Food Chemistry 276 (2019) 15–21

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Physicochemical properties and mineral content of honey samples from T

Vojvodina (Republic of Serbia)
⁎
Marijana B. Sakača, , Pavle T. Jovanova, Aleksandar Z. Marića, Lato L. Pezob, Žarko S. Kevrešana,
Aleksandra R. Novakovića, Nataša M. Nedeljkovića
a
University of Novi Sad, Institute of Food Technology, 21000 Novi Sad, Bulevar cara Lazara 1, Serbia
b
University of Belgrade, Institute of General and Physical Chemistry, 11000 Belgrade, Studentski trg 12/V, Serbia

A R T I C LE I N FO A B S T R A C T

Keywords: Forty-five honey samples of three honey types – two monofloral (acacia and sunflower) and one polyfloral
Honey (meadow) were collected from the Autonomous Province of Vojvodina (Republic of Serbia) and analysed for
Physicochemical parameters their physicochemical parameters (moisture, acidity, pH, ash, electrical conductivity, glucose, fructose, hydro-
Minerals xymethylfurfural (HMF), CIE L∗a∗b∗ colour and mineral content). All tested physicochemical parameters were in
Botanical origin
agreement with the EU regulation except for moisture (4 samples) and HMF content (1 sample).
Vojvodina
Principal component analysis (PCA) was used for assessing the effects of three honey types on all investigated
Chemical compounds studied in this article: physicochemical parameters. According to PCA, acacia honey samples can be clearly isolated and form a cluster,
Sodium hydroxide (PubChem CID: 14798)
while, regarding other honey types, PCA can offer the possibility to distinguish sunflower and meadow honey
Potassium chloride (PubChem CID: 4873)
samples regarding examined physicochemical parameters and mineral content.
Acetonitrile (PubChem CID: 6342)
Methanol (PubChem CID: 887)
Zinc acetate dihydrate (PubChem CID:
2724192)
Formic acid (PubChem CID: 284)
Nitric acid (PubChem CID: 944)
Hydrochloric acid (PubChem CID: 313)
Tripotassium hexacyanoferrate III trihydrate
(PubChem CID: 71306920)

1. Introduction
Honey is a supersaturated aqueous solution of different carbohy-
drates, including fructose, glucose, maltose, sucrose and other oligo-
and polysaccharides. Glucose and fructose represent the major honey
sugars (85–95%), while sucrose is present in a small amount (Al-Farsi
et al., 2018). It also contains other minor components, such as proteins,
enzymes, amino and organic acids, vitamins, polyphenols, minerals and
other phytochemicals (Amiry, Esmaiili, & Alizadeh, 2017; Čanadanović-
Brunet et al., 2014). Furthermore, some papers reported that honey
contains more than 200 substances (Ferreira, Aires, Barreira, &
Estevinho, 2009). Due to the presence of functional components honey
represents a potent therapeutic agent with bactericidal, antiviral and
antiinflammatory activity (Liu, Ye, Lin, Wang, & Peng, 2013).
Botanical origin is one of the most important properties of honey,
being crucial for consumers’ commitment. Its determination is based on
pollen analysis, but its limitation leads to the development of new
methods which enable an easier approach in determination of honey
botanical and geographical origin. This implies utilization of a range of
quality parameters for honey characterization.
The quality of honey is usually assessed by physicochemical, sen-
sorial and microbiological parameters. The physicochemical properties
depend on the type of nectar (botanical origin), geographical origin
(climate conditions and soil composition) and handling (storage and
transport). There are many papers reporting the physicochemical
parameters of honey, such as moisture, aw, sugar composition, pH,
acidity, ash, electrical conductivity, 5-hydroxymethylfurfural (HMF),
and colour (Boussaid et al., 2018; Lazarević et al., 2012), which are
used to distinguish different honey types. Also, adulteration of honey
can be determined using the quality parameters (da Silva, Gauche,
Gonzaga, & Costa, 2016).
Honey is characterised by a tendency to crystallize spontaneously at

⁎
Corresponding author.
E-mail address: marijana.sakac@fins.uns.ac.rs (M.B. Sakač).

