Characteristic Features of Kolludere Valley (Bitlis-Hizan) Honey

: This study was carried out to reveal the characteristic features of honey produced in the Kolludere Valley, which is located within the borders of the Hizan district of Bitlis province. This area is isolated as there are no seasonal migratory beekeepers. In 2022, a flora study was carried out in the area and 133 plant taxa belonging to 19 families from which bees receive nectar or pollen were identified. 23 of these taxa are endemic. Content analysis of honey samples taken from the study area was carried out. Proline value, which is an important parameter of honey quality, was determined as 809.41 mg kg -1 , diastase number 28.9, HMF 2.9 mg kg -1 , and sucrose 0.2 g/100g. All other parameters (humidity, acidity, pH, fructose+glucose, fructose/glucose, saccharose, maltose, electrical conductivity) were also met standart according to the values of the Food Codex Honey Communiqué and European Union Standards. In addition, pollen analysis of honey samples was made and the data were compared with the flora. In honey samples, pollen is generally minor or rare, and pollen of a dominant taxon was found in only one sample. For this reason, most of the honey produced in the Kolludere Valley was evaluated as flower honey.


Introduction
With the rapidly increasing world population, it is getting harder to reach clean and safe food.Basic foods contaminated with toxic compounds (pesticides, artificial fertilizers, food additives, etc.) are among the most important factors threatening human health (Demir and Ayaz, 2022).One of the foods that has an important place in human nutrition is honey.Honey, with its vitamins, minerals, organic acids, amino acids, enzymes, and compounds such as flavonoids, is an important food that is nutritious, easily digestible, and has protective properties against various diseases (Ozmen and Akalın, 2006;Mutlu et al., 2017).Sugar, moisture, elements, HMF, enzyme, organic acids, vitamins, etc. content, glucose and fructose ratios, pH, acidity, electrical conductivity, etc. parameters determine the value of honey (Acquarone et al., 2007).One of the factors affecting honey quality is plant diversity.
The producers of natural products are decreasing day by day.Therefore, the sustainability of these products is important.Hizan district of Bitlis is one of the best examples of traditional crop cultivation.Honey is an important product for the province of Bitlis.In terms of its geographical structure and vegetation, Hizan is a district that has an advantageous position in terms of beekeeping activities (Ozdemir et al., 2016).Hizan's geographical location and rich plant diversity provide advantages for honey production.
The aim of this study is to reveal the quality of the Kolludere Valley (Hizan) honey with a scientific approach so that it can get the value it deserves.For this purpose, honey flora of the region, pollen analysis, and honey content analysis were revealed.

Research Area
Kolludere Valley is located in the northeast of Hizan and the west of Gevaş district (Figure 1).

Distinctive Features of Kolludere Valley Honey
Kolludere Valley is a geographically isolated place surrounded by high mountains.In addition, it has a rich plant diversity as it has different habitats such as deep valleys, streams, forest areas, and steppe areas.Because of these properties, it is very suitable for honey production (Behçet, 1994).Beekeeping activity starts in April in the spring and continues until the end of August.Beekeeping starts in June, as the flowering period is a little late in high places.However, the natural comb honey harvest is expected until October.
Beekeepers carry out all their beekeeping activities in Hizan.It is not possible to move it to another place depending on the season.It is also an isolated area as there is no entrance for itinerant beekeepers.Traditional honeycomb is mostly preferred for honey production.(Figure 2).Caucasian honey bees are preferred in Hizan.
Figure 2.An image from the research area.

Flora study
Between May and August of 2022, a total of 12 field studies were carried out and plant samples visited by bees were collected.The collected samples were identified and preserved in the Herbarium of the Biology Department of the Faculty of Arts and Sciences at Bitlis Eren University.Flora of Türkiye (Davis, 1965 and1985;Davis et al., 1988) was used to define plant taxa.Families are listed alphabetically, and species according to their importance.The phrase "END" has been added to the end of the endemic taxa.

Honey analysis
Ten honey samples were taken from different locations in the research area.Honey analyses were carried out at Ordu Province Apiculture Research Institute, Food Technology Department Laboratory.The following parameters were determined for each sample.

