MOISTURE AND ACIDITY AS INDICATORS OF THE QUALITY OF HONEY ORIGINATING FROM VOJVODINA REGION

Th e color, aroma and fl avor are major sensory characteristics of honey, which are mainly determined by the botanical origin of honey as well as by processing and storage conditions. Increased moisture content above the maximum permitted level could result in honey spoilage, which aff ects its sensory properties. Th e higher water-in-honey content, the greater possibility of yeast fermentation and thus the change of the fl avor and color of honey. Fermentation process results in alcohol formation and, in the presence of oxygen, the alcohol will break down to acetic acid and water, which causes honey to have sour taste. Th us, moisture content of honey is a critical parameter for its quality as it aff ects the stability of honey and its resistance to microbial spoilage during storage. Physicochemical analysis of moisture content and acidity of honey play an important role in determining the overall characteristic of honey and fi nal assessment of its quality. In this study, the investigation of aforementioned parameters resulted in positive quality assessment for 48 of 50 examined honey samples produced in 2013 in the territory of Vojvodina.


INTRODUCTION
What is it that makes honey so special foodstuff ? An answer to this question is highly complex, same as its extraordinary taste is. Honey is durable food, it never spoils and has virtually unlimited shelf life. Th e production of honey involves a wide range of factors acting together in perfect harmony.
Probably the most important property describing the chemical composition of honey is its diversity, that is, one could not fi nd even two fully identical honey samples (Rogulja et al., 2009). However, huge body or information available to modern consumers results in their increased expectations and demands in view of the safety and quality of food (Prica et al., 2009). Th erefore, there is a need to fi nd the answer to the question: What is the quality of honey, how is it assessed and graded? One of the possible approaches to evaluate the quality of honey includes passing of relevant regulations establishing minimum and maximum levels of particular substances and ingredients in foodstuff s. In Serbia, the quality requirements for honey are stipulated in the Regulation on quality and other requirements for honey, other bee products, products based on honey and other bee products (Sl. list, 2003).
According to the Regulation (Sl. list SCG, 2003), honey is defi ned as "sweet, dense, crystallized, viscous product produced by honeybees from the nectar of honeyplant fl owers or from secretions of living parts (conifer or hardwood species), which the bees collect, transform by combining with specifi c substances of their own, and deposit in honeycombs to mature". In Codex standard (2001), honey is defi ned as "natural sweet substance produced by honey bees from the nectar of plants or from secretions of living parts of plants or excretions of plant sucking insects on the living parts of plants, which the bees collect, transform by combining with specifi c substances of their own, deposit, dehydrate, store, and leave in the honey comb to ripen and mature". Chemical composition of honey implicates highly complex mixture of more than 200 diff erent substances (Ferreira et al., 2009). Some of these substances are produced by honeybees, some originate from honeyplants, whereas some are produced during the maturation process in the honeycomb (Krell, 1996).
Honey types, as well as the individual samples within particular type, diff er by their composition according to their fl oral and geographic origin, climatic conditions, honeybee species as well as processing and storage conditions (Škenderov and Ivanov, 1986).
Th e average composition of honeys includes some 17% water, 38.19% fructose, 31.28% glucose, 1.31% saccharose, 7.31% maltose, 7.11% lactose, 0.04% nitrogen and some 0.169% ash. Aft er the carbohydrates, water is the second most important component of honey. Its content ranges between 15 and 23% (Krell, 1996). Th e moistur content substantially aff ects some physical properties of honey (crystallization, viscosity, specifi c weight) and is infl uenced by climatic factors, bee species, bee-colony's strength, humidity and air temperature in the hive, processing and storage conditions as well as by the honeyplant species. However, there are no substantial diff erences in water content between individual honey types (Škenderov and Ivanov, 1986).
