Food Research International 64 (2014) 634646

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Steady, dynamic and creep rheological analysis as a novel approach to

detect honey adulteration by fructose and saccharose syrups:
Correlations with HPLC-RID results
Mustafa Tahsin Yilmaz a,, Nevruz Berna Tatlisu a, Omer Said Toker a, Safa Karaman b, Enes Dertli c,
Osman Sagdic a,d, Muhammet Arici a
a

Yildiz Technical University, Chemical and Metallurgical Engineering Faculty, Food Engineering Department, 34210 Istanbul, Turkey
Erciyes University, Engineering Faculty, Food Engineering Department, 38039 Kayseri, Turkey
c
Bayburt University, Engineering Faculty, Food Engineering Department, 69000, Bayburt, Turkey
d
'TBTAK MAM, Food Engineering Institute, 41470, Gebze-Kocaeli, Turkey
b

a r t i c l e

i n f o

Article history:
Received 10 February 2014
Accepted 20 July 2014
Available online 27 July 2014
Keywords:
Honey
Adulteration
Saccharose and fructose syrups
Rheology
HPLC-RID

a b s t r a c t
In this study, natural honey was adulterated with the addition of adulterants, namely saccharose and fructose
syrups at a ratio of 0%, 10%, 20%, 30%, 40% and 50% by weight. Steady, dynamic and creep tests were conducted,
revealing that the changes in the ow, viscoelastic and creep behavior of natural honey were clear and remarkable. Syrup addition decreased viscosity (), storage (G) and loss modulus (G) values of the control honey samples. Deformation represented by the compliance (J(t)) values was more prominent in the adulterated honey
samples. In addition, HPLC-RID analysis was conducted to determine major sugar composition of the adulterated
samples. Pearson's correlation test indicated that there were signicant (P b 0.05; 0.01) correlations between
sugar composition and rheology parameters, (viscosity), K, K (intercepts for G and complex modulus (G),
respectively) and 0 (viscosity of Maxwell dashpot), suggesting that K, K, K and 0 could be prominent indicators for presence of saccharose or fructose syrups added in natural honey within the studied concentration levels.
These results suggested that use of steady, dynamic and creep analysis would be a novel and potential approach
to detect honey adulteration by fructose and saccharose syrups.
2014 Published by Elsevier Ltd.

1. Introduction
Honey, with its high nutritional and benecial properties, is the
oldest natural sweetening agent (Ozdemir, Dagdemir, Ozdemir, &
Sagdic, 2009). Honey is a valuable source of rich nutritious compounds
for the human body such as sugars, macro and micro elements and
biologically active substances (Smanalieva & Senge, 2009). Phenolic
compounds, minerals, proteins, organic acids (gluconic acid, acetic
acid, etc.), free amino acids, enzymes (invertase, glucose oxidase, catalase, phosphatases) and vitamins (ascorbic acid, niacin, pyridoxine,
etc.) are among other minor constituents present in natural honey
(Alvarez-Suarez, Gonzales-Paramas, Santos-Buelga, & Battino, 2010;
Marghitas et al., 2009). Although fructose and glucose, as the predominant monosaccharides, exist in honey with a percentage of 6085, it also
contains maltose and sucrose at lower levels (Doner, 1977; Doner &
Hicks, 1982). It is accepted as a valuable product because of its multiple
benets such as prebiotic (Sanz et al., 2005) as well as nutritional and
antioxidant characteristics (Tornuk et al., 2013). Honey still attracts a

Corresponding author. Tel.: +90 212 383 4575; fax: +90 212 383 4571.
E-mail address: mtyilmaz@yildiz.edu.tr (M.T. Yilmaz).

http://dx.doi.org/10.1016/j.foodres.2014.07.009
0963-9969/ 2014 Published by Elsevier Ltd.

great attention due to its anticarcinogenic (Al-Waili, 2004), antiviral

(Zeina, Othman, & Al-Assad, 1996), anti-fungal (Molan, 1997), antibacterial and anti-inammatory activities (Doner, 1977; Doner &
Hicks, 1982). It is also used as an apitherapy agent due to these characteristics (Ozdemir et al., 2009).
The cost of natural honeybee honey is much greater than that of any
other sweeteners because of its high nutritional value and unique avor
characteristics; therefore producers tended to adulterate honey with
less expensive substances in order to decrease the cost of honey
(Sivakeseva & Irudayaraj, 2002). The most common adulteration
methods are by overfeeding of bees with sugar and other types of sucrose or by adding saccharose (Guler, Bakan, Nisbet, & Yavuz, 2007).
In addition, the natural carbohydrate prole of honey is simulated by
using some of the simple and complex sugars such as corn syrups,
high fructose corn syrups and invert syrups, which are comparatively
inexpensive sweetening products (Swallow & Low, 1994). Addition of
fructose or industrial glucose results in a change of the fructose/glucose
ratio, which has to be 11.2 in natural honey (Puscas, Hosu, & Cimpoiu,
2013). The ratio differing from this value can mean that the honey is
adulterated. However, it is still difcult to understand and evaluate
the adulterations in honey because of variations in honey carbohydrates
and their similarities with sugar syrup composition (Kushnir, 1979),

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

Nomenclature
f
G
G
G
G0
G1
J
JMAX
JSM

K
K
K
R2
tan

50
R (%)

0
1

frequency [Hz]
storage modulus [Pa]
loss modulus [Pa]
complex modulus [Pa]
instantaneous shear modulus of the Maxwell element
(Pa)
shear modulus of KelvinVoigt element (Pa)
creep compliance (Pa1)
compliance at the end of the creep test (Pa1)
compliance pertaining to the Maxwell spring (Pa1)
viscosity [Pa s]
angular frequency [rad s1]
intercept for complex modulus [Pa]
intercept for storage modulus [Pa]
intercept for loss modulus [Pa]
coefcient of determination
loss tangent [dimensionless]
shear rate [s1]
viscosity at 50 s1 (Pa s)
percentage recovery
shear rate (s1)
shear stress (Pa)
viscosity of liquid lling the dashpot of the Maxwell element (Pa s)
viscosity of liquid lling the dashpot of the KelvinVoigt
element (Pa s)

