Food Chemistry 155 (2014) 155–160

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Food Chemistry

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Detection of adulterated honey produced by honeybee (Apis mellifera L.)colonies fed with different levels of commercial industrial sugar (C3 andC4 plants) syrups by the carbon isotope ratio analysis

http://dx.doi.org/10.1016/j.foodchem.2014.01.0330308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +90 362 312 1919.E-mail address: aguler@omu.edu.tr (A. Guler).

Ahmet Guler a,⇑, Hasan Kocaokutgen b, Ali V. Garipoglu a, Hasan Onder a, Deniz Ekinci c, Selim Biyik a

a Faculty of Agriculture, Animal Science Department, Ondokuzmayis University, 55139 Atakum, Samsun, Turkeyb Faculty of Arts and Science, Department of Chemistry, Ondokuzmayis University, 55139 Atakum, Samsun, Turkeyc Faculty of Agriculture, Department of Biotechnology, Ondokuzmayis University, 55139 Atakum, Samsun, Turkey

a r t i c l e i n f o

Article history:Received 15 August 2013Received in revised form 12 January 2014Accepted 14 January 2014Available online 24 January 2014

Keywords:HoneySugarsColonyFeedingAdulterated13C/12C isotopePatterns

a b s t r a c t

In the present study, one hundred pure and adulterated honey samples obtained from feeding honeybeecolonies with different levels (5, 20 and 100 L/colony) of various commercial sugar syrups including HighFructose Corn Syrup 85 (HFCS-85), High Fructose Corn Syrup 55 (HFCS-55), Bee Feeding Syrup (BFS), Glu-cose Monohydrate Sugar (GMS) and Sucrose Sugar (SS) were evaluated in terms of the d13C value of honeyand its protein, difference between the d13C value of protein and honey (Dd13C), and C4% sugar ratio.Sugar type, sugar level and the sugar type * sugar level interaction were found to be significant(P < 0.001) regarding the evaluated characteristics. Adulterations could not be detected in the 5 L/colonysyrup level of all sugar types when the d13C value of honey, Dd13C (protein–honey), and C4% sugar ratiowere used as criteria according to the AOAC standards. However, it was possible to detect the adultera-tion by using the same criteria in the honeys taken from the 20 and 100 L/colony of HFCS-85 and the100 L/colony of HFCS-55. Adulteration at low syrup level (20 L/colony) was more easily detected whenthe fructose content of HFCS syrup increased. As a result, the official methods (AOAC, 978.17, 1995;AOAC, 991.41, 1995; AOAC 998.12, 2005) and Internal Standard Carbon Isotope Ratio Analysis couldnot efficiently detect the indirect adulteration of honey obtained by feeding the bee colonies with the syr-ups produced from C3 plants such as sugar beet (Beta vulgaris) and wheat (Triticium vulgare). For this rea-son, it is strongly needed to develop novel methods and standards that can detect the presence and thelevel of indirect adulterations.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Honey, which is accepted as a functional food, is sold at highrates due to its excellent nutritional value. Honey should be unadul-terated, hygienic and pure (Codex, 2001; Council Directive 2001/110/EC). However, this valuable product is vulnerable to variousadulterations at each stage of production and processing (Anklam,1998; Bogdanov et al., 2005; Ruiz-Matute, Weiss, Sammatora,Finely, & Sanz, 2010; White & Winters, 1989). The adulteration ofhoney can be done by direct addition of sucrose syrups that are pro-duced from sugar beet or by the addition of industrial sugar (glu-cose and fructose) syrups obtained from starch by heat, enzymeor acid treatment (Martin, Macias, Sanchez, & Rivera, 1998;Padovan, De Jong, Rodrigues, & Marchini, 2003; Ruiz-Matute, Soria,Martinez-Castro, & Sanz, 2007) to the honey and by feeding the bee

colonies with excessive amounts of these syrups during the mainnectar period (Guler, Bakan, Nisbet, & Yavuz, 2007).