Pollen analysis
Pollen analysis is generally used to determine the botanical source of honey (Almeida-Muradian et al., 2005;Bastos et al., 2004).The following method was used for pollen analysis in honey (Sorkun 2008).10 g of honey is poured into a test tube and diluted with 20 ml of pure water.For this process, a water bath with a temperature of 45 °C is used for 10-15 m.After the mixture is homogenized, the test tube is centrifuged at 3500 rpm for 45 m.After this stage, the particle at the bottom of the tube is transferred to a slide with the help of a needle.
The prepared slides were examined with an Olympus CX21 light microscope.In the diagnosis of pollen, reference pollen microphotographs from various sources (Sawyer, 1981;Pehlivan, 1995;Sorkun 2008), the pollen slides of the plants collection of Kafkas University Biology Department, and plants collected from the research area were used.
The results obtained from the examinations of the preparations were evaluated according to the criteria used by Louveaux et al. (1978).According to these criteria; 45% or more pollen are dominant, 16-44% secondary, 3-15% minor, and 3% or less trace pollen were accepted.Pollen analyses were carried out by pollen experts from the Department of Biology of Kafkas University.

Bee flora of the research area
In the plant samples collected from the research area, 133 taxa belonging to 19 families were determined.23 of these taxa were endemic (Table 1).

Honey Analysis
The amino acid profile can give an idea about the botanical origin of honey samples (Anklam, 1998).Besides proline, honey contains 26 amino acids and their amount depends on the source of the honey (nectar or honey extract).The proline content of honey constantly decreases during storage, so proline is an indicator of honey maturity (Von der Ohe et al., 1991).A minimum value of 180 mg kg -1 for proline is internationally accepted (Hermosín et al., 2003).
Many parameters such as proline and HMF content, electrical conductivity, and enzyme activities are important in sugar-added honey.Proline has been proposed as a quality criterion for honey in terms of sugar adulteration (Bogdanov and Martin, 2002).The proline results of the samples taken from different locations were well above the standards.Proline values were in the range of 366-1286, with an average proline value of 809 mg kg -1 .In the study, fructose + glucose values of flower honey ranged between 65.7 and 72.8 and it was determined that it was 68.7% on average.Fructose+glucose values were expected to be at least 60 (g 100g -1 ).The fructose/glucose ratio was determined as 1.3 on average.The expected value in this parameter should be between 0.9 and 1.4.As the fructose/glucose ratio increases, the tendency to crystallize in honey decreases.Another sugar component value examined was sucrose.Very low sucrose was detected in only two of the samples.The average sucrose was 0.2.This value was found below 5%, which is the highest value determined by the Turkish Food Codex Honey (Official newspaper; Communiquity No: 2020/7) Communique and European Union Standards (Bogdanov et al., 2002).Maltose average was 1.8.The moisture content of honey is the most important criterion in evaluating the maturity and shelf life of honey.The moisture average detected in the samples was 15.3.The standard water ratio should be less than 20%.
The sum of free acids, lactones, and esters determines the total acidity in honey (Kahraman et al., 2010).Free acidity contributes to flavor, provides resistance to microorganisms, increases chemical reactions, antibacterial and antioxidant properties, and also gives some information about the source of honey.The amount of free acid should not be more than 50 meq in 1000 g honey according to the standards.In this study, the average free acid value was 21.5 meq.
Enzyme content is one of the quality criteria of honey.The enzyme invertase, which converts nectar into honey, converts sucrose into glucose and fructose.Diastase enzyme converts starch into small sugars.Although there are different levels in honey depending on the plant source and flora, the diastase rate being more or less than the expected level can give clues during the quality determination in honey.However, the diastase activity may differ depending on the protein amount of the pollen in honey and other substances (Artık, 2004).According to the analysis results, the average diastase number of honey samples was 29.This rate is far above the standards.
The average value of hydroxymethylfurfural (HMF) was 2.9 mg kg -1 .HMF is a substance that is formed as a result of heating carbohydrates in honey or storing them in unsuitable environments in terms of heat and is unsuitable for human health.HMF ratio in honey is a maximum 40 mg kg -1 .The fact that the HMF value is above this value indicates that the honey may have been stored in a hot environment or subjected to heat treatment and that honey with this feature cannot be sold legally.5-HMF may be formed by dehydration of sugar at low temperatures under acidic circumstances (Lee and Nagy, 1990).Its concentration rises dramatically as the temperature of the thermal treatment and storage rises (Capuano and Fogliano, 2011).According to Turhan et al. (2008), there is no direct association between the 5-HMF level of honey and its composition.