which triggers the importance of method development for quality control of honey and detection of its adulteration.
A great number of efforts have been exerted so far to detect adulteration in honey based on electrochemical analysis (Gritzapis &
Timotheou-Potamia, 1989), enzymatic methods (Le Marec & Lesgards,
1991), thin-layer chromatography (Pukl & Prosek, 1990; Reiffov &
Nemcov, 2006), carbon isotopy (White, 1992), ow injection analysis
(Peris-Tortajada, Puchades, & Maquieira, 1992), gas-chromatography
(Carlsson, Karlsson, & Sandberg, 1992), high-performance liquid chromatography (Antoov, Polakovi, & Ble, 1999; Bugner & Feinberg,
1992), anion-exchange liquid chromatography (Goodall, Dennis,
Parker, & Sharman, 1995; Swallow & Low, 1994), Fourier transform infrared spectroscopy (Sivakesava & Irudarayaj, 2001; Sivakeseva &
Irudayaraj, 2002), differential scanning calorimetry (Cordella et al.,
2002), mid-infrared near infrared transectance spectroscopy (Kelly,
Petisco, & Downey, 2006; Sivakeseva & Irudayaraj, 2002), gas chromatographymass spectroscopy (Ruiz-Matute, Soria, Martinez-Castro, &
Sanz, 2007), high-performance anion exchange chromatography with
pulsed amperometric detection method (Cordella, Militao, Clement, &
Carbol-Bass, 2003; Morales, Corzo, & Sanz, 2008), high-performance
thin-layer chromatography (Puscas et al., 2013), isotope ratio mass
spectrometry in combination with an elemental analyzer (Tosun,
2013) and low eld nuclear magnetic resonance (Ribeiro et al., 2014).
Most of the aforementioned methods are based on time-consuming
chemical or enzymatic reactions requiring long preparation steps and
laborious preliminary experiments as well as expert operators.
Therefore, alternative methods that would allow faster and easier
detection of honey adulteration should be continuously developed
and tested. Accordingly, the EU Commission has also tended to encourage development of harmonized analytical methods to permit
the verication for different honeys (Puscas et al., 2013). In this respect, detection of adulteration based on the changes in physical
and rheological properties of honey may be an alternative and a
very different approach considering the aforementioned methods.

635

Accordingly, the fructose/glucose ratio in honey is a factor determining the crystallization rate of honey, thus directly affecting the rheological, namely physical properties of honey. Therefore, the use of
rheological methods can be a novel and potential approach for detection of honey adulteration by fructose and saccharose syrups. Glucose tended to crystallize more due to its lower solubility (Venir,
Spaziani, & Maltini, 2010). Glucose may crystallize as -D-glucose
monohydrate at temperature ranges lower than 50 C (Venir et al.,
2010). The other two forms, namely, -D-glucose anhydrous and
anhydrous forms, are stable at the temperature range of 5080 C
and at temperatures above 80 C (Young, 1957). The transition temperature of glucose from its monohydrate to anhydrous form is
found to be lower than 30 C when saturated with fructose. In addition, natural honeys exhibit Newtonian behavior and their rheological properties are strongly inuenced by temperature (Gmez-Diaz,
Navaza, & Quintans-Riveiro, 2006; Kumar & Mandal, 2009; Yoo,
2004). However, crystallized honeys show non-Newtonian ow behavior with yield stress and thixotropy (Chen, Lin, Wu, & Chen,
2009; Smanalieva & Senge, 2009). From these reports, it is clear
that the rheological properties of honey are greatly inuenced by
storage temperature and so resultant crystallization. Accordingly,
storage temperature and fructose/glucose (F/G) ratio are regarded
to be determinants for crystal size formed in the product (Lupano,
1997). Honey samples having F/G ratios more than 1.33 do not crystallize for a long time (White, 1978), while those having less than
1.11 ratio crystallizes quickly (Smanalieva & Senge, 2009). These reports also reveal a necessity to detect such adulterants in honeys
stored at different temperature levels. Therefore, temperature
sweep tests should be also conducted to determine temperature dependency of adulterated honey samples.
In the literature, no study has been conducted so far on detection
of adulteration in honey based on its rheological changes. This study
was undertaken to detect adulteration in natural honey by saccharose and fructose syrups at different ratios (0, 10, 20, 30%, 40 and
50%) on the basis of steady, dynamic and creep/recovery rheological
analysis. In addition, HPLC-RID analysis followed to determine the
sugar composition of the adulterated honey samples in order to
conrm the rheological test results by nding possible correlations
between sugar composition and rheological parameters of the adulterated honey samples.
2. Materials and methods
2.1. Materials
Control (natural) honey samples were collected from a local market
in stanbul, Turkey. Saccharose and fructose were obtained from Merck
(Merck, Darmstadt, Germany). The adulterants, namely, saccharose or
fructose syrups, were prepared by slowly adding 150 g of saccharose
or fructose powder to 100 g of water, followed by mixing the mixtures
with a magnetic stirrer at a constant speed. Both syrup types were concentrated to approximately 75 brix at 60 C. For preparation of adulterated honey samples, the prepared syrups were added to natural honey
samples in relevant concentrations (0, 10, 20, 30, 40 and 50%, w/w).
The adulterated honey samples were stirred in a temperaturecontrolled water bath for 30 min at room temperature. Then, the samples were centrifuged for 3 min at 2500 rpm to remove impurities and
were stored at room temperature until the analyses.
2.2. Physicochemical analyses
Color was analyzed by using an automatic colorimeter (Konica
Minolta, model CM-5, Mississauga, ON, Canada) and they were recorded
as the values of L, a, and b. L values measure the level brightness
(0100), a red to green (+ = red and = green), and b yellow to
blue (+ = yellow and = blue). All analyses were carried out in

636

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

triplicate. To measure the turbidity of the samples, a turbidimeter

(HACH, 2100 N, USA) was used and the results were stated as NTU
(nephelometric turbidity unit). The pH values were measured with a
pH meter (WTW-Inolab, Weilheim, Germany) in a solution of 10% (w/
v) honey in distilled water at 25 C. An Aqualab water activity (aw)
meter (Decagon, Pullman, WA) was used for the determination of
water activity of the samples at 20 C. The brix values were determined
using an automatic refractometer (Reichert AR 700, USA) at 20 C. Dry
matter contents were measured by conventional drying method as described (AOAC, 2000). Ash content was determined by incinerating the
samples at 625 C in a mufe oven (Protherm, Ankara, Turkey).

Complex modulus, G, was used to characterize the overall response

of the sample against the sinusoidal strain (Gunasekaran & Ak, 2000).





