Research ArticlePollen Physicochemical and Mineral Analysis ofCroatian Acacia Honey Samples Applicability for Identificationof Botanical and Geographical Origin

Natalija Uršulin-Trstenjak1 Dinko PuntariT2 Davor LevaniT1

Vlatka GvozdiT3 Celjka Pavlek4 Ada PuntariT5 Eda PuntariT6 Ida PuntariT7

Domagoj VidosavljeviT8 Dario LasiT4 and Marina VidosavljeviT9

1University North 104 Brigade 3 42000 Varazdin Croatia2Catholic University of Croatia Ilica 242 10000 Zagreb Croatia3Department of Chemistry Josip Juraj Strossmayer University of Osijek Kuhaceva 20 31000 Osijek Croatia4Andrija Stampar Teaching Institute of Public Health Mirogojska 16 10000 Zagreb Croatia5Faculty of Food Technology and Biotechnology University of Zagreb Pierottijeva 6 10000 Zagreb Croatia6Croatian Environment and Nature Protection Agency Radnicka 80 10000 Zagreb Croatia7Health Centre of the Zagreb County Ljudevita Gaja 37 10430 Samobor Croatia8Faculty of Medicine Josip Juraj Strossmayer University of Osijek Cara Hadrijana 10e 31000 Osijek Croatia9General Hospital Vinkovci Zvonarska 57 32000 Vinkovci Croatia

Correspondence should be addressed to Natalija Ursulin-Trstenjak natalijaursulin-trstenjakuninhr

Received 5 July 2017 Revised 17 October 2017 Accepted 5 December 2017 Published 21 December 2017

Academic Editor Anca Ioana Nicolau

Copyright copy 2017 Natalija Ursulin-Trstenjak et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The aim of the study was to investigate health safety and quality of the Croatian acacia honey the selected elements in the soil andwhether multivariate methods can provide identification of the origin of honey The study included 200 acacia honey samples and100 soil samples from East Northwest Croatia and Istria The proportion of acacia in honey was determined by conducting pollenanalysis Water free acids electric conductivity reducing sugars saccharose diastase and HMF were determined No significantdifferences were found using Kruskal-Wallis test regarding the physicochemical parameters (119901 = 09190) the mineral content ofhoney (119901 = 08955) or themineral composition of the soil (119901 = 08789) No significant correlation was found between the analyzedelements in honey and soil Multivariate methods indicated that East Croatia honey samples have higher concentrations of waterHMF and higher concentrations of measured elements except for Al Honey samples from Northwest Croatia are characterizedby low concentrations of elements and a higher concentration of saccharose The Istria samples are richer in reducing sugars freeacids diastase higher conductivity higher content of the acacia pollen grains and lower concentrations of most metals Honeyfrom Northwest Croatia and Istria shares the high concentration of Al in honey

1 Introduction

Honey has been used for centuries as a natural food [1] Thecomposition of honey depends on the plant species climateconditions environmental conditions and beekeeping prac-tice Mostly it consists of carbohydrates water protein freeamino acids enzymes vitamins minerals and another 200or so compounds [2]

Biodiversity of melliferous plant species from differentclimatic and geographic regions of Croatia provides greatpotential for honey production with acacia honey beingone of the most common Therefore to determine the geo-graphical origin and composition of honey many analyticaltechniques and parameters have been used in combinationwith statistical methods Physicochemical parameters inhoney can point to fermentation sugar adding heating

HindawiJournal of Food QualityVolume 2017 Article ID 8538693 11 pageshttpsdoiorg10115520178538693

2 Journal of Food Quality

honey adulteration attempts inappropriate storage and soforth [3 4] The water content in honey ranges from 15to 23 [5ndash7] Honey which is placed on the market mustnot have water content higher than 20 [3 4] Permittedvalue of free acids in honey is lt50mEq of acid1000 g ofhoney [3] Their presence tells us whether the honey is older[8] Electrical conductivity (mScm) is a physical propertywhich greatly depends on the content of mineral substancesand acids in honey [9] Electrical conductivity of floral andblended honey needs to be lower than 08mScm [3 4] Thesum of glucose and fructose or reducing sugars (g100 g) inhoney must be at least 60 g100 g [3] Carbohydrates are themain ingredient of honey and their proportion is 73ndash83[7 10] Determining the amount of saccharose (le10 g100 g)is important for confirming possible adulteration of honeyfeeding the bees sugar (saccharose) or directly adding sugarin honey [11] Diastase activity (DN) represents one of themain parameters in determining the intensity of heating thehoney during its processing and storage [12 13] Neverthelessthe proportion of HMF in the fresh sieved honey usually doesnot exceed 10mgkg [14 15]

Macro and micro elements expressed as a percentage ofash can be found in floral honey in the amount up to 02Honey usually contains the following minerals K Ca NaMg Cl P Fe Mn and others [7] Mineral content primarilydepends on the botanical origin and climatic conditions butusually also on the type of soil where the honey plant grows[16]

Minerals from the soil are transported through the rootsystem to the honey plant moving on to nectar and fromthere to honey therefore the proportion of macro elementsparticularly Ca Mg Mn and Na is influenced by thecomposition of soil which is geochemically and geologicallydetermined [17]

The aim and purpose of the study was to provide areview of health safety and quality of the differences in thepollen analysis physicochemical parameters and compo-sition and concentration of twelve elements in the acaciahoney from three regions in Croatia (East Northwest andIstria) Also an overview of differences with regard to thecomposition and concentration of identical elements fromthe soil from the same locations was given Moreover theauthors tried to identify to what extent multivariate statisticalmethodsmdashespecially the cluster and PCA analysesmdashcan havea predictive significance for the composition and geographi-cal origin of honey

2 Materials and Methods

21 Materials The study included 200 samples of acaciahoney collected from beekeepers in 2009 and 2010 Thesamples originated from three Croatian regions NorthwestCroatia represented by the samples from the VarazdinKrapina-Zagorje and Bjelovar-Bilogora Counties with a totalof 120 samples East Croatia represented by the samples fromthe Pozega-Slavonia Virovitica-Podravina Osijek-Baranjaand Vukovar-Srijem Counties with a total of 40 samplesand Istria region in the county of the same name also witha total of 40 samples Moreover 100 soil samples (Figure 1)

SH Northwest CroatiaIH East CroatiaI Istria

Figure 1 Diagram of the Republic of Croatia with three regions(East Croatia Northwest Croatia and Istria) and locations of acaciahoney and soil sampling

were sampled on the same locations (according to the GPSpoints of the apiary locations) of which there are 60 samplesinNorthwest and 20 in East Croatia and in Istria respectively

22 Methods

221 Pollen Grains Pollen grains were identified and quan-tified by applying microscopy (400x magnification) to prepa-rations taken from the honey samples The proportion ofeach type of pollen was calculated as a percentage of totalpollen The collection of reference pollen preparations wasused for the determination of pollen grains from the samplesaccording to the principle of comparison Complying withthe provisions of the EU directive on the quality of honey [4]of the criteria of expert publications [18] and of the currentlegislation in the Republic of Croatia [17] the requirementof marking the botanical origin of honey is the presence ofthe prevailing pollen The term ldquounifloral honeyrdquo is used todescribe those types of honey which originate mainly fromone dominant plant species Unifloral honey can be labeledbased on a particular plant type if it contains at least 45of the pollen grains of the same plant species with someexceptions therefore for acacia honey the amount is 20(Robinia pseudoacacia L) [18 19]

222 Physicochemical Parameters As it regards physico-chemical parameters water content () in honey was deter-mined by a method based on refractometry Free acids inhoney were determined so that the prepared sample wastitrated using the solution of 01M of sodium hydroxide topH 830 Electric conductivity in honey is defined as theconductivity of a 20 aqueous solution of honey at 20∘Cwhere 20 refers to the dry weight of honey The resultsare expressed in mScm The method for the determinationof reducing sugars and saccharose in honey is based onreducing Fehlingrsquos solution by titration with a sugar solution

Journal of Food Quality 3

from honey and using methylene blue as the indicator Theproportion of saccharose is obtained from the differencein the results obtained before and after the inversion [20]When determining the diastase activity in honey the methodused is based on the hydrolysis of 1 starch solution from1 g of honey in one hour at a temperature of 40∘C Whendetermining hydroxymethylfurfural (HMF) in honey usingtheWinklermethod the procedure is based on the reaction ofhydroxymethylfurfural with barbituric acid and p-toluidinewhich results in pink the intensity of which is measured at awavelength of 550 nm [21 22]

223 Determination of Mineral Substances in Honey Uponthe homogenization of honey the organic matter undergoesa wet chemical decomposition using concentrated nitric acidand hydrogen peroxide in a microwave apparatus (AntonPaar Multiwave 3000) The method of mass spectrome-try for detecting metals in honey is based on inductivelycoupled plasma (ICP-MS) which leads to ionization Massspectrometer is used to detect and identify ions based on theirmasscharge ratios [23 24]

224 Determination of Mineral Substances in Soil Soil sam-pling is defined by a specific procedure Each sample wastaken from a specific point previously defined (according tothe Gauss-Kruger coordinates) The samples were extractedfrom the soil profile at a depth of 05 meters and then storedon the ground in PVC containers In the laboratory priorto sieving the samples were dried at ambient temperatureand homogenized in an agate mill and then sieved to 2mmfractions