Honey in its natural form is characterized by extremely low moisture content, thus very few bacteria and microorganisms can survive in such environment, which is essential for its resilience (Geiling, 2013). Yet, honey is highly hygroscopic substance and its moisture content may vary depending on air humidity during storage. Th e higher moisture-in-honey content, the greater is the possibility that the yeasts will ferment and change the fl avor. Namely, fermentation process results in alcohol formation and, in the presence of oxygen, the alcohol will break down to acetic acid and water, which causes honey to have sour taste and to spoil (Rogulja et al., 2009).
It is well established that molasses, a byproduct of cane sugar, is similar to honey by its properties, yet-although it has a long shelf life molasses can eventually spoil. Th e durability of honey is partly to be attributed to the bees themselves. Nectar, the fi rst component collected by bees to make honey, is by its nature highly humid with a moisture content ranging from 60-80%. Th roughout the process of making honey, the bees dry out much of this moisture by fl apping their wings. Th e chemical composition of bees' stomach significantly contributes to honey's resilience to spoilage. Bees' stomach produces an enzyme called glucose oxidase, which mixes with the nectar, breaking it down into two by-products: gluconic acid and hydrogen peroxide, the latter one being of crucial importance for the maintenance of quality of honey (Geiling, 2013).
Honey is naturally highly acidic. Its pH is extremely low, ranging between 3 and 4.5, which inhibits the growth of bacteria and other spoil-ready organisms (Geiling, 2013). During a long time, formic acid has been considered major (if not the only one) acid in the honey. Nowadays, it is well established that honey contains a wide range of organic acids. Besides the formic acid, honey contains oxalic acid, butyric acid, citric acid, 2,3-dihydroxybutanedioic acid, malic acid, pyroglutamic acid, lactic acid, benzoic acid, maleic acid, gluconic acid, isobutyric acid, succinic acid, pyruvic acid, α-ketoglutaric acid and glycolic acid. Out of these, gluconic acid, a byproduct of enzymatic activity of glucose oxidase, predominates. According to the data from the literature, the content of organic acids in honey ranges between 0.17 and 1.17% (average range 0.57%). Most of organic acids are present in honey in the form of esters, which contributs to its characteristic fl avor and aroma. Some of the acids are introduced into honey via the nectar, i.e., their contents depends on the type of the honey, whereas some are produced during storage process and are infl uenced by storage temperature and processing conditions. Th e acidity of honey can range from 8.7 to 59.5 meq/kg, with an average of 29.1 meq/kg. Increased acidity of honey is an indicator for a fermentation process and transformation of alcohol into organic acid (Rogulja et al., 2009). It is believed that moisture content less than 18% will prevent the fermentation. However, this possibility cannot be absolutely excluded even in honeys with moisture content below 17.1% since the potential eff ects of yeast content and temperature of honey as well as distribution and availability of water aft er crystallization have to be taken into consideration (Krell, 1996).
Moisture content can be considered the most important parameter of honey quality as it determines its stability and resistance towards microbial spoilage (fermentation) during storage (Bogdanov et al., 1999). Th e infl uence of acid content on fermentation processes, fl avor and aroma as well as bactericidal properties of honey make the total acidity an important indicator of quality of honey. To that end, the objective of this study was to investigate these quality parameters in honey samples collected during 2013 in the territory of Vojvodina.

