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Article history: Botanical origin of the nectar predominantly affects the chemical composition of honey. Analytical
Received 27 March 2014 techniques used for reliable honey authentication are mostly time consuming and expensive. Addition-
Received in revised form 8 August 2014 ally, they cannot provide 100% efficiency in accurate authentication. Therefore, alternatives for the
Accepted 10 August 2014
determination of floral origin of honey need to be developed. This study aims to discriminate character-
Available online 20 August 2014
istic Anatolian honey samples from different botanical origins based on the differences in their molecular
content, rather than giving numerical information about the constituents of samples. Another scope of
Keywords:
the study is to differentiate inauthentic honey samples from the natural ones precisely. All samples were
Honey
Botanical origin
tested via unsupervised pattern recognition procedures like hierarchical clustering and Principal Compo-
ATR-FTIR spectroscopy nent Analysis (PCA). Discrimination of sample groups was achieved successfully with hierarchical cluster-
Multivariate analysis ing over the spectral range of 1800–750 cm 1 which suggests a good predictive capability of Fourier
Hierarchical Cluster Analysis Transform Infrared (FTIR) spectroscopy and chemometry for the determination of honey floral source.
Principal Component Analysis Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2014.08.040
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
S. Gok et al. / Food Chemistry 170 (2015) 234–240 235
resonance (NMR) (Lindner, Bermann, & Gamarnik, 1996), gas chro- which have ‘‘organic’’ certificate. Taurus flower honey is collected
matography (Low & South, 1995) and carbon isotope ratio analysis from the Taurus Mountains located at the south Mediterranean
(White, Winters, Martin, & Rossmann, 1998). These techniques region of Anatolia.
used for reliable honey authentication are mostly time consuming Rhododendron honey, locally called as ‘‘mad honey’’ or ‘‘toxic
and expensive. Additionally, they cannot provide 100% success in honey’’, is made up of spring flowers of Rhododendron ponticum
authentication. Infrared (IR) spectroscopy has been preferred as a (rhododendron plant). Rhododendrons mainly grow in the eastern
rapid, non-destructive, reagent-free, operator independent and Black Sea Region of Turkey. Their phenolic content and antimicro-
cheap technique in food industry for the quantification of various bial activities are quite different from the other honey plant
food samples (Chalmers & Griffiths, 2002; Li-Chan, Chalmers, & species. Nectar contains andromedotoxin, which causes various
Griffiths, 2010). IR spectroscopy was applied in different honey physiological effects in humans (Onat, Yegen, Lawrence, Oktay, &
samples for the determination of botanical or geographical origin, Oktay, 1991).
detection of adulteration and for the quantification of fructose, glu- Chestnut honey is produced from both nectar and secretum
cose, sucrose, maltose, pH value and electrical conductivity (Chung, collection by honey bee. These are collected from various regions
Ku, & Lee, 1999; Lichtenberg-Kraag, Hedtke, & Bienefeld, 2002; of Anatolia.
Ruoff, 2006; Tewari & Irudayaraj, 2004). Chemometric methods Pine honey is a kind of honeydew honey. It is produced via
based on Fourier Transform Infrared (FTIR) spectroscopy were also using the secretum of an insect (Marchalina hellenica) living in
used for honey adulteration (Rios-Corripio, Rojas-López, & the trunk of pine tree and collected by bees. Pine honey is a specific
Delgado-Macuil, 2012; Subari, Saleh, Shakaff, & Zakaria, 2012) endemic product, and can be found only in Turkey and Greece.
and characterisation with limited number of Mexican honeys Cedar honey, used in this study, was collected from the Taurus
(Rios-Corripio, Rios-Leal, Rojas-López, & Delgado-Macuil, 2011). Mountains in the Mediterranean region of Turkey, and is mainly
Etzold and Lichtenberg-Kraag (2008) have developed FTIR based originated from Cedar trees.