Determination of minerals in the soil was conductedby using the samples prepared by extracting the mixture ofconcentrated acids Sample preparation was performed inTeflon containers with 10mL of a solution consisting of amixture of concentrated acids (HF-hydrofluoric acid HCl-hydrochloric acid HNO3-nitric acid and HClO4-perchloricacid) The solution is evaporated to dryness at 200∘C Thenthe residue is dissolvedwith 4mL of 50HCl and the sampleis heated in amicrowave oven After cooling the solutions arepoured into the polypropylene vials and filled up to reach thevolume of 10mL with 5 HCl Preparation of the sample inaqua regia extraction 3mL of distilled water 75mL of 6MHCl and 25mL of 14M HNO3 are added in 1 g of the soilsample at 20∘C and it is left to react overnight After that thesample is heated to boiling for two hours and the solution iscooled and filtered Determination of metal concentrations isalso carried out using inductively coupled plasma and massspectrometry (ICP-MS) [23 25]

225 Statistical Analysis For the purposes of processing anddescribing the data descriptive statistics Kruskal-Wallis testand multivariate techniques (Cluster Analysis and PrincipalComponent Analysis) were used Data analysis was carriedout using the statistical software Statistica version 12Cluster Analysis Clustering or cluster analysis is used toclassify data objects characterized by the values of a setof variables into groups The Euclidean distance of two

Table 1 Pollen analysis of acacia honey from Northwest CroatiaEast Croatia and Istria (119909 = acacia proportion-Robinia pseudoa-cacia L)

NorthwestCroatia

119909 4288Minndashmax 22ndash68

East Croatia 119909 3911Minndashmax 11ndash70

Istria 119909 5520Minndashmax 33ndash71

Total 119909 4355Minndashmax 22ndash71

representing objects can be taken as an indicator of similar-itydissimilarity of the investigated variablesThe equation forthe Euclidean distance between objects 119894 and 1198941015840 is

1198631198941198941015840 = radic 119898sum119895=1

(119909119894119895 minus 1199091198941015840 119895)2 (1)

where119898 is the number of variablesAn agglomerative single-linkage method was used and

the results are presented in a form of dendrogramPrincipal Component Analysis (PCA) Principal componentanalysis reduces a set containing a large number of mea-sured variables to a data set containing fewer new variablesThese new variables (principal components PCs) are linearcombinations of the original onesThese linear combinationsare chosen to represent a maximum possible proportion ofthe variability contained in the original data The results areusually presented in a form of biplots biplots allow us toinvestigate a trend in a data table and show relationships orcontrasts between objects and between variables and finallyrelationships between variables and objectsThrough variablereduction and visual display PCA allows us to observe thesources of variation in an analyzed data matrix

In general

PC119894 = 11989711198941198831 + 11989721198941198832 + sdot sdot sdot + 119897119899119894119883119899 (2)

where PC119894 is 119894-th principal component (PCs) and 119897119895119894 is theloading of the observed variable119883119895 [26ndash28]3 Results and Discussion

Pollen analysis showed that out of 200 analyzed samplesof acacia honey 199 of them (995) had at least a 20proportion of acacia pollen grains therefore according tothe legislation it can be called unifloral honey [19] Theproportion of acacia pollen grains ranged from 3911 in EastCroatia over 4288 in Northwest Croatia and to 5520 inIstria which is an average of 4355 with a range from 11(one sample) to 70 (Table 1)

The results of determining the physicochemical param-eters are shown in Table 2 The results have shown thatall the samples of honey according to the physicochemical

4 Journal of Food Quality

Table2Ph

ysicochemicalindicatorsin

acaciaho

neyfro

mthreeC

roatianregion

s(119909120590

range)

Analysis

Water

()

Free

acids

(mEq

1000

g)Electricalcond

uctiv

ity(m

Scm

)Re

ducing

sugars

(g10

0g)

Saccharose

(g10

0g)

Diastase

(DN)

HMF

(mgkg)

Northwest

Croatia

1199091678

1045

015

6893

062

1038

356

12059010

3220

003

191

052

194

211

EastCr

oatia

1199091694

1050

015

6846

049

1133

592

12059012

419

5004

238

041

296

291

Istria

1199091701

1102

018

7062

055

1375

354

12059010

6115

003

091

051

234

297

Total

1199091691

1065

016

6933

055

1182

434

120590111

177

003

173

048

241

266

Minndashm

ax1678ndash1701

1045ndash1102

015ndash0

186846ndash

7062

049ndash0

62

1038ndash1375

356ndash592

MPC

le20lt50

08

ge60le10

ge8le40

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 5

parameters are safe and of high quality [3] Kruskal-Wallistest has shown no significant differences among the regionswith regard to the physicochemical parameters of honey (119901 =092)

The water content in our study ranged between 1678and 1701 which is in accordance with legislation thatallows up to 20 The permitted value of free acid in honeyis lt50mEq of acid1000 g of honey This means that thatthe average values obtained in this study ranging from1045ndash1102mEq of acid1000 g are within the limits Averagevalues of electrical conductivity in the samples of this researchrange from 015 to 018mScm which meets the generalrequirements of a maximum of 08mScm In honey thesum of the proportions of glucose and fructose or reducingsugars has to be at least 60 g100 g We estimated the averageof 6933 g100 g in the range of 6846ndash7062 g100 g All thetested samples havemet the requirement for saccharose (up to10 g100 g of the sample) and ranged from 049 to 062 g100 gGeneral requirements for the value of diastase activity are8DN whereas our values ranged from 1038 to 1375DNThe proportion of HMF was low (an average of 356 to592mgkg) which is less than 10mgkg the upper limit forthe first class honey [3]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) in three Croatian regions was examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg (Table 3) The totalproportion of all the elements in honey was an averageof 56555mgkg (ranging from 42404mgkg in NorthwestCroatia to 76555mgkg in Istria) which makes an averageof less than 006 (expressed as a percentage of ash) andis within the normal proportion of elements in floral honeyto 02 [3] Macro elements (K Ca Na and Mg) therebyaccounted for an average of 55464mgkg or 9807 of all theproportion of elements Potential contaminants (Pb and Cd)accounted for a total average of insignificant le006mgkg orle002 of the content

The percentage of K ranged from 5123 to 7029 (119909 =5756) The determined concentrations were in the rangeof 25869mgkg to 36080mgkg (119909 = 32554mgkg) Theproportion of Ca in the tested samples of honey ranged from1481 to 2104 compared to other minerals The lowestconcentration of Ca expressed in mgkg was 7437mgkg andthe highest 18439mgkg (119909 = 11124mgkg)The proportionof Na in the samples tested in this study was expressed asa percentage from 1021 to 2166 The highest averagevalue of Na was 16886mgkg in East Croatia and the lowestvalue of 5162mgkg was recorded in Northwest Croatia (119909 =9585mgkg) The average value of Mg proportion in thesamples of acacia honey in this study ranged from 367to 413 (119909 = 389) while the average concentrationranged from 1682mgkg to 3068mgkg (119909 = 2201mgkg)Among micro elements only Zn had an average proportionexceeding 1 (119909 = 126 119909 = 712mgkg) and Al (119909 =027 119909 = 155mgkg) and Fe (119909 = 022 119909 = 123mgkg)greater than 02 The proportions of all the other elementsthat had been tested ranged between 006 (Cu) and le001

(Cd) Kruskal-Wallis test did not show significant differencesbetween the regions with regard to concentrations of twelveelements measured in the honey samples (119901 = 089)

A total of twelve elements in the soil (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in three Croatian regionswere examined as a percentage of an individual macro ormicro element as well as the range and the average value of anelement expressed inmgkg (Table 4)The total proportion ofelements in the soil was an average of 5130688mgkg (rangeof 4120511mgkg in Northwest Croatia to 6437118mgkgin Istria) Kruskal-Wallis test did not show significant differ-ences between regionswith regard to concentrations of twelvemetals measured in the soil samples (119901 = 0845)

Fe had the highest average concentration in soil (119909 =24 98550mgkg range of 2135523 to 3084230mgkg)and the highest average proportion (119909 = 4870) incomparison to the other elements It is followed by Alwith the concentration in the range of 10275mgkg to2335070mgkg (119909 = 15 86478mgkg) and with the pro-portion of 2829ndash3628 (119909 = 3092) Ca is in the thirdplace with an average concentration of 440621mgkg andthe average proportion of 858 It is followed by Mgwhich had an average proportion of 659 and the averageconcentration of 337896mgkg Then there is K with anaverage concentration of 133139mgkg with an average pro-portion of 260 followed by an average proportion of Mnof about 118 and an average concentration of 60601mgkgand an average concentration of Na of 58592mgkg and aproportion of 114The proportion of all the other elementsincluding potential contaminants (Cd and Pb) was in therange of 015 (Zn) to le001 (Cd)

Table 5 shows the values of the correlation coefficientsbetween the concentrations of minerals (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in acacia honey and soilfor three regions in the Republic of Croatia Although strongpositive correlation coefficients were obtained among theconcentrations of twelve elements in certain types of honey(eg 119903 = 099 between the Istrian honey and the honey ofNorthwest Croatia or 119903 = 092 between the Istrian honeyand the honey of East Croatia 119901 lt 005) as well as strongpositive correlations between the concentrations of the twelveelements in soils no significant correlations were foundbetween the concentrations of the twelve elements in honeyand soil

