Research Paper

Characterization of virgin olive oil from Southern Tunisia

Wissem Zarrouk1, Faouzia Mahjoub Haddada1, Bechir Baccouri1, Imen Oueslati1, Wael Taamalli1,Xavier Fernandez2, Louisette Lizzani-Cuvelier2, Douja Daoud1, Mokhtar Zarrouk1

1 Laboratoire Caractérisation et Qualité de l’Huile d’Olive, Centre de Biotechnologie deBorj Cedria, BP 901, Hammam-Lif, Tunisia

2 Laboratoire de Chimie des Molécules Bioactives et des Arômes, UMR CNRS 6001, Faculté des Sciences deNice Sophia-Antipolis, Nice, France

Fruits from three Tunisian cultivars of Olea europea L. grown in the southeast of Tunisia were harvested atthe maturity stage of ripeness and immediately processed with a laboratory mill. There are as yet no dataon the chemical composition of virgin olive oils from the southeast of Tunisia, an area characterized by anarid condition of growth for olive trees. Our results showed significant differences in the analytical pa-rameters examined for the three cultivars such as fatty acid composition, total phenols and o-diphenols,and the content of chlorophylls and carotenoids, confirming the importance of genetic factors in thechemical characteristics of the oil. Headspace solid-phase microextraction (HS-SPME) was applied to theanalysis of volatile compounds of virgin olive oils. Forty-eight compounds were isolated and characterizedby GC-RI and GC-MS, representing 94.1–98.1% of the total amount. (E)-Hex-2-enal, the main com-pound extracted by SPME, characterized the olive oil headspace for all samples. So, it was clearly shownthat there were qualitative and quantitative differences in the proportion of volatile constituents from oilsof the various cultivars.

Keywords: Cultivars, virgin olive oil, chemical characteristics, lipoxygenase pathway, volatile compounds.

Received: March 14, 2007; accepted: June 29, 2007

DOI 10.1002/ejlt.200700085

Eur. J. Lipid Sci. Technol. 2008, 110, 81–88 81

1 Introduction

Virgin olive oil, one of the main components of the Med-iterranean diet, is highly appreciated all over the world forits delicious taste and aroma, as well as for its beneficialnutritional properties. The nutritional benefits are pri-marily related to the fatty acid composition, mainly due tothe high content of oleic acid and also to the balancedratio of saturated and polyunsaturated fatty acids [1]. Inaddition, olive oil presents considerable amounts of naturalantioxidants.

This product is also characterized by a distinctive aro-matic quality. Flavor and aroma of virgin olive oil are gen-erated by a number of volatile constituents that are presentat extremely low concentrations [2]. Many compounds,

mainly carbonyl compounds, alcohols, esters and hydro-carbons were found in the volatile fraction of virgin olive oil.The C6 and C5 compounds [3], especially C6 linear unsa-turated and saturated aldehydes, represent the most impor-tant fraction of volatile compounds of high-quality virginolive oils from a quantitative point of view [4]. However, C6and C5 compounds are enzymatically produced from poly-unsaturated fatty acids through the so-called lipoxygenase(LOX) pathway, and their concentrations depend on thelevel and the activity of each enzyme involved in this LOXpathway [5, 6]. Other factors that influence the volatiles areclimate and soil type [7] and the ripening cycle of the fruit[8]. The effect of processing equipment has also been stud-ied [9].

In the Tunisian region, the presence of typical varieties,the peculiar microclimatic conditions and precise oliveorchard management lead to the production of very valuableolive oils with a distinctive taste. Thus, we propose, in thiswork, the global characterization of three olive varieties,cultivated in the southeast of Tunisia, by means of analysesof the volatiles obtained from their virgin olive oils. To avoid

Correspondence: Mokhtar Zarrouk, Laboratoire Caractérisation et Qua-lité de l’Huile d’Olive, Centre de Biotechnologie de Borj Cedria, BP 901,2050 Hammam-Lif, Tunisia.E-mail: mokhtar.zarrouk@cbbc.rnrt.tnFax: 1216-7-9412638

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82 W. Zarrouk et al. Eur. J. Lipid Sci. Technol. 2008, 110, 81–88

the influence of other factors in the characterization, olive treeswere cultivated under the same pedoclimatic conditions, olivefruits were picked at the maturity stage of ripeness, and theiroils were extracted with the same processing system.

