is increasing, and diastase activity decreases [
13]. Honey’s phenolic compounds are secondary plant metabolites represented by flavonoids and phenolic acids [
14], originating from flower nectar, propolis and pollen [
15]. The total phenolic content indicates the content of phenolic substances that are important from a nutritional point of view due to their antiviral, antimicrobial, antioxidant, anti-inflammatory, anti-atherogenic, and anticancer properties [
11,
15].
The chemical composition of honey affects the physicochemical properties of honey such as electrical conductivity, color, and pH. The electrical conductivity of honey correlates with the content of mineral substances in honey, which ranges from 0.04% to 0.2% [
11]. Due to higher mineral content, honeydew honeys usually have higher electrical conductivity, in comparison with nectar honeys [
16]. Color properties such as honey color and color intensity are variable depending on the content of different coloring substances, such as phenolic acids, flavonoids, carotenoids, Maillard reaction products, mineral substances, and pollen grains [
17,
18]. The pH value reflects acid content of honey and indicate honey fermentation. Honey naturally contains numerous organic and inorganic acids and pH value of honey varies from 3.4 to 6.2, depending on botanical origin of honey [
13].
In the European Union is the quality of honey regulated by Council Directive of the EU 110/2001 [
19] related to honey. This EU legal act specifies composition criteria for saccharide content, hydroxymethylfurfural content, moisture content, electrical conductivity, free acidity, diastase activity, and water-insoluble content in honeys. In Kurdistan, there are no standard specifications for evaluating the quality of produced honey, and there are no specialized laboratories [
3].
Due to the scarcity of published data that comprehensively evaluates the properties of honeys directly from Kurdish beekeepers from Iraqi Kurdistan, the main objectives were to (1) analyze specific physicochemical and bioactive parameters relating to honey quality and biological value in honeys collected directly from Kurdish beekeepers from Dohuk and Erbil Governorates of Iraqi Kurdistan, and (2) to compare the quality of Kurdistan honeys with the requirements of European Union law and (3) to compare the parameters of Kurdistan honeys with honeys directly from Czech and Slovak beekeepers.
2. Materials and Methods
2.1. Honey Samples from Kurdish Beekeepers
Ten honey samples were obtained directly from Kurdish beekeepers from Iraqi Kurdistan. Honey samples were produced in different areas of the Dohuk and Erbil Governorates in 2018. Eight samples of honey came from beekeeping and two samples from wild bees. All honey samples were nectar honeys. The samples were stored in the dark at room temperature (21 ± 2 °C) in original packaging from beekeepers until the analysis. A detailed specification of the analyzed honey samples from Kurdish beekeepers is given in the
Table 1.
2.2. Honey Samples from Czech and Slovak Beekeepers
Totally twenty honey samples of different botanical and geographical origin were collected directly from Czech (n = 10) and Slovak beekeepers (n = 10). Honeys were produced in various regions of Czech (Vlkaneč (n = 3); Lupůvka (n = 1); Valtice (n=1); Brno (n = 5)) and Slovak Republic (Borský Mikuláš (n = 1); Hradište pod Vrátnom (n = 1); Podbranč (n = 2), Senica, Kunov (n = 4); Lehota (n = 1); Piešťany (n = 1)) in 2018. All honey samples were nectar honeys. The samples were stored in the dark at room temperature (21 ± 2 °C) in their original packaging until the analysis.
2.3. Legislative Composition Criteria and pH
All standards and chemicals used in this study were of analytical grade. Diastase activity, hydroxymethylfurfural content, moisture content, electrical conductivity, pH, and free acidity were determined according to the Harmonized Methods of the International Honey Commission [
20].
Diastase activity in honey samples was determined spectrophotometrically based on the Phadebas method (Phadebas Honey Diastase Test, Phadebas AB, Kristianstad, Sweden) using Specord 200 Plus spectrophotometer (Analytic Jena AG, Jena, Germany) at 620 nm. The diastase activity was expressed as the diastase number (DN) in Schade units.
Hydroxymethylfurfural content in honey samples was analyzed using the HPLC-UV method. The analysis was performed using an HPLC system (Alliance 2695, PDA detector 2996, Waters, Milford, Massachusetts, USA), a column (Zorbax Eclipse XDB-C18-5 μm, Agilent, Santa Clara, California, USA), with water-methanol (90:10) mobile phase. The analysis conditions were as follows: isocratic elution, flow rate 1.0 mL/min, sample injection 20 μL and column temperature 35 °C. HMF was detected and quantified in the UV at 285 nm using external standard and expressed in mg/kg of honey.
Moisture content in honey samples were determined using the refractometric method with the Abbé refractometer (AR 4, A.Krüss Optronic GmbH, Hamburg, Germany). Moisture content was expressed in g/100 g after the conversion of determined refractive index.
Electrical conductivity was determined using the conductometric method on an inoLab Cond 730 conductometer (WTW, Weilheim, Germany). Electrical conductivities were expressed in milli Siemens per meter (mS/m).
pH and free acidity were determined using pH meter MP230 (Mettler-Toledo, Greifensee, Switzerland) and an HC 113 electrode (Theta 90, Prague, Czech Republic). Three-point calibration was used at pH 4.0, 7.0 and 10.0. The honey samples were dissolved in distilled water without carbon dioxide and titrated with 0.1 mol/L sodium hydroxide (Penta, Czech Republic) solution to pH 8.3. Free acidity of honey was expressed in milliequivalents per kg of honey.
2.4. Bioactive Compounds
The total phenolic contents were determined by the Folin-Ciocalteu method described by Silici et al. [
21]. The absorbance was measured at 765 nm using a Specord 200 Plus spectrophotometer (Analytic Jena AG, Jena, Germany). Standard calibration solutions were diluted from a gallic acid (Penta, Czech Republic) stock solution at a concentration range of 0–900 mg/10 mL (R2 = 0.9990). The results were expressed as mg of gallic acid equivalent (GAE) per 100 g of honey.
Honey color intensity was analyzed according to the method described by Beretta et al. [
17]. The absorbance was measured at two different wavelengths (450 nm and 720 nm) using Specord 200 Plus spectrophotometer (Analytic Jena AG, Jena, Germany) and the difference in absorbance was expressed as mAU.
The color of the honey samples was measured using Honey Color Photometer Hanna HI96785 (Hanna Instruments, Jud. Salaj, Romania). Colorimeter was calibrated with glycerol standard reference (Hanna Instruments, Jud. Salaj, Romania) and honey color was expressed in millimeter (mm) Pfund. The analyzed honey samples were categorized using the United States Department of Agriculture (USDA) approved color standards [
22].
2.5. Statistical Analysis
All assays were performed in duplicate, and the results were expressed as a mean value ± standard deviation (SD). The statistical analysis of the results was performed with the Unistat (6.5) software and the Microsoft Excel 2016. The significant differences were obtained by a Shapiro-Wilk Normality Test followed by Mann-Whitney U Test. The differences at a 95 % (p < 0.05) confidence level were considered statistically significant.