The chemical composition and sensory characteristics vary significantly within different honey types. In order to determine botanical origin of honey, it is necessary to conduct rather complicated and time consuming analytical methods. IR spectroscopy has not yet been experimentally explored for honey analysis in Croatia, so the aim of this study was to determine claimed botanical origin of honey using both, standard and alternative (IR spectroscopy) methods, for the purpose of their comparison, Altogether 144 samples of nine different unifloral honey types (black locust, sweet chestnut, lime, sage, heath, rosemary, lavender, mandarin and strawberry tree) were collected from different Croatian regions directly from the beekeepers. In order to confirm claimed botanical origin of collected honey samples, melissopalinological analysis, moisture and electrical conductivity measurements were conducted. Infrared spectra of honey samples were recorded using the ABB Bomem MB102 Fourier-transform infrared spectrometer (FT-IR spectrometer). Selected IR spectral regions were analyzed by multivariate data analysis, principal components analysis (PCA). Preliminary PCA of IR spectra showed significant clustering of the analyzed samples by botanical origin. The results of this study showed that IR spectroscopy provides reliable results, but also represents rapid and cheap analytical tool in comparison to commonly used standard analytical methods. This research has also provided the first insight in infrared spectra of Croatian honeys

Domagoj Barišić

Dragan Bubalo

Lidija Svečnjak

Nikola Biliškov

English

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191Agriculturae Conspectus Scientifi cus | Vol. 76 (2011) No. 3 (191-195)ORIGINAL SCIENTIFIC PAPERSummaryTh e chemical composition and sensory characteristics vary signifi cantly within dif-ferent honey types. In order to determine botanical origin of honey, it is necessary to conduct rather complicated and time consuming analytical methods. IR spectros-copy has not yet been experimentally explored for honey analysis in Croatia, so the aim of this study was to determine claimed botanical origin of honey using both, standard and alternative (IR spectroscopy) methods, for the purpose of their com-parison, Altogether 144 samples of nine diff erent unifl oral honey types (black locust, sweet chestnut, lime, sage, heath, rosemary, lavender, mandarin and strawberry tree) were collected from diff erent Croatian regions directly from the beekeepers. In or-der to confi rm claimed botanical origin of collected honey samples, melissopalino-logical analysis, moisture and electrical conductivity measurements were conducted. Infrared spectra of honey samples were recorded using the ABB Bomem MB102 Fourier-transform infrared spectrometer (FT-IR spectrometer). Selected IR spectral regions were analyzed by multivariate data analysis, principal components analysis (PCA). Preliminary PCA of IR spectra showed signifi cant clustering of the analyzed samples by botanical origin. Th e results of this study showed that IR spectroscopy provides reliable results, but also represents rapid and cheap analytical tool in com-parison to commonly used standard analytical methods. Th is research has also pro-vided the fi rst insight in infrared spectra of Croatian honeys.Key wordshoney, botanical origin, standardized analytical methods, infrared spectroscopyApplication of Infrared Spectroscopy in Honey Analysis Lidija SVEČNJAK 1 ( )Nikola BILIŠKOV 2Dragan BUBALO 1Domagoj BARIŠIĆ 11 University of Zagreb, Faculty of Agriculture, Department of Fisheries, Beekeeping and Special ZoologySvetošimunska 25, 10000 Zagreb, Croatia e-mail: lsvecnjak@agr.hr2 Rudjer Boskovic Institute, Division of Organic Chemistry and BiochemistryBijenička 54, 10000 Zagreb, CroatiaReceived: May 12, 2011 | Accepted: July 11, 2011Agric. conspec. sci. Vol. 76 (2011) No. 3192 Lidija SVEČNJAK, Nikola BILIŠKOV, Dragan BUBALO, Domagoj BARIŠIĆAimTh e proportion of nectar or honeydew of certain plant spe-cies in honey varies depending on the type of vegetation, the period of plant blooming or the production of honeydew by the insects, as well as on the time when the beekeepers have pro-duced honey. Absolutely unifl oral honeys do not exist because the honey bees never visit only one plant species, even though it dominates in a given area. Consequently, the chemical composi-tion and sensory characteristics within diff erent honey types vary signifi cantly. In order to determine botanical origin of honey, it is necessary to conduct melissopalynological analysis, as well as sensory and certain physico-chemical analysis, because only on the basis of aggregate