CHARACTERIZATION OF SERBIAN SUNFLOWER HONEYS BY THEIR PHYSICOCHEMICAL CHARACTERISTICS

Five physicochemical parameters (water content, electrical conductivity, total acidity (pH), ash mass fraction and concentration of free acids) were analyzed in 15 sunflower honeys collected from several localities in Vojvodina, Serbia. The mean values of analyzed honeys were: water content 16.87%; concentration of free acids 27.43 mEq/kg; electrical conductivity 0.34 mS/cm; pH 3.64 and ash mass fraction 0.13%. The selected physicochemical characteristics of all honey samples from Serbia analyzed in this research can be considered to be within the parameters expected for sunflower honey in general. The values for ash mass fraction, electrical conductivity and concentration of free acids in all sunflower honey samples showed similar trends. High correlation was established between electrical conductivity and ash mass fraction. Statistically significant difference (p≤0.05) was established only for average values of free acids concentration between honey samples originating from the localities Kanjiža and Čelarevo. All of the analyzed honey samples were found to meet national and European legislation for investigated parameters

The electrical conductivity (EC), total acidity (pH), and ash mass fraction are related to the concentration of mineral salts, organic acids and proteins (Acquraone et al., 2007;Lazarević, Jovetić, & Tešić, 2017). The EC is very often used in routine quality control of honey. This parameter is defined as that of a 20% weight in volume solution in water at 20° C, where the 20% refers to honey dry matter (International Honey Commission Methods, 2009). It is used to distinguish between floral and honeydew honeys according to the standard. In accordance with the regulation concerning the quality of honey in the Republic of Serbia (Pravilnik, 2015), the minimum EC in honeydew placed on the market is set at 0.8 mS/cm. For other types of honey, the maximum permitted value of EC is 0.8 mS/cm. This parameter is also important for the authentication of unifloral honeys (Kaškonienė et al., 2010). Other factors such as floral source, amount of organic acids and proteins, and storage time can also influence the electric conductivity of honey (Karabagias et al., 2014). High electric conductivity values do not necessarily correspond to higher amounts of ash in the honey (Escuerdo et al., 2014). The EC value depends on the ash and acid content in honey: the higher their content, the higher the resulting EC (International Honey Commission Methods, 2009). The ashes of honey give an indication of environmental pollution and hence also an indication of geographical origin. The reference value for the ash mass fraction is not prescribed by the regulations.
The acidity of honey causes the presence of organic acids (tartaric, citric, oxalic, acetic, etc.), nectar or secretions of bees (Yadata, 2014). The acidity of honey can be determined by titration with sodium hydroxide (free acidity) or directly measuring the pH value (pH value). The natural acidity of honey can be increased by the storage and ripening of honey, as well as during the fermentation of honey.
Honey that is adulterated with sugar syrup has very low acidity (<1), while honey that is adulterated with invert sugar has a pronounced high acidity (Yadata, 2014). The acidity value related to the balance of organic acids naturally present in honey varies according to the floral source and the bee species (Sousa et al., 2016). The maximum value of free acidity in all types of honey (except in baker's honey) is 50 mEq/kg (Pravilnik, 2015). The reference value for the total acidity is not prescribed by the national regulations.
Sunflower honey is bright yellow, fragrantsmelling, dry, with an aroma of pollen, slightly herbaceous. This is a traditional honey with exceptional healing properties and nutritional value. It is discovered that sunflower pollen consistently reduced a protozoan pathogen (Crithidia bombi) infection in bumble bees and also reduced a protozoan pathogen (Nosema ceranae) of the honey bee (Apis mellifera) indicating the potential for broad antiparasitic effects (Giacomini et al., 2018). Sunflower honey contains small percentage of sucrose and therefore gets quickly crystallized (Srinual & Pilairuk, 2009;Sari, & Ayyildiz, 2012). Sunflower is cultivated only in the southern regions, where there is a lot of sun and where the climate is favorable for growing this plant. In Serbia, honey production is well developed thanks to a suitable climate and geographic location. The beekeeping, which has been sig-nificantly developed and maintained in Serbia since the 10 th century, is an important agricultural activity (Lazarević, 2016).
The purpose of this study was to determine water content, electrical conductivity, ash and acidity of sunflower honey samples collected from beekeepers in different regions of Vojvodina (northern Serbia), in order to obtain information about the honey quality and safety. In our research, which is part of a study of the physicochemical and sensory characteristics of Serbian unifloral and mixed honey, the behavior of honey was specifically examined with respect to the electrical conductivity and free acidity.

Honey samples
A total of 15 samples of sunflower honey harvested in 2019 were collected from beekeepers in different regions of the Autonomous Province of Vojvodina, Serbia. The sampling included locations in municipalities Kikinda (3 samples), Čelarevo (7 samples) and Kanjiža (5 samples). All collected samples were in their original packaging (jars) and transferred to the laboratory of Scientific Veterinary Institute "Novi Sad" for examination. Manufacturers used field observations for botanical origin determination. Our research included only the samples with confirmed botanical origin stated on the manufacturing specification label. All selected samples were produced by Apis mellifera. Honey analyses were carried out immediately after sampling. The honey analysis procedures were performed in two replicates.