2 Materials and methods

2.1 Oil sample extraction

The oil samples studied were obtained from three olivevarieties: Jemri Ben Guerdane, Chemlali Zarzis and Zal-mati, grown in the southeast of Tunisia (region of Mede-nine). All olive varieties are grown in the locality of BenGuerdane. Olives were harvested at a known ripeningdegree (Tab. 1) and the same laboratory mill was used toprepare the olive oil samples. Collection, carriage andprocessing of the olive samples were carried out rapidlyand with care. Therefore, only drupes not damaged, freshand healthy were selected. The collected olive sampleswere washed and deleafed, crushed with a hammercrusher, and the paste mixed at 25 7C for 30 min, cen-trifuged at 3500 rpm over 3 min without addition of warmwater and then transferred into dark glass bottles, andstored in the dark at 4 7C prior to analysis.

2.2 Oil sample analysis

Free acidity, peroxide value, and UV absorption character-istics were determined following the analytical methodsdescribed in Regulations EEC/2568/91 and latter modifica-tions of the European Union Commission [10].

The fatty acid composition of the oils was determined bygas chromatography (GC) as fatty acid methyl esters(FAME). FAME were prepared by saponification/methyla-tion with sodium methylate according to the European UnionCommission modified Regulation EEC 2568/91 [11].

Total phenols and ortho-diphenols were quantifed color-imetrically [12]. Phenolic compounds were isolated by tripleextraction of a solution of oil in hexane with a methanic watermixture (60 : 40). The absorbance data were obtained at 725and 370 nm wavelength [13, 14]. Results are as mg caffeicacid/kg oil.

To evaluate the oil stability, the induction time for oxida-tion was measured by the Rancimat apparatus (Metrohm Co.,Basel, Switzerland). A flow of air (10 L/h) was bubbledthrough the oil (3.5 g) heated at 100 7C.

Chlorophyll and carotenoid contents (mg/kg) were deter-mined at 670 and 470 nm in cyclohexane, using the specificextinction values, by the method of Mínguez-Mosquera et al.[15].

Table 1. Analytical and compositional characteristics of virgin olive oil from the three studied cultivars. Data are expressed as meanvalues 6 SD of three independent experiments. Duncan’s test has been used to assess significance (Duncan’s test, p = 0.05). Valuesfollowed by the same symbol are not significally different.

Analytical oil parameters Cultivars

Jemri Ben Guerdane Chemlali Zarzis Zalmati

Olive ripening degree 3.20 6 0.10{ 3.40 6 0.10{ 3.50 6 0.20{

Acidity [as oleic acid, g/100 g] 0.41 6 0.02{ 0.38 6 0.05{ 0.40 6 0.01{

Peroxide index [meq O2/kg] 2.00 6 0.10§ 4.50 6 0.50{ 3.50 6 0.50{

K232 1.50 6 0.03{ 1.59 6 0.11{ 1.05 6 0.02§

K270 0.11 6 0.04{ 0.11 6 0.00{ 0.12 6 0.02{

Total phenols [as caffeic acid, mg/kg] 364.12 6 15.21§ 431.13 6 17.94{ 506.62 6 22.72{

o-Diphenols [as caffeic acid, mg/kg] 199.20 6 8.42{{ 213.24 6 9.21{ 188.12 6 6.53{