Direct adulteration creates unfairness not only for the consum-ers but also for the pure honey producers (Anklam, 1998;Bogdanov et al., 2005; Guler et al., 2007). This issue has beeninvestigated by many scientists for many years (Cabanero, Recio,& Ruperez, 2006; Elflein & Raezke, 2008; Kerkvliet & Meijer,2000; Padovan et al., 2003; White & Winters, 1989). In the begin-ning, the scientists concentrated on different physicochemicalparameters like melissopalynological pattern, botanic origin (Kropfet al., 2010; Oddo et al., 2004). sensory analysis (Guler, Bek, &Kement, 2008) sugar profile, amino acid profile, enzyme activi-ties (diastase, invertase), hydroxymethylfurfural and proline(Bogdanov et al., 2005; Cotte, Casabianca, Chardon, Leheritier, &Grenier-Loustalot 2003; Cotte et al., 2004; Guler et al., 2007;Kerkvliet & Meijer, 2000; Oddo et al., 2004; Padovan, Rodriquez,Leme, Jong David, & Marchini, 2007; Raezke & Elflein, 2007).Although each property has its own importance on the quality

156 A. Guler et al. / Food Chemistry 155 (2014) 155–160

determination of the honey, their analyses are time consuming andnot sensitive enough for determination of professional adultera-tion. To solve the problems discussed above, scientists concen-trated on development of new analytical methods like stablecarbon isotope ratio mass spectrometry analysis (Doner & White,1977; White & Doner, 1978). The principle of this method is basedon the differences between the 13C/12C ratio of C4 originating frommonocotyledonous species of cane sugar and corn, when comparedto dicotyledon species (C3 plants) (Bender, 1971; Elflein & Raezke,2008; Padovan et al., 2003; Simsek, Bilsel, & Goren, 2012). The iso-topic ratio of 13C/12C indicates the way of CO2 fixation and utiliza-tion by plants. While the C3 plants, which are the main nectarproviding sources of the honeybees, utilize the Calvin and Bensoncycle, the C4 plants from, which invert sugar syrups are produced,use both the Calvin and Benson cycle and the Hatch–Slack cycle. Itis known that the C4 plants use more CO2 than the C3 plants. Thed13C values of the C3 and the C4 plants show variability between�23‰ to �28‰ and �9‰ to �15‰, respectively (Cabanero et al.,2006; Ruiz-Matute et al., 2010). There is a general agreement thatthe difference between the d13C value of protein and honey (Dd13C)should not be greater than 1‰, which, above this value, corre-sponds to 7% adulteration. In addition, the d13C value of honeyshould be more negative than �23.5‰ in order to be classified aspure (Cabanero et al., 2006; Padovan et al., 2003; Ruiz-Matuteet al., 2010).

Indirect adulteration carried out by excessive supplementaryfeeding of the bee colonies in the main nectar flow period also cre-ates injustice for both the consumers and the pure honey produc-ers (Bogdanov et al., 2005; Guler et al., 2007; Oddo et al., 2004).The problems on detection of adulteration are also valid for indi-rectly adulterated honey. In addition, the latter issue has not beeninvestigated in detail. Furthermore, it is not known how honeybeesuse different levels of these commercial sugar syrups. Also, it is notknown which sugars are converted into which type and level ofsugars in adulterated honey by honeybees. We do not have ade-quate knowledge as to whether indirect adulteration (overfeedingbee colonies with industrial sugar syrups) can be determined byusing official methods (AOAC 978.17, 1995, AOAC 991.41, 1995;AOAC 998.12, 2005). There have been no published findingsregarding the d13C value of honey and its protein, difference be-tween the d13C value of protein and honey (Dd13C), and C4% sugarratio in the indirectly adulterated honey. Furthermore, many scien-tists (Ozcan, Arslan, & Ceylan, 2006; Ruiz-Matute et al., 2010) havesuggested that all these sugars should be tried with bee colonies inthe field. Therefore, we decided to use different commercial indus-trial sugar syrups and levels as bee food during the main nectarflow period in order to answer all the questions discussed above.

Thus, the present study aimed to determine indirect adultera-tion in the honey produced by feeding honeybees with differentlevels (5, 20 and 100 L/colony) of various industrial sugars includ-ing HFCS-85 (Zea mays), HFCS-55 (Zea mays), glucose monohydrate(GMS, Triticium vulgare), sucrose (SS, Beta vulgaris) and bee foodsyrup (BFS) by using official methods (AOAC 978.17, 1995, AOAC991.41, 1995, and AOAC 998.12, 2005). We investigated whetherthese values change with sugar types and levels, and whether indi-rect adulteration can be detected using the above criteria for differ-ent types and levels of industrial sugars.