0 2  2 1=2
G G

Non-linear regression was applied to the plots of G and G versus

data and the magnitudes of intercepts K, K and K, and R2 were computed using the following equations (Kang & Yoo, 2008; Yoo & Rao,
1996).
0

G K

2.3. HPLC analysis

The major sugar (fructose, glucose and saccharose) compositions of
the samples were determined according to the method described by
Jahanbin, Moini, Gohari, Emam-Djomeh, and Masi (2012). For this purpose, 1 g of honey was dissolved in 9 mL of distilled water and the mixture was ltered using a 0.45 m syringe lter. The ltrate was injected
to the HPLC (Agilent 1100, USA) equipped with a refractive index detector (RID). An Agilent Zorbax carbohydrate analysis column (5 m and
4.6 mm 150 mm) was used and HPLC conditions were set as follows:
mobile phase, 80% acetone and 20% water; ow rate, 1.4 mL/min; injection volume, 20 L and the column temperature was set to be 25 C.
Sugars were identied according to their retention times by comparing
with sugar standards. The sugar concentration was calculated by using
the calibration curve of each sugar.
2.4. Rheological analysis
Steady shear, dynamic shear and creep and recovery analyses were
carried out using a stress or strain controlled rheometer (Anton Paar,
MCR 302, Austria) equipped with a peltier system. All of the experiments were performed by a parallel plate conguration (diameter
50 mm, gap 0.5 mm).
2.4.1. Steady shear analysis
The prepared samples were sheared in the range of 0.1100 s1 at
25 C. A total of 25 data points were recorded at 10 s intervals during
the shearing. Each measurement was replicated three times in two different samples (each 1 mL). The apparent viscosity was determined as a
function of shear rate. The ow curves, shear stress versus shear rate,
were plotted by increasing shear rate. Obtained data were tted to a
Newtonian model. The related parameters for this model were
calculated using the following equation:
n

the shear rate (s1) and is the viswhere is the shear stress (Pa),is
cosity of the sample.
2.4.2. Dynamic shear analysis
The amplitude sweep test was performed at 1 Hz in the strain range
of 0.1100% to determine the linear viscoelastic region (LVR). Frequency sweep test was performed at 1% strain (determined by amplitude
sweep test) over a frequency range of 0.110 Hz at 25 C. Each measurement was repeated three times with three replications.
The viscoelastic parameters of G (elastic or storage modulus) and G
(viscous or loss modulus) are calculated using the following equations
(Steffe, 1996).
0

G G cos

G G sin

G K

G K

In order to observe the variation in the steady and dynamic shear parameters by temperature, a temperature sweep test was conducted at a
shear rate of 50 s1 and 1 Hz, respectively, at temperature levels ranging between 5 and 50 C. Briey, temperature sweep test was carried
out to determine dependency of the viscoelastic parameters on
temperature.
2.4.3. Creep and recovery analysis
These tests were conducted at constant stress (0.1 Pa within the
LVR). Deformation of the viscoelastic materials approaching a steady
state in the time when the deformation rate remains constant was the
critical point; after this time, the stress was applied and then suddenly
removed and analyzed for recoverable shear. In this time, the stress
was instantly applied and maintained for 150 s, and then released to
allow sample recovery for a further 150 s. Each measurement was repeated three times with three replications.
Creep parameters were obtained from calculating a constant stress
() over time (t) and expressed using the creep compliance (J) function
as represented by Eq. (8) in terms of shear deformation ():
J t t =

where () was the shear deformation.

The Burger model, consisting of Maxwell and KelvinVoigt models
associated with series, is widely used in the food industry to provide information about the internal structure of a product (Dolz, Hernandez, &
Delegido, 2008). The system deformation per unit stress, namely compliance (J), is a function of time and calculated using the following equation (Eq. (9)) (Steffe, 1996):
J t




1
1
tG1

1 exp

1
G0
G1
|{z}
|{z}
Elastic

Viscoelastic behavior

t
0
|{z}

Viscous flow

where J(t) is the overall compliance at any time t in the creep phase, G0
is the elastic modulus of the Maxwell unit, 0 is the viscosity of the liquid
lling the dashpot of the Maxwell element (Pa s), G1 is the shear modulus of the KelvinVoigt unit, and 1 is the viscosity of the liquid lling
the dashpot of the KelvinVoigt element (Pa s) (Barry, 1983). The values
G0, G1, 0 and 1 can be used to understand the internal structure of a
product (Dolz et al., 2008).
2.5. Method validation
Different honey samples were selected to analyze and test the method validation parameters. Some samples were marked as control in
order to compare the results. The following parameters, namely,

0.13
0.08
0.01
0.02
0.02
0.01

1.64
1.59
1.74
1.41
1.44
0.92
0.42
0.56
0.32
0.33
0.48
0.44
29.77
28.72
25.63
22.66
19.06
15.34
0.09
0.62
0.37
0.65
0.56
0.92

0.206
0.211
0.186
0.164
0.122
0.104

3.1. Physicochemical properties

Table 1 shows the physicochemical properties of adulterants
(saccharose and fructose syrups), HAS (adulterated honey samples
with saccharose syrup) and HAF (adulterated honey samples with fructose syrup). As can be seen, the adulterants were brighter (L values)
than the control honey sample, which was expected since saccharose
and fructose syrups were brighter than honey and the L value generally
increased with the addition of these syrups to honey. On the other hand,
the control honey sample was redder (a values) and yellower (b values)
than the adulterants. Expected results were generally observed for the L,
a and b values that were between those values of the adulterants and
the control honey sample. However, such trends were not observed in
the turbidity, but these values generally decreased depending on the addition of the adulterants. In pH values, a consistent trend was observed,
decreasing linearly with adulterant addition. Expected results were observed in the aw values which increased with the addition of adulterants
having higher aw values. For brix, dry matter and ash content values, no
clear trend was observed with the adulterant addition.
HAS and HAF were the adulterated honey samples with saccharose and fructose syrups, respectively.
a

81.99
77.11
78.06
79.87
81.09
82.73

0.01
0.01
0.01
0.01
0.01
0.01

8.29
5.63
4.47
3.05
1.94
0.79

0.01
0.01
0.02
0.02
0.02
0.01

77.02
42.13
41.13
39.79
37.86
35.21

0.02
0.02
0.01
0.01
0.01
0.01

72.70
38.30
24.10
22.37
17.70
16.53

2.21
1.47
0.10
0.47
0.00
0.41

0.01
0.02
0.04
0.05
0.02
0.01

0.539
0.567
0.593
0.607
0.619
0.624

4.13
3.93
3.81
3.81
3.74
3.59

0.01
0.01
0.01
0.01
0.02
0.02

81.69
80.66
79.85
78.41
78.46
77.63

0.31
0.08
0.59
0.39
0.21
0.25

85.17
83.93
82.46
82.54
81.59
84.17

0.18
0.22
0.19
0.39
0.19
0.12

0.01
0.01
0.05
0.08
0.01
0.01
2.21
2.65
1.79
2.59
0.36
1.58

72.70
104.0
89.37
53.07
25.70
21.80
0.02
0.01
0.01
0.01
0.01
0.01

77.02
73.05
67.66
61.71
56.34
47.83
0.01
0.01
0.01
0.01
0.01
0.02

8.29
6.21
4.34
2.63
1.47
0.17
0.01
0.01
0.01
0.01
0.01
0.02

81.99
81.33
82.70
84.66
85.81
87.32

0.04 0.00
0.66 0.00

Adulterants
Saccharose syrup
Fructose syrup
Adulterated honey samples
HASa
0% (control honey)
10%
20%
30%
40%
50%
HAFa
0% (control honey)
10%
20%
30%
40%
50%