MATERIAL AND METHODS
To the purpose of determining the moisture content and total acidity, 50 samples of diff erent honeys originating from Vojvodina region were collected. All samples were in their original packages and were transferred to the laboratory and stored in a cold and dark place. Th e investigated samples included 12 samples of meadow honey, 14 samples of acacia honey, 14 samples of linden honey, 4 samples of multifl ower honey, 5 samples of sunfl ower honey and 1 sample of forest honey.
Moisture content was determined by the refractometric method (Sl. list SFRJ, 1985), using an Abbe refractometer (Model RMT, Optech, Italy). All measurements were performed at 20 º C aft er equilibrium. Th e corresponding % moisture from the refractive index of the honey sample was calculated by consulting a standard table for this purpose.
Th e acidity of honey was determined by volumetric method (Sl. list SFRJ, 1985). Ten grams of honey were dissolved in 75 ml of distilled water and alcoholic solution of phenolphthalein was added. Th e solution was titrated with 0.1 mol/dm 3 NaOH. Acidity (milimol of formic acid per kg of honey) was determined as 10 times the volume of NaOH used in titration.

RESULTS AND DISCUSSION
Th e obtained results on moisture content and total acidity in the examined honey samples are displayed in Table 1.
Moisture content in the examined samples ranged between 14.2 and 20.2%, with an average of 16.5±1.01%. Pursuant to relevant Regulation in Serbia (Sl. list SCG, 2003), which is harmonized with the EU Directive (EU Council 2002), maximum moisture content in honey put in the market is fi xed to 20%. According to the obtained results, moisture content exceeded maximum permitted value in only one sample of sunfl ower honey.
Th e acidity value in the same samples ranged from 7.75 mmol/kg to 44 mmol/kg, with an average of 17.38±6.79 mmol/kg. Pursuant to EU Council (2002), the maximum permitted acidity of honey is 50 meq/kg (the unit meq/ kg is identical with mmol/kg since the acidity is expressed as the content of formic acid). Maximum value permitted by Serbian Regulation (Sl. list SCG, 2003) is somewhat lower, being 40 mmol of formic acid per 1000 g of the sample. Our results revealed that acidity was higher than the maximally permitted level (according to Serbian Regulation) in only one sample of linden honey. Analysis of the results obtained for the investigated parameters in honey samples revealed the lowest average values for water content and acidity in samples of forest honey and acacia honey, respectively. Th e highest average values for both parameters were established in multifl ower honey samples.
Th e composition of organic acids in honey has not yet been adequately investigated; however, some evidence (Rogulja et al., 2009) suggest that acacia, chestnut and meadow honeys are characterized by particularly low contents of organic acids, whilst darker honeys in general appear to be higher in acidity. Our results also demonstrated low acidity of acacia honey as compared with other examined honey types. Th e results obtained for meadow honey do not correspond with the aforementioned evidence, yet the acidity was within the proper range.
Determination of physicochemical parameters in diff erent honeys has been the topic of numerous researches both in Serbia and worldwide. Th e investigation of diff erent quality parameters in 226 honey samples originating from Braničevo and Podunavlje regions during 2010-2012 revealed that honey in this region of Serbia is of good quality. Namely, all samples were characterized by adequate moisture content, and only one sample of acacia honey demonstrated increased acidity (Milošević et al., 2013). Examination of 201 honey samples originating from the entire territory of Serbia (acacia, sunfl ower and linden) was performed during 2009. Th e average moisture content ranged from 16.12% in acacia honey samples to 17.98 in sunfl ower honey samples. Free acidity diff ered widely among the three studied botanical samples, ranging from 11.20 in acacia honey samples to 25.65 meq/kg in sunfl ower honey samples (Lazarević et al., 2012). Th e investigation of the quality of diverse honeys produced in Montenegro (Đuričković et al., 2012) revealed moisture contents ranging from 17.0% in acacia honey to 19.2 in sage honey. Th e lowest and highest total acidity was determined in acacia honey (10 mmol/kg) and sage honey (40.0 mmol/kg), respectively.
Moisture content reported for fi ve honey samples from Portugal ranged from 15.9 to 17.2%, whereas free acidity was within the range 16.0-32.0 meq/ kg (Gomes et al., 2010). Determination of moisture content in 70 honey samples in Turkey revealed as much as 10% of inadequate samples, whereas the acidity values ranged between 6.94 and 29.6 meq/kg (Kahraman et al., 2010). In honey samples originating from India, the highest average values for water content were obtained for mustard honey (21.75 %), whereas eucalyptus and clover honeys had somewhat lower moisture contents (19.4 and 18.7 %, respectively) (Singh and Bath, 1997). Th e acidity level of the examined samples ranged between 29.5 and 41.5 meq/kg. By analyzing the samples of multifl oral honey collected in Venezuela during rainy and dry seasons, De Rodriguez et al. (2004) concluded that climatic conditions are of no importance for moisture content in honey. Namely, one of two honey samples with moisture content above 20% originated from dry season. Th e authors are of the opinion that increased moisture content is more likely associated with insuffi cient maturity of honey rather than with climatic conditions (De Rodriguez et al., 2004). Similar rates of moisture content in honey were reported in Argentina. Th e moisture content in 143 analyzed samples was within a range 16 (Popek, 2002).
As obvious from a brief review of quality control of honey in Serbia and worldwide, the analysis of physicochemical parameters is of vital importance in quality assessment. Although the aforementioned researches encompassed diff erent types of honey, our research demonstrated that the quality of honey from Vojvodina corresponds to that of honeys available in international market.

CONCLUSION
Th e moisture content exceeded the maximum level permitted by the Serbian Regulation in only one of 50 analyzed honey samples. Moreover, in only one sample, the acidity was above the upper limit of 40 mmol of acid per 1000 g of sample (Sl. list SCG, 2003). We can conclude that 96% of investigated samples corresponded with the prescribed quality parameters, which may be taken as indicative of freshness of all honey samples. Nevertheless, potential eff ects of storage conditions on the quality of honey strongly suggest the necessity of continuous monitoring of the aforementioned parameters throughout the year.

AKNOWLEDGMENTS
Th is work is supported by a grant from the Ministry of Education, Science and Technological Development, Republic of Serbia, Project number TR 31084 and IPA Project "PANONIAN BEE" -Increasing of competitiveness in the Cross-border Area No 2012/307-813.