PCA calibration models with German honeys. Although, there have Fake (adulterated) honey used in this study was collected from
been many attempts for searching alternative methods for authen- Apis mellifera. Hives were fed with sugar (sucrose) syrup thus sugar
tication of honey, some of these studies have been limited with was incorporated into the honey via bee-feeding. Study in this field
certain number of unifloral honey sources and have not been has shown that adulteration is also possible via bee-feeding syrups
tested sufficiently with polyfloral samples (Ruoff, 2006) and with and this can cause chemical modifications of the honey quality
Anatolian honeys specifically. similar to artificial adulteration via direct syrup incorporation to
In the current study, it was aimed to estimate botanical origin of honey (Cordella, Militão, Clément, Drajnudel, & Cabrol-Bass, 2005).
honey samples that are specific to Anatolia by applying two differ- Maple syrup produced from the xylem sap of maple tree and
ent multivariate analysis techniques to the Attenuated Total contains primarily sucrose and water. Maple syrups retrieved to
Reflectance (ATR)-FTIR spectroscopic data. With this work, it was the study are Canadian origin and were used as a non-honey con-
intended to exemplify the usage and success of ATR-FTIR spectros- trol group. Fructose syrup was directly purchased from the market.
copy coupled with multivariate analysis in botanical origin assign- Grape molasses (pekmez) is traditional syrup produced by boiling
ment with a high number of sample groups. This work will also of the pressed grape juice and special grape soil mixture or cream
provide basis to honey adulteration determination studies. of lime. It is rich in both carbohydrates and minerals.
2. Experimental
2.2. Instrumentation and sample analysis
2.1. Samples
Spectra from all samples were collected in the one-bounce ATR
mode in a Spectrum 100 FTIR spectrometer (Perkin-Elmer Inc.,
A total of 144 honey samples were collected from different geo-
Norwalk, CT, USA) equipped with a Universal ATR accessory.
graphical regions of Turkey. The majority of samples used in this
Samples were placed on Diamond/ZnSe crystal plate (Perkin-
study were procured from well known certificated honey brands
Elmer) and scanned from 4000 to 650 cm 1 for 50 scans with
which have BRC (British Retail Consortium) certificate and
resolution of 4 cm 1 at room temperature. Each sample was repli-
officially declare that honeys are subjected to all chemical and
cated three times. Identical spectra were obtained in each case. This
physical analysis to detect quality and purity in addition to
process was done to see the accuracy of the absorbance values,
descriptive organoleptic and microscopic analysis to determine
which might be affected from intra-sample variability and from
floral and regional origins. Some samples were collected directly
variation in experimental conditions. Average spectra were used
from the primary producers. The region and origin of production
for further analysis. Data manipulations were carried out via
were known for all samples.
Spectrum 100 software (Perkin-Elmer).
Flower originated (polyfloral (n = 30), anzer (n = 3), organic
(n = 13), Taurus flower (n = 6)), tree originated (pine (n = 22),
chestnut (n = 10), cedar (n = 6)) and rhododendron honey (n = 30) 2.3. Chemometrics
samples in addition to fake (adulterated) honey (n = 6), maple
syrup (n = 6), fructose syrup (n = 6) and grape molasses (n = 6) Cluster and Principal Component Analysis were applied to clas-
samples were included in the study. The sample size of each group sify the samples based on spectral differences. For the determina-
is indicated as ‘‘n’’. tion of spectral differentiation among studied groups, cluster
Honey samples were grouped as tree and flower originated ones analysis was performed via OPUS 5.5 software (Bruker Optics,
basically. Flower originated group is composed of polyfloral hon- GmbH). Vector normalised, first derivative of each spectrum in
eys, collected from different regions of Turkey, which are Anzer the range of 1800–750 cm 1 was used as an input data. Spectral
honey, organic flower honey and Taurus flower honey. Anzer distances were calculated between pairs of spectra as Pearson’s
honey, composed of nectar, mainly collected from Anzer plant correlation coefficients and Euclidean distance was used to calcu-
(thymus species) in the narrow region located in Rize/Ikizdere/ late the sample similarities and to indicate the complete linkage
Anzer. It has been largely studied in terms of its medicinal proper- clustering by Ward’s algorithm.