Cluster analysis of the chemical composition of honeyfrom the three regions of the Republic of Croatia revealed theexistence of differences between East Croatia and the othertwo regions (Figure 2)

The next step was the principal component analysis(PCA) (Figure 3) which ldquoconfirmedrdquo the diversity of thecomposition of honey from East Croatia when compared tothe other two regions primarily in higher concentrations ofall the tested elements except for Al and in higher valuesof water and HMF and lower values of pollen proportionelectrical conductivity free acids reducing sugars saccha-rose diastase activity and Al In contrast acacia honey fromNorthwest Croatia was characterized by higher concentra-tions of saccharose and lower concentrations of almost allof the other remaining parameters while honey from Istria

6 Journal of Food Quality

Table3Micro

andmacro

elementsin

acaciaho

neyfro

mthreeC

roatianregion

saccordingto

thep

ropo

rtionpercentage

()averagec

oncentratio

ns(119909120590)an

drang

e(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

11990925869

7495

6707

1682

331

166

092

020

016

022

004

le001

42404

12059064

99

4230

3760

808

123

103

072

009

009

010

002

001

119909(

)6236

1713

1479

413

077

043

025

005

004

005

001

le001

EastCr

oatia

11990936080

18439

16886

3068

1646

122

138

051

019

098

008

le001

76555

12059016454

97570

9757

1176

1233

085

097

035

010

065

005

001

119909(

)5123

2104

2166

367

184

017

020

006

003

011

001

le001

Istria

11990935715

7437

5162

1852

161

176

138

029

015

016

005

le001

5070

6120590

9613

5602

2384

1200

127

071

118

022

011

013

003

001

119909(

)7029

1481

1021

367

032

035

028

006

003

004

001

le001

Total

11990932554

1112

49585

2201

712

155

123

033

017

045

005

le001

56555

12059010855

6768

5300

1062

495

086

095

022

010

030

004

001

119909(

)5756

1967

1695

389

126

027

022

006

003

008

001

le001

Minndashm

ax25869ndash36080

7437

ndash18439

5162ndash16886

1682ndash3068

161ndash1646

122ndash17

6092ndash138

020ndash0

51

015ndash0

19016ndash0

98

004

ndash008le001ndash0

01

Minndashm

ax

5123ndash7029

1481ndash2104

1021ndash2166

367ndash4

13032ndash184

017ndash0

43

020ndash0

26

005ndash0

06

003ndash0

04

004

ndash011

001ndash0

01le001ndash0

01

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

Journal of Food Quality 3

from honey and using methylene blue as the indicator Theproportion of saccharose is obtained from the differencein the results obtained before and after the inversion [20]When determining the diastase activity in honey the methodused is based on the hydrolysis of 1 starch solution from1 g of honey in one hour at a temperature of 40∘C Whendetermining hydroxymethylfurfural (HMF) in honey usingtheWinklermethod the procedure is based on the reaction ofhydroxymethylfurfural with barbituric acid and p-toluidinewhich results in pink the intensity of which is measured at awavelength of 550 nm [21 22]

223 Determination of Mineral Substances in Honey Uponthe homogenization of honey the organic matter undergoesa wet chemical decomposition using concentrated nitric acidand hydrogen peroxide in a microwave apparatus (AntonPaar Multiwave 3000) The method of mass spectrome-try for detecting metals in honey is based on inductivelycoupled plasma (ICP-MS) which leads to ionization Massspectrometer is used to detect and identify ions based on theirmasscharge ratios [23 24]

224 Determination of Mineral Substances in Soil Soil sam-pling is defined by a specific procedure Each sample wastaken from a specific point previously defined (according tothe Gauss-Kruger coordinates) The samples were extractedfrom the soil profile at a depth of 05 meters and then storedon the ground in PVC containers In the laboratory priorto sieving the samples were dried at ambient temperatureand homogenized in an agate mill and then sieved to 2mmfractions

Determination of minerals in the soil was conductedby using the samples prepared by extracting the mixture ofconcentrated acids Sample preparation was performed inTeflon containers with 10mL of a solution consisting of amixture of concentrated acids (HF-hydrofluoric acid HCl-hydrochloric acid HNO3-nitric acid and HClO4-perchloricacid) The solution is evaporated to dryness at 200∘C Thenthe residue is dissolvedwith 4mL of 50HCl and the sampleis heated in amicrowave oven After cooling the solutions arepoured into the polypropylene vials and filled up to reach thevolume of 10mL with 5 HCl Preparation of the sample inaqua regia extraction 3mL of distilled water 75mL of 6MHCl and 25mL of 14M HNO3 are added in 1 g of the soilsample at 20∘C and it is left to react overnight After that thesample is heated to boiling for two hours and the solution iscooled and filtered Determination of metal concentrations isalso carried out using inductively coupled plasma and massspectrometry (ICP-MS) [23 25]

225 Statistical Analysis For the purposes of processing anddescribing the data descriptive statistics Kruskal-Wallis testand multivariate techniques (Cluster Analysis and PrincipalComponent Analysis) were used Data analysis was carriedout using the statistical software Statistica version 12Cluster Analysis Clustering or cluster analysis is used toclassify data objects characterized by the values of a setof variables into groups The Euclidean distance of two

Table 1 Pollen analysis of acacia honey from Northwest CroatiaEast Croatia and Istria (119909 = acacia proportion-Robinia pseudoa-cacia L)

NorthwestCroatia

119909 4288Minndashmax 22ndash68

East Croatia 119909 3911Minndashmax 11ndash70

Istria 119909 5520Minndashmax 33ndash71

Total 119909 4355Minndashmax 22ndash71

representing objects can be taken as an indicator of similar-itydissimilarity of the investigated variablesThe equation forthe Euclidean distance between objects 119894 and 1198941015840 is

1198631198941198941015840 = radic 119898sum119895=1

(119909119894119895 minus 1199091198941015840 119895)2 (1)

where119898 is the number of variablesAn agglomerative single-linkage method was used and

the results are presented in a form of dendrogramPrincipal Component Analysis (PCA) Principal componentanalysis reduces a set containing a large number of mea-sured variables to a data set containing fewer new variablesThese new variables (principal components PCs) are linearcombinations of the original onesThese linear combinationsare chosen to represent a maximum possible proportion ofthe variability contained in the original data The results areusually presented in a form of biplots biplots allow us toinvestigate a trend in a data table and show relationships orcontrasts between objects and between variables and finallyrelationships between variables and objectsThrough variablereduction and visual display PCA allows us to observe thesources of variation in an analyzed data matrix

In general

PC119894 = 11989711198941198831 + 11989721198941198832 + sdot sdot sdot + 119897119899119894119883119899 (2)

where PC119894 is 119894-th principal component (PCs) and 119897119895119894 is theloading of the observed variable119883119895 [26ndash28]3 Results and Discussion

Pollen analysis showed that out of 200 analyzed samplesof acacia honey 199 of them (995) had at least a 20proportion of acacia pollen grains therefore according tothe legislation it can be called unifloral honey [19] Theproportion of acacia pollen grains ranged from 3911 in EastCroatia over 4288 in Northwest Croatia and to 5520 inIstria which is an average of 4355 with a range from 11(one sample) to 70 (Table 1)

The results of determining the physicochemical param-eters are shown in Table 2 The results have shown thatall the samples of honey according to the physicochemical

4 Journal of Food Quality

Table2Ph

ysicochemicalindicatorsin

acaciaho

neyfro

mthreeC

roatianregion

s(119909120590

range)

Analysis

Water

()

Free

acids

(mEq

1000

g)Electricalcond

uctiv

ity(m

Scm

)Re

ducing

sugars

(g10

0g)

Saccharose

(g10

0g)

Diastase

(DN)

HMF

(mgkg)

Northwest

Croatia

1199091678

1045

015

6893

062

1038

356

12059010

3220

003

191

052

194

211

EastCr

oatia

1199091694

1050

015

6846

049

1133

592

12059012

419

5004

238

041

296

291

Istria

1199091701

1102

018

7062

055

1375

354

12059010

6115

003

091

051

234

297

Total

1199091691

1065

016

6933

055

1182

434

120590111

177

003

173

048

241

266

Minndashm

ax1678ndash1701

1045ndash1102

015ndash0

186846ndash

7062

049ndash0

62

1038ndash1375

356ndash592

MPC

le20lt50

08

ge60le10

ge8le40

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 5

parameters are safe and of high quality [3] Kruskal-Wallistest has shown no significant differences among the regionswith regard to the physicochemical parameters of honey (119901 =092)

The water content in our study ranged between 1678and 1701 which is in accordance with legislation thatallows up to 20 The permitted value of free acid in honeyis lt50mEq of acid1000 g of honey This means that thatthe average values obtained in this study ranging from1045ndash1102mEq of acid1000 g are within the limits Averagevalues of electrical conductivity in the samples of this researchrange from 015 to 018mScm which meets the generalrequirements of a maximum of 08mScm In honey thesum of the proportions of glucose and fructose or reducingsugars has to be at least 60 g100 g We estimated the averageof 6933 g100 g in the range of 6846ndash7062 g100 g All thetested samples havemet the requirement for saccharose (up to10 g100 g of the sample) and ranged from 049 to 062 g100 gGeneral requirements for the value of diastase activity are8DN whereas our values ranged from 1038 to 1375DNThe proportion of HMF was low (an average of 356 to592mgkg) which is less than 10mgkg the upper limit forthe first class honey [3]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) in three Croatian regions was examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg (Table 3) The totalproportion of all the elements in honey was an averageof 56555mgkg (ranging from 42404mgkg in NorthwestCroatia to 76555mgkg in Istria) which makes an averageof less than 006 (expressed as a percentage of ash) andis within the normal proportion of elements in floral honeyto 02 [3] Macro elements (K Ca Na and Mg) therebyaccounted for an average of 55464mgkg or 9807 of all theproportion of elements Potential contaminants (Pb and Cd)accounted for a total average of insignificant le006mgkg orle002 of the content