https://doi.org/10.1016/j.foodchem.2018.09.149
Received 18 July 2018; Received in revised form 21 September 2018; Accepted 24 September 2018
Available online 25 September 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
M.B. Sakač et al. Food Chemistry 276 (2019) 15–21

room temperature. This process leads to problems in processing and Table 1

handling. The way to avoid crystallization during honey storage and Location, type and number of honey samples.
handling is to apply thermal processing not only to dissolve the crystals Location Town/District Type Number of samples
but to suppress microbial contamination of honey (Kabbani, Sepulcre, &
Wedekind, 2011). However, thermal treatment can be followed by a Subotica/Bačka acacia 1
Novi Sad/Bačka 1
number of undesirable reactions, among which browning and colour
Sombor/Bačka 1
changes as well as the development of some food contaminants, e.g. Kula/Bačka 1
formation of HMF occur (Bulut & Kilic, 2009). HMF can be converted Vršac/Banat 3
into cytotoxic and mutagenic compound 5-sulfoxymethyfurfural Zrenjanin/Banat 1
(Capuano & Fogliano, 2011). The generation of HMF also happens Kikinda/Banat 2
Bela Crkva/Banat 1
under inadequate handling and storage conditions (temperature, pH,
Bogatić/Srem 1
moisture) of honey (Önür et al., 2018). Šid/Srem 2
Colour of honey is one of the most important sensorial parameters Sremska Mitrovica/Srem 1
for consumers, which varies widely. Lightly coloured honeys are more Vrbas/Bačka meadow 1
Zemun/Bačka 1
attractive, but brownish tones are also accepted in some regions (Saudi
Apatin/Bačka 1
Arabia, Germany). The colour depends on the type of honey and some Srbobran/Bačka 1
chemical characteristics (mineral content) (González-Miret, Terrab, Bačka Topola/Bačka 1
Hernanz, Fernandéz-Recamales, & Heredia, 2005). It is possible that Vršac/Banat 1
transition elements existing in honey form the complexes with honey Bela Crkva/Banat 1
Plandište/Banat 2
organic compounds which tend to be highly colourful and, therefore,
Pančevo/Banat 1
affect colour of honey (Harris, 2014). Colour changes of honey may Sremska Mitrovica/Srem 1
occur slowly during storage or rapidly during thermal treatment as a Ruma/Srem 2
result of Maillard reaction (da Silva et al., 2016). Šid/Srem 2
Mineral content of different types of honey reflects the botanical Odžaci/Bačka sunflower 1
Vrbas/Bačka 1
origin and the type of soil on which plant and nectar were found Apatin/Bačka 1
(Alqarni, Owayss, Mahmoud, & Hannan, 2014). The mineral content of Bačka Topola/Bačka 1
honey is in the range of 0.04–0.2% (da Silva et al., 2016), although Kula/Bačka 1
Alqarni et al. (2014) reported some types containing more than 1%. Vršac/Banat 1
Alibunar/Banat 2
Potassium is the most abundant element, representing approximately
Bela Crkva/Banat 2
one-third of the total mineral content (Bogdanov, Haldimann, Pančevo/Banat 1
Luginbühl, & Gallmann, 2007) or even more (> 70%) according to Zrenjanin/Banat 1
some other authors (Fernández-Torres et al., 2005; Vanhanen, Bečej/Banat 1
Emmertz, & Savage, 2011). Sremska Mitrovica/Srem 2
Total 45
The Republic of Serbia is characterised by a long-lasting tradition in
apiculture with great honey annual production of 4000–6000 t and an
exceptional average annual export growth rate in quantity (32.8%) in beyond the reach of predators. The honey was extracted by hand from
the period 2006–2015 (Pocol, Ignjatijević, & Caviccioli, 2017). Ac- the hives using pressure or wooden presses. Approximately 0.5 kg of
cording to the Statistical Office of the Republic of Serbia (2018) the each honey sample was purchased directly from the collectors to carry
honey annual production was over 7000 t in 2017 in which the Au- out the present study. The samples were stored at room temperature in
tonomous Province of Vojvodina participated with more than 2000 t a dark place until analyses.
(nearly 30%). The increasing honey production and increasing trend in
export growth rate suggest that honey from Serbia has to fulfil re-
2.2. Moisture content
quirements for honey defined in the EU.
The main types of monofloral honey produced in the Republic of
The moisture content was measured using a refractometer
Serbia are acacia, sunflower and linden honey (Lazarević et al., 2012)
(Schmidt/Haensch ATR-ST, Labexchange – Die Lab orgerätebörse
as well as polyfloral honey (meadow), and this production profile is
GmbH, Burladingen, Germany). A few drops of honey were placed on
similar to neighbouring Romania for the last decade (Isopescu,
the refractometer prism and a reading was taken directly from the
Josceanu, Colta, & Spulber, 2017). Due to the increasing potential of
display (AOAC, 2000).
Serbia for exporting honey and the necessity of ensuring its quality and
authenticity, the aim of this study was to evaluate the physicochemical
parameters of honey samples collected from the Autonomous Province 2.3. Acidity
of Vojvodina (Republic of Serbia). In this work, 45 honey samples from
22 different geographical origins, produced in 2016, were examined The free acidity was determined by a titrimetric method using a
regarding following physicochemical parameters: moisture, acidity, pH, solution containing 10 g honey in 75 mL of CO2-free distilled water
ash, electrical conductivity, glucose, fructose, hydroxymethylfurfural (AOAC, 2000). The titration was carried out using 0.1 M NaOH and
(HMF), colour (CIE L*a*b*) and mineral content. stopped at pH 8.5. The acidity was calculated as 10 times the volume of
0.1 M NaOH used for neutralization of 10 g of honey and expressed in
2. Materials and methods milliequivalents of acid per kg of honey (meq/kg).