Pollen Analysis
As a result, pollens of 43 genera belonging to 20 families were determined.One out of six honey samples were defined as unifloral and five honey samples were defined polyfloral honey.In a previous study on Bitlis honey, five honey samples were investigated and all of them were found to be multifloral (Kızılpınar Temizer et al., 2020).As a result of pollen analysis of 24 honey samples collected from Siirt province, it was determined that eight of them were unifloral (Gürbüz et al., 2019b).According to honey pollen analysis conducted in Şırnak province, which is one of the regions close to Bitlis province, two of 23 honey samples were determined to be unifloral (Gürbüz et al., 2019a).Unifloral honey was determined at the fifth station.The dominant pollen taxa of fifth station honey is Taraxacum.Taraxacum was the most densely pollinated genus.It was present in all samples.It was secondary in the first, second, fourth locations, minor in the sixth location, and a trace in the second location.Especially Taraxacum kurdiciforme was the most common species in the region.Pollen analysis was carried out on 67 different honey samples in Hakkari province, and it was determined that Taraxacum pollen was dominant in one sample and mostly in minor and trace amounts in the other samples (Sarısu, 2011).According to melissopalynological examinations made on 100 honey samples of Kars province and its district, Taraxacum pollen was detected as minor and trace in eight honey samples (Gençay Çelemli et al., 2018).Taraxacum (dandelion) honey is a honey produced and characterized in Europe (Oddo and Piro, 2004).Rapid complete granulation with finely ordered crystals, cream to yellow color sometimes with a grayish tint, and an intense pungent ammonia persistent odor and taste are other typical characteristics of Italian dandelion honey (Oddo et al., 1995).Dandelion honey, which is a new record in terms of Turkish monofloral honey, was obtained from Bingöl province, and melissopalynological and chemical content analyses of the honey were performed (Ozenirler et al., 2018).In Bingöl province, one of the eight honey samples was determined to be unifloral and this honey was determined to be Quercus honey (Soyer, 2018).The pollens in polyfloral honey were rarely secondary (Astragalus, Hypericum, Plantago, and Taraxacum), mostly in minor or trace amounts.Eryngium, Taraxacum, Astragalus, Rosa, and Verbascum were observed in all samples, Prangos, Arctium, Tragopogon, Vicia, Mentha, Salvia, and Rumex were observed in five samples, varying in dominant, secondary, minor or trace rates.These results were compatible with the results obtained in the flora determination.According to another study conducted on Bitlis Hizan honey, Rosaceae, Fabaceae, Boraginaceae, and Brassicaceae taxa were observed in all honey samples (Kılıç et al., 2016).
Hypericum was the group that was intensely detected in the research area and preferred by bees.Especially Hypericum triquetrifolium was concentrated in the research area.Hypericum pollens were detected as secondary in third and sixth locations, and as minor or trace pollen in other locations.In honey research conducted in Hakkari province, Hypericum pollen was determined to be dominant in one honey sample (Sarısu, 2011).According to the research conducted by Tosunoğlu et al. (2023) on 44 different honey samples in Gümüşhane province, Hypericum pollen was determined as an indicator for altitudes above 1500 m and was also detected as trace, minor, and secondary pollen taxon in 24 pollen samples.
Arctium tomentosum was among the plants that bees showed great interest in the research area.The pollen of this plant was detected in five of the six locations sampled.It was mostly in the minor or trace pollen group.Asteraceae pollen is one of the most abundant pollen taxa in honey samples (Sarısu, 2011;Bakoğlu et al., 2014;Gençay Çelemli et al., 2018;Gürbüz et al., 2019b).Asteraceae pollen also was determined as an indicator for altitudes above 1500 m (Tosunoglu et al., 2023).Astragalus (geven) was one of the leading plants in beekeeping.In fact, the name of the honey found in most of Anatolia is associated with this plant.It was one of the dominant plants of the steppe areas in the search area.Astragalus pollen was found in all honey samples.Secondary pollen was detected in two samples (first and third), minor in two of the other locations, and trace pollen in two of them.This means that Kolludere Valley honey was not "geven" honey.It was determined dominantly in honey samples from most places in the Eastern Anatolia region.In the research conducted on five honey samples in Bingöl province, it was determined that two honey samples contained Astragalus honey.The taxon whose pollen is most frequently found in Hakkari honey is Astragalus, one of the natural plants of the region, and was determined to be the main nectar and pollen source for the local honey (Sarısu, 2011).Astragalus honey has antioxidant and antimicrobial properties.The Astragalus honey sample from Erzurum has the best antioxidant activity (Küçükaydın et al., 2023).