The percentage of K ranged from 5123 to 7029 (119909 =5756) The determined concentrations were in the rangeof 25869mgkg to 36080mgkg (119909 = 32554mgkg) Theproportion of Ca in the tested samples of honey ranged from1481 to 2104 compared to other minerals The lowestconcentration of Ca expressed in mgkg was 7437mgkg andthe highest 18439mgkg (119909 = 11124mgkg)The proportionof Na in the samples tested in this study was expressed asa percentage from 1021 to 2166 The highest averagevalue of Na was 16886mgkg in East Croatia and the lowestvalue of 5162mgkg was recorded in Northwest Croatia (119909 =9585mgkg) The average value of Mg proportion in thesamples of acacia honey in this study ranged from 367to 413 (119909 = 389) while the average concentrationranged from 1682mgkg to 3068mgkg (119909 = 2201mgkg)Among micro elements only Zn had an average proportionexceeding 1 (119909 = 126 119909 = 712mgkg) and Al (119909 =027 119909 = 155mgkg) and Fe (119909 = 022 119909 = 123mgkg)greater than 02 The proportions of all the other elementsthat had been tested ranged between 006 (Cu) and le001

(Cd) Kruskal-Wallis test did not show significant differencesbetween the regions with regard to concentrations of twelveelements measured in the honey samples (119901 = 089)

A total of twelve elements in the soil (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in three Croatian regionswere examined as a percentage of an individual macro ormicro element as well as the range and the average value of anelement expressed inmgkg (Table 4)The total proportion ofelements in the soil was an average of 5130688mgkg (rangeof 4120511mgkg in Northwest Croatia to 6437118mgkgin Istria) Kruskal-Wallis test did not show significant differ-ences between regionswith regard to concentrations of twelvemetals measured in the soil samples (119901 = 0845)

Fe had the highest average concentration in soil (119909 =24 98550mgkg range of 2135523 to 3084230mgkg)and the highest average proportion (119909 = 4870) incomparison to the other elements It is followed by Alwith the concentration in the range of 10275mgkg to2335070mgkg (119909 = 15 86478mgkg) and with the pro-portion of 2829ndash3628 (119909 = 3092) Ca is in the thirdplace with an average concentration of 440621mgkg andthe average proportion of 858 It is followed by Mgwhich had an average proportion of 659 and the averageconcentration of 337896mgkg Then there is K with anaverage concentration of 133139mgkg with an average pro-portion of 260 followed by an average proportion of Mnof about 118 and an average concentration of 60601mgkgand an average concentration of Na of 58592mgkg and aproportion of 114The proportion of all the other elementsincluding potential contaminants (Cd and Pb) was in therange of 015 (Zn) to le001 (Cd)

Table 5 shows the values of the correlation coefficientsbetween the concentrations of minerals (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in acacia honey and soilfor three regions in the Republic of Croatia Although strongpositive correlation coefficients were obtained among theconcentrations of twelve elements in certain types of honey(eg 119903 = 099 between the Istrian honey and the honey ofNorthwest Croatia or 119903 = 092 between the Istrian honeyand the honey of East Croatia 119901 lt 005) as well as strongpositive correlations between the concentrations of the twelveelements in soils no significant correlations were foundbetween the concentrations of the twelve elements in honeyand soil

Cluster analysis of the chemical composition of honeyfrom the three regions of the Republic of Croatia revealed theexistence of differences between East Croatia and the othertwo regions (Figure 2)

The next step was the principal component analysis(PCA) (Figure 3) which ldquoconfirmedrdquo the diversity of thecomposition of honey from East Croatia when compared tothe other two regions primarily in higher concentrations ofall the tested elements except for Al and in higher valuesof water and HMF and lower values of pollen proportionelectrical conductivity free acids reducing sugars saccha-rose diastase activity and Al In contrast acacia honey fromNorthwest Croatia was characterized by higher concentra-tions of saccharose and lower concentrations of almost allof the other remaining parameters while honey from Istria

6 Journal of Food Quality

Table3Micro

andmacro

elementsin

acaciaho

neyfro

mthreeC

roatianregion

saccordingto

thep

ropo

rtionpercentage

()averagec

oncentratio

ns(119909120590)an

drang

e(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

11990925869

7495

6707

1682

331

166

092

020

016

022

004

le001

42404

12059064

99

4230

3760

808

123

103

072

009

009

010

002

001

119909(

)6236

1713

1479

413

077

043

025

005

004

005

001

le001

EastCr

oatia

11990936080

18439

16886

3068

1646

122

138

051

019

098

008

le001

76555

12059016454

97570

9757

1176

1233

085

097

035

010

065

005

001

119909(

)5123

2104

2166

367

184

017

020

006

003

011

001

le001

Istria

11990935715

7437

5162

1852

161

176

138

029

015

016

005

le001

5070

6120590

9613

5602

2384

1200

127

071

118

022

011

013

003

001

119909(

)7029

1481

1021

367

032

035

028

006

003

004

001

le001

Total

11990932554

1112

49585

2201

712

155

123

033

017

045

005

le001

56555

12059010855

6768

5300

1062

495

086

095

022

010

030

004

001

119909(

)5756

1967

1695

389

126

027

022

006

003

008

001

le001

Minndashm

ax25869ndash36080

7437

ndash18439

5162ndash16886

1682ndash3068

161ndash1646

122ndash17

6092ndash138

020ndash0

51

015ndash0

19016ndash0

98

004

ndash008le001ndash0

01

Minndashm

ax

5123ndash7029

1481ndash2104

1021ndash2166

367ndash4

13032ndash184

017ndash0

43

020ndash0

26

005ndash0

06

003ndash0

04

004

ndash011

001ndash0

01le001ndash0

01

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

4 Journal of Food Quality

Table2Ph

ysicochemicalindicatorsin

acaciaho

neyfro

mthreeC

roatianregion

s(119909120590

range)

Analysis

Water

()

Free

acids

(mEq

1000

g)Electricalcond

uctiv

ity(m

Scm

)Re

ducing

sugars

(g10

0g)

Saccharose

(g10

0g)

Diastase

(DN)

HMF

(mgkg)

Northwest

Croatia

1199091678

1045

015

6893

062

1038

356

12059010

3220

003

191

052

194

211

EastCr

oatia

1199091694

1050

015

6846

049

1133

592

12059012

419

5004

238

041

296

291

Istria

1199091701

1102

018

7062

055

1375

354

12059010

6115

003

091

051

234

297

Total

1199091691

1065

016

6933

055

1182

434

120590111

177

003

173

048

241

266

Minndashm

ax1678ndash1701

1045ndash1102

015ndash0

186846ndash

7062

049ndash0

62

1038ndash1375

356ndash592

MPC

le20lt50

08

ge60le10

ge8le40

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 5

parameters are safe and of high quality [3] Kruskal-Wallistest has shown no significant differences among the regionswith regard to the physicochemical parameters of honey (119901 =092)

The water content in our study ranged between 1678and 1701 which is in accordance with legislation thatallows up to 20 The permitted value of free acid in honeyis lt50mEq of acid1000 g of honey This means that thatthe average values obtained in this study ranging from1045ndash1102mEq of acid1000 g are within the limits Averagevalues of electrical conductivity in the samples of this researchrange from 015 to 018mScm which meets the generalrequirements of a maximum of 08mScm In honey thesum of the proportions of glucose and fructose or reducingsugars has to be at least 60 g100 g We estimated the averageof 6933 g100 g in the range of 6846ndash7062 g100 g All thetested samples havemet the requirement for saccharose (up to10 g100 g of the sample) and ranged from 049 to 062 g100 gGeneral requirements for the value of diastase activity are8DN whereas our values ranged from 1038 to 1375DNThe proportion of HMF was low (an average of 356 to592mgkg) which is less than 10mgkg the upper limit forthe first class honey [3]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) in three Croatian regions was examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg (Table 3) The totalproportion of all the elements in honey was an averageof 56555mgkg (ranging from 42404mgkg in NorthwestCroatia to 76555mgkg in Istria) which makes an averageof less than 006 (expressed as a percentage of ash) andis within the normal proportion of elements in floral honeyto 02 [3] Macro elements (K Ca Na and Mg) therebyaccounted for an average of 55464mgkg or 9807 of all theproportion of elements Potential contaminants (Pb and Cd)accounted for a total average of insignificant le006mgkg orle002 of the content