Water content
Water content was determined by refractometry, measuring the refractive index (

Electrical conductivity
Electrical conductivity was measured at 20° C in solutions of honey samples (20.0 g dry matter of honey in volume solution in 100 ml dis-tilled water) using a conductometer Crison (Type Basic 30). The method of measuring is prescribed by International Honey Commission Methods (2009).

Free acidity and pH
The acidity of honey was determined by volumetric method (International Honey Commission Methods, 2009). Ten grams of honey were dissolved in 75 ml of distilled water, the pH is measured and solution was titrated with 0.1 M NaOH to pH 8.30. Acidity is expressed in milliequivalents/kg honey (mEq/kg).

Ash mass fraction
The ash content of honey means the residue which is obtained by a defined procedure and expressed as a percentage by weight (International Honey Commission Methods, 2009). The honey is ashed at 600 °C and the residue is weighed.

Statistical analysis
Statistically significant differences between the individual parameters at different locations were determined using one-way ANOVA (PAST, Version 2.12, Oslo, Norway). Statistical linear regression analysis was performed by the PAST software package, version 2.12, Oslo, Norway. Data were presented as mean, standard deviation, minimum and maximum value.

RESULTS AND DISCUSSION
Average values of water content, electrical conductivity, acidity, pH and ash mass fraction of honey obtained in this study are shown in Table 1. The obtained values were compared with the values that are prescribed by the Regulation on the quality of honey in the Republic of Serbia (Pravilnik, 2015). Also, the results were compared with the results from other authors from both Serbia and other countries. ANOVA one way statistical analysis revealed statistically significant difference between the localities Kanjiža and Čelarevo at p≤0.05 only for free acidity of honey.
The water content values in all investigated honey samples ranged between 14.60 -18.60% (average 16.87 ± 1.23%). The water content in all investigated honey samples was below the maximum permitted level (20%) set by regulations for honeys (European Commission, 2002;Pravilnik, 2015). The honey moisture in depends on the production season, floral source, abundance of nectar flow, soil, ventilation of the beehive, colony strength, and meteorological conditions in the area of honey production, primarily air humidity (Kirs, Pall, Martverk, & Laos, 2011;Escuerdo et al., 2014;Sousa et al., 2016;Lazarević et al., 2017). An important factor that could affect the water content is honey maturation and harvest time.
The ash mass fraction, electrical conductivity and free acidity values of all sunflower honey samples showed similar trends (Figure 1). Since investigated properties are a function of the concentration of the ions present in the honey, their correlation was examined (Table  2). It was found that there is a correlation between electrical conductivity and ash mass, acidity and ash mass, acidity and electrical conductivity. Between other examined parameters, the correlation coefficients were insignificant. Correlation was established between electrical conductivity and ash mass fraction (R 2 =0.611). Correlation between free acidity and electrical conductivity calculated for sunflower honey samples was R 2 =0.476. In our earlier study that included 55 honey samples (16 samples of acacia honey, 18 samples of meadow honey, 9 samples of multifloral honey, 6 samples of linden honey, and 6 samples of honeydew honey), significant linear correlation between free acidity and electrical con- Figure 1. Relations between ash content, electrical conductivity and free acidity values of sunflower honeys ductivity was established only for meadow and multifloral honey samples (R 2 = 0.8210 and R 2 = 0.7688). A similar correlation coefficient to that obtained for sunflower honey was found for honeydew honey (R 2 =0.4996) (Živkov Baloš et al., 2018).

CONCLUSIONS
The water content, electrical conductivity, total acidity, ash mass fraction and concentration of free acids of sunflower honey collected from beekeepers in different regions of the Autonomous Province of Vojvodina, Serbia was determined. Our results demonstrated that Serbian sunflower honey is characterized by good quality since the examined parameters closely related to the quality of honey, showed values in agreement with legal limits established by the national and European legislation. The hypothesis about potential existence of a correlation between the parameters tested was confirmed for electrical conductivity/ash mass, acidity/ash mass fraction, as well as for acidity/electrical conductivity. Likewise, we found that these parameters show similar trends. By observing the geographical characteristics of Serbian sunflower honey, we found statistically significant differences only between average values of free acidity of honey originating from localities of Kanjiža and Čelarevo.
Therefore, further research on physicochemical properties of sunflower honey is required to confirm the quality and authenticity of this product. The importance of such research lies in its contribution to better understanding the benefits of this type of honey.

АCKNOWLEDGEMENTS
This research was funded by the Ministry of Education, Science and Technological development of Republic of Serbia by the Contract of implementation and financing of scientific research work of NIV-NS in 2020, Contract No: 451-03-68/2020-14/200031 from January 24, 2020.