2.1. Collection of samples 2.4. pH measurement

Forty-five honey samples (15 acacia, 15 sunflower, and 15 meadow)
harvested in 2016 from the Autonomous Province of Vojvodina in the
Republic of Serbia were used in this study. The samples were collected
from several locations in Vojvodina (Table 1). Honey samples were
obtained from traditional beehives placed in trees or other places
The pH was measured on 20% w/v honey:water solutions using the
same method of dilution as in the conductivity measurements. A
SevenEasy™ pH meter (Mettler Toledo, Urdorf, Switzerland) and InLab
427 electrode (Mettler Toledo) was used to measure the pH. The probe
was calibrated with pH standards, 4.01 ± 0.02 and 7.01 ± 0.02, prior

16
M.B. Sakač et al.
to each measurement (Bogdanov, 2002).
2.5. Ash content
The ash content was determined according to the methods of AOAC
(2000). Five grams of honey were placed in combustion pots, which
required preheating to darkness with a gas flame to prevent honey
foaming. Thereafter, the samples were incinerated at high temperature
(550 °C) in a burning muffle for 5 h. After cooling at room temperature,
the obtained ash was weighed.
2.6. Electrical conductivity
The electrical conductivity was measured on a 20% w/v honey:-
water solution, on a dry matter basis (Bogdanov, 2002). The moisture
content of the individual honeys, as determined by refractometry, was
used to calculate the correct dilutions. The conductivity measurements
were conducted using a RIAC conductivity meter CM 100/E with an YSI
3418 electrode (Yellow Springs Instruments Inc., Yellow Springs, OH).
The meter was calibrated with a 1413 μS/cm (0.1 M KCl) standard so-
lution at 20 °C.
2.7. Glucose and fructose analysis
For determination of sugar composition (glucose, fructose) honey
sample was dissolved in 3-fold higher volume of demineralized water
and an aliquot was diluted with 2-fold higher volume of acetonitrile
and kept at −18 °C until analysis. Prior to HPLC analysis prepared
samples were filtered through 0.45 µm pore size PTFE filters (Rotilabo-
Spritzenfilter 13 mm, Roth, Karlsruhe, Germany) before injection into
the HPLC system. A high-performance liquid chromatography method
(obtained with Agilent, Zorbax Carbohydrate 4.6 × 250 mm, 5 μm
column, Agilent Technologies Inc., USA) was applied to quantify sugars
(glucose and fructose) in honey samples.
HPLC analysis was performed using a liquid chromatograph
(Agilent 1200 series), equipped with an evaporative light scattering
detector (ELSD), on an Agilent, Zorbax Carbohydrate 4.6 × 250 mm,
5 μm column (Agilent Technologies, USA) with acetonitrile and water
(75:25, v/v) as a mobile phase at a flow-rate of 1.100 mL/min. Total
run time was 12 min. The column temperature was 30 °C, and 10 μL of
the sample was injected using autosampler.
Identification of glucose and fructose was achieved by comparing
their retention time values with those of standards: D-(+)-glucose
monohydrate (≥99.5%, Sigma-Aldrich) and D-(-)-fructose (≥99.5%,
Sigma-Aldrich). The external standard method was a technique used for
quantification.
2.8. Hydroxymethylfurfural (HMF) analysis
Sample preparation: The extraction procedure was performed ac-
cording to the method proposed by Rufián-Henares and De La Cueva
(2008) with the modifications which were done by Petisca, Henriques,
Pérez-Palacios, Pinho, and Ferreira (2014). Ten grams of sample were
suspended in 5 mL water:methanol (70:30). The mixture was thor-
oughly stirred during 1 min and then 2.0 mL Carrez I and Carrez II
solutions (Carl Roth GmbH, Germany) were added and centrifuged at
5000 rpm (4 °C) during 15 min, recovering the supernatant to a 15 mL
flask. Two more consecutive extractions were made with 2 mL of wa-
ter:methanol (70:30) until collecting 10 mL of supernatant. Two milli-
liters of this solution were centrifuged at 8000 rpm for 15 min before
being analysed.
HPLC-DAD analysis: The chromatographic separation and quantifi-
cation of HMF was performed using the HPLC method described by
Ariffin, Ghazali, and Kavousi (2014) and Tomasini et al. (2012) with
some modifications.