92.00 0.14
95.81 0.00

0.539
0.574
0.581
0.634
0.643
0.692

4.13
4.01
3.93
3.92
3.78
3.18

0.01
0.01
0.01
0.01
0.02
0.02

81.69
79.77
80.33
77.69
78.26
77.55

0.31
0.87
0.29
0.39
0.25
0.32

85.17
84.56
84.26
83.31
83.40
82.37

0.18
0.41
0.43
1.04
0.58
2.24

0.206
0.283
0.220
0.202
0.193
0.142

0.09
0.23
0.13
0.71
0.06
0.49

29.77
32.31
28.69
27.81
24.04
21.73

75.36 0.00

75.36 0.01
74.72 0.01
0.759 0.01
0.661 0.01
3.80 0.51
2.17 0.01

90.63 0.15
23.30 0.10

5.08 0.01
4.13 0.01

75.36 0.62
74.72 0.13

32.47
35.71
37.54
40.55
43.16
44.44
0.01
0.03
0.01
0.01
0.01
0.00

1.64 0.13
9.49 0.26
12.21 0.07
16.72 0.22
22.65 0.01
33.07 0.08

3. Results and discussion

0.42
0.25
0.06
0.42
0.22
0.27
32.47
33.34
30.42
29.45
26.45
23.13
0.01
0.03
0.02
0.01
0.01
0.01

74.72 0.00

Glucose (%)

Sugar composition

repeatability, sensitivity and linearity, were used to validate the analytical methods.

SPSS Statistics (SPSS Statistics 17.0, Armonk, NY, USA) was used
to conduct ANOVA to show the effect of adulterant levels on steady
and dynamic shear parameters as well as to perform validation tests
(P b 0.05; 0.01). Bivariate correlations between sugar composition
and rheology parameters of adulterated honey samples were analyzed
by Pearson's test using Minitab 14.0 software. Principal component
analysis (PCA) was performed using XLSTAT software (XLSTAT, 2008,
Addinsoft, New York, NY) to categorize the honey samples based on
their sugar composition and rheological parameters.

aw

Chemical properties

Turbidity (NTU)
b
a
L

Physical properties
Samples

Table 1
Physicochemical properties and sugar composition of samples.

637

2.6. Statistical analysis

pH

Brix

Dry matter (%)

Ash (%)

Fructose (%)

Saccharose (%)

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

3.2. HPLC analysis

Fig. 1 shows the chromatograms of the standard mixture of
sugars and adulterated honey samples with different levels of
saccharose/fructose syrups using an Agilent Zorbax carbohydrate
analysis column operating with 80% acetone and 20% water as
mobile phase at 25 C. The method allowed separation of all analytes
that could be detected by an RID detector. The internal standards
(fructose, glucose and sucrose) were eluted at 6.32, 7.91 and 14.99 min,
respectively, without interfering with the elution of the other standards.
For each compound, a linear regression was performed and the regression equations were y = 0.007x 224.5, y = 0.0069x + 2398.8
and y = 0.0072x 534.2 for fructose, glucose and sucrose standards,
respectively. The determination coefcients (R2) were N0.993, indicating that there was a linear relationship between the chromatographic
response areas and the concentrations for all the compounds. The instrument detection limit (IDL) for each compound was measured
based on the signal to noise ratio of 3 and ranged between 20 and
160 mg/L.
Table 1 shows the major sugar composition of adulterants and adulterated honey samples. As can be seen from the table, the HPLC results
reected the expected trends in the change of sugar composition. The
saccharose content of the control honey sample increased with the increase in added saccharose level and the saccharose content of HAS linearly increased as the saccharose level increased. This was also the case
for the HAF with the fructose content linearly increased with fructose
addition. Regarding the fructose content of HAS and the saccharose content of HAF, they were observed to decrease with increase in saccharose
or fructose level, respectively. However, it should be also noted here

638

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

Fig. 1. HPLC-RID chromatograms for peaks of standards (fructose, glucose and saccharose) and adulterated honey samples with different levels of saccharose and fructose syrups.

that no clear trend was observed in the fructose contents of HAS and
saccharose contents of HAF although these contents were determined
to show a generally decreasing trend. The possible reason could be attributed to the fact that the saccharose might have been inverted with
the help of acids and enzymes (Tosun, 2013) naturally present in
honey in the course of time in both cases, namely HAS and HAF.
3.3. Steady shear properties
Table 2 shows the Newtonian model parameters for adulterants and
adulterated honey samples with different levels of saccharose and
fructose. All samples (including control honey) had Newtonian ow
behavior. It is well known that natural honeys exhibit Newtonian ow
behavior (Juszczak & Fortuna, 2006; Karaman, Yilmaz, & Kayacier,
2011; Kumar & Mandal, 2009; Lazaridou, Biliaderis, Bacandritsos, &
Sabatini, 2004; Yoo, 2004). From the table, it is also clear that saccharose
and fructose syrup addition signicantly (P b 0.05) decreased the
viscosity of the control (natural) honey sample and viscosity decreased
(P b 0.05) as the levels of these adulterants increased. These results
were also evident from those presented in Fig. 2 which shows the
shear stress data as a function of shear rate for adulterants and adulterated honey samples. Shear stress values of all the samples linearly increased with increase in shear rate, indicating that all samples showed
Newtonian ow behavior (Rao & Tattiyakul, 1999; Sikora, Kowalski,
Tomasik, & Sady, 2007; Steffe, 1996). The results in Fig. 2 also proved
that shear stress values of the adulterated honey samples decreased as
the level of adulterants, namely, saccharose and fructose syrups, increased, revealing that syrup addition decreased the viscosity of natural
honey. This result was expected since viscosity of saccharose and fructose syrups was found to be 0.297 Pa s and 1.265 Pa s, respectively,
which were lower than that of the control honey sample (6.531 Pa s).
These results clearly suggest that adulteration in natural honey can be
detected by steady shear rheological analysis.