Stability [h] 65.52 6 3.37{ 80.54 6 0.45{ 51.07 6 3.65§

Chlorophyll content [mg/kg] 12.99 6 1.07{ 6.04 6 1.58{ 7.18 6 1.96{

Carotenoid content [mg/kg] 9.18 6 0.15{ 4.43 6 1.42§ 6.62 6 0.74{

Palmitic acid [%] 18.07 6 1.21{ 15.01 6 0.51{ 18.23 6 0.44{

Palmitoleic acid [%] 2.37 6 0.18{ 1.80 6 0.17{ 1.88 6 0.06{

Stearic acid [%] 2.67 6 0.25{ 3.22 6 0.40{ 2.68 6 0.24{

Oleic acid [%] 63.22 6 1.26{ 69.54 6 0.78{ 58.83 6 0.16§

Linoleic acid [%] 12.29 6 0.31{ 9.17 6 0.26§ 17.09 6 0.40{

Linolenic acid [%] 0.69 6 0.24{ 0.69 6 0.22{ 0.66 6 0.10{

Arachidic acid [%] 0.60 6 0.24{ 0.54 6 0.06{ 0.58 6 0.18{

Unsaturated fatty acids 78.66 6 1.26{ 80.99 6 0.49{ 78.46 6 0.69{

Monounsaturated fatty acids 65.69 6 1.08{ 71.34 6 0.68{ 60.71 6 0.22§

Monounsaturated fatty acids/polyunsaturatedfatty acids ratio

5.06 6 0.12{ 7.24 6 0.19{ 3.42 6 0.10§

Oleic acid/linoleic acid ratio 5.15 6 0.04{ 7.59 6 0.14{ 3.44 6 0.07§

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Eur. J. Lipid Sci. Technol. 2008, 110, 81–88 Olive oil composition 83

2.3 Volatile compound analyses

The fiber was obtained from Supelco Company (Bellefonte, PA,USA). The fiber used for the extraction of the volatile compo-nents was divinylbenzene/carboxen/polydimethylsiloxane(DVB/CAR/PDMS) 50/30 mm. Before use, the fiber was condi-tioned as recommended by the manufacturer (1 h at 270 7C).The olive oil sample (5 g) was placed in a 20-mL vial closed by aPTFE/silicone septum (Interchim). Before extraction, the stabi-lization of the headspace in the vial was accomplished by equili-bration for 60 min at 25 7C. The extraction was carried out at25 7C (room temperature) with magnetic stirring (900 trs/min)[16].

To determine the optimal adsorption time of the fiber tothe sample headspace, the DVB/CAR/PDMS fiber wasexposed for time periods of 10, 30, 60, 90 and 120 min. Asampling time of 90 min was chosen to perform the analysis[16, 17].

After exposure, injections were performed using an SPMEautosampler (CTC Analytics, Switzerland). The fiber wasthermally desorbed into a gas chromatograph and left in theinjection port (equipped with a 0.75 mm i.d. inlet liner) for4 min. The injector was set at 250 7C and operated in thesplitless mode for 2 min unless otherwise stated. Before sam-pling, the fiber was reconditioned for 5 min in a washing portat 250 7C and blank runs were realized periodically during thestudy.

GC analyses were carried out using two Agilent 6890Ngas chromatographs, one equipped with an FID and onecoupled to an Agilent 5973N system mass-selective detector(quadrupole). Both were equipped with fused-silica capil-lary columns HP-1 (PDMS, 50 m60.2 mm i.d., film thick-ness 0.33 mm for GC-FID and 0.5 mm for GC-MS). Thecarrier gas was helium for GC-FID and GC-MS; the oventemperature was programmed from 60 7C (5 min) to 250 7Cat 2 7C/min and then held isothermal (20 min). The FIDtemperature was set at 250 7C, and the temperatures of theion source and the transfer line were 170 and 280 7C,respectively. The ionization energy was 70 eV; electronionization mass spectra were acquired over the mass rangeof 35–350 atomic mass unit.