The percentage of K ranged from 5123 to 7029 (119909 =5756) The determined concentrations were in the rangeof 25869mgkg to 36080mgkg (119909 = 32554mgkg) Theproportion of Ca in the tested samples of honey ranged from1481 to 2104 compared to other minerals The lowestconcentration of Ca expressed in mgkg was 7437mgkg andthe highest 18439mgkg (119909 = 11124mgkg)The proportionof Na in the samples tested in this study was expressed asa percentage from 1021 to 2166 The highest averagevalue of Na was 16886mgkg in East Croatia and the lowestvalue of 5162mgkg was recorded in Northwest Croatia (119909 =9585mgkg) The average value of Mg proportion in thesamples of acacia honey in this study ranged from 367to 413 (119909 = 389) while the average concentrationranged from 1682mgkg to 3068mgkg (119909 = 2201mgkg)Among micro elements only Zn had an average proportionexceeding 1 (119909 = 126 119909 = 712mgkg) and Al (119909 =027 119909 = 155mgkg) and Fe (119909 = 022 119909 = 123mgkg)greater than 02 The proportions of all the other elementsthat had been tested ranged between 006 (Cu) and le001

(Cd) Kruskal-Wallis test did not show significant differencesbetween the regions with regard to concentrations of twelveelements measured in the honey samples (119901 = 089)

A total of twelve elements in the soil (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in three Croatian regionswere examined as a percentage of an individual macro ormicro element as well as the range and the average value of anelement expressed inmgkg (Table 4)The total proportion ofelements in the soil was an average of 5130688mgkg (rangeof 4120511mgkg in Northwest Croatia to 6437118mgkgin Istria) Kruskal-Wallis test did not show significant differ-ences between regionswith regard to concentrations of twelvemetals measured in the soil samples (119901 = 0845)

Fe had the highest average concentration in soil (119909 =24 98550mgkg range of 2135523 to 3084230mgkg)and the highest average proportion (119909 = 4870) incomparison to the other elements It is followed by Alwith the concentration in the range of 10275mgkg to2335070mgkg (119909 = 15 86478mgkg) and with the pro-portion of 2829ndash3628 (119909 = 3092) Ca is in the thirdplace with an average concentration of 440621mgkg andthe average proportion of 858 It is followed by Mgwhich had an average proportion of 659 and the averageconcentration of 337896mgkg Then there is K with anaverage concentration of 133139mgkg with an average pro-portion of 260 followed by an average proportion of Mnof about 118 and an average concentration of 60601mgkgand an average concentration of Na of 58592mgkg and aproportion of 114The proportion of all the other elementsincluding potential contaminants (Cd and Pb) was in therange of 015 (Zn) to le001 (Cd)

Table 5 shows the values of the correlation coefficientsbetween the concentrations of minerals (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in acacia honey and soilfor three regions in the Republic of Croatia Although strongpositive correlation coefficients were obtained among theconcentrations of twelve elements in certain types of honey(eg 119903 = 099 between the Istrian honey and the honey ofNorthwest Croatia or 119903 = 092 between the Istrian honeyand the honey of East Croatia 119901 lt 005) as well as strongpositive correlations between the concentrations of the twelveelements in soils no significant correlations were foundbetween the concentrations of the twelve elements in honeyand soil

Cluster analysis of the chemical composition of honeyfrom the three regions of the Republic of Croatia revealed theexistence of differences between East Croatia and the othertwo regions (Figure 2)

The next step was the principal component analysis(PCA) (Figure 3) which ldquoconfirmedrdquo the diversity of thecomposition of honey from East Croatia when compared tothe other two regions primarily in higher concentrations ofall the tested elements except for Al and in higher valuesof water and HMF and lower values of pollen proportionelectrical conductivity free acids reducing sugars saccha-rose diastase activity and Al In contrast acacia honey fromNorthwest Croatia was characterized by higher concentra-tions of saccharose and lower concentrations of almost allof the other remaining parameters while honey from Istria

6 Journal of Food Quality

Table3Micro

andmacro

elementsin

acaciaho

neyfro

mthreeC

roatianregion

saccordingto

thep

ropo

rtionpercentage

()averagec

oncentratio

ns(119909120590)an

drang

e(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

11990925869

7495

6707

1682

331

166

092

020

016

022

004

le001

42404

12059064

99

4230

3760

808

123

103

072

009

009

010

002

001

119909(

)6236

1713

1479

413

077

043

025

005

004

005

001

le001

EastCr

oatia

11990936080

18439

16886

3068

1646

122

138

051

019

098

008

le001

76555

12059016454

97570

9757

1176

1233

085

097

035

010

065

005

001

119909(

)5123

2104

2166

367

184

017

020

006

003

011

001

le001

Istria

11990935715

7437

5162

1852

161

176

138

029

015

016

005

le001

5070

6120590

9613

5602

2384

1200

127

071

118

022

011

013

003

001

119909(

)7029

1481

1021

367

032

035

028

006

003

004

001

le001

Total

11990932554

1112

49585

2201

712

155

123

033

017

045

005

le001

56555

12059010855

6768

5300

1062

495

086

095

022

010

030

004

001

119909(

)5756

1967

1695

389

126

027

022

006

003

008

001

le001

Minndashm

ax25869ndash36080

7437

ndash18439

5162ndash16886

1682ndash3068

161ndash1646

122ndash17

6092ndash138

020ndash0

51

015ndash0

19016ndash0

98

004

ndash008le001ndash0

01

Minndashm

ax

5123ndash7029

1481ndash2104

1021ndash2166

367ndash4

13032ndash184

017ndash0

43

020ndash0

26

005ndash0

06

003ndash0

04

004

ndash011

001ndash0

01le001ndash0

01

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

Journal of Food Quality 5

parameters are safe and of high quality [3] Kruskal-Wallistest has shown no significant differences among the regionswith regard to the physicochemical parameters of honey (119901 =092)

The water content in our study ranged between 1678and 1701 which is in accordance with legislation thatallows up to 20 The permitted value of free acid in honeyis lt50mEq of acid1000 g of honey This means that thatthe average values obtained in this study ranging from1045ndash1102mEq of acid1000 g are within the limits Averagevalues of electrical conductivity in the samples of this researchrange from 015 to 018mScm which meets the generalrequirements of a maximum of 08mScm In honey thesum of the proportions of glucose and fructose or reducingsugars has to be at least 60 g100 g We estimated the averageof 6933 g100 g in the range of 6846ndash7062 g100 g All thetested samples havemet the requirement for saccharose (up to10 g100 g of the sample) and ranged from 049 to 062 g100 gGeneral requirements for the value of diastase activity are8DN whereas our values ranged from 1038 to 1375DNThe proportion of HMF was low (an average of 356 to592mgkg) which is less than 10mgkg the upper limit forthe first class honey [3]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) in three Croatian regions was examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg (Table 3) The totalproportion of all the elements in honey was an averageof 56555mgkg (ranging from 42404mgkg in NorthwestCroatia to 76555mgkg in Istria) which makes an averageof less than 006 (expressed as a percentage of ash) andis within the normal proportion of elements in floral honeyto 02 [3] Macro elements (K Ca Na and Mg) therebyaccounted for an average of 55464mgkg or 9807 of all theproportion of elements Potential contaminants (Pb and Cd)accounted for a total average of insignificant le006mgkg orle002 of the content

The percentage of K ranged from 5123 to 7029 (119909 =5756) The determined concentrations were in the rangeof 25869mgkg to 36080mgkg (119909 = 32554mgkg) Theproportion of Ca in the tested samples of honey ranged from1481 to 2104 compared to other minerals The lowestconcentration of Ca expressed in mgkg was 7437mgkg andthe highest 18439mgkg (119909 = 11124mgkg)The proportionof Na in the samples tested in this study was expressed asa percentage from 1021 to 2166 The highest averagevalue of Na was 16886mgkg in East Croatia and the lowestvalue of 5162mgkg was recorded in Northwest Croatia (119909 =9585mgkg) The average value of Mg proportion in thesamples of acacia honey in this study ranged from 367to 413 (119909 = 389) while the average concentrationranged from 1682mgkg to 3068mgkg (119909 = 2201mgkg)Among micro elements only Zn had an average proportionexceeding 1 (119909 = 126 119909 = 712mgkg) and Al (119909 =027 119909 = 155mgkg) and Fe (119909 = 022 119909 = 123mgkg)greater than 02 The proportions of all the other elementsthat had been tested ranged between 006 (Cu) and le001

(Cd) Kruskal-Wallis test did not show significant differencesbetween the regions with regard to concentrations of twelveelements measured in the honey samples (119901 = 089)

A total of twelve elements in the soil (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in three Croatian regionswere examined as a percentage of an individual macro ormicro element as well as the range and the average value of anelement expressed inmgkg (Table 4)The total proportion ofelements in the soil was an average of 5130688mgkg (rangeof 4120511mgkg in Northwest Croatia to 6437118mgkgin Istria) Kruskal-Wallis test did not show significant differ-ences between regionswith regard to concentrations of twelvemetals measured in the soil samples (119901 = 0845)

Fe had the highest average concentration in soil (119909 =24 98550mgkg range of 2135523 to 3084230mgkg)and the highest average proportion (119909 = 4870) incomparison to the other elements It is followed by Alwith the concentration in the range of 10275mgkg to2335070mgkg (119909 = 15 86478mgkg) and with the pro-portion of 2829ndash3628 (119909 = 3092) Ca is in the thirdplace with an average concentration of 440621mgkg andthe average proportion of 858 It is followed by Mgwhich had an average proportion of 659 and the averageconcentration of 337896mgkg Then there is K with anaverage concentration of 133139mgkg with an average pro-portion of 260 followed by an average proportion of Mnof about 118 and an average concentration of 60601mgkgand an average concentration of Na of 58592mgkg and aproportion of 114The proportion of all the other elementsincluding potential contaminants (Cd and Pb) was in therange of 015 (Zn) to le001 (Cd)