Fig. 3 shows the temperature sweep test results indicating the effect
of temperature (from 5 C to 50 C) on apparent viscosity at shear rate
50 s 1 (50) values of adulterants and adulterated honey samples
with different levels of fructose and saccharose syrups. As can be seen,
50 values of all the samples linearly decreased as the temperature
level increased. The thermal energy of the molecules and the intermolecular distances between them increased with increasing temperature,
which results in reduction of intermolecular forces; therefore viscosity
of the samples decreases (Arslan, Yener, & Esin, 2005; Hassan &
Hobani, 1998; Holdsworth, 1971). But, it should be noted here that

Table 2
Newtonian model parameters dening ow behavior of samples.
Samples
Adulterants
Saccharose syrup
Fructose syrup
Adulterated honey samples
HAS
0% (control honey)
10%
20%
30%
40%
50%
HAF
0% (control honey)
10%
20%
30%
40%
50%

(Pa s)

R2

0.297
1.265

0.999
0.999

6.531a
5.650b
3.704c
2.972d
2.239e
2.019f

0.998
0.996
0.998
0.998
0.998
0.999

6.531a
4.028b
2.462c
2.067d
1.598e
1.085f

0.998
0.998
1.000
0.997
0.997
0.999

Different lowercase letters show differences (P b 0.05) between the adulteration levels.
HAS and HAF were the adulterated honey samples with saccharose and fructose syrups,
respectively.

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

639

Fig. 3. Temperature sweep tests indicating the effect of temperature on apparent viscosity
values (50) at a shear rate of 50 s1 of adulterants and adulterated honey samples (Control, natural honey; F, fructose syrup; S, saccharose syrup).

Fig. 2. Shear stress versus shear rate data for adulterants and adulterated honey samples
(Control, natural honey; F, fructose syrup; S, saccharose syrup).

temperature did not inuence the trend of 50 values to decrease as the

adulterant level increased, suggesting that honey adulteration can be
detected in honeys within a temperature range between 5 C and
50 C. Furthermore, steady shear analysis showed the great deviations
of the ow curves from those of control and adulterated honey samples
with 10% of saccharose and fructose syrups (Fig. 3). This result revealed
that it was possible to detect adulteration in honey even at the 10% level
within a temperature range of 520 C.
3.4. Dynamic shear properties
G (storage modulus) versus G (loss modulus) values of adulterants
and adulterated honey samples are shown in Fig. 4. As can be seen the G
and G values of all the samples increased with frequency. But, attention
should be drawn to the fact that the G values showed non-linear increment while the G values exhibited a linear increment. This means that,
in dynamic rheological characterization, G values should be taken into
consideration to reach a conclusion for viscoelastic properties of adulterated honey samples. Another point that should be taken into account
was that magnitudes of the G values were remarkably higher than
those of the G values, indicating that both adulterants and adulterated

honey samples had viscous nature rather than elastic. In addition, no

cross-point of G and G was observed along the whole frequency
range studied. From a structural point of view, it can be stated that the
honey samples exhibited liquid-like behavior because G values were
higher than G values.
G, G and G values were subjected to non-linear regression as a
function of frequency (Eqs. (5), (6) and (7)) to calculate the magnitudes
of intercepts K, K, and K along with their R2 values. Based on the high
R2 values (Table 3), it was possible to say that the Newtonian model was
successful in modeling the dynamic shear behavior of adulterated
honey samples. Table 3 also shows the effect of adulterants on
the dynamic shear behavior of samples. It is seen that K and K values
linearly decreased (P b 0.05) as the adulterant level increased. This decrease (P b 0.05) was also the case for the K values, but it was nonlinear. Given that the indices for complex modulus, K, represent total
resistance to deformation of a material considered to be elastic solid, it
is possible to say that the control honey had the highest total resistance
to deformation and this resistance decreased with adulterant addition.
These results suggested that K would potentially be a good indicator
to detect adulteration at the levels ranging between 10 and 50%.
Detection of honey adulteration is of great concern to the food industry; therefore, numerous techniques have been developed and applied
so far. Previously, traditional methods were used to detect impurities,
but they are now rarely used because of their poor specicity
(Hudson, 1942; Seoane, Moresco, & Sansn, 2008). For this purpose, in

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M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

Fig. 4. G (storage modulus) and G (loss modulus) values of adulterants and adulterated honey samples (Control, natural honey; F, fructose syrup; S, saccharose syrup) as a function of
frequency.

recent years, many analytical methods and techniques have been used
to detect honey adulteration. These methods were based on high performance liquid chromatography (HPLC) with mass spectrometry
(MS) (Cheng, Tsai, & Chang, 2006), coupled with several systems such
as refractive index (RI) detection (Park, Yang, Kim, & Kim, 2012), pulsed
amperometric detection (PAD) (Morales et al., 2008), evaporative light
scattering detection (ELSD) (Zhou et al., 2014) and UV detection (Yan &
Evenocheck, 2012). Other chromatographic techniques including gas
chromatographymass spectrometry (GCMS) (Ruiz-Matute et al.,
2007) and high-performance thin layer chromatography (HP-TLC)
(Puscas et al., 2013) have been tested and used in this purpose. However, some problems have been faced; for example, the specicity of detectors is limited by their high sensitivity to ambient temperature,
pressure and ow-rate changes, and poor signal-to-noise ratio, which
might lead to false results. In our study, detection of adulteration was

not based on any sensitive detector; so any risk stemming from such
changes was not the case, which would enable the analyst to avoid
from such problems. Another advantage is that the rheological tests
are not time-consuming, are not expensive and do not require remarkable analytical skills.
Detection of honey adulteration has also been achieved by stable
carbon isotopic ratio by mass spectrometry (SCIR) (Cengiz, Durak, &
Ozturk, 2014; inar, Eki, & Cokun, 2014; Guler et al., 2014; Simsek,
Bilsel, & Goren, 2012). However, despite some potential advantages,
some problems have also been reported by Cengiz et al. (2014) who
pointed out the homogeneity problem of the sample. They also stated
that even if the honey samples could be ltered to achieve homogeneity
before analysis by an IRMS system, in this time, the homogeneity of the
extracted protein would be a common problem. Therefore, this technique will require effective clean-up procedures in order to obtain a

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

Table 3
Newtonian model parameters describing dynamic shear properties of samples.
Samples

Adulterants
Saccharose syrup
Fructose syrup
Adulterated honey samples
HAS
0% (control honey)
10%
20%
30%
40%
50%
HAF
0% (control honey)
10%
20%
30%
40%
50%

G = K()

G = K()

G = K()