Retention indices were determined with C5–C26 alkanestandards as reference (retention times determined forSPME experiment: 20 s at 50 7C). Relative amounts of in-dividual compounds are based on peak areas obtainedwithout FID response factor correction. Three replicateswere performed for each sample. The average of thesevalues and the standard deviation were determined for eachcomponent identified.

The identification of the constituents was based on com-parison of the retention times with those of authentic samples,on computer matching against commercial (NIST 1998,Wiley N, MassFinder 2.1 Library 2001) libraries and outlaboratory-made spectral library built up from pure sub-stances and MS literature data [18–20], and then confirmed

by comparison of retention indices with published index data(Davies, 1990, BACIS, ESO 2000, 1999). Several structureswere also confirmed by standard compound injection. Allchemicals were purchased from Fluka or Sigma-Aldrich(Saint Quentin Fallavier, France).

2.4 Statistical analysis

All analyses were carried out in triplicate and the results arereported as the mean values. Data of analytical characteristicsof oils were analyzed by ANOVA/MANOVA using the Statis-tica 5.0 package (StatSoft ‘97 edition). Duncan’s multiplerange tests were used to determine significant differencesamong data.

3 Results and discussion

3.1 Free acidity, UV spectrophotometric indices,peroxide index

In all studied samples, the free acidity did not exceed the limitof 0.8% established for the best-commercial-quality olive oil,designated extra virgin [21]. The peroxide values of the stud-ied oils were below the limit of 20 meq oxygen/kg of oil, whichis accepted as the limit for ‘extra-virgin’ olive oil quality. K232

and K270 do not exceed the limit of 2.5 and 0.22, respectively,limits established for the extra-virgin olive oil (Tab. 1).

Cultivars had no significant influence on these analyticalparameters, which are basically affected by factors causingdamage to the fruits (e.g. olive fly attacks or improper systemsof harvesting, carriage and storage of olives).

3.2 Fatty acid composition

The fatty acid composition, and noticeably the oleic acid/linoleic acid ratio, affects the taste of virgin olive oil, a condi-ment which is largely responsible for the sapidity and healthfuleffects of the Mediterranean diet [22]. The mean FAMEcomposition of the oils from the analyzed cultivars is shown inTab. 1. The major fatty acids were oleic, palmitic, linoleic,stearic and palmitoleic. As can be seen, the fatty acid compo-sition of the studied oils is variable, depending on the cultivar.The differences were mainly remarkable for oleic and linoleicacids (p �0.05). In fact, the oleic acid percentage varied from58.8% (Zalmati) to 69.5% (Chemlali Zarzis) of total fattyacids. The current biochemical evidence indicates that, inolive and other plant species, the polyunsaturated fatty acids(C18:2 and C18:3) are produced by the consecutive desa-turation of oleic acid. Accordingly, Zalmati and ChemlaliZarzis oils had the highest and the lowest linoleic acid levels,respectively. Linolenic acid, which presents the highest level ofunsaturation of olive oil, shows similar contents for the threestudied cultivars (<0.7%). Palmitic acid, the major saturatedfatty acid in olive oil, varied between 15% (Chemlali Zarzis)

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84 W. Zarrouk et al. Eur. J. Lipid Sci. Technol. 2008, 110, 81–88

and 18.23% (Zalmati). Another important saturated fatty acidis stearic acid; its content is within the range of 2.67–3.22% forJemri Ben Guerdane and Chemlali Zarzis oils. The variabilityof the fatty acid composition, from all tested samples, coveredthe normal range expected for olive oils [21].

The oleic/linoleic acid ratio (O/L) of Chemlali Zarzis oilwas higher than those calculated for Jemri Ben Guer and Zal-mati (7.6 against 5.1 and 3.4, respectively).