Liquid chromatograph (Agilent 1200 series, Agilent Technologies,
17
Food Chemistry 276 (2019) 15–21
Santa Clara, CA, USA), equipped with a DAD detector and an Eclipse
XDB-C18, 1.8 μm, 4.6 × 50 mm column (Agilent) was used for quanti-
fication of hydroxymethylfurfural in the obtained extracts. Separation
of the analyte was achieved with a column temperature of 30 °C and
sample injection volume of 2 µL. The mobile phase consisted of two
eluents, H2O (0.1% HCOOH) (A) and methanol (B), delivered at a flow
rate of 0.75 mL/min. The isocratic elution was employed with the ratio
A:B (90:10, v/v). The DAD wavelength was set at 284 nm. The total run
time of the analysis was 5 min.
2.9. Colour
The colour of honey samples was measured using a Minolta
Chromameter (Model CR-400, Minolta Co., Osaka, Japan). The samples
were placed in 20-mm-thick holders and measured against a black-and-
white background. The weight of honey sample was 20 g and the
measurement was done at room temperature (23 ± 1 °C).
CIE L*a*b* coordinates were measured, where L* is the luminance
component (ranging from 0 to 100), while a* and b* are colour co-
ordinates related respectively with the red/green and yellow/blue
spectral ranges, with values varying from −120 to +120.34.
Colour measurements were taken from each sample at five points (1
central and 4 corner points). Measurements were made in 25 replica-
tions.
2.10. Mineral analysis
The mineral content in honey was determined by AAS after two-
phase dry ashing. Around 5 g of honey was weighted in a ceramic
crucible and put on a hot plate. The temperature of the hot plate was
gradually increased and when no fume was observed the crucible was
transferred into a muffle furnace, which was preheated at 550 °C. After
4 h of ashing the crucible was removed from the furnace and left to
cool. In the next phase 1:1 HNO3 (5 mL) was added to the crucible.
Thereafter, it was put on the hot plate (120–150 °C) in order to eva-
porate HNO3 and transferred into the muffle furnace (550 °C) for 4 h. If
no clean gray-whitish ash was obtained, the addition of HNO3 and
heating was repeated. After final cooling ash was dissolved following
the procedure: after careful addition of 1:1 HCl (10 mL) the crucible
was heated at 100–110 °C until 5 mL was evaporated. The rest 5 mL was
transferred into a 50-mL flask which contained appropriate amount of
Cs and La and water was added to the mark.
Content of K, Na, Ca, Mg, Fe, Cu and Zn was measured using an
atomic absorption spectrophotometer (Varian SPECTRAA-10), with all
parameters (wavelength, slit, flame stoichiometry) set by the manu-
facturer recommendation. Thousand ppm standards were purchased
from AccuStandard (USA) and all calibration curves used were in the
linear range (R = 0.999).
2.11. Statistical analysis
Results were expressed as mean ± standard deviation of triplicate
analyses for all measurements, except the colour determination of the
samples which was performed in 25 repetitions. Analysis of variance
(ANOVA) for comparison of sample means was used to analyse varia-
tions in observed parameters among the samples. All data were pro-
cessed statistically using the software package STATISTICA 10.0
(StatSoft Inc., Tulsa, OK, USA).
Principal component analysis (PCA) was used to discover the pos-
sible correlations among measured variables.
3. Results and discussion
Physicochemical parameters of three types of honey (acacia, sun-
flower, and meadow) harvested in 2016 from the Autonomous Province
of Vojvodina (Republic of Serbia) were investigated. Table 2 presents
M.B. Sakač et al. Food Chemistry 276 (2019) 15–21

Table 2
Physicochemical parameters of different types of honey (acacia, sunflower, and meadow) from Vojvodina (Republic of Serbia).
Type Moisture Acidity pH Ash Conductivity Glucose Fructose HMF
(%) (meq/kg) (%) (mS/cm) (%) (%) (mg/kg)

Acacia (n = 15) Mean value ± SD 16.6 ± 1.82a 9.77 ± 2.08a 3.99 ± 0.14a 0.08 ± 0.02a 0.25 ± 0.03a 30.1 ± 1.56a 34.3 ± 2.08a 2.57 ± 1.96b
Min 13.2 6.55 3.74 0.05 0.20 27.8 31.7 0.44
Max 21.3 13.4 4.19 0.11 0.28 33.1 39.4 8.20
Samples exceeding limits 1 0 – – 0 – – 0