R2

R2

R2

0.026
0.027

0.937
0.890

1.268
0.291

0.999
0.999

1.269
0.292

0.999
0.999

0.046b
0.041c
0.039d
0.033e
0.051a
0.003f

0.969
0.962
0.905
0.918
0.941
0.054

6.367a
5.811b
4.397c
3.594d
2.837e
2.234f

0.999
0.999
0.999
0.999
0.999
0.999

6.367a
5.811b
4.397c
3.594d
2.838e
2.234f

0.999
0.999
0.999
0.999
0.999
0.999

0.046b
0.052a
0.042c
0.028d
0.018e
0.021f

0.969
0.939
0.967
0.919
0.951
0.958

6.367a
4.382b
2.696c
2.039d
1.567e
1.111f

0.999
0.999
0.999
0.998
0.999
0.999

6.367a
4.382b
2.697c
2.039d
1.567e
1.111f

0.999
0.999
0.999
0.998
0.999
0.999

Different lowercase letters show differences (P b 0.05) between the adulteration levels.
HAS and HAF were the adulterated honey samples with saccharose and fructose syrups,
respectively.

pure protein extract. The second problem is that the SCIRA technique is
time-consuming, destructive, and expensive and requires considerable
analytical skills that are hard to meet in routine monitoring analysis

641

(Li, Shan, Zhu, Zhang, & Ling, 2012). On the other hand, in detection of
adulteration based on rheological analysis techniques, neither any
clean-up procedure nor extension time, expensive systems and great
analytical skills are required, which would facilitate and accelerate the
detection procedure.
In addition, other methods have been developed to detect honey
adulteration based on thermal analysis (Cordella et al., 2002), capillary
electrophoresis (CE) (Khandurina & Guttman, 2005) and nuclear magnetic resonance (Cotte et al., 2007). Although it has been demonstrated
that these methods could be used to assess the adulteration of honey,
similar disadvantages as SCIR would also be the case. Therefore, faster,
user-friendly and cost-effective analytical techniques should be developed to detect adulteration in honey. In this respect, several other techniques based on spectroscopy have also been recently offered for faster
detection of honey adulteration. Among them are middle infrared (MIR)
(Gallardo-Velzquez, Osorio-Revilla, Zuiga-de, & Rivera-Espinoza,
2009) and near infrared (NIR) spectroscopy (Chen et al., 2011; Zhu
et al., 2010), high-resolution nuclear magnetic resonance (HR-NMR)
(Bertelli et al., 2010), Raman spectroscopy (Li et al., 2012) and
Fourier-transform Raman spectroscopy using canonical variate analysis
(CVA) (Paradkar & Irudayaraj, 2001). Although the spectroscopic
methods have some advantages with respect to speed, simplicity and
cost-effectiveness, the targeted compounds to be detected have identical molecular structures, which may result in unsatisfactory results in
terms of reaching clear decisions (Cengiz et al., 2014). On the other
hand, detection of adulteration by rheological tests is not directly
based on detection of molecular structure, which will provide a great
advantage to an analyst in case adulteration would be detected in
honey samples adulterated with sugars having molecules with identical
structures.
Given all the pros and cons of the reported techniques, some chemometric approaches seem to be useful in detection of honey adulteration
(Cai et al., 2013). Therefore, as reported by Bogdanov and Martin
(2002), the chemometric analysis will not be useful for detection of
adulteration in unioral honeys using routine quality parameters as
there is a great variation of parameters in polyoral honeys. Chemometric detection is based on different parameters such as water, proline, ash
content, electrical conductivity, acidity (free and lactone), pH, HMF, diastase and sugars. However, these parameters change depending on the
botanical origin of honeys, which makes the method practically questionable. In addition, the fact that some parameters such as HMF,

Table 4
Burger model parameters dening creep behavior of samples.
Samples

Burger model parameters

G0 106 (Pa)

Adulterants
Saccharose syrup
Fructose syrup
Adulterated honey samples
HAS
0% (control honey)
10%
20%
30%
40%
50%
HAF
0% (control honey)
10%
20%
30%
40%
50%

Fig. 5. Compliance values (J(t)) as a function of time for adulterants and adulterated honey
samples (Control, natural honey; F, fructose syrup; S, saccharose syrup).

0.7
0.4

0 (Pa s)

G1 (Pa)

1 (Pa s)

R2

1.3
0.3

0.5
193

34
5757

0.999
0.999

190
43 106
247
194
73
70 106

0.999
0.999
0.999
0.999
0.999
0.999

190
56
80
254
2234
11,340

0.999
0.999
0.999
0.999
0.999
0.999

5
25
7
11
7
31

7.0a
6.0b
5.0c
4.0d
4.0d
2.0e

19
29
26
14
8
30

5
0.0003
1
12
0.002
0.006

7.0a
5.1b
3.0c
2.0d
1.6e
1.1f

19
1
2
14
212
1152

Different lowercase letters show differences (P b 0.05) between the adulteration levels.
HAS and HAF were the adulterated honey samples with saccharose and fructose syrups,
respectively.

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diastase and content of individual sugars are storage- and heatdependent (Bogdanov & Martin, 2002) should be taken into account.
In summary, majority of the aforementioned methods show
good precision, accuracy, and reliability; however, they are timeconsuming, subjective, expensive and component-dependent and require complicated pretreatments and long experimental steps as well
as considerable analytical skills. Therefore, it is essential to develop
fast, simple and cost-effective analytical methods to detect and quantify
adulterations in honey. In this respect, detection of adulteration based
on rheological methods might be a promising approach in terms of
avoiding such disadvantages and problems.
3.5. Creep and recovery properties
In this study, creep and recovery measurements were conducted because highly concentrated sucrose solutions like saccharose/fructose
syrup and honey might be in a metastable state, having a tendency to
crystallize (Quintas, Brando, Silva, & Cunha, 2006). Therefore, a collision between the molecules is promoted by shearing. These results in
nucleation and subsequent crystal growth (Hartel, 1993; Shastry &
Hartel, 1996), which limit the use of steady-state ow measurements
conducted to characterize rheological properties of such solutions. To
be more precise, a faster crystallization occurs, leading a change in the
rheological behavior due to the increasing shear applied during the
measurements (Quintas et al., 2006). Therefore, in addition to steady
and dynamic shear measurements, creep and recovery tests were also
followed in this study to conrm the other rheological test results.
3.5.1. Creep phase
The creep test results for the values of compliance J = / as a function of time were displayed in Fig. 5 where the effects of adulterant level
on the creep behavior of adulterated honey samples can be seen in a
time interval between 0 and 150 s. The recovery phase in Fig. 5 corresponding to the time interval of 150 t 300 s will be discussed
later in the recovery phase section. Table 4 indicates the values of G0,
G1, 0 and 1 and the related determination coefcients (R2 values).
The R2 values higher than 0.999 in all cases indicated that the tting of
J = f(t) in the interval 0 t 150 s could be successfully done based
on the Burger model (Eq. (9)) for the adulterants and adulterated
honey samples as affected by different levels of adulterants.
G0, G1, 0 and 1 values reect the structure of any food system and
decrease in these values shows its weakened structure; namely a decrease in the shear moduli and viscosity of the elements present in the
Burger model. G0, the instantaneous shear modulus, represents a measure of elastic strength on the bonds making up the interfacial network
structure (Lobato-Calleros, Aguirre-Mandujano, Vernon-Carter, &
Snchez-Garca, 2000). As can be seen in Table 4, G0 values of the