Table 5 shows the values of the correlation coefficientsbetween the concentrations of minerals (K Ca Na Mg ZnAl Fe Cu Mn Ni Pb and Cd) in acacia honey and soilfor three regions in the Republic of Croatia Although strongpositive correlation coefficients were obtained among theconcentrations of twelve elements in certain types of honey(eg 119903 = 099 between the Istrian honey and the honey ofNorthwest Croatia or 119903 = 092 between the Istrian honeyand the honey of East Croatia 119901 lt 005) as well as strongpositive correlations between the concentrations of the twelveelements in soils no significant correlations were foundbetween the concentrations of the twelve elements in honeyand soil

Cluster analysis of the chemical composition of honeyfrom the three regions of the Republic of Croatia revealed theexistence of differences between East Croatia and the othertwo regions (Figure 2)

The next step was the principal component analysis(PCA) (Figure 3) which ldquoconfirmedrdquo the diversity of thecomposition of honey from East Croatia when compared tothe other two regions primarily in higher concentrations ofall the tested elements except for Al and in higher valuesof water and HMF and lower values of pollen proportionelectrical conductivity free acids reducing sugars saccha-rose diastase activity and Al In contrast acacia honey fromNorthwest Croatia was characterized by higher concentra-tions of saccharose and lower concentrations of almost allof the other remaining parameters while honey from Istria

6 Journal of Food Quality

Table3Micro

andmacro

elementsin

acaciaho

neyfro

mthreeC

roatianregion

saccordingto

thep

ropo

rtionpercentage

()averagec

oncentratio

ns(119909120590)an

drang

e(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

11990925869

7495

6707

1682

331

166

092

020

016

022

004

le001

42404

12059064

99

4230

3760

808

123

103

072

009

009

010

002

001

119909(

)6236

1713

1479

413

077

043

025

005

004

005

001

le001

EastCr

oatia

11990936080

18439

16886

3068

1646

122

138

051

019

098

008

le001

76555

12059016454

97570

9757

1176

1233

085

097

035

010

065

005

001

119909(

)5123

2104

2166

367

184

017

020

006

003

011

001

le001

Istria

11990935715

7437

5162

1852

161

176

138

029

015

016

005

le001

5070

6120590

9613

5602

2384

1200

127

071

118

022

011

013

003

001

119909(

)7029

1481

1021

367

032

035

028

006

003

004

001

le001

Total

11990932554

1112

49585

2201

712

155

123

033

017

045

005

le001

56555

12059010855

6768

5300

1062

495

086

095

022

010

030

004

001

119909(

)5756

1967

1695

389

126

027

022

006

003

008

001

le001

Minndashm

ax25869ndash36080

7437

ndash18439

5162ndash16886

1682ndash3068

161ndash1646

122ndash17

6092ndash138

020ndash0

51

015ndash0

19016ndash0

98

004

ndash008le001ndash0

01

Minndashm

ax

5123ndash7029

1481ndash2104

1021ndash2166

367ndash4

13032ndash184

017ndash0

43

020ndash0

26

005ndash0

06

003ndash0

04

004

ndash011

001ndash0

01le001ndash0

01

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

6 Journal of Food Quality

Table3Micro

andmacro

elementsin

acaciaho

neyfro

mthreeC

roatianregion

saccordingto

thep

ropo

rtionpercentage

()averagec

oncentratio

ns(119909120590)an

drang

e(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

11990925869

7495

6707

1682

331

166

092

020

016

022

004

le001

42404

12059064

99

4230

3760

808

123

103

072

009

009

010

002

001

119909(

)6236

1713

1479

413

077

043

025

005

004

005

001

le001

EastCr

oatia

11990936080

18439

16886

3068

1646

122

138

051

019

098

008

le001

76555

12059016454

97570

9757

1176

1233

085

097

035

010

065

005

001

119909(

)5123

2104

2166

367

184

017

020

006

003

011

001

le001

Istria

11990935715

7437

5162

1852

161

176

138

029

015

016

005

le001

5070

6120590

9613

5602

2384

1200

127

071

118

022

011

013

003

001

119909(

)7029

1481

1021

367

032

035

028

006

003

004

001

le001

Total

11990932554

1112

49585

2201

712

155

123

033

017

045

005

le001

56555

12059010855

6768

5300

1062

495

086

095

022

010

030

004

001

119909(

)5756

1967

1695

389

126

027

022

006

003

008

001

le001

Minndashm

ax25869ndash36080

7437

ndash18439

5162ndash16886

1682ndash3068

161ndash1646

122ndash17

6092ndash138

020ndash0

51

015ndash0

19016ndash0

98

004

ndash008le001ndash0

01

Minndashm

ax

5123ndash7029

1481ndash2104

1021ndash2166

367ndash4

13032ndash184

017ndash0

43

020ndash0

26

005ndash0

06

003ndash0

04

004

ndash011

001ndash0

01le001ndash0

01

119909arithm

eticmean120590stand

arddeviation

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

Journal of Food Quality 7

Table4

Micro

andmacro

elementsin

thesoilfro

mthreeCr

oatia

nregion

saccordingto

theprop

ortio

npercentage

()averageconcentrations(119909120590)an

dtherangeof

concentrations

(mgkg)

Elem

ents

K(m

gkg)

Ca(m

gkg)

Na

(mgkg)

Mg

(mgkg)

Zn(m

gkg)

Al

(mgkg)

Fe(m

gkg)

Cu(m

gkg)

Mn

(mgkg)

Ni

(mgkg)

Pb(m

gkg)

Cd

(mgkg)

Total

(mgkg)

NorthwestC

roatia

1199096310

7361290

47059

275672

7450

1027543

2275897

1756

56678

2779

1257

023

4120511

12059020401

171293

1392

459539

2062

349090

7446

24

555

27032

1192

262

018

119909(

)15

1875

114

661

018

2472

5556

004

138

007

003

0001

EastCr

oatia

119909187705

420375

79487

554313

6970

1396822

2135523

1462

48537

1810

1386

020

4834411

12059074689

222766

46675

233962

3714

4766

93

549727

479

18605

506

692

009

119909(

)388

870

164

1147

0001

2889

4417

003

100

004

003

000

Istria

119909148606

539599

49229

183702

8860

2335070

3084230

3759

76587

5083

2364

031

6437118

1205909095

6657305

13249

6510

43662

180219

78740

09

1597

23271

1840

915

036

119909(

)231

838

077

285

014

3628

4791

006

119

008

004

000

Summary

119909133139

4404

21

58592

337896

7760

1586478

2498550

2326

60601

3224

1669

025

5130680

12059062015

3504

55

24616

119535

3146

875993

722787

877

2296

91179

623

021

119909(

)260

858

114

659

015

3092

4870

005

118

006

003

000

Minndashm

ax6310

7ndash187705

361290ndash

539599

47059ndash79487

183702ndash554313

6970ndash

8860

1027543ndash2335070

2135523ndash3084230

1462ndash3759

48537ndash76587

1810

ndash5083

1257ndash2364

020ndash0

31Minndashm

ax

151ndash388

838ndash875

077ndash164

285ndash114

70001ndash018

2829ndash

3628

4417

ndash5556

003ndash0

06

100ndash

138

004

ndash008

003ndash0

04

0001ndash0001

119909arithm

eticmean120590stand

arddeviation

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

8 Journal of Food Quality

Table 5 An overview of the correlation coefficient between the concentrations of minerals (elements) in acacia honey and soil from threeCroatian regions

NWC-soil EC-soil IS-soil NWC-honey EC-honey IS-honeyNWC-soil 0979145 0966227 minus0161628 minus0178466 minus0147623EC-soil 0979145 0980134 minus0127430 minus0148898 minus0112774IS-soil 0966227 0980134 minus0157446 minus0180116 minus0140965NWC-honey minus0161628 minus0127430 minus0157446 0967384 0991399EC-honey minus0178466 minus0148898 minus0180116 0967384 0926812IS-honey minus0147623 minus0112774 minus0140965 0991399 0926812NWC Northwest Croatia EC East Croatia IS Istria

Istria

East Croatia

NW Croatia

6000 8000 10000 12000 140004000Linkage distance

Figure 2 Dissimilarities between the three types of honey inCroatia according to the analyzed parameters and regions

was characterized by higher values of pollen proportion freeacids electrical conductivity reducing sugars and diastaseactivity with lower concentrations of water saccharoseHMFand the majority of examined metals The ldquolinkrdquo betweenhoney from Northwest Croatia and Istria is the higherconcentration of Al

The study included 200 samples of acacia honey fromthree Croatian regions and 100 soil samples from the samelocationsWe studied the proportion of acacia pollen physic-ochemical parameters and macro and micro elements inhoney and the same selected elements in the soil togetherwiththe relationship between them to give an overview of foodsafety and quality of acacia honey in Croatia In addition byapplying multivariate statistical methods we tried to make acontribution to the research of the combination of optimumindicators of the status and quality of honey and tests thatcould have a predictive effect with regard to determining thecomposition and the botanical and geographical origin ofhoney