Meadow (n = 15) Mean value ± SD 17.8 ± 1.55a 19.3 ± 1.88b 3.95 ± 0.12a 0.13 ± 0.03b 0.71 ± 0.08b 30.6 ± 1.44a 33.7 ± 1.24a 3.37 ± 2.77b
Min 15.6 16.0 3.79 0.08 0.43 28.4 31.8 0.58
Max 19.7 22.5 4.19 0.17 0.76 33.8 36.2 69.5
Samples exceeding limits 0 0 – – 0 – – 1

Sunflower (n = 15) Mean value ± SD 17.7 ± 3.18a 19.1 ± 3.19b 3.88 ± 0.25a 0.15 ± 0.04c 0.73 ± 0.07b 31.2 ± 1.52a 35.9 ± 1.25b 1.19 ± 1.12a
Min. 10.2 8.30 3.38 0.08 0.57 29.4 31.8 0.50
Max. 24.1 21.5 4.16 0.22 0.83 34.1 36.2 5.14
Samples exceeding limits 3 0 – – 0 – – 0

SD – standard deviation; Min – minimum value; Max – maximum value.

Means in the same column with different superscript are statistically different (p ≤ 0.05).

the quality parameters (moisture, acidity, pH, ash, electrical con-
ductivity, glucose, fructose, and HMF) of 15 acacia, 15 meadow and 15
sunflower honey samples.
The moisture content depends on the botanical origin of honey,
relative humidity in the region and the processing and storage condi-
tions (da Silva et al., 2016; Yücel & Sultanoglu, 2013). Lower moisture
content can lead to the development of caramelisation and Maillard
reaction during honey storage, while higher water content might cause
honey fermentation and formation of acetic acid (Chirifie, Zamora, &
Motto, 2006), both considered undesirable. Many different parameters
of honey can be affected by moisture content – crystallization, viscosity,
solubility, taste, and colour (Escuredo, Míguez, Fernández-González, &
Seijo, 2013).
The moisture content is within the range of 13.2–21.3%,
15.6–19.7% and 10.2–24.1% for acacia, meadow and sunflower honey,
respectively, meaning that some samples (1 in the group of acacia
honey and 3 in the group of sunflower honey) exceeded the limit
(> 20%) (Codex Alimentarius Commission, 2001). The honey samples
which did not meet the criterion for moisture content could be unstable
during storage and susceptible to spoilage by yeast fermentation and
bacteria found in honey. Although variation in moisture content be-
tween different honey botanical origins was noted in the literature
(Gallina, Stocco, & Mutinelli, 2010), there were no significant differ-
ences (p ≤ 0.05) in this parameter between examined honey types.
The obtained results are close to those previously reported by
Vanhanen et al. (2011) and Boussaid et al. (2018). Since Lazarević et al.
(2012) analysed honey samples from Serbia it is noteworthy to point
out that moisture content obtained by the mentioned authors (acacia
16.48 ± 1.3% and sunflower honey 18.07 ± 1.5%) did not differ
from the results obtained in this study (Table 2). In addition, moisture
content of honey samples from Croatia is in line with our results (Šarić,
Matković, Hruškar, & Vahčić, 2008).
Acidity is related to the presence of organic acids and some other
compounds in honey (lactones, esters and inorganic ions) (da Silva
et al., 2016). Phenolic acids, vitamin C and proteins existing in honey
act as H donors and consequently also contribute to honey acidity
(Halliwell & Gutteridge, 1989). During honey deterioration the fer-
mentation of sugars into organic acids occurs leading to the increased
free acidity. The maximum level of acidity in honey is 50.00 meq/kg
(Codex Alimentarius Commission, 2001) and regarding this defined
level all of the investigated honey samples met the requirement
(Table 2). Honey with the lowest acidity was acacia with the mean
value of 9.77 ± 2.08 meq/kg, while meadow honey was characterised
by the highest mean value (19.3 ± 1.88 meq/kg), which did not differ
from sunflower honey (19.1 ± 3.19 meq/kg). Lazarević et al. (2012)
also found the lowest free acidity in acacia honey (11.6 meq/kg), while
the highest was detected in sunflower honey (27.2 meq/kg). Similar
results for honey acidity were published by Šarić et al. (2008), who
found the significant difference in acidity between acacia and meadow
honey. Acidity of acacia honey is positively correlated to moisture
content (r = 0.563) and negatively correlated to colour parameter L*
(r = -0.612), while acidity of sunflower honey samples is negatively
correlated to ash content (r = -0.574).
The pH is an indicator of possible microbial growth, because pH
between 7.2 and 7.4 is the optimum for most microorganisms.
Therefore, pH values ranging from 3.2 to 4.5 consider acceptable for
honey samples (da Silva et al., 2016). The pH mean values of honey
samples from Vojvodina were acidic and relatively close, ranging be-
tween 3.88 ± 0.25 for sunflower honey and 3.99 ± 0.14 for acacia
honey (Table 2). Although differences in acidity between examined
honey types existed there were no variations in pH values (p ≤ 0.05)
(Table 2). Alqarni, Owayss, and Mahmoud (2016) also found that these
two parameters are not directly related to each other because of the
buffering actions of various acids and presence of minerals. Namely, the
acidity of honey depends on the presence of organic acids in equili-
brium with their corresponding lactones, internal esters and some in-
organic ions, such as phosphates, sulphates and chlorides (Alvarez-
Suarez et al., 2010). The range of pH values (3.49–4.56) reported by
Lazarević et al. (2012) is consistent with our results.
The ash content of all honey samples varied between 0.05% and
0.22% with the significant differences (p ≤ 0.05) between honey types
(Table 2), meaning that mineral content can serve to distinguish honey
types from investigated plant origins. In addition, if the mineral content
of all analysed honey samples was calculated by adding together each
of mineral determined in honey (Table 3) the obtained mean of total
mineral content ranged from 297 mg/kg (acacia honey) to 793 mg/kg
(sunflower honey), and the differences between honey types were also
statistically significant (p ≤ 0.05). The obtained values are comparable
with those obtained by Vanhanen et al. (2011), who found the range of
126–4060 mg/kg for the mean of total mineral content for 18 de-
termined elements in monofloral honey from New Zealand. The lowest
mean total mineral content in acacia honey was previously confirmed
by Šarić et al. (2008).
The predominant mineral in all honey samples was K, followed by
Ca, Mg, Zn and Fe, while Na and Cu were under limits of quantification
for those minerals (< 50 and < 1, respectively) (Table 3). Similar mi-
neral hierarchy order was identified by Alqarni et al. (2014). Potassium
was reported to be the most abundant mineral in honey (da Silva et al.,
2016) and this finding was confirmed in our study, being the highest in
sunflower honey (mean value 590 mg/kg) and the lowest in acacia
honey (mean value 239 mg/kg). Contrary to our findings, Alqarni et al.
(2014) discovered that acacia honey from Saudi Arabia had the highest