ties that cause its market price to be 10 times higher than other Principle Component Analyses (PCA) was used as a data reduc-
honeys. Organic honeys are collected from the well known brands tion method where each spectrum, which consists of hundreds of
236 S. Gok et al. / Food Chemistry 170 (2015) 234–240
absorbance values, is represented by a point in a multidimensional classification of different honey samples has been proposed in pre-
space using a linear transformation. vious researches. In 1960, discriminant functions of monosaccha-
In this work, PCA was conducted on the ATR-FTIR spectra ride and ash content in addition to pH values were used for
over 4000–650 cm 1 1700–1600 cm 1, 1175–940 cm 1 and 940– classification of honey samples (Kirkwood, Mitchell, & Smith,
700 cm 1 range using by ‘‘Kinetics’’, a custom made program 1960). Linear discriminant analysis was employed to select most
running under MATLAB (Matlab, Mathworks Inc.). useful measurands by evaluating different sugars, water, pH value,
colour, diastase enzyme activity conductivity and hyroxymethyl-
3. Results & discussion furfural content. Later, by using pH value, free acidity, electrical
conductivity, fructose, glucose and raffinose contents, botanical
In the current study, ATR-FTIR spectroscopy has been used to origins of honeys were estimated perfectly (Devillers, Morlot,
compare honey samples based on their spectral differences in the Pham-Delegue, & Dore, 2004). In addition, flower honey was char-
4000–650 cm 1 spectral region. A representative ATR-FTIR spec- acterised by high concentration values for glucose and fructose and
trum of honey is given in Fig. 1. Table 1 presents the band assign- low free acidity, polyphenol content, lactone quantity and electri-
ments along with the corresponding modes of vibrations in the cal conductivity; whereas honeydew honeys have low concentra-
ATR-FTIR spectrum of honey, based on the literature (Gallardo- tion glucose and fructose while showing high free acidity,
Velázquez, Osorio-Revilla, Loa, & Rivera-Espinoza, 2009; Kelly, polyphenol content, lactone quantity and electrical conductivity
Downey, & Fouratier, 2004; Movasaghi, Rehman, & Rehman, (Sanz, Gonzalez, de Lorenzo, Sanz, & Martinez-Castro, 2005).
2008; Sivakesava & Irudayaraj, 2001; Subari et al., 2012; Tewari The algorithms behind the cluster and Principal Component
& Irudayaraj, 2004, 2005). Analysis that were used in the current study are quite different.
Fig. 2 shows comparative infrared spectra of all samples in the PCA-like techniques can be preferred primarily for the determina-
4000–650 cm 1 region. In this figure, spectral differences between tion of general relationship among data (Gasper et al., 2010). How-
the groups were clearly seen. ever, if one wants to show the grouping of similar data gathered
Based on the spectral differences Hierarchical Cluster Analysis from different samples, cluster analysis must be performed
(HCA) and PCA have been applied to different spectral regions. (Wang & Mizaikoff, 2008). Similar samples tend to be classified
Use of chemometrics together with classical methods for the in the same cluster and the level of difference between the clusters
1
Fig. 1. Representative ATR-FTIR spectrum of honey in the 4000–650 cm spectral region.
Table 1
General band assignment of ATR-FTIR spectrum of honey. The related references are indicated in the parenthesis.
1
Region 1 3000–2800 cm C–H stretching (carbohydrates) (Gallardo-Velázquez et al., 2009)
O–H stretching (carboxylic acids) (Movasaghi et al., 2008)
NH3 stretching (free amino acids) (Gallardo-Velázquez et al., 2009; Sivakesava & Irudayaraj, 2001)
1
Region 2 1700–1600 cm O–H stretching/bending (water) (Cai & Singh, 2004; Stuart, 1997)
C@O stretching (mainly from carbohydrates) (Gallardo-Velázquez et al., 2009)
N–H bending of amide I (mainly proteins) (Philip, 2009)
1
Region 3 1540–1175 cm O–H stretching/bending (Gallardo-Velázquez et al., 2009; Tewari & Irudayaraj, 2004)
C–O stretching (carbohydrates) (Tewari & Irudayaraj, 2004)
C–H stretching (carbohydrates) (Tewari & Irudayaraj, 2005)
C@O stretching of ketones (Tewari & Irudayaraj, 2004)
1
Region 4 1175–940 cm C–O & C–C stretching (carbohydrates) (Subari et al., 2012; Tewari & Irudayaraj, 2005)
Ring vibrations (mainly from carbohydrates) (Gallardo-Velázquez et al., 2009; Tewari & Irudayaraj, 2004)
1
Region 5 940–700 cm Anomeric region of carbohydrates (Mathlouthi & Koenig, 1986; Subari et al., 2012)
C–H bending (mainly from carbohydrates) (Gallardo-Velázquez et al., 2009; Kelly et al., 2004; Tewari & Irudayaraj, 2004)
Ring vibrations (mainly from carbohydrates) (Tewari & Irudayaraj, 2004)
S. Gok et al. / Food Chemistry 170 (2015) 234–240 237
1 1
Fig. 2. Comparative ATR-FTR spectra of all samples in the 4000–650 cm spectral region. Spectra were normalised to the band located at 3300 cm .