3.3 Total phenols and o-diphenols

Phenolic compounds, the most bioactive components of vir-gin olive oils, are one of the bases of the nutritional propertiesof this product. For these compounds, Tab. 1 shows changesin total phenols and o-diphenols in the studied oils accordingto cultivar. In fact, Zalmati oil showed the highest value in totalphenol concentration (507 mg/kg) whereas Jemri Ben Guer-dane oil recorded the lowest (364 mg/kg); Chemlali Zarzis oilhad an intermediate value (431 mg/kg). Thus, we observe thatour samples were relatively rich in total phenols (.300 mg/kg). It has long been known that the level of phenolics in planttissues can be influenced by environmental factors such asambient temperature and water availability [23]. With regardto the latter factor, a water deficit tends to generate a stresssituation that induces the production of phenolics [24], andthis factor could be related to the increase in the phenol con-tent of the oils from Southeastern Tunisia which is character-ized by a very low rainfall accumulation in the year (150 mm/year).

o-Diphenol concentrations were also studied and differ-ences were found among o-diphenol contents in the analyzedsamples. Contrarily to phenol concentrations, the highestcontent of o-diphenols was observed in Chemlali Zarzis oil(213 mg/kg), followed by Jemri Ben Guerdane (about199 mg/kg), while Zalmati showed the lowest value (188 mg/kg).

These observations are in favor of the existence of a gen-otype effect, as the extraction system, the olive ripeness andthe pedoclimatic and agronomic conditions were the same forall the cultivars.

3.4 Oxidative stability

Oxidative stability is a central parameter in the estimation ofextra-virgin olive oil quality, as it gives a reliable evaluation ofthe susceptibility to oxidative degeneration, which is the maincause of its adulteration [25].

The oxidative stability of the tested olive oils variedaccording to varieties. It ranged from a minimum of 51.1 h forZalmati to 80.5 h for Chemlali Zarzis, while the mean stabilityof Jemri Ben Guerdane oil was about 65.5 h. According to theseresults and previously published data for other Tunisianmonovarietal olive oils, our samples seemed to have a high oxi-dative stability. All the data were obtained under the same

Rancimat conditions using an oil sample of 3.5 g warmed to100 7C and an air flow of 10 L/h.

One notices that, in spite of the highest value in total phe-nols, Zalmati oil showed the lowest stability value, which is dueto the lower level in o-diphenols (188 mg/kg) and to the loweroleic/linoleic ratio (3.4). The highest stability observed inChemlali Zarzis oil can be explained by its richness in o-diphenols (213 mg/kg) and also by its high oleic/linoleic ratio(7.6).

3.5 Chlorophylls and carotenoids

The total pigment content of olive oils is an important qualityparameter because it correlates with color, which is the firstattribute of virgin olive oil evaluated by consumers. Thus,virgin olive oil has a color from green-yellow to gold, depend-ing on the variety and the stage of maturity. Tab. 1 shows thatthe content of chlorophylls and carotenoids varied accordingto cultivar. However, Jemri Ben Guerdane oil was dis-tinguished from the others for its higher level of chlorophyllsand carotenoids (13 and 9.2 mg/kg, respectively). Pigmentswere also found in Zalmati and Chemlali Zarzis oils atremarkable concentrations. In fact, the content of chlorophyllsand carotenoids in Zalmati oil were 7.2 and 6.6 mg/kg,respectively, while Chemlali Zarzis oil presented 6 and 4.4 mg/kg, respectively.

3.6 Volatile components

Extra-virgin olive oils are characterized by a delicate and fra-grant aroma which is related to a complex mixture of volatilecompounds. The detection of aromatic volatiles emitted byolive oils is of key importance in the quality control of thisproduct [26]. Components identified from the three cultivarsare listed as means of three replicates in Tab. 2. Forty-eightvolatile compounds were isolated and characterized by GC-RIand GC-MS analysis, representing 94.1–98.1% of the totalpeak area percentage.

From a qualitative point of view, one can see that alde-hydes, ketones, hydrocarbons, alcohols and esters are the mainchemical families present in the analyzed headspaces. Alde-hydes, in particular (E)-hex-2-enal, coming from the LOXpathway, represent the predominant chemical family presentin the headspace.