Table 6
Results of the PCA analysis using data obtained from physicochemical and rheological
analyses of the samples.
Principal component

Eigenvalues

PC1
PC2
PC3
PC4
PC5
PC6
PC7
PC8
PC9
PC10

Explained variance

For PC

Cumulative

Variance (%)

Cumulative

11.257
5.002
1.373
1.145
0.513
0.406
0.154
0.066
0.064
0.021

11.257
16.259
17.632
18.777
19.290
19.696
19.850
19.916
19.980
20.001

56.283
25.008
6.864
5.724
2.565
2.031
0.772
0.330
0.320
0.103

56.283
81.291
88.155
93.879
96.444
98.474
99.247
99.576
99.897
100.00

adulterated honey samples showed no consistent trend with increasing

adulterant level, which means no clear effect of adulterants on elastic
strength on the bonds making up the interfacial network structure of
honey. This was also the case for the G1 and 1 values, exhibiting great
uctuations with adulterant level. These results revealed that G0, G1,
and 1 cannot be clear indicators to understand if honey would be deformed by addition of the adulterants and how the internal structure
of adulterated honey would be. In other words, these creep parameters
could not be used to detect the potential presence of adulterants, namely saccharose and fructose syrups in honey. However, the effect of adulterant level on the viscosity represented by the Maxwell dashpot (0)
parameter was clear; namely, adulterant addition could be revealed
by the 0 values which decreased (P b 0.05) as the adulterant level increased (Table 4). In addition, Fig. 5 shows the variation of the shear
creep and recovery compliance J(t) with respect to the adulterant
level, indicating that the adulterated honey samples with higher saccharose and fructose syrups exhibited higher J(t) values during creep and
recovery. It can be said based on these results that adulterant addition
induced large deformation in the viscoelastic nature of honey; thus,
weakening its internal structure for the same applied stress. These results suggested that viscosity represented by the Maxwell dashpot
(0) can be a good indicator of saccharose or fructose adulteration in
honey.
3.5.2. Recovery phase
The recovery analysis results indicated that the samples reached the
maximum deformation (JMAX) after 150 s of stress application. The
stress applied was removed at the time when was equal to 0, and
then, the compliance values J = f(t) were measured at a duration of
150 s (Yilmaz, Karaman, Cankurt, Kayacier, & Sagdic, 2011). Fig. 5 also
indicates the experimental results for the recovery phase of the adulterants and adulterated honey samples in a time interval between 150 and

Table 5
Pearson correlation coefcients (r) between sugar composition (HPLC results) and rheology (steady, dynamic and creep) parameters of adulterated honey samples.
Adulterated honey samples

HAS

HAFd

Sugar composition

Fructose
Glucose
Saccharose
Fructose
Glucose
Saccharose

Rheology parameters
Steady shear parametera

Dynamic shear parametersb

0.684
0.587
0.750
0.970
0.971
0.886

0.938
0.907
0.953
0.959
0.896

0.938
0.907
0.953
0.959
0.896

0.683

0.683

0.881
0.848
0.909
0.939
0.864
0.622

Burger model parametersc

G0

G1

0.367
0.238
0.544
0.137
0.168
0.013

0.934
0.873
0.980
0.815
0.871
0.810

0.071
0.179
0.041
0.675
0.798
0.824

0.425
0.314
0.552
0.684
0.806
0.829

In bold, correlations between the sugar composition (HPLC results) and rheology (steady, dynamic and creep) parameters were signicant.
a
: apparent viscosity.
b
K, K and K: magnitudes of intercepts for G (storage modulus), G (loss modulus) and G (complex modulus), respectively.
c
G0: elastic modulus of Maxwell unit; 0: viscosity of Maxwell dashpot; G1: shear modulus of KelvinVoigt unit; 1: viscosity of KelvinVoigt dashpot.
d
HAS and HAF were the honey samples adulterated with saccharose and fructose syrups, respectively.
P b 0.05.
P b 0.01.

M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

643

Fig. 6. Score plots of established PCs (Control, natural honey; F, fructose syrup; S, saccharose syrup).

300 s with changing adulterant levels. As can be seen in Fig. 5, the adulterants showed a Newtonian behavior, with a linear response of strain
during the force application and no recovery was observed after the
force was removed, which is typical of Newtonian uids. Consistent results were reported by Quintas et al. (2006) who observed no recovery
for an 82.90% sucrose solution. As far as the adulterated honey samples
were concerned, similar situation was the case; namely, no recovery
was observed. Furthermore, this situation did not change with increased level of adulterants; however, the recovery start point increased
as the adulterant level increased (Fig. 5). From these results, it can be
deduced that the effect of adulteration was clear, changing the creep
recovery behavior of natural honey, and easily deforming the honey
structure that could be immediately detected by creeprecovery analysis. These results would be promising in developing an alternative approach for detection of such adulterants in honey.

3.6. Correlations between sugar composition and rheology parameters

Pearson's test was used to analyze correlations between sugar composition and rheology parameters of adulterated honey samples. In
Table 5, the analysis results were presented. Signicant (P b 0.05;
0.01) negative and positive correlations were found between sugar
composition and rheology parameters. These parameters were (viscosity), K, K (magnitudes of intercepts for G and G, respectively)
and 0 (viscosity of Maxwell dashpot), proving that , K, K and 0
could be indicators for the presence of saccharose or fructose syrups in
natural honey within the studied concentration levels ranging between
10 and 50%.