2. Materials and methods

2.1. Materials

Honey samples were taken from the colonies in the ApiculturalResearch and Application Unit of the Agriculture Faculty of Ond-okuzmayis University, Samsun, Turkey. Colonies were retained in

Samsun (41.2�N, 36.20�E) in the Black Sea Region during the winterand spring, and in the vicinity of Gulacar Valley, near Gumushane(40.274�N, 39.29�E), during the nectar flow period (June, July andAugust). The main sources of nectar-producing plants in the valleywere identified by the Department of Biology of the Faculty of Artsand Science, Ondokuzmayis University. The Gulacar valley is rich innectar-producing plant species (Guler et al., 2007). The main plantresources are Satureja thybra, Lamium album, Trifilium ambigum,Salvia forskahler, Astragalus microcehalus, Astragalus asciocalyx,Cichorium intybus, Thymus leucotrichus, Coronilla varia subsp. Varia,Salvia amasiaca, Melilotus officinalis, Medicago varia, Teucrium cha-maedrys, Anchusa azurea, Galium verum and Veronica peduncularis.

2.2. Sugar sources

Types, origins, compositions, forms, proportions and company’snames of the industrial sugars used in the study are summarized inTable 1.

2.3. Methods

2.3.1. Maintenance and preparation of the colonyThe colonies with two aged queen bees of the same genetic ori-

gin were used in the study. All of the environmental factors (framescovered with adult bees, frames covered with brood, foundationcomb, drugs, transport) were standardized, and all of the mainte-nance and control procedures were performed by the same staffs.

The colonies were settled in empty beehives with bees, broodand honey frames. Standard bee-feeding methods were appliedin the early spring (Guler, 2008; Sammataro & Avitabile, 1998).Twelve kilograms of sucrose syrup (1:1.5 w/w, water:sugar) weregiven to each colony in March and April to ensure the growthand strength of the forage worker bee population of colonies forthe main nectar flow season. The honey and honeycomb framesof all colonies were taken at the end of May (Guler, 2008). Aftersettling bees in the hives, cake and syrup were not further providedto the colonies. Transporting and shaking processes were donewith standard beehives (Langstroth), and veterinary drugs werenot used for any honeybee diseases. The processes were appliedto all groups on the same day. Ten days after all these arrange-ments, colonies were transported (470 km) to another region(Gulacar Valley, Gumushane) in order to obtain rich and unpol-luted nectar sources.

In the study, pure blossom honey (PBH), which was produced inthe same environmental conditions by applying the shaking meth-od (Guler, 2008), was used as the control. As with the other com-mercial industrial sugar groups, feeding, development andmaintenance of the bee colonies were conducted up to a predeter-mined stage in the spring season, and after that point, the queenand worker bees together were shaken to the empty hives. Afterthis process, no syrup, cake or chemicals were given to the colo-nies. Only comb foundations were given to the colonies. The honeyproduced in this study was regarded as a polifloral honey as therewere many plant species in the region in which this study was con-ducted. In total, 130 colonies (ten colonies for the pure or controlgroup and eight colonies for each of the three levels of five sugarsyrup types; 10 + 5 * 3 * 8 = 130) were used in the study.

2.3.2. Syrup levels and preparationThese industrial commercial sugars and levels were used for the

first time as a bee food supplement during the main nectar flowperiod in this study. In all treatments, syrups were prepared daily,were mixed often, left for one day and finally were given to thecolonies at 6:00–7:00 pm. Levels of 5, 20 and 100 L/colony ofHFCS-85, HFCS-55, GMS, SS and BFS were used in the study. Theselevels were selected on the basis of syrup amounts that are used by

Table 1Types, origins, compositions, forms, proportions, supplied company names of the industrial sugars used in the experiment.

Sugar type Origin Form Composition Usage proportion Company name

HFCS-85 Corn (Zea mays) Liquid 84.9% fructose12.8% dextrose

1:3 (water:sugar, w:w) Cargill

HFCS-55 Corn (Zea mays) Liquid 55.6% fructose39.6% dextrose

1:3 (water:sugar, w:w) Cargill

GMS Wheat (Triticum vulgare) Powder 99.0% glucose 70% water + 30% sugar Syral company (Belgium)SS Beet sugar (Beta vulgaris) Crystalline 99.5% sucrose 1:1.5 (water:sugar, w:w) Turhal Sugar Company (Turkey)BFS Beet sugar (Beta vulgaris) Liquid pasteurised 30–36% sucrose

27–30% glucose37–40% fructose

1:3 (water: BFS, w:w) Konya Sugar Company (Turkey)

Table 2� 13

A. Guler et al. / Food Chemistry 155 (2014) 155–160 157

beekeepers in Turkey (Guler et al., 2007). Syrup was given tothe experimental colonies at different intervals (twice for the5 L/colony, eight times for the 20 L/colony and forty times for the100 L/colony). Before new syrup application, the amount of uncon-sumed syrup (g/colony) was recorded on each colony’s card.

2.3.3. Honey harvest and honey sample preparationHoney was harvested at the end of August 2012 by centrifuge

and filtered through a 0.2-mm sieve into lactin. Honey sampleswere taken from six colonies, which were chosen randomly fromamong all available groups, and a total of 100 samples (five indus-trial sugars * three syrup levels * six repetitions = 90 adulteratedsamples + 10 pure honey or control samples) were analysed usingthe analytical methods described below.

2.3.4. Analytical methodsOne hundred (100) honey samples were analysed using the

same methods during the same time period to ensure uniformconditions and comparability (Raezke & Elflein, 2007; White &Winters, 1989). The following compositional properties weredetermined for pure blossom honey (control) and adulterated hon-ey samples: d13C values were determined by Elemental Analyzer–Isotope Ratio Mass Spectrometry (EA–IRMS) after complete samplecombustion to carbon dioxide, as described by AOAC (1998) meth-od 991.41. The C4% sugar contents in honey samples were deter-mined by using the AOAC standard (998.12) and the equationgiven below (Elflein & Raezke, 2008; Padovan et al., 2003; White,1992; White & Winters, 1989; White, Winters, Martin, &Rossmann, 1998).

%C4 ¼ d13Cprotein � d13Choney=d13Cprotein � �9:7ð Þ

Means (X) and standard errors (�S�X ) related to the d C value of the 100 adulteratedand pure honeys and theirs protein.

Sugars Syruplevels

d13C protein d13C honey Honeyquality

HFCS-85 5a �25.02 ± 0.03c �25.15 ± 0.05f Pure20 �24.82 ± 0.03b �21.70 ± 0.10c Adulterated100 �23.38 ± 0.12a �15.87 ± 0.11a Adulterated

HFCS-55 5 �25.4 ± 0.07fgh �25.53 ± 0.11gh Pure20 �25.07 ± 0.04cd �24.52 ± 0.15d Pure100 �23.38 ± 0.04a �17.2 ± 0.07b Adulterated

GMS 5 �25.53 ± 0.03ghi �25.88 ± 0.04ik Pure20 �25.35 ± 0.03efg �25.53 ± 0.06gh Pure100 �25.25 ± 0.06def �25.73 ± 0.04hi Pure

BFS 5 �25.17 ± 0.07cde �25.42 ± 0.05g Pure20 �25.33 ± 0.08ef �25.62 ± 0.05gh Pure100 �25.27 ± 0.06ef �24.88 ± 0.05e Pure

SS 5 �25.72 ± 0.07I �25.95 ± 0.04ik Pure20 �25.4 ± 0.10fgh �25.75 ± 0.10hi Pure100 �25.57 ± 0.05hi �25.75 ± 0.06hi Pure

PBH n = 10 �25.97 ± 0.03nk �26.07 ± 0.03k PureSignificance <0.000 <0.000

HFCS = High Fructose Corn Syrup, GMS = Glucose Monohydrate Sugar, BFS = BeeFeeding Syrup, SS = Sucrose Sugar, PBH = Pure Blossom Honey (n = 10).Values within rows with different letters differ significantly (P < 0.001).

a For each syrup level n = 6.

2.4. Statistical analysis

The study was carried out according to the Randomised Facto-rial Plot Design. Pairwise Permutation Test was used for compari-son. Homogeneity of variance was performed by using LeveneVariance Homogeneity Test and One-Sample Kolmogorov–SmirnovTest (Onder, 2007). The variance analysis was performed by usingSAS pocket programme and Duncan test was used for comparisonof averages (SAS, 1988).

3. Results and discussion

All syrup levels of the HFCS-85, HFCS-55, BFS and SS sugarswere consumed eagerly by bees in a short time. However, noneof the three syrup levels of Glucose Monohydrate Sugar (GMS)were consumed heartily. In this group, only 61 L of syrup at the100 L/colony level were consumed for each of the eight colonies.This showed that bees were not able to consume GMS syrup. How-ever, the SS and BFS syrups were consumed eagerly and depositedas honey. All of the combs were glazed in these two groups.

The mean �X� �

and standard errors �S�Xð Þ values for d13C protein,d13C honey, Dd13C (protein–honey), and C4% ratios are given inTables 2 and 3. There were highly significant (P < 0.001) differencesbetween sugar types and sugar levels in terms of the investigatedcharacteristics. Furthermore, the sugar type/syrup level interactionwas found to be significant (P < 0.001). Each of these characteristicsis discussed further below.

3.1. d13C value of honey and its protein (‰)

The d13C value of protein was lowest in the 100 L/colony ofHFCS-85 (�23.38 ± 0.12‰) and HFCS-55 (�23.38 ± 0.04‰). It was�25.97 ± 0.03‰ for the pure honey (PBH). The d13C value of honeyranged between �15.87 ± 0.11‰ and �26.07 ± 0.03‰ in the 100adulterated and pure honey samples. This ratio was �26.07 ±0.03‰ for pure honey (PBH). The lowest and the highest negatived13C values of honey were found for the 100 L/colony syrup levelof HFCS-85 and the PBH group (�15.87 ± 0.11‰ and �26.07 ±0.03‰, respectively). Of the 100 samples, eighteen were identifiedas fraudulent at the 20 and 100 L/colony syrup levels of HFCS-85and at the 100 L/colony syrup level of HFCS-55 due to the fact thattheir d13C values were below �23.5‰ (AOAC 978.71 standard).However, adulteration was not detected for the 5 L/colony ofHFCS-85, the 5 and 20 L/colony of HFCS-55, and all syrup levelsof GMS, SS and BFS when their d13C values were taken into account.The d13C value of honey taken from the 5 L/colony level of SS was�25.95 ± 0.04‰, which was closest to that of the PBH (control). Itwas observed that the d13C value of adulterated honeys decreasedin a negative way depending on increasing amounts of HFCS-85

Table 3Means ð�XÞ and standard errors ð�S�XÞ related to the d13C value of the 100 adulterated and pure honeys and difference between the d13C value of protein and honey, C4% ratio.

Sugars Syrup levels Dd13C (protein–honey) Honey quality C4% Honey quality

HFCS-85 5a 0.13 ± 0.08b* Pure 0.2 ± 0.13f Pure20 �3.12 ± 0.08d Adulterated 20.62 ± 0.54c Adulterated100 �7.52 ± 0.09f Adulterated 54.77 ± 0.71a Adulterated

HFCS-55 5 0.20 ± 0.04b Pure 0 ± 0f Pure20 �0.55 ± 0.15c Pure 3.67 ± 0.86d Pure100 �6.18 ± 0.09e Adulterated 45.2 ± 0.58b Adulterated

GMS 5 0.35 ± 0.05ab Pure 0 ± 0f Pure20 0.18 ± 0.05b Pure 0 ± 0f Pure100 0.48 ± 0.03a Pure 0 ± 0f Pure

BFS 5 0.25 ± 0.11ab Pure 0.22 ± 0.22f Pure20 0.28 ± 0.09ab Pure 0.1 ± 0.1f Pure100 �0.38 ± 0.06c Pure 2.47 ± 0.39e Pure

SS 5 0.23 ± 0.1ab Pure 0.1 ± 0.1f Pure20 0.35 ± 0.09ab Pure 0 ± 0f Pure100 0.18 ± 0.07b Pure 0 ± 0f Pure

PBH n = 10 0.10 ± 0.04b Pure 0.09 ± 0.09f PureSignificance <0.000

HFCS = High Fructose Corn Syrup, GMS = Glucose Monohydrate Sugar, BFS = Bee Feeding Syrup, SS = Sucrose Sugar, PBH = Pure Blossom Honey (n = 10).a =For each syrup level n = 6.