Comparing the three cultivars, we can observe that therewere some differences in the constituents of the volatile frac-tion. However, the products of the LOX cascade were themajor components of the volatile fraction of the analyzed oils,and the sum of the areas of their peaks accounted for 81.6, 81,and 59.9% of the total areas of Zalmati, Jemri Ben Guerdaneand Chemlali Zarzis oils, respectively.

The percentage of C6 compounds – also called “greenvolatiles” [27] – differed according to the cultivar. In fact,Jemri Ben Guerdane oil showed the highest percentages(72.1%) of C6 compounds identified in its volatile fraction

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Eur. J. Lipid Sci. Technol. 2008, 110, 81–88 Olive oil composition 85

Table 2. Volatile compounds, extracted by HS-SPME, in the three Tunisian monovarietal virgin olive oils. Data expressed as meanvalues 6 SD of three independent experiments.

Compounds{ RI{ Jemri Ben Guerdane§ Chemlali Zarzis Zalmati

Ethanol 577 0.38 6 0.10 2.59 6 0.18 1.04 6 0.09Propan-2-one 580 3.31 6 0.29 5.53 6 0.59 2.34 6 0.03Acetic acid 592 1.45 6 0.16 5.73 6 2.64 0.37 6 0.042-Methylpentane 593 0.11 6 0.01 nd 3.71 6 0.823-Methylpentane 596 0.34 6 0.03 1.45 6 0.98 1.13 6 0.101-Penten-3-one 656 3.33 6 0.54 nd 3.01 6 0.821-Penten-2-one 658 nd 4.54 6 1.50 ndPentan-3-one 670 0.76 6 0.25 1.53 6 0.59 0.83 6 0.24Heptane 700 tr{{ 0.20 6 0.02 0.11 6 0.00(E)-Pent-2-enal 723 0.30 6 0.01 0.40 6 0.01 0.35 6 0.01(Z)-Pent-2-enol 745 1.02 6 0.05 1.30 6 0.08 1.07 6 0.03Toluene 750 3.36 6 0.21 7.66 6 0.64 4.26 6 0.15(Z)-Hex-3-enal 770 nd nd 0.41 6 0.02Hexanal 772 4.73 6 0.28 5.34 6 0.35 6.09 6 0.00Octene 785 nd 0.15 6 0.02 ndOctane 800 1.00 6 0.07 2.59 6 0.13 1.08 6 0.04(Z)-Hex-2-enal 817 0.84 6 0.01 0.47 6 0.06 0.78 6 0.02(E)-Hex-2-enal 825 60.40 6 0.63 35.30 6 2.73 58.90 6 0.36(Z)-Hex-3-enol 833 nd nd 1.65 6 0.28(E)-Hex-2-enol 843 1.09 6 0.04 nd 0.52 6 0.15Hexanol 846 1.27 6 0.08 0.47 6 0.11 0.37 6 0.08p-Xylene 852 0.22 6 0.01 0.18 6 0.10 0.11 6 0.01Heptan-2-one 864 tr 0.10 6 0.01 ndHeptanal 875 0.54 6 0.05 0.39 6 0.04 0.24 6 0.083,4-Diethylhexa-1,5-diene{{ 892 0.37 6 0.00 0.40 6 0.03 0.42 6 0.003,4-Diethylhexa-1,5-diene{{ 896 0.27 6 0.03 0.24 6 0.03 0.29 6 0.00(E)-Hept-2-enal 925 0.24 6 0.03 0.60 6 0.10 0.42 6 0.073-Ethylocta-1,5-diene{{ 931 1.88 6 0.11 2.02 6 0.25 2.28 6 0.043-Ethylocta-1,5-diene{{ 937 1.46 6 0.10 1.40 6 0.18 1.58 6 0.02Hexanoic acid 956 nd 0.22 6 0.00 0.13 6 0.04Octan-2-one 964 nd 0.15 6 0.03 ndOctanal 976 0.12 6 0.02 0.35 6 0.04 0.20 6 0.01(Z)-Hex-3-enyl acetate 983 0.97 6 0.09 3.22 6 0.49 trDeca-3,7-diene{{ 985 1.09 6 0.09 1.21 6 0.16 1.12 6 0.05Deca-3,7-diene{{ 987 1.89 6 0.67 1.78 6 0.15 1.63 6 0.06Deca-3,7-diene{{ 989 nd nd trHexyl acetate 991 2.85 6 0.26 2.88 6 0.45 ndn-Decane 996 0.16 6 0.01 0.15 6 0.01 0.10 6 0.01Limonene 1016 0.81 6 0.28 0.64 6 0.12 tr(E)-b-Ocimene 1032 0.14 6 0.01 0.34 6 0.06 0.26 6 0.01Nonanal 1074 0.67 6 0.18 0.91 6 0.09 tr(Z)-4,8-Dimethylnona-1,3,7-triene 1100 0.17 6 0.01 0.23 6 0.04 nd(E)-Allocimene 1122 nd nd 0.22 6 0.01Octanoic acid 1147 nd 0.23 6 0.13 trBenzoic acid 1168 0.46 6 0.05 0.74 6 0.06 trDodecane 1196 0.10 6 0.00 0.13 6 0.00 trNonanoic acid 1253 tr tr 0.29 6 0.04a-Farnesene 1494 tr 0.38 6 0.06 trTotal 98.10 6 0.85 94.14 6 1.73 97.31 6 0.91