18
M.B. Sakač et al. Food Chemistry 276 (2019) 15–21

Table 3 decomposition of monosaccharides or during the Maillard reaction.

Mineral content of different types of honey (acacia, sunflower, and meadow) Codex Alimentarius Commission (2001) defined a maximum level of
from Vojvodina (Republic of Serbia). 40.00 mg/kg for HMF in honey originated from nontropical regions.
Type of honey K Na Mg Zn Ca Cu Fe Most of acacia and sunflower honey samples could be considered
fresh as their HMF mean values (Table 2) were lower than 4.12 mg/kg
(mg/kg) reported as a limit for honey freshness by Tornuk et al. (2013) and they
fulfilled the above mentioned requirement (max 40.00 mg/kg).
Acacia (n = 15) Mean 239a < 50 6.20a 0.78a 32.8a <1 1.24a
value Sunflower honey samples differed in HMF from two other honey
Min 23.8 2.50 0.10 1.67 0.50 types, but there was no significant difference (p ≤ 0.05) in HMF content
Max 763 22.6 4.50 172 10.8 between acacia and meadow honey samples. This finding confirms that
Meadow Mean 468b < 50 20.1b 1.84a 137b <1 0.79a HMF serves as an indicator for honey freshness, but not for determi-
(n = 15) value nation of honey botanical origin. Among meadow honey samples one
Min 136 2.50 0.10 8.81 0.50 had HMF of 69.5 mg/kg and did not meet the criterion defined by the
Max 755 35.5 6.69 219 1.59
Codex Alimentarius Commission (2001). Since the amount of 10 mg/kg
Sunflower Mean 590c < 50 24.5b 1.73a 162b <1 0.89a HMF naturally presents in honey (Alqarni et al., 2016) this significant
(n = 15) value
increase might be addressed to overheating or inadequate storage
Min. 217 14.8 0.49 97.1 0.50
Max. 1192 30.8 9.04 269 5.19 (Yücel & Sultanoglu, 2013) or could be a result of adulteration by
adding invert sugar syrup (Al-Farsi et al., 2018).
Min – minimum value; Max – maximum value. The mean value of glucose ranged from 30.1% to 31.2% for all in-
Means in the same column with different superscript are statistically different vestigated honey samples with no significant differences (p ≤ 0.05)
(p ≤ 0.05). between honey types, while fructose varied between 33.7% and 35.9%
being different in the case of sunflower honey (Table 2). According to
K content among other tested honey types, which indicates the influ- the Codex Standard for Honey (Codex Alimentarius Commission, 2001)
ence of type of soil on the mineral content of honey. the total value of glucose and fructose should be above 60%. The mean
Our K values are in agreement with amounts of K measured in of total glucose and fructose content was 64.4% in acacia, 64.3% in
monofloral honey from New Zealand (Vanhanen et al., 2011), re- meadow, and 67.1% in sunflower honey samples, and, therefore, all
presenting 74% of the total mineral content in meadow and sunflower examined honey samples fulfilled the requirement. Šarić et al. (2008)
honey, while this ratio was even 86% in acacia honey. Vanhanen et al. found similar values for total glucose and fructose content in acacia
(2011) found that 73% of the total mineral content is addressed to (65.0–75.7%) and meadow (62.1–76.1%) honey samples collected in
potassium, Fernández-Torres et al. (2005) reported 72%, but Bogdanov Croatia.
et al. (2007) revealed that K corresponds to only one-third of the total Colour of honey is sensorial parameter important for the consumers.
mineral content. Natural colour of honey derives from various factors such as botanical
Sunflower honey possessed not only the highest K content, but Ca origin, mineral composition, climate, storage conditions, etc. and can