* Values within rows with different letters differ significantly (P < 0.001).

158 A. Guler et al. / Food Chemistry 155 (2014) 155–160

and HFCS-55. This finding is compatible with the findings of previ-ous studies (Elflein & Raezke, 2008; Padovan et al., 2003; Simseket al., 2012; Tosun, 2013) that aimed to detect adulteration byaddition of various levels of HFCS syrup to honey. The smallestchange in the d13C ratio of honey occurred in the GMS group. Thiswas attributed to the fact that the GMS sugar was produced fromwheat, which is a C3 plant. Another reason might be the fact thatonly 61 kg of 100 kg of GMS syrup was consumed by the colonies.Thus, all values for the GMS group really belong to the 61 L/colonylevel of GMS sugar. This might be attributed to the fact that beescannot hydrolyze this sugar (glucose) into honey sugars (fructoseand glucose). It is known that bees invert glucose into fructose(Ruiz-Matute et al., 2010). However, it is obvious from the presentstudy that bees cannot use glucose given in excessive and amounts.This might be attributed to a deficiency in enzymes (especially dia-stase) that are used to invert glucose into fructose. The d13C valuesof the adulterated honeys produced by using the 5, 20 and 100 L/colony of GMS, SS and BFS were very similar (ranging from�24.88 ± 0.05‰ to �25.95 ± 0.04‰), although they were statisti-cally different. However, there were great differences in the d13Cvalues of honey taken from the 5, 20 and 100 L/colony of theHFCS-85 and HFCS-55 (Table 2). The d13C value of honey was lowerand more variable when compared to the d13C value of its protein.

The d13C value of honey in the 100 L/colony of HFCS-85(�15.87 ± 0.11‰), in which the highest fraudulence was detected,was more positive than those reported by Tosun (2013) (50% hon-ey + 50% HFCS; �18.95 ± 0.21‰), Cabanero et al. (2006) (90% hon-ey + 10% HFCS; �23.8‰), Elflein and Raezke (2008) (50%honey + 50% HFCS; �22.3‰) and Padovan et al. (2003) (10% hon-ey + 90% HFCS; �17.49 ± 0.11‰). We thought that there might bethree reasons for the difference. The first reason is that we usedHFCS as honeybee food and therefore it was metabolized or pro-cessed by honeybees, whereas other authors added HFCS directlyto the honey and it was not metabolized by honeybees. The secondreason is that we used a higher amount of HFCS (100 L HFCSsyrup per colony) when compared to the other authors. The thirdreason is that there have been differences in the studies in relationto fructose proportion of the used HFCS. While our compared value(�15.87 ± 0.11‰) belonged to HFCS-85, the value reported byPadovan et al. (2003) belonged to HFCS-55. There has been noinformation on the amount of fructose in the HFCS used byCabanero et al. (2006), Elflein and Raezke (2008), and Tosun(2013). The closest value to our finding (�17.49 ± 0.11‰) was

reported by Padovan et al. (2003). Although they used HFCS-55as a sugar source, the proportion of the HFCS in the honey and su-gar mixture was the same as in the present study.

In fact, it may be thought that this type of comparison is notcorrect due to the fact that the compared values were obtainedfrom different applications. Namely, our values belong to the hon-eys containing sugar metabolized by honeybees, whereas othervalues belong to the honeys containing sugar or syrup not metab-olized by honeybees. However, this comparison may give thechance to see whether there are any differences between two pro-cesses or not.