{ Order of elution and percentages of components are given on apolar column (HP-1).{ Retention indices as determined on HP-1 column using the homologous series of n-alkanes.§ Peak area % (percent normalized areas) determined by HS-SPME–GC/FID analysis.# Compound not detected.{{ Trace (,0.1%).{{ Correct isomer not characterized.

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86 W. Zarrouk et al. Eur. J. Lipid Sci. Technol. 2008, 110, 81–88

with respect to Zalmati (68.7%) and Chemlali Zarzis (47.7%).The differences in the concentrations of these compoundsshould be mostly related to the variety as there were no exter-nal variables that might unequally affect the enzyme activity ofa particular variety (e.g. climate, soil, harvesting and extrac-tion conditions).

In all oil samples, the most representative C6 compoundsare aldehydes, which accounted for 96.3, 91.4 and 86.2% ofthe whole C6 fractions of Zalmati, Jemri Ben Guerdane andChemlali Zarzis oils, respectively (Fig. 1). The percentages ofalcohols and esters differed for each variety, evidencing a strictdependence on the enzymatic store [28].

The percentages of (E)-hex-2-enal (the most abundantaroma compound extracted by HS-SPME) in Jemri BenGuerdane and Zalmati oils were still higher than thoseobserved in Chemlali Zarzis oil (60.4 and 58.9 against35.3%). (E)-Hex-2-enol was present in the aroma of JemriBen Guerdane and Zalmati oils at low amounts (1.09 and0.52%, respectively) and was completely absent in Chem-lali Zarzis oil, pointing out the absence of alcohol dehy-drogenase (ADH) activity. However, the different amountsof (E)-hex-2-enol detected in Jemri Ben Guerdane andZalmati oils were depending on the genetic levels of ADHof the two cultivars [28]. The high level of (E)-hex-2-enalin the analyzed oils shows the pre-eminence of the (E)-hex-2-enal/(E)-hex-2-enol pathway compared to the hex-anal/hexanol pathway in the three studied varieties [29].(Z)-Hex-3-enal and (Z)-hex-3-enol were present only inZalmati oil.