3.7. PCA analysis

PCA was applied to classify the control and adulterated honey samples based on physicochemical and all of the rheological results, namely
steady, dynamic and creep/recovery results. According to the PCA results, four different PCs were established to explain the total variability
of physicochemical and rheological properties of the samples. Table 6
shows the Eigen values and variance value of each PC. As seen, four
PCs were adequate for explanation of variability due to their Eigen
value higher than unity. PC1, PC2, PC3 and PC4 accounted for 56.283%,
25.008%, 6.864% and 5.724% of the total variability, respectively, in the
data set. In other words, 93.879% of the total variance in the data set
can be satisfactorily described by these four PCs. Larrigaudiere,
Lentheric, Puy, and Pinto (2004) reported that the percentage higher
than 70% is considered as sufcient for explanation of variability; therefore, in the present study established PCs were adequate to classify control honey and adulterated honey samples with respect to their
physicochemical and rheological properties.
Score plots of the PCs are presented in Fig. 6 in which PC1PC2, PC1
PC3 and PC1PC4 plots are shown. As seen from the gure, the control
honey sample and the sample adulterated with 10% fructose were clustered on the bottom right quadrant of the PC1PC2 plot due to their a,
brix, pH and K values, indicating that these were among the rheological
parameters which could not be used for detection of honey adulteration
with 10% concentration of fructose. The other fructose adulterated
honey samples were located on the bottom left quadrant of the PC1
PC2 plot, which might have resulted from the fructose concentration
and G1 value calculated from the creep data. As seen also in Fig. 6,
honey samples adulterated with saccharose syrups in concentrations

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M.T. Yilmaz et al. / Food Research International 64 (2014) 634646

Table 7
Validation of rheological analysis to detect honey adulteration by repeatability and sensitivity parameters.
Parameters tested

Control

(Pa s)

G (Pa)

G (Pa)

(Pa s)

JMAX (Pa1)

Sensitivity

Repeatability

Mean
SD
RSD%
Mean
SD
RSD%
Mean
SD
RSD%
Mean
SD
RSD%
Mean
SD
RSD%

7.267
0.176
0.02
0.369
0.170
0.46
52.346
1.588
0.03
8.331
0.253
0.03
16.913
4.687
0.28

Saccharose

Fructose

10%

50%

10%

50%

6.592
0.257
0.04
0.990
1.184
1.20
0.990
1.184
1.20
8.139
1.401
0.17
14.097
6.479
0.46

3.645
0.211
0.06
5.169
3.366
0.65
3.093
3.366
1.09
8.068
1.153
0.14
26.491
10.899
0.41

5.905
0.263
0.04
0.246
0.025
0.10
0.246
0.025
0.10
7.318
0.865
0.12
20.715
6.814
0.33

2.339
0.115
0.05
0.100
0.047
0.47
0.102
0.046
0.45
2.855
0.110
0.04
45.431
6.488
0.14

Control

Saccharose
2%

4%

6%

8%

7.253
0.171
a
0.337
0.065
a
53.420
1.303
a
8.502
0.207
a
13.296
1.562
b

6.718
0.348
a
0.365
0.223
a
49.566
0.857
ab
7.889
0.136
ab
22.162
8.834
a

6.596
0.288
a
0.276
0.137
a
45.650
1.103
bc
7.266
0.176
bc
22.384
2.906
a

5.737
0.479
b
0.223
0.011
a
44.670
1.746
c
7.110
0.278
c
18.456
0.800
ab

5.402
0.437
b
0.247
0.045
a
45.466
4.353
bc
7.236
0.693
bc
16.490
1.846
ab

Control

Fructose
2%

4%

6%

8%

7.253
0.171
ab
0.337
0.065
a
53.420
1.303
a
8.502
0.207
a
13.296
1.562
b

7.812
0.241
a
0.315
0.065
a
50.778
3.329
a
8.082
0.530
a
18.936
2.703
a

6.327
0.631
cd
0.282
0.029
a
48.140
5.391
ab
7.662
0.858
ab
13.162
0.944
b

6.520
0.195
bc
0.245
0.085
a
42.724
1.968
b
6.800
0.313
b
12.548
0.477
b

5.523
0.699
d
0.258
0.039
a
43.526
1.546
b
6.927
0.246
b
16.740
1.814
a

ad

For each parameter tested and sugar type, different lower case letters show differences between the concentrations (P b 0.01).

of 10%, 20% and 30% were clustered on the top right quadrant of the
PC1PC2 plot. Turbidity and b values, ash and glucose contents,
among the other rheological parameters K, K, 0 and values are responsible for this clustering. According to the PCA results, it was seen
that the magnitudes of the K, K, 0 and rheological parameters
could be used for detection of adulteration in saccharose adulterated
honey samples. Honey samples adulterated with saccharose in concentrations of 40% and 50% were clustered on the left quadrant of the PC1
PC2 plot due to their saccharose content and L and 1 values.

3.8. Method validation

In addition to conrmation of the rheological methods with HPLCRID results by Pearson correlation analysis, the methods were also validated with the following validation parameters.

3.8.1. Repeatability
The repeatability of honey samples was calculated using twelve successive measurements and expressed as the percent relative standard
deviation (RSD%). The RSD% values were calculated (1) to range between 0.04 and 0.06 for values in 10 and 50% saccharose and fructose
adulteration, (2) to be 0.10 for G and G values in 10% fructose adulteration, (3) to range between 0.04 and 0.17 for values in 10 and 50%
saccharose and fructose adulteration and (4) to be 0.33 for JMAX values
in 10% fructose adulteration (Table 7). Such low RSD% values indicated
the repeatability of the rheological parameters in detection of honey
adulteration at such concentrations. However, the data obtained for
the and parameters were more repeatable.

3.8.3. Linearity
In this study, the rheological method linearity was based on four
concentration levels between 20% and 50% of sugar adulteration. The
linearity was determined by preparing honey samples adulterated
with different saccharose and fructose concentrations. The determination coefcients (R2) and linear regression equations are presented in
Fig. 7. For , G and parameters, high determination coefcients
(Fig. 7) were obtained, indicating that there was exact linearity between
the determined adulteration ratios and these parameters. However, this
was not case for the G and JMAX parameters, as can be seen by their relatively low R2 values. Based on these results, it was possible to say that ,
G and parameters were appropriate for determining adulteration in
honey samples.
4. Conclusion
In this study, natural honey was adulterated with different levels of
saccharose and fructose syrups at a ratio of 0%, 10%, 20%, 30%, 40% and
50% by weight. Steady, dynamic and creep tests were conducted to detect such adulterations at specied ratios. The rheological analysis test
results revealed that adulteration at these levels could be clearly detected by remarkable changes in the ow, viscoelastic and creep behavior of
natural honey. Signicant correlations found between the rheology parameters and sugar composition of adulterated honey samples suggested that these parameters could be a combination of indicators for
detection of such adulterations in honey at specied ratios. This was
also demonstrated by our validation data which indicated that , G
and parameters could be used to precisely determine the adulteration status of honey samples, resulting from saccharose/fructose.
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3.8.2. Sensitivity (LOD)

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detected by , G and parameters at 4%. Thus, the LOD was generally
determined to be 4%. Based on our results, these rheological parameters
can detect adulteration ratio greater than 4%.

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