3.2. Difference between the d13C value of honey and its protein(Dd13C, ‰)

The Dd13C value ranged from �7.52 ± 0.09‰ to 0.48 ± 0.03‰ inthe pure and adulterated honeys produced by feeding the honey-bee colonies with 5, 20 and 100 L/colony levels of different sugars(Table 3). The highest positive Dd13C value was determined in GMS(0.48 ± 0.03‰) and the highest negative Dd13C value was found inthe 100 L/colony syrup level of HFCS-85 (�7.52 ± 0.09‰). Accord-ing to the AOAC 991.4 standard, the difference between the d13Cvalue of honey and its protein should not be greater than 1‰, orthis value should be more positive than �1. Adulteration up to7% can be detected using this standard. This rule is also acceptedas the most significant criterion of purity (Padovan et al., 2003).When this standard was taken into consideration, in the presentstudy, of the 100 honey samples, eighteen were found as adulter-ated, which came from the 20 and 100 L/colony of HFCS-85(�3.12 ± 0.08‰ and �7.52 ± 0.09‰, respectively) and the 100 L/colony of HFCS-55 (�6.18 ± 0.09‰). Although the 20 L/colony ofHFCS-55 and the 100 L/colony of BFS had negative values(�0.55 ± 0.15‰ and �0.38 ± 0.06‰, respectively), they were notconsidered as fraudulent due to the fact that the values were morepositive than �1. However, this still indicates the presence of thesugars produced from C4 plants in these honey samples. Likewise,all of the adulterated samples were associated with the sugars(HFCS-85 and HFCS-55) produced from C4 plants (corn). Therefore,as stated in previous studies (Cabanero et al., 2006; Elflein &Raezke, 2008; Padovan et al., 2003; Tosun, 2013; White & Winters,1989), our study also showed that it is not possible to detect adul-terations performed by feeding the bee colonies with syrup pro-duced from C3 plants by using the difference between the d13C

Table 4Relationships between the d13C value of honey and its protein, and C4% ratio.

d13C honey d13C P–d13C H C4%

d13C protein 0.932 �0.890 0.917d13C honey �0.995 0.997d13C P–d13C H 0.995

D13C P–d13C H: difference between the d13C value of protein and honey.

Fig. 1. Residue graphic related to the regression equation.

A. Guler et al. / Food Chemistry 155 (2014) 155–160 159

value of honey and its protein. The results of the BFS sugar groupconfirm this finding, since BFS contains the invert sugars producedfrom both C3 plants (sugar beet) and small amount of C4 plants (su-gar cane and corn). The Dd13C value (�0.38 ± 0.06‰) and the C4%ratio (2.47 ± 0.39%) in the 100 L/colony syrup level of this sugarindicated that it contained a low amount of invert sugar. In theprospectus of this sugar, inverted fructose was reported at a rateof 37–40% (Table 3).

The difference between the d13C value of honey and its proteinin the 100 L/colony HFCS-85 (�7.52 ± 0.09‰) was found to bemore negative than those reported by Tosun (2013) (50% HFCSaddition; �6.00 ± 0.28‰) and Raezke and Elflein (2007) (50% HFCSaddition; �2.00‰). The more negative ratio indicates a higher levelof adulteration. This might be attributed to the application of thehigher syrup level (100 L/colony) in the present study.

3.3. C4% ratio

The C4% ratio changed between 0.0 ± 0.0% and 54.77 ± 0.71%.The highest C4% sugar ratio was found in the honey taken fromthe 100 L/colony level of HFCS-85 (54.77 ± 0.71%). This ratio was20.62 ± 0.54% for the 20 L/colony level of HFCS-85 and45.2 ± 0.58% for the 100 L/colony level of HFCS-55. At the 5 L/col-ony level of the HFCS-85 and HFCS-55 sugars and at all syrup levelsfor GMS and SS and PBH (pure honey) had similar C4% values. TheC4% value at the 100 L/colony syrup level of BFS was found to be2.47 ± 0.39% (Table 3).