The trace levels of (Z)-hex-3-enyl acetate and theabsence of hexyl acetate in Zalmati oil indicated a very lowcontent of alcohol acetyl transferases (AAT) catalyzing theproduction of these esters [28] (Table 2). A higher level ofAAT was evidenced in Chemlali Zarzis oil since the percent-

age of (Z)-hex-3-enyl acetate is more important than in JemriBen Guerdane oil (3.22% against 0.97%).

All these results indicate that olive oil aroma compoundsaccumulate differently according to the cultivar. In fact,accumulation of these metabolites has a close dependence onthe enzymatic store, which is genetically determined [28].

Considerable amounts of C5 volatile compounds reportedelsewhere as significant contributors to the volatile profile [5,6] were found in the aroma of the three Tunisian virgin oliveoils. Evidence indicates that C5 compounds are the results ofactivity of an additional branch of the LOX pathway [6]. Forthese compounds, we observed that 1-penten-2-one wasregistered only in Chemlali Zarzis oil with remarkable content(4.54%). 2-Methylpentane and 1-penten-3-one, absent inChemlali Zarzis, were present in Jemri Ben Guerdane andZalmati oils in different amounts (0.11 and 3.33% in JemriBen Guerdane and 3.71 and 3.01% in Zalmati, respectively).

As far as hydrocarbons are concerned, recently severalauthors have extensively studied the hydrocarbon fraction ofvirgin olive oils and its possible usefulness for their character-ization. In our headspace oil samples, two aromatic hydro-carbons (toluene and p-xylene) were detected. However, it hasbeen reported that virgin olive oil could contain higher levelsof volatile contaminants due to the absence of a refining pro-cess that leads to a decrease of total volatile compounds [30].For this reason, the assessment of volatile contaminants inolive oil deserves a special attention for some researchers.

In addition, several terpenic hydrocarbons (mono- andsesquiterpenes) were found in small percentages of the detec-ted volatiles. Monoterpenic hydrocarbons, such as limoneneand (E)-b-ocimene were detected in all the analyzed samples,but in different amounts from one cultivar to another; whereas(E)-allocimene was present exclusively in Zalmati oil (0.22%).a-Farnesene, a tetra-unsaturated acyclic sesquiterpene, was

Figure 1. Distribution of C6 aldehydes, alcoholsand esters in relation to total C6 compounds in thethree Tunisian olive oils.

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Eur. J. Lipid Sci. Technol. 2008, 110, 81–88 Olive oil composition 87

present at higher level in Chemlali Zarzis oil (0.38%) com-pared to Jemri Ben Guerdane and Zalmati oils (trace levels forboth samples).

These results showed that each cultivar can be dis-tinguished from the other by a comparison of the constituentsof their volatile fractions.

4 Conclusion

All the described results have shown a wide variability in thechemical characteristics of the studied virgin olive oils becauseof the diversity of the varieties. The differences in the fattyacid composition were mainly remarkable for oleic and linoleicacids. Our samples were relatively rich in total phenols(.300 mg/kg), and the Rancimat test showed that, wherever ahigh amount of o-diphenols was found, the time of resistanceto forced oxidation was greater. Jemri Ben Guerdane oil isdistinguishable from the others for its higher content ofchlorophylls and carotenoids (13 and 9 mg/kg, respectively).

The analysis of the three Tunisian monovarietal virginolive oils has shown that there were qualitative and quantitativedifferences between the volatile profiles of the oils from thetested varieties, in spite of having been cultivated under iden-tical environmental conditions. As the harvesting period andextraction conditions were similar for all studied samples, theresults indicate that genetic factors related to the cultivars areone of the most important aspects of the volatile compositionof olive oil.

Acknowledgments

This work has been done as a part of a National Research Project.We thank the Ministry of Higher Education, Scientific Researchand Technology for financially supporting this program. Part ofthis work was carried out at the Laboratoire de Chimie des Molé-cules Bioactives et des Arômes, Equipe Analyse d’Extraits Natur-els, Université de Nice Sophia-Antipolis, France.

Conflict of interest statement

The authors have declared no conflict of interest.

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