By using pollen analysis it was found that the vast major-ity of the samples of acacia honey from Croatia (995) hadat least a 20 proportion of pollen grains of acacia (Robiniapseudoacacia L) therefore according to the legislation it canbe called unifloral honey [3 10] Characterization of honeyaccording to the proportion of pollen is already known in theEuropean countries such as Spain Italy and Poland [29ndash31]and even in other parts of the world such as India [32] Saudi

Ca

Na

K

Mg

Zn

Fe

Cu

Mn

AlNi

Pb

Cd

Water

AcidConductivity

Sugar

Saccharose

Diastase

Pollen

Factor 1 (6192)

minus10

minus05

00

05

10

Fact

or 2

(30

08

)

East Croatia

Istria

North West Croatia

100500minus05minus10

C6H6O3

Figure 3 An overview of the PCA (Principal Component Analysis)of the chemical composition of acacia honey from three regions ofthe Republic of Croatia Water water Aci free acids (mEq100 g)Conductivity electrical conductivity (mScm) Sugar reducingsugars (g100 g) Saccharose saccharose (g100 g) Diastase diastase(DN) and HMF HMF (mgkg)

Arabia [33] and Morocco [34] There have been a relativelysmall number of studies of acacia honey on the Croatianterritory [35ndash37] and our research has contributed to theexisting knowledge and data that characterize the Croatianacacia honey

Physicochemical parameters were determined accordingto the criteria laid down in the Croatian legislation andconsistent with the directives and recommendations of theEU [3 4] All determined physicochemical parameters werewithin the permitted values and thus speak in favor ofhigh quality and good selection of the acacia honey on theCroatian market taken for this research and of course ofgood beekeeping practices

Thus the water content in all the samples was within20 and our results are consistent with the results of otherresearchers on the samples of acacia honey from Croatia(1540ndash1630) Romania (1790) and Italy (1710) [36ndash39]

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

Journal of Food Quality 9

The literature shows various values of free acids in thesamples of acacia honey The average values obtained inthe study (1045 to 1102mEq1000 g) are lower than inSlovenia (24mEq1000 g) are similar to a Croatian study(101ndash107mEq1000 g) and a study conducted by Italianauthors (112mEq1000 g) and are moderately higher than inthe results of other Croatian researches and one in Romania(65ndash84mEq1000 g) [36 38ndash40]

The average values of electrical conductivity in the sam-ples of this study that range from 015 to 018mScm complywith the results obtained on 513 samples of the Europeanacacia honey (016mScm) and the results of the Romanianand Croatian researchers (011mScm-022mScm) [36 3739 41] but are lower than the average values of the Slovenianacacia honey (026mScm) [40]

We found the average sum of glucose and fruc-tose (reducing sugars) is 6933 g100 g in the range of6846ndash7062 g100 g On the European research level con-ducted on 454 samples of acacia honey the obtained averagevalue is 692 g100 g [35] Other literature sources fromCroatia and Romania also show the results that match thisresearch (674ndash715 g100 g) [37 39 42]

All the tested samples have met the requirement regard-ing saccharose level ranging from 049 to 062 g100 g whichis moderately lower than the samples of acacia honey fromRomania (155 g100 g) Croatia (from 24 to 49 g100 g) andItaly (21 g100 g) [37ndash39 41]

The obtained average values of diastase activity in ourstudy ranged from 1038 to 1375DN and are in accordancewith the European and previous Croatian results for acaciahoney [37 38]

The proportion of HMF was low (an average of356ndash592mgkg) which is less than 10mgkg However itcan be said that according to this category honey used inthe study is of the first class In contrast Saric et al who alsostudied samples of the Croatian acacia honey obtained valuesin a wider range (47ndash365mgkg) [37] The proportion ofHMF in the Moroccan honey ranged from 32 to 526mgkg[43]

A total of twelve micro and macro elements in acaciahoney (K Ca Na Mg Zn Al Fe Cu Mn Ni Pb andCd) from three Croatian regions were examined through apercentage () of each macro and micro element as wellas through the range and the average value of each macroand micro element expressed in mgkg The total proportionof all the elements in honey was 56555mgkg on averagewhich makes the average less than 006 (expressed as ashproportion) and is within the normal proportion of elementsin floral honey to 02 [3] Macro elements (K Ca Na andMg) thereby accounted for an average of 55464mgkg or9807 of all the elements Potential contaminants (Pb andCd) accounted for a total average of negligiblele006mgkg orle002As expectedKhad the largest proportion and its per-centage ranged from 5123 to 7029 (119909 = 5756) Thedetermined concentrations were in the range of 25869mgkgto 36080mgkg (119909 = 32554mgkg) It was followed by Cawith an average proportion of 1967 then byNawith 1695and Mg with 389 Our results are consistent with theresults of the mineral composition research of the Slovenian

and Romanian honey [39 40] Kruskal-Wallis test did notshow significant differences between the regions with regardto concentrations of the twelve elements measured in thesamples of honey (119901 = 089)

Even though the mineral substances in honey are poorlyrepresented with regard to quantity (an average of 01ndash02or less in floral honey) honey contains a number of mineralsubstances of which some are very important for the properfunctioning of the human body The most common is Kwhich usually makes 25ndash50 of the total proportion ofmineral substances More often it is common for Na Caand P to appear (1058) Accordingly in the Croatian andEuropean regulations the values are prescribedmdashthey allowthe maximum proportion of ash to be 06 for floral honey[43] These values are prescribed because the increasedproportion of ash can be a sign of honey adulteration withsugar molasses [44] It is only necessary to point out that theregulations regarding maximum levels of certain contami-nants in foodstuff in Croatia prescribe maximum levels forcertain contaminants that may be present in certain foodincluding heavy metals and nonmetals however maximumpermitted levels of Pb andCd or other contaminants in honeyare not prescribed [3 4]

In addition to honey the same twelve elements weredetermined in the soil (K Ca Na Mg Zn Al Fe Cu MnNi Pb and Cd) sampled from the apiary sites where thesamples of honey were taken for this research The totalproportion of all the elements in the soil was an averageof 5130688mgkg (range of 4120511mgkg in NorthwestCroatia to 6437118mgkg in Istria) Kruskal-Wallis test didnot show significant differences between the regions withregard to the concentrations of the twelve metals measuredin the soil samples (119901 = 0845)

The order is as follows Fe had the highest averageproportion in the soil (119909 = 4870) it was followed byAl with an average proportion of 3092 Ca is in the thirdplace with 858 then the following elements follow Mgwhose average proportionwas 659 thenK with an averageproportion of 260 then Mn with around 118 and Nawith an approximate proportion of 114 The proportion ofall the other elements including potential contaminants (Cdand Pb) ranged between 015 (Zn) and le001 (Cd) Theseresults are with some exceptions more or less consistentwith the results of the research conducted by the GeologicalSurvey when creating the Geological Atlas of Croatia andare evidently geochemically and geologically specified [45]Generally it can be said that the preparations and analyses ofthe soil samples for mineral composition are a contributionto the obviously correctly conducted procedures of samplingIn addition the results reveal a good environment condition(in this case its component the soil) in locations where beesare bred in the Republic of Croatia

It is considered as it has already been pointed out that theproportion ofmineral substances in honey largely depends onits botanical origin but also on the climatic conditions andthe composition of soil onwhich the honey plantwas growing[16] Moreover it is also believed that the characteristic soilcomposition of a particular region can be seen in the mineralcomposition of honey plants or the mineral composition of

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

10 Journal of Food Quality

its nectar and pollen [46] Nevertheless in our study wehave not confirmed the aforementioned since no significantcorrelation between the concentrations of the twelve elementsin honey and soil was found This lack of correlation shouldcertainly point to the fact that the concentrations of elementsin honey were not dependent on their concentration in thesoil and this may shed new light on the theory regarding theconnection between the concentration of minerals in the soiland honey which should be studied in the future

Multivariate analyses (cluster and PCA) of the chemicalcomposition of acacia honey from three Croatian regionssurprisingly showed good separation of the characteristicfeatures of honey in individual Croatian regions First thecluster analysis showed the separation of the characteristicsof honey in East Croatia in relation to the other two regionsAfter that the PCA analysis confirmed the diversity ofthe composition of honey from East Croatia with higherconcentrations ofmost elements (all except Al) higher valuesof water and HMF and lower values of the proportion ofpollen electrical conductivity free acids reducing sugarssaccharose diastase activity andAl Conversely acacia honeyfrom Northwest Croatia has been characterized by higherconcentrations of saccharose and lower concentrations ofalmost all of the other remaining parameters while honeyfrom Istria has been characterized by higher values ofthe proportion of pollen free acids electrical conductivityreducing sugars and diastase activity and with the lowerconcentrations of water saccharose HMF and a majority ofexamined metals The ldquolinkrdquo between honey from NorthwestCroatia and that from Istria is the higher concentration of Al

4 Conclusion

In conclusion we could say that by analyzing pollen physic-ochemical parameters and mineral composition of acaciahoney from three Croatian regions and the mineral compo-sition of the soil from the same locations we presented thehigh quality of honey and the lack of correlation between themineral composition of honey and soil By using multivariatestatistical methods we may have made a contribution tothe attempts of optimizing analytical and statistical methodswhich could have predictive significance for the botanical andgeographical composition of honey

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] M T Iglesias CDe LorenzoMDC Polo P JMartın-Alvarezand E Pueyo ldquoUsefulness of Amino Acid Composition toDiscriminate between Honeydew and Floral Honeys Appli-cation to Honeys from a Small Geographic Areardquo Journal ofAgricultural and Food Chemistry vol 52 no 1 pp 84ndash89 2004