The C4% ratio shows the presence of C4 sugars in honey; this ratioshould not be greater than 7% (AOAC 998.12). Above this percentageindicates an adulteration. As in the d13C value of honey and Dd13C(protein–honey), the honeys coming from the 20 and 100 L/colonyof HFCS-85 (20.62 ± 0.54% and 54.77 ± 0.71%) and the 100 L/colonyof HFCS-55 (45.2 ± 0.58%) were identified as adulterated when theC4% value was taken into consideration. These C4% ratios were inaccordance with the results of Raezke and Elflein (2007) that wereobtained from the addition of 20% ISS (Invert Sugar Syrup) to purehoney. However, C4% ratios of the adulterated honeys were higherthan those reported by Raezke and Elflein (2007) with the additionof 20% and 50% invert syrup (5.5% and 13.7%, respectively). In addi-tion, C4% ratio of the honey in the 20 L/colony of HFCS-85(20.62 ± 0.54%) was higher compared to the result of Tosun (2013)obtained by the addition of 20% HFCS to honey (12.55 ± 0.21%).However, the C4% ratio of the honey in the 20 L/colony of HFCS-55(3.67 ± 0.86%) was lower than the value reported by the latterauthor for the same honey and HFCS mixture. These differencesmay be attributed to not only the nature of the honey, i.e. we usedHFCS as bee food while the others added it to the honey, but alsothe differences in the fructose rate of HFCS. While adulterationwas detected in the 20 L/colony of HFCS-85, it was not found inthe 20 L/colony of HFCS-55. This indicated the effect of the fructosecontent of the used HFCS on determination of the adulteration.

3.4. Relationships between the investigated characteristics

The relationships between the d13C value of honey and its pro-tein, Dd13C (protein–honey), and C4% ratio are presented in Table 4.

There were highly significant relationships between the investi-gated characteristics. The most important positive relationship(P < 0.001) was found between the d13C value of honey and C4%(r = 0.997). The quadratic model was determined to be the bestmodel for curve estimation, which is required for determiningexplanatory ability of the d13C value of honey for prediction ofC4%. In light of this information, quadratic regression equation[(C4% = 215.045 + 12.879 * d13C honey + 0.176 * (d13C honey)2] wasformed. The significance level and coefficient of determination(R2) for this model was found to be P < 0.001 and R2 = 0.995,respectively. The obtained findings indicate that the success ofthe d13C value of honey for predicting the C4% was very high. Fur-thermore, the absence of extreme values of residues can be consid-ered as an indicator of efficiency of this equation (Fig. 1).

4. Conclusion

The nutrition of bees is essential in certain periods of the year,especially during brood rearing, growth and strengthening of theworker and forage bee populations, queen rearing and comb foun-dation, as well as during uniting and establishing the nuclei of col-onies to meet nutrient requirements. However, giving excessiveamounts of syrup during the main nectar flow period (indirectadulteration) leads to a reduction in honey quality and honeybeebiology.

It is possible to detect the adulteration of honey produced byusing strong syrups of sugars derived from C4 plants such as corn(Zea mays) and sugar cane (Saccharum officinarium) by using thed13C value of honey and d13C value of its protein, difference be-tween the Dd13C value of honey and its protein, and the C4% ratio.Adulteration at low syrup levels (20 L/colony) can be more easilydetected when the fructose (monosaccharide) content of HFCS syr-ups increased. However, honey C4% sugar analysis (AOAC 998.12,2005) was more efficient in detecting adulteration compared tothe other parameters. According to the findings of the presentstudy, we suggest that 10 and 15 L/colony of HFCS-85 and 30and 40 L/colony of HFCS-55 sugar levels should be investigatedto determine adulteration. On the other hand, it can be concludedthat the official methods (AOAC, 978.17, 1995; AOAC, 991.41,1995; AOAC 998.12, 2005) and EA-IRMS cannot efficiently detectthe indirect adulteration of honey made by feeding bee colonieswith various excessive levels of syrups originating from C3 plantssuch as sugar beet (B. vulgaris) and wheat (T. vulgare). For this rea-son, it is strongly needed to develop novel methods and standardsthat can detect the presence and the level of such adulterations.

Acknowledgements

This study was funded by The Scientific and Technological Re-search Council of Turkey (TUBITAK) (Project number 111 O 268

160 A. Guler et al. / Food Chemistry 155 (2014) 155–160

TOVAG, with the title ‘‘Determination of the effect of some indus-trial sugars on honeybee (Apis mellifera L.) colony productivity,behavior and honey quality’’. The authors are grateful to TUBITAKfor financial support, to Professor Dr. H. Guray Kutbay for the iden-tification of nectar source plants and Dr. Semiha Guler for her con-tribution to the manuscript.

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