results of mentioned analyses, the fi nal confi rmation of the claimed botanical origin can be given. Th e analytical methods used for honey classifi cation are mostly the same as the ones used for routine quality control (Bogdanov et al., 2004). Th ese methods have been harmonized and de-scribed by the International Honey Commission (Bogdanov et al., 1997). However, these methods, especially melissopalyno-logical analysis, require specialised knowledge and expertise, and involve a long counting procedure. Th us, the identifi cation of the botanical origin of honey represents rather diffi  cult and time-consuming process.It is clear that the diff erences between distinctive types of honey are not confi ned only to their sensory qualities, physico-chemical characteristics and the botanical origin (pollen spec-trum). One of the methods that provide information on the total chemical composition of any sample is infrared spectroscopy (IR), a type of vibrational spectrosocpy, where molecular vibrations are being analyzed. Infrared spectrum of an organic compound provides a unique fi ngerprint, which is readily distinguished from the absorption patterns of all other compounds; only op-tical isomers absorb in exactly the same way (Skoog and Leary, 1992). Although this method is rather simple and undemanding from the experimental point of view, it has been recognized as very eff ective in honey analytics only a few years ago. Until re-cently, IR was considered to be completely inapplicable to sam-ples containing higher water content. However, the development of new methods for spectra recording, of which the most impor-tatnt is attenuated total refl ectance (ATR), allowed the obtain-ing of quantitative information on the chemical composition of water-rich samples. Th e advantages of this method over com-monly used standard ones are that it is fast, reliable and easy to use. Furthermore, vibrational measurements require minimal or no sample preparation, and only a very small amount of the sample, thus allowing in situ monitoring of any sample. Based on comparison of several spectroscopic methods used for honey analysis, Ruoff  et al. (2006, 2007) have presented comparative advantages of IR spectroscopy over other used methods. In these studies, a large sample of diff erent honey types originating from diff erent European countries, was analyzed using contempo-rary chemometrics and was compared with standard methods. Th e results have shown that IR spectroscopy can be considered as a reliable for distinguishing the botanical origin of honey. Th ere are also few other papers indicating that IR spectroscopy can be used for authetication of the botanical origin of honey (Lichtenberg-Kraag et al., 2002; Bertelli et al, 2007; Woodcock et al., 2009) but this method has still not been standardized nor implemented in regulations related to honey quality control. Furthermore, there are still certain aspects of this method that need to be additionally explored, repeated and verifi ed in order to make it outright reliable and applicable as an analytical tool in general practice. IR spectroscopy has not been experimental-ly explored yet for honey analysis in Croatia, so the aim of this study was to confi rm claimed botanical origin of honey samples collected from the beekeepers from diff erent Croatian regions, using both, standard and alternative (IR spectroscopy) meth-ods, for the purpose of their comparison. Moreover, the results of this research will provide the fi rst insight in infrared spectra of Croatian honeys.Material and methodsAltogether 144 samples of nine diff erent unifl oral honey types (black locust, sweet chestnut, lime, sage, heath, rosemary, lav-ender, mandarin and strawberry tree) were collected from dif-ferent Croatian regions directly from the beekeepers (Table 1). In order to confi rm claimed botanical origin of collected honey samples, melissopalinological analysis was conducted ac-cording to the method of Louveaux et al. (1978) where the iden-tifi cation and counting of pollen grains in prepared insoluble honey sediment was performed at 400x magnifi cation under light microscope. Th e identifi cation was carried out using reference  Honey type / botanical species N Black locust (Robinia pseudoacacia L.) 35 Sweet chestnut (Castanea sativa M.) 38 Lime (Tilia spp.) 19 Sage (Salvia officinalis L.) 20 Lavander (Lavandula angustifolia M.) 3 Mandarin (Citrus unshiu Marcovitch) 14 Heath (Erica spp.) 9 Rosemary (Rosmarinus officinalis L.) 3 Strawberry tree (Arbutus unedo L.) 3 Total 144 Table 1. Th e number of analyzed samples (N) by honey typeFigure 1. IR spectra of saturated aqueous solutions of D-(-)-fructose and D-(+)-glucoseAgric. conspec. sci. Vol. 76 (2011) No. 3193Application of Infrared Spectroscopy in Honey Analysissamples of pollen in the form of native preparations (reference collection of the University of Zagreb, Faculty of Agriculture) and literature (Von der Ohe and Von der Ohe, 2000). A stand-ard sample of 500 pollen grains was used for pollen and honey type determination (Von der Ohe et al., 2004). Unifl oral types of honey were determined in accordance with Croatian regulations given in Ordinance on the quality of unifl oral honey (Ministry of Agriculture, Fisheries and Rural Development, 2009). Physico-chemical parameters (moisture, electrical conductivity) were determined according the methods offi  cially prescribed by the Croatian Regulations (Ministry of Agriculture and Forestry, 2000) and in accordance with Harmonised methods of the European Honey Commission (Bogdanov et al.1997). Moisture (water content) was determined from the refractive index of the honey obtained by Abbé refractometer (AOAC 969.38). Electrical conductivity was measured by a multirange conductivity meter HI 8733 (Hanna Instruments) (Bogdanov et al., 1997).Infrared spectra of honey samples were recorded using the ABB Bomem MB102 Fourier-transform infrared spectrometer (FT-IR spectrometer) with a DTGS detector and CsI optics. Single refl ection attenuated total refl ection (ATR) technique was used to obtain spectra. For this purpose, a MKII Golden GateTM ATR accessory (Specac Inc., Woodcock GA, USA) was used. Each spectrum was recorded as the ratio of the spectrum of ATR element containing sample to the spectrum of the empty ATR plate. Since model MB102 is a single-beam spectrometer, a background was taken with the empty ATR plate before meas-uring each sample. Each spectrum represents an average of 100 Fourier-transformed interferograms. Th e nominal resolution was 4 cm-1, which gives a distance between two points in the resulting spectrum % 1= 1.2 cm  . Th e honey samples were re-corded as obtained. Th e spectral range from 1200 to 700 cm-1  (maximum ab-sorbance established at 1026 cm-1) was subjected to further sta-tistical analysis since the greatest alterations in honey have been identifi ed in this particular spectral region (Figure 1). Selected IR spectral regions were analyzed by multivariate data analysis, principal components analysis (PCA). Statistical analy-sis was performed using the statistical soft ware package Matlab 6.0. Preparation of spectra for PCA was performed as follows: all spectra were smoothed using the Savitzky-Golay procedure by fi tting 4th order polynomial to every 13 spectral points. First and second derivative spectra were calculated. Aft er that, each absorbance was multiplied by corresponding overall standard deviation. In this way spectral ranges that change signifi cantly were accented. Spectra were cut to 1200 – 700 cm-1 range and they were normalised to unit area. Principal component analysis was applied to in this way prepared spectra (Figure 2). Results and discussionTh e results of conducted standardized methods have con-fi rmed analyzed samples as unifl oral, for each honey type. Average values of electrical conductivity of analyzed honey types were ranged within the chemical quality criteria for unifl oral honeys (Table 2), established for each honey type (Accorti et al., 1986; Figure 2. Input IR spectra as prepared for PCAAgric. conspec. sci. Vol. 76 (2011) No. 3194 Lidija SVEČNJAK, Nikola BILIŠKOV, Dragan BUBALO, Domagoj BARIŠIĆPersano Oddo and Piro, 2004; Kenjerić et al., 2006). Th e authen-tication of the botanical origin was also conformed by melis-sopalynological analysis. Pollen spectrum was determined on the basis of the share of specifi c pollen grains in the insoluble honey sediment. Honey is mostly consisted of hydrated glucose and fructose, together with higher carbohydrates in smaller amount. Th us, typical IR spectrum of honey is dominated by the features corre-sponding to hydrogen-bonded hydroxyl groups of carbohydrates (Figure 3), together with the bands due to the skeletal vibrations of carbohydrates. Th e absorption due to the other, less abundant, honey components is mainly overlapped.Preliminary principal component analysis (PCA) of IR spectra showed reliable and signifi cant clustering of the analyzed sam-ples by botanical origin. Th e samples were grouped close together creating uniform clusters for each of nine analyzed honey types in the PCA scatter plot (Figure 4). Applied PCA of IR spectra showed that IR spectroscopy provides results equally reliable to those achieved by standard analytical methods. Clusters of nine diff erent unifl oral honey types obtained by PCA correspond to the results of their clasiffi  cation confi rmed by standardized physico-chemical and melissopalynological analysis.ConclusionsBeing very sensitive to the overall chemical composition of the sample, infrared spectroscopy, especially when coupled with multivariate statistical analysis, provides reliable results in this regarding determination of botanical origin of honey. Vibrational spectroscopy enables a non-destructive chemical characterization of honey based on identifi able spectral features. Th e authentication of the botanical origin of nine unifl oral honey types was confi rmed using both, standard and alternative (FT-IR spectroscopy) methods. Th e results of this study showed that IR spectroscopy provides equally reliable results, but also represent rapid and cheap analytical tool in comparison to commonly used standard analytical methods. Th erefore, it is expected that IR spectroscopy will contribute to authentication and quality con-trol of Croatian honey.References Accorti M., Persano Oddo L., Giola Plazza M., Sabatini A.G. (1986). Schede di caratterizzazione delle principali qualitadi miele Italiano. Apicoltora, 2:20-21. AOAC (1996). Offi  cial methods of analysis (16th ed.). Washington, DC: Association of Offi  cial Analytical Chemists. Methods: Offi  cial method 969.38.  Bertelli D., Plessi M., Sabatini A.G. Lolli M. Grillenzoni F. (2007). Classifi cation of Italian honeys by mid-infrared diff use refl ec-tance spectroscopy (DRIFTS). Food Chemistry 101:1565-1570.  Bogdanov S., Martin P., Lullman C. (1997) Harmonised methods of the European honey commision. Apidologie, extra issue, 1-59.  Bogdanov S., Ruoff  K., Persano Oddo L. (2004). Physico-chemical methods for the characterisation of unifl oral honeys: a review. Apidologie 35:S4-S17.  Kenjerić D.,Primorac Lj., Mandić M.L., Bubalo D., Perl Pirički A., Flanjak I. (2006). Dalmatian sage (Salvia offi  cinalis L.) honey characterization. Deutsche Lebensmitte-Rundschau 10:479-484. Lichtenberg-Kraag B., Hedtke C., Bienefeld K. (2002). Infrared spectroscopy in routine quality analysis of honey. Apidologie 33:327–337. Louveaux J., Maurizio A., Vorwohl G. (1978). Methods of melisso-palynology. Bee World. 59: 139-157. Ministry of Agriculture and Forestry (2000). Quality standard of honey and other bee products. Ministry of Agriculture, Fisheries and Rural Developement (2009). Ordinance on the quality of unifl oral honey. Persano Oddo L.,  Piro R. (2004). Main European unifl oral honeys: descriptive sheets. Apidologie 35:S38–S81. Ruoff  K., Luginbühl W., Künzli R., Iglesias M. T., Bogdanov S., Bosset J. O., von der Ohe K., von der Ohe W., Amadò R. (2006). Authentication of the Botanical and Geographical Origin of Honey by Mid-Infrared Spectroscopy. J. Agric. Food Chem. 54:6873-6880. Ruoff  K., Luginbühl W., Bogdanov S., Bosset J.O., Estermann  B., Ziolko T., Kheradmandan S., Amadò R. (2007). Quantitative determination of physical and chemical measurands in honey by near-infrared spectrometry. Eur Food Res Technol. 225:415-423. Skoog D.A., Leary J.J. (1992). Introduction to Instrumental Analysis, 4thed., Saunders College Publishing, New York. Von der Ohe K., Von der Ohe W. (2000). Celler Melissopalynologische Sammlung, Niedersächsisches Lamdesinstitut für Bienenkunde, Celle. Von der Ohe W., Persano Odo L., Piana M.L., Morlot M., Martin P.. (2004). Harmonized methods of melissopalinology. Apidologie 35: 18-25. Woodcock T., Downey G., O’Donnell C.P. (2009). Near infrared spectral fi ngerprinting for confi rmation of claimed PDO prov-enance of honey. Food Chemistry 114:742-746.Table 2. Electrical conductivity range (min, max) for unifl oral honey types and average values of analyzed samplesFigure 3. Typical FT-IR spectrum of honey with arbitrary assignation of the IR absorption bands to corresponding vibrations Honey type / botanical species Min/max (mS/cm) x  (mS/cm) Black locust (Robinia pseudoacacia L.) 0.09-0.23 0.15 Sweet chestnut (Castanea sativa M.) 0.86-1.91 1.22 Lime (Tilia spp. 0.37-0.87 0.65 Sage (Salvia officinalis L.) 0.15-0.52 0.36 Lavander (Lavandula angustifolia M.) 0.12-0.31 0.29 Mandarin (Citrus unshiu Marcovitch) 0.11-0.31 0.27 Heath (Erica spp.) 0.50-0.85 0.64 Rosemary (Rosmarinus officinalis L.) 0.08-0.23 0.23 Strawberry tree (Arbutus unedo L.) 0.60-0.90 0.84 Agric. conspec. sci. Vol. 76 (2011) No. 3195Application of Infrared Spectroscopy in Honey AnalysisFigure 4. (a) Clustering of 9 unifloral honey types by botanical origin obtained using principal component analysis (PCA) of IR spectra; (b) increased region of clusters of all honey types except lavenderacs76_33

Application of Infrared Spectroscopy in Honey Analysis

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