[2] L R Silva R Videira A P Monteiro P Valentao and P BAndrade ldquoHoney from Luso region (Portugal) physicochem-ical characteristics and mineral contentsrdquo Microchemical Jour-nal vol 93 no 1 pp 73ndash77 2009

[3] Republic of Croatia Honey Regulations Narodne novine532015 (in Croatian)

[4] European Union (EU) ldquoCouncil Directive 2001110EC of 20December 2001 relating to honeyrdquo Official Journal of theEuropean Communities L pp 47ndash52 2010

[5] R Krell ldquoValue-added products from beekeepingrdquo in FAOAgricultural Services Bulletin Chapter 2 p 124 1996

[6] M C Zamora and J Chirife ldquoDetermination of water activitychange due to crystallization in honeys from Argentinardquo FoodControl vol 17 no 1 pp 59ndash64 2006

[7] S Skenderov and C Ivanov Pcelinji proizvodi i njihovokoriscenje translation by Stamenovic B Ivanova K Petrov JTehnologija Hrane Knjizara Nolit Beograd Serbia 1986

[8] National Honey Board Carbohydrates and the Sweetness ofHoney 2004 httpwwwnhborgdownloadfactshtcarbpdf

[9] L S M Costa M L S Albuquerque L C Trugo et al ldquoDeter-mination of non-volatile compounds of different botanicalorigin Brazilian honeysrdquo Food Chemistry vol 65 no 3 pp 347ndash352 1999

[10] R S Barhate R Subramanian K E Nandini and H UHebbar ldquoProcessing of honey using polymeric microfiltrationandultrafiltrationmembranesrdquo Journal of FoodEngineering vol60 no 1 pp 49ndash54 2003

[11] N Vahcic and D Matkoavic ldquoKemijske fizikalne senzorskeznacajke medardquo Prehrana I Biotehnologija vol 15 2009(Croatian) httpwwwpcelinjakhrOLDindexphpPrehrana-i-biotehnologijakemijske-fizikalne-i-senzorske-znaajke-medhtml

[12] S Karabournioti and P Zervalaki ldquoThe effect of heating onhoney HMF and invertaserdquo Apiacta vol 36 pp 177ndash181 2001

[13] J S Bonvehı M S Torrento and J M Raich ldquoInvertase activityin fresh and processed honeysrdquo Journal of the Science of Foodand Agriculture vol 80 no 4 pp 507ndash512 2000

[14] N Spano L Casula A Panzanelli et al ldquoAn RP-HPLC deter-mination of 5-hydroxymethylfurfural in honey the case ofstrawberry tree honeyrdquo Talanta vol 68 no 4 pp 1390ndash13952006

[15] M A Ramirez Cervantes S A Gonzales Novelo and E SauriDuch ldquoEffect of temporary thermic treatment of honey onvariation of the quality of the same during storagerdquoApiacta vol35 pp 162ndash170 2000

[16] P Przyby and A Wilczynska ldquoHoney as an environmentalmarkerrdquo Food Chemistry vol 74 no 3 pp 289ndash291 2001

[17] E Anklam ldquoA review of the analyticalmethods to determine thegeographical and botanical origin of honeyrdquo Food Chemistryvol 63 no 4 pp 549ndash562 1998

[18] Codex Alimentarius Commission Revised Codex Standard forHoney Codex Stand12-1981

[19] Republic of Croatia Regulations on the Quality of UnifloralHoney Narodne Novine 1222009

[20] International Honey Commission ldquoHarmonised methods ofthe International (European)HoneyCommissionrdquo 2009 httpwwwbee-hexagonnetfilesfilefileEIHC-methods 2009pdf

[21] Schweizerisches Lebensmittelbuch - SLMB Kapitel 23A HonigBestimmung der Amylactivitat (nach Phadebas) Bern EDMZ1995

[22] S Bogdanov K Ruoff and L PersanoOddo ldquoPhysico-chemicalmethods for the characterisation of unifloral honeys a reviewrdquoApidologie pp 4ndash17 2004

[23] Perkin Elmer Sciex 30-minute Guide to ICPMS 2004

Journal of Food Quality 11

[24] RThomas Practical Guide to ICP-MS A Tutorial for BeginnersCRC Press New York NY USA 2nd edition 2008

[25] Croatian Standards Institute Quality of Soil - Extraction of theElements Soluble in Aqua Regia 2004 HRN ISO 114662004

[26] V Gvozdic V Tomisic V Butorac and V Simeon ldquoAssociationof nitrate ion with metal cations in aqueous solution a UV-Visspectrometric and factor analysis studyrdquoCroatica ChemicaActavol 82 pp 553ndash559 2009

[27] I Jolliffe ldquoPrincipal Component Analysisrdquo in Encyclopedia ofStatistics in Behavioral Science University of Aberdeen UK2005

[28] N Sakac VGvozdic andM Sak-Bosnar ldquoDetermination of thebotanical origin of starch using direct potentiometry and PCArdquoCarbohydrate Polymers vol 87 no 4 pp 2619ndash2623 2012

[29] R Fernandez-Torres J L Perez-Bernal M A Bello-Lopez MCallejon-Mochon J C Jimenez-Sanchez and A Guiraum-Perez ldquoMineral content and botanical origin of Spanish hon-eysrdquo Talanta vol 65 pp 686ndash691 2005

[30] M E Conti J Stripeikis L Campanella D Cucina and M BTudino ldquoCharacterization of Italian honeys (Marche Region)on the basis of their mineral content and some typical qualityparametersrdquo Chemistry Central Journal vol 1 no 1 article no14 2007

[31] M Madejczyk and D Baralkiewicz ldquoCharacterization of Polishrape and honeydew honey according to their mineral contentsusing ICP-MS and F-AASAESrdquo Analytica Chimica Acta vol617 no 1-2 pp 11ndash17 2008

[32] V Nanda B C Sarkar H K Sharma and A S Bawa ldquoPhysico-chemical properties and estimation ofmineral content in honeyproduced from different plants in Northern Indiardquo Journal ofFood Composition and Analysis vol 16 no 5 pp 613ndash619 2003

[33] K A Osman M A Al-Doghairi S Al-Rehiayani and M I DHelal ldquoMineral contents and physicochemical properties ofnatural honey produced in Al-Qassim region Saudi ArabiardquoJournal of Food amp Environment vol 5 pp 142ndash146 2007

[34] H Belouali M Bouaka and A Hakkou ldquoDetermination ofsome major and minor elements in the east of Moroccohoneys through Inductively Coupled Plasma optical EmissionSpectrometryrdquo Apiacta vol 43 pp 17ndash24 2008

[35] D Bubalo M Draic M L Mandic et al ldquoKezic N Botanickopodrijetlo meda s podrucja jadranske regijerdquo in Proceedings ofthe 41st Croatian and 1st International Symposium of AgricultureFaculty of Agriculture pp 549-50 Osijek 2006

[36] D Kenjeric M L Mandic L Primorac D Bubalo and A PerlldquoFlavonoid profile of Robinia honeys produced inCroatiardquoFoodChemistry vol 102 no 3 pp 683ndash690 2007

[37] G Saric D Matkovic M Hruskar and N Vahcic ldquoCharacter-isation and classification of croatian honey by physicochemicalparametersrdquo Food Technology and Biotechnology vol 46 pp355ndash367 2008

[38] L PersanoOddo L Piana S Bogdanov A Bentabol P GotsiouJ Kerkivliet et al ldquoBotanical species giving unifloral honey inEuroperdquo Apidologie vol 35 pp 82ndash93 2004

[39] L A Marghitas D S Dezmirean C B Pocol M Ilea O Bobisand I Gergen The Development of a Biochemical Profile ofAcacia Honey by Identifying Biochemical

[40] T Golob and A Plestenjak ldquoQuality of Slovene honeyrdquo FoodTechnology and Biotechnology vol 37 pp 195ndash201 1999

[41] S Popek ldquoA procedure to identify a honey typerdquo Food Chem-istry vol 79 no 3 pp 401ndash406 2002

[42] Lj Primorac I Flanjak D Kenjeric D Bubola and Z Topol-njak ldquoSpecific rotation and carbohydrate profile of croatianunifloral honeysrdquo Czech Journal of Food Sciences vol 29 pp515ndash519 2011

[43] A Terrab M J Dıez and F J Heredia ldquoCharacterisation ofMoroccan unifloral honeys by their physicochemical character-isticsrdquo Food Chemistry vol 79 no 3 pp 373ndash379 2002

[44] OMHernandez JM G Fraga A I Jimenez F Jimenez and JJ Arias ldquoCharacterization of honey from the Canary Islandsdetermination of the mineral content by atomic absorptionspectrophotometryrdquo FoodChemistry vol 93 no 3 pp 449ndash4582005

[45] J Halamic S Miko and J Halamic Eds Geochemical Atlas ofthe Republic of Croatia Zagreb Croatian Geological Survey (inCroatian English full text Geochemical Atlas of the Republic ofCroatia Zagreb Croatian Geological Survey 2009

[46] C Porrini A G Sabatini S Girotti S Ghini P Medrzycki andF Grillenzoni ldquoHoney bees and bee products as monitors ofthe environmental contaminationrdquo Apiacta vol 38 pp 63ndash702003

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology