沙漠研究 19-1, 371- 374 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Derivatives of the UV Absorption Spectra, ECN Groups, and Phytosqualene
Contents in Virgin Olive Oils from Varieties Grown in the Extreme Conditions ofTataouine Region (Southern Tunisia)
Imen OUESLATI1, Mokhtar GUERFEL1, Hédia MANAI1, Douja DAOUD1,Jacinto SANCHEZ2, Mokhtar ZARROUK1
Abstract: This study was carried out on the analysis of the physicochemical composition ofsome Tunisian virgin olive oils (VOOs) from varieties grown in the harsh pedoclimaticconditions of the region of Tataouine. According to the UV absorption spectra and therespective derivative ones, all the studied samples belong to the category “extra virgin”. HPLC
analysis of triacylglycerols permitted the identification and quantification of four
triacylglycerol groups: ECN 44, 46, 48, and 50, which are present at the highest levels in thestudied VOOs. Small amounts of some other groups (ECN 38, 40 and 42), were also observedin all samples. The analysis of phytosqualene, and its related alcohol composition, showed that
all the studied samples contained levels expected for good quality VOO. The Principal
component analysis applied to the analytical data reveal that the great variability in oilcomposition are influenced exclusively by the genetic factor.
Keywords: ECN groups, Phytosqualene, UV derivative spectrophotometry, Virgin olive oil

1. Introduction
The beneficial effect of the consumption of the virgin olive oil (VOO) on human health is well known
and related to the characteristic of the fatty acid composition and the presence of minor components, suchas phytosqualene, phytosterols and antioxidant molecules as tocopherols and other phenolic compounds(Owen et al., 2000). Some recent researches have shown the influence of the geographic regions (BenTemime et al., 2006) and the fruit maturity (Baccouri et al., 2007) on the chemical composition of theVOO, but, to our knowledge, no references can be found regarding the influence of the severe pedoclimaticconditions on the quality of VOO. This study are focused on the characterization of VOOs from varieties
grown under the harsh pedoclimatic condition of Tataouine (chalky grounds, very high temperature, lowrainfall and the presence of the Sahara which covers more than 70% of the Tataouine region) (Bonvallot,1986). Many analytical parameters, such as quality parameters, ECN groups, phytosqualene content andrelated alcohol composition, have been considered in the characterization of the Tataouine varieties.
2. Materials and Methods
2.1. Oil Samples
Four samples of VOOs, grown in the locality of Douirat in the region of Tataouine (southern Tunisia),
were analyzed from the following varieties: Chemlali Tataouine, Fakhari Douirat, Zarrazi Douirat, andDhokar Douirat. Oil extraction was performed using an Abencor laboratory oil mill, kneading the olive
paste at 28 ˚C for 30 min. All samples were stored at -20 ˚C in darkness until analysis.
2.2. ECN groups Composition
The analysis of triacylglycerols was performed according to the official chromatographic method of
the European Economic Community Regulations no. 2568/91 (EEC, 1991). A Hewlett Packard HPLC
(HP 1050, Agilent Technology) quaternary pump instrument equipped with a detector of refraction index(IR) model HP 1040 was employed using a Lichrosorb RP18 column (250 × 4.6 mm, 5 μm particle size;
Teknocroma, Barcelona, Spain). Settings were: column oven, 45 ˚C; elution solvent: acetone-acetonitrile
1

Laboratoire Caractérisation et Qualité de l’Huile d’Olive, Centre de Biotechnologie, Technopole de Borj-Cédria,
B.P. 901, 2050 Hammam-Lif, Tunisia; Fax no. 0021679412948, tel. no. 0021679412948, E-mail:mokhtar.zarrouk@cbbc.rnrt.tn
2
Instituto Tecnológico Agroalimentario. Dirección General de Infraestructuras y Desarrollo Tecnológico. Junta deExtremadura. Carretera de Cáceres s/n. 06071 Badajoz. Spain.

372(60:40, v/v) at a rate of 1.2 mL/min.
The injected volume was 10 μL.
2.3. Quantification of phytosqualene
Phytosqualene determination wasaccomplished on a reverse phase
Nucleosil C18 column (particle size 5μm, 125 × 4.0 mm i.d.,
Macherey-Nagel, Düren, Germany).
The elution solvent was 100%
acetonitrile; the flow rate, 1.2 mL/min; and the injection volume, 10 μL.
Detection was achieved at 208 nm.

Fig. 1. Example of calculation of the Δh value from the first derivativeof the UV spectrum of a VOO sample (Abs, absorbance).

2.4. Determination of Phytosterols.
The content of phytosterols (%) was determined according to procedures described by Sanchez-Casas
et al. (2004). The apparatus was a Hewlett-Packard instrument model HP 6890 gas chromatograph,equipped with an FID, an HP-5MS capillary column (30 m × 0.25mm × 0.25μm), and a 6890 Agilent
automatic injector. The working conditions of the chromatograph were: injector, 300 ˚C; isothermalcolumn, 260 ˚C; and detector, 325 ˚C.
2.5. Determination of Quality parameters.
Quality of the samples was evaluated based on measurements of acidity, peroxide value andabsorbance at 232 and 270 nm (EEC, 1991).
Concerning the first derivatives, the oil solutions (0.5%, wt/vol) were prepared in iso-octane, and theUV spectrum was recorded between 190 and 350 nm. Quantitative data for the first derivatives wereexpressed as Δh values, calculated as the vertical distance between the two minima at 239 and 246 nm of
the first derivative spectrum in the side towards longer wavelengths, divided by the concentration of thesample solution (%, wt/vol) (Fig. 1): Δh = h239 − h246c2.6. Statistical analysis
Principal components analysis is performed with XLSTAT software, Version 2007. Significant
differences among clones were determined using an analysis of variance, followed by a Duncan’s multiple
range tests, using SPSS 16.0 for Windows (SPSS Inc., 2007).
3. Results and Discussion
3.1. Free Acidity, Peroxide Index and Specific UV Extinction Coefficient determinations
For all olive oil samples analysed, the values of the physicochemical parameters evaluated (acidity ≤1%1% 270 ≤ 0.22, and0.8%, PV ≤ 20 mequiv of O2 kg-1, E1cm
232 ≤ 2.5) (Table 1) fell within the ranges
E1cm
established for the highest quality category of “extra virgin” olive oil, as stated by Regulation
EC/1989/2003.
3.2. Derivatives of the UV Absorption Spectra
Concerning the first derivatives, the results of the four samples showed no significant differences (p <0.05) between varieties. All the results showed a Δh which did not exceed 0.6. Based on the work of
1%
232 values had negative ΔhGrigoriadou and Tsimidou (2006), oils with higher peroxide index and E1cmvalues. High positive values are expected for more recent oils whereas high negative values are expected foroils stored for long or under inappropriate conditions. Values around zero are indicative of oils that only
marginally fall within the upper commercial category “extra”. Our observations showed that all the studied
oils belong to the category extra virgin and are in line with those of Grigoriadou and Tsimidou (2006), who
21% 232 and the Δh (y = –0.032x + 0.0866, R = 0.91, p < 0.05).
showed a good correlation between the E1cm373

Table 1. Means and standard deviations of the quality parameters and some minor compounds evaluated in thestudied olive oil samples from the Tataouine varieties.
Chemlali Tataouine
Fakhari Douirat
Zarrazi Douirat
Dhokar Douirat
Phytosqualene (mg kg-1)
2605±4a‡6048±6d3933±3c2363±2b-1
caa
Phytosterol (mg kg )1717±21040±31063±21415±3b
Δ5 Avenasterol (%)
19.54±1.45d
16.14±1.32c
9.55±1.54a
11.83±0.56b
0.25±0.03a
0.30±0.05b
0.45±0.03bc
0.64±0.04c
FFA (as OA, g kg-1)-1aab
PI (meq O2 kg )4.00±0.54
5.00±0.38
15.46±0.55
16.03±0.69baaa
1% 270 nm
0.16±0.00
0.14±0.00
0.14±0.02
0.12±0.07b
E1cm
baa
1% 232 nm
2.30±0.03
1.95±0.02
1.88±0.06
2.27±0.07b
E1cm
1% 232 /1% 270E1cm
E1cm

ΔK x 10
Δh

3

14.4a
-6.0±0.0a
0.03±0.005a

13.9a-2.0±0.0b
0.04±0.000a

13.4a
7.0±0.1c
0.004±0.001a

18.92b
-3.0±0.1b
0.06±0.007a

FFA, free fatty acids; OA, Oleic acid; PI, Peroxide index. ΔK = K270–(K266–K274)/2; Δh = (h239–h246)/C [%, wt/vol]‡Different letters for the same quality parameter indicate significant differences among varieties (p < 0.05).

3.3. Phytosqualene content and related alcohol composition
All of the analysed samples contained levels expected for good quality VOOs. The analysis ofDhokar Douirat and Chemlali Tataouine oils showed the lowest phytosqualene content (2363 and 2605
mg/kg oil, respectively), whereas, Zarrazi Douirat and Fakhari Douirat showed an amount of suchcompound two fold-higher than that of Dhokar Douirat and Chemlali Tataouine VOOs (3933 and 6048 mg
kg-1 oil, respectively). Our results confirm that the variability in the phytosqualene content of the studied
oils can be attributed to varietal factor.
Table 1 reports the amounts of the precursor of phytosqualene, the phytosterols. Only the total
phytosterol and the Δ5-avenasterol were presented due to their importance in the in authentication and thestability of the VOO during heat treatment. A wide variability of the phytosterols components from one
variety to another was observed (Table 1). All of the olive oil samples contained more than 1000 mg/kg,
the minimum value established by EU Regulations for the category ‘extra virgin’ olive oil. Moreover, avery high Δ5-avenasterol content of the samples was observed, which reached 19.53% of total sterol
content in the case of Chemlali Tataouine oil. This amount was almost twice high that found for bothSpanish VOOs from the variety “Cornicabra” (Salvador et al., 2001) and Tunisian varieties.
3.4. ECN TAG group compositions
ECN TAG group contents, expressed inpercentage of total TAG are shown in Figure 2.
The predominant ECN TAG groups in all
samples are ECN 44, 46, 48, and 50. Together
these groups accounted for more than 90 % of thetotal TAGs in almost all the analyzed VOOsamples.
Other minor ECN TAG groups, such as
ECN 38, 40 and 42, are also present in almost all
samples with an amount less than 2%. TheFig. 2. ECN TAG group compositions in the studied olive
ECN 48 TAG group, was predominant inoil sample from the Tataouine varieties. WithECN, equivalent carbon number; ECN=CN-2DB,
Chemlali Tataouine, Fakhari Douirat, and Zarrazi
where CN is the number of carbon atoms in all acyls,Douirat (65 % on average). Its level was higher
and DB is the number of double bonds.
than that in Dhokar Douirat (38.76%). TAG ofECN 42, 46, and 44 were observed to be highest
in Dhokar Douirat (3.17, 37.28, and 16.29%, respectively). Some negligible ECN TAG groups, such as
ECN 38, are also detected in the studied VOOs with an amount < 1%. Except in Dhokar Douirat VOO,
ECN 40 was not detected in any VOO samples. Chemlali Tataouine, Fakhari Douirat, and Zarrazi Douirat
showed small differences in the proportion of ECN 44 and ECN 50 TAG group proportions, with mean valuesof 4.27 and 7.30%, respectively.

374

3.5. Chemometrics.
In order to study how the studied parameters are useful in chemometric analysis to discriminate between
varieties, a principal component analysis (PCA) was performed. The first and the second principal
components were sufficient to display the data structure, since they explained 74.70% of the total variance.
By examining the scores-plot (Fig. 3) in the area defined by the first and the second principal components,the samples were separated into four groups based on the studied parameters. Group I, which is locatedon the left side of the scores-plot, is composed of Dhokar Douirat. Group II, which is located in thesymmetrical position of the scores-plot, consists of Zarrazi Douirat. Group III, is located at the top of thescores-plot, and is formed by Chemlali Tataouine. Group IV is located on the right side of the scores-plot
and includes Fakhari Douirat. Since the samples were well described by the scores-plot, the loading plot(Fig. 3) was analysed in order to show which variables influenced the group separation. Group I wascharacterised by the highest content of ECN 40, 42, 44, and 46. Group II was characterised by a highlevel of phytosqualene, acidity and ΔK. Group III was characterised by the highest level of phytosterol.
Finally, group IV was characterised by the lowest ECN 38, 48, and 50.

Fig. 3. Loading plot (Projection of the variables) and Scores-plot (Projection of the samples studied) bydimensions F1 and F2 from principal component analysis (PCA) applied to all the studied parameters.

4. Conclusions
All the obtained results showed that the great variability in oil composition among the studiedvarieties are influenced exclusively by the genetic factor.
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