and Mg content (mean values 162 mg/kg and 24.5 mg/kg, respectively) be useful in classifying monofloral honeys (Gonzales, Burin, & del Pilar
(Table 3). The determined mean values of Ca in sunflower and meadow Buera, 1999). Natural honeys with higher mineral content tend to be
honey samples were higher than those reported by Vanhanen et al. darker (González-Miret et al., 2005) due to the formation of colourful
(2011) and Alqarni et al. (2014), who found 7.21–94.3 mg/kg and complexes between transition elements and some organic compounds
63.8–99.9 mg/kg, respectively. Contrary, Mg content in our honey existing in honey (Harris, 2014). The data in Table 4 illustrate the
samples was significantly lower than those published by Alqarni et al. colour parameters of investigated honey samples from Vojvodina. The
(2014). lightness mean value of acacia honey samples was the highest
In addition, local and imported honey samples from Saudi Arabia (73.9 ± 8.90) representing the brightest honey, while sunflower and
were more abundant in Fe (67–98 mg/kg) (Alqarni et al., 2014) in meadow honey were darker, with L* of 64.5 ± 7.11 and 57.0 ± 6.10,
comparison with our results (mean values 0.79 mg/kg for meadow and respectively. The green tone was present in all samples (negative a*
1.24 mg/kg for acacia honey), while the differences between Zn content value), dominantly in sunflower honey. The yellow hue (b*) was the
were smaller if the results obtained by Alqarni et al. (2014)
(3.44–5.72 mg/kg) and these obtained in our study (mean values
Table 4
0.78 mg/kg for acacia and 1.84 mg/kg for meadow honey) were com-
Colour parameters (L*, a* and b*) of different types of honey (acacia, sunflower,
pared. and meadow) from Vojvodina (Republic of Serbia).
Electrical conductivity is a common parameter in honey quality
Type of Colour parameters
control being in a positive correlation with ash content and acidity due
honey
to the presence of ions, organic acids and proteins (Yücel & Sultanoglu, L* a* b*
2013). Since minerals are brought into honey primarily with pollen,
electrical conductivity correlates with pollen content of monofloral Acacia Mean 73.9 ± 8.90c −2.24 ± 0.63a 13.9 ± 4.15a
honeys (Kaškonienė, Venskutonis, & Čeksterytė, 2010) and can serve in (n = 15) value ± SD
Min 51.6 −2.86 8.45
determination of botanical origin (Terrab, Gonzales, Díez, & Heredia, Max 82.4 −0.67 24.9
2003). Acacia honey samples were characterised with the lowest elec-
Meadow Mean 57.0 ± 6.10a −1.68 ± 1.15a 31.0 ± 4.65b
trical conductivity (0.25 ± 0.03 mS/cm) (Table 2), which was useful
(n = 15) value ± SD
in determination of this honey type. Nevertheless, this parameter could Min 46.3 −4.28 24.6
not be used to distinguish meadow and sunflower honey types. The Max 68.1 −0.12 40.1
obtained mean values of electrical conductivity were lower than the Sunflower Mean 64.5 ± 7.11b −3.48 ± 1.15b 29.4 ± 5.30b
recommended maximum value of 0.800 mS/cm (Codex Alimentarius (n = 15) value ± SD
Commission, 2001) and close to the data previously published by Min. 52.8 −5.47 23.2
Lazarević et al. (2012) and Šarić et al. (2008). Max. 76.9 −1.86 40.1

The HMF content indicates a degree of honey deterioration caused

SD – standard deviation; Min – minimum value; Max – maximum value.
by a) strong and/or prolonged thermal treatment and b) inadequate or Means in the same column with different superscript are statistically different
prolonged storage conditions (Önür et al., 2018). It is formed by the (p ≤ 0.05).

19
M.B. Sakač et al.
Fig. 1. Principal component analysis (PCA) ordination of physicochemical parameters based on component correlations for the honey samples from Vojvodina
(Republic of Serbia).
lowest in acacia honey, while meadow and sunflower honey belonged
to the same group concerning that colour parameter.
In this paper, principal component analysis (PCA) was applied to
analyse the similarities of the various honeys types collected from the
different locations in Vojvodina (Table 1). PCA was used to allow easy
representation of observed parameters and different types of honey
samples in a two-dimensional diagram. According to the results pre-
sented in Fig. S1, the first five principal components have eigenvalues
greater than 1. These five components explain 75.53% of the data
variation. However, the scree plot shows that the eigenvalues start to
form a straight line after the second principal component, which leads
to conclusion that only the first two principal components (explaining
50.75% of the variation) could be used for the adequate explanation of
the data. Loadings were considered to evaluate correlations between
variables.
The PCA of the presented physicochemical data explained that the
first two principal components accounted for 50.75% of the total var-
iance (36.30% and 14.45%, respectively) in the sixteen variables
(moisture, acidity, pH, ash, conductivity, glucose, fructose, HMF, L*, a*,
b*, K, Mg, Zn, Ca, and Fe) (Fig. 1). Considering the map of the PCA
performed on the data, the negative contribution to the first principal
component calculation (PC1) was observed for several variables: acidity
(11.49%), ash (8.77%), conductivity (14.52%), b* (12.43%), K (8.54%),
Mg (13.86%) and Ca (13.19%). None of the observed variables showed
the significantly positive contribution to the PC1 (Table S1).
The colour parameter a* (17.9%) and moisture (15.9%) showed the
positive influence on the second component calculation (PC2), while
the colour parameter L* (16.0%), glucose (13.1%) and fructose (16.8%)
showed negative score values according to the second principal com-
ponent calculation. Chakir, Romane, Marcazzan, and Ferrazzi (2016)
investigated different types of honey and reported that variables which
loaded highly to the PC1 were based on acidity, colour, HMF and dia-
stase activity, while electrical conductivity contributed markedly to the
PC2. Terrab, Diez, and Heredia (2002) established water content,
acidity, and proline as classification factors. The PCA evaluation of the
physicochemical and phenolic parameters of different types of honey
showed that colour coordinate a*, ash content and electrical con-
ductivity were crucial for distinguishing honey types (Oroian &
Ropciuc, 2017). The same authors revealed that sunflower honey
samples and polyfloral honey groups were closed, because polyfloral
20
Food Chemistry 276 (2019) 15–21
honeys have a great variability of pollen types and cannot be well de-
fined.
PCA analysis shows that the diversity between different honey types
could be determined by L* colour parameter (negative contribution to
the PC2), increased mineral content (Ca, Mg and K) (negative con-
tribution to the PC1), increased moisture content (positive contribution
to the PC1) and colour parameter a* (positive influence on the PC2).
Acacia honey samples are placed on the right side of the PCA graph,
and they are characterised by the highest L* value. At the opposite left
side of the graph, sunflower honey samples can be observed, which are
characterised by increased Ca, Mg and K content. The increased b*
value and enhanced levels of acidity and electrical conductivity are also
specific for sunflower honey samples in comparison with acacia honey
samples. Meadow honey samples are positioned between sunflower and
acacia group, placed at the upper part of the PCA graph. Meadow honey
samples are characterised by the highest moisture content and in-
creased a* colour parameter compared to other honey samples.
4. Conclusions
The physicochemical characterisation of honey samples (acacia,
sunflower and meadow) collected from the Autonomous Province of
Vojvodina (Republic of Serbia) during 2016 was conducted in order to
a) examine the quality of honey samples and b) determine any simila-
rities and differences that may be attributed to honey botanical origin.
All investigated honey samples (45) met the examined quality cri-
teria except 4 samples in the case of moisture content and one for HMF
content meaning that 11% of all samples were not in accordance with
EU regulative.
PCA resulted in splitting all examined honey samples into three
groups, corresponding to specific honey types. The acacia honey sam-
ples are clearly isolated to form a cluster, while, regarding sunflower
and meadow honey types, PCA represents a good tool for their se-
paration based on examined physicochemical parameters.
Acknowledgements
This paper is a result of the research within the project TR31029,
entitled Functional bakery products for the people with metabolic disorders,
financed by the Ministry of Education, Science and Technological
M.B. Sakač et al.
Development, Republic of Serbia.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2018.09.149.
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