1
Fig. 3. Hierarchical clustering of all samples in the 1800–750 cm (fingerprint) spectral region.
is indicated with heterogeneity values (Ward, 1963). Ward’s algo- that only two groups, which show the smallest growth in heteroge-
rithm was previously reported to give one of the best predictions, neity factor H, are merged. Detailed information about this method
among the different methods used in cluster analysis (Lasch, was reported in Severcan et al. (2010).
Haensch, Naumann, & Diem, 2004; Severcan, Bozkurt, Gurbanov, In this study, in order to reduce the number of variables prior to
& Gorgulu, 2010). As opposed to other methods, algorithm tries performing cluster analysis, we used the PCA. This was conducted
to find groups which are as homogeneous as possible. This means on four different regions. Depending on the PCA outputs, the
238 S. Gok et al. / Food Chemistry 170 (2015) 234–240
1 1 1 1
Fig. 4. PCA scatter plots for all of the samples over 4000–650 cm (A), 1700–1600 cm (B), 1175–940 cm (C), and 940–700 cm (D) spectral region. (Ellipses have a
confidence factor of 0.8.)
regions having the highest principal component (PC) values were 1800–750 cm 1 spectral region was selected for successful
selected for HCA. Other spectral regions, used for PCA analysis, discrimination of clusters.
were also tried for hierarchical clustering of the sample groups. For the calculation of sample similarities, the Euclidean distance
However, the best differentiation was achieved only in the was used indicating the complete linkage clustering values. The
1800–750 cm 1 region. This region includes the anomeric region results obtained are represented in Fig. 3 in the form of dendro-
at 950–750 cm 1 which was frequently preferred for the spectral grams. Clear cut classes were gathered over the range of 1800–
analysis of carbohydrates in IR spectroscopy. Analysis in this range 750 cm 1 with high heterogeneity values (up to 10). All of the tree
makes it possible to distinguish bands characteristic for a and b originated samples (chestnut, cedar, pine) are aggregated in one
conformers or pyranoid and furanoid ring vibrations of mono cluster on the left arm. As the maple syrup is also the maple tree
and polysaccharides (Mathlouthi & Koenig, 1986). In addition to originated sample, it shows more similarity to tree originated
alpha and beta conformers, the fingerprint region (1800– group than to the flower originated ones. One arm of the second
750 cm 1) contains other contributions that arise from different cluster is composed of flower originated honey samples including
molecules. Especially water (around 1640 cm 1) and minute polyfloral, anzer, organic, Taurus flower and rhododendron honeys.
amount of protein molecules give bands in the indicated region. Anzer, organic and Taurus flower honeys are region specific sam-
Also the differences among honey samples can be related not only ples, it is known that the purity of their botanical origins is higher
to different water content in the different honeys but also to the than polyfloral honey group. So they were clustered in the same
interaction between water molecules and carbohydrates, depend- arm. As the rhododendron honey is collected from the Black Sea
ing on their structure. The precise assignment of bands in this Region mountains, it was clustered closer to that group than
region cannot be stated unequivocally. However fingerprint region polyfloral ones. Fructose syrup, grape molasses and the fake
provides a unique spectrum for each compound where the (adulterated) honey were agglomerated on the far right arm of
position and intensity of the bands are specific for every the second cluster in that they differ from the natural samples in
polysaccharide (Filippov, 1992; Li-Chan et al., 2010). Therefore, terms of their carbohydrate content significantly.
S. Gok et al. / Food Chemistry 170 (2015) 234–240 239
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