Verbascum is one of the genus with the most species in the Flora of Türkiye.There are more than 340 species, most of which are endemic in our country, with approximately 360 species worldwide.Endemic species are mostly found in Eastern, Southern, and Central Anatolian regions.Pollen of Verbascum was detected in all honey samples taken in the search area.Verbascum murbeckianum Hub.-Mor, and Verbascum songaricum subsp.subdecurrens Hub.-Purple.are endemic taxa, and also, the type specimen was collected from Bitlis.Therefore, this group has an important place in search area honey.Among the honey samples obtained from Diyarbakır and Bingöl regions which are close to the study area, it was determined that Verbascum pollen was dominant at a rate of 97% in mullein honey, while other pollen types (Asteraceae and Campanula ) were found in trace amounts below 3% (Ozkök, 2019).
Prangos pabularia is one of the plant groups that are found in wide areas in Hizan.According to the statement of beekeepers, it is one of the plants most preferred by bees.Pollens of this plant were detected in five of six samples of Hizan honey.It is generally in the minor or trace pollen group.Brassicaceae pollen was found in all locations except the fifth location.Most members of this family bloom in early spring and bees do not benefit much in short-term flowering.However, especially the Lepidium latifolium is very dense and remains flowering for a long time.
Lamiaceae is one of the most important plant families in terms of beekeeping.However, the pollen of the members of this family was not found in Hizan honey.Salvia and Mentha pollen were detected in trace or minor amounts in five locations.Ozler (2018) claimed that Fabaceae, Rosaceae, Eucalyptus, and Centaurea were determined melliferous plants (Ozler, 2018).Sorkun et al. (1989) determined that pollen grains belonging to the families of Asteraceae, Fabaceae, Fagaceae, Myrtaceae, Malvaceae, Brassicaceae, Scrophulariaceae, Lamiaceae, and Oleaceae are the important source of Turkish flower honey (Sorkun et al., 1989).
In the samples from all locations, it was defined as Taraxacum honey, because Taraxacum was dominant in the fifth sample, and because there was no dominant pollen in the other samples (generally below 45%), the honeys were determined as polyfloral honey (multifloral origin) (Table 4).This shows that the plant biodiversity is high in Hizan, so bees collect pollen from a large number of plants.Since single dominant pollen cannot be determined in polyfloral honey, these honeys are generally named according to the geographical region where they are obtained and offered for sale in this way.Especially in our country, this practice is one of the most important criteria in determining the price of honey.
Honey pollen analysis can be used to determine the botanical origin of honey.This study, which was carried out in Hizan, was also carried out for this purpose.Pollen analysis is also an important parameter in determining the quality of honey.Evaluation of pollen together with other parameters (Flora, proline, HMF, etc.) reveals the characteristic features of Hizan honey.
Honey is classified by pollen analysis.Which plant has the most pollen in honey, is called by the name of that plant (Sorkun, 1985).According to the results of the pollen analysis performed on honey samples from the Kemaliye-Erzincan region, only one of 29 samples was identified as unifloral (Yurtsever, 2004).As a result of the pollen analysis study conducted in the Antalya region, Apiaceae, Raphanus raphanistrum, Cirsium, Eucalyptus, Plantago, and Ulmus pollens were determined as dominant (Silici, 1995).In their pollen analysis in the Rize region, they found Castanea sativa pollen to be dominant (Sorkun et. al., 1989).In the study conducted in Bingöl province, 46.14% of the pollen was composed of Astragalus, and the others were composed of Thymus, Tribulus, and Lamium (Bakoğlu et al., 2004).

Conclusion
Field studies and honey analyses (chemical, pollen) conducted in the Kolludere Valley, where intensive beekeeping activities are carried out, show that this place has suitable conditions in terms of beekeeping.The rich flora affects the quality of honey in a very important way (Karakaya et al., 2023).The suitability of values such as proline, diastase, and HMF, which determine the quality of honey, reveals the honey quality of the region.When the results obtained are compared with the results of the provinces that obtained geographical indication certificates in the Eastern Anatolia Region, it was determined that better results were obtained.Thus, the results obtained in this scientific study support the necessity of applying for geographical indication of Hizan Honey and its usability for situations such as the promotion and marketing of honey.

Figure 1 .
Figure 1.Map of Research area.

Table 3 .
Analysis results of honey samples taken from the research area N.D*: not detected.

Table 4 .
Pollen analysis results of honey samples