Chemical composition of virgin olive oils from the Chemlali cultivar with regard to the method of the olive tree propagation

The chemical composition of virgin olive oil (VOO) depends on many factors: olive tree cultivation, harvesting, pedoclimatic conditions, olive cultivar, plant density and number of processing steps required; mainly crushing, malaxation and centrifugation (Salvador et al., 2003; Cerretani et al., 2005; Torres and Maestri, 2006; Guerfel et al., 2010). Tunisia is a very important country in the olive oil producing world, the largest African exporter and fourth worldwide after Spain, Italy and Greece. The olive tree (Olea europaea L.) is present in practically every region of the country, up to the border of the southern dessert. The conventional method of olive tree propagation in Tunisia is based on vegetative multiplication using cuttings, grafting, or suckers. These methods have been frequently used for the propagation of some highly valued cultivars, particularly the Chemlali cultivar. The Chemlali cultivar is ubiquitous in the Tunisian arable land and contributes to up to 80% of the national olive oil production. Chemlali olive oil is characterized by the relatively low levels of oleic acid (53-55%) and high levels of palmitic and linoleic acids (Manaï et al., 2007). Recently, a major effort has been made to improve the quality of olive oil produced in Tunisia. Perhaps, production improvement can be made once the main drawbacks are known (cultivar type and ⁄ or the origin of olives used for oil extraction). This is the first evaluation of the chemical composition of Chemlali virgin olive oils in relation to the method used for the olive tree propagation (cuttings, suckers). Because of the importance of this cultivar for Tunisian oil production, the aim of this work was to characterize virgin olive oils of Chemical composition of virgin olive oils from the Chemlali cultivar with regard to the method of the olive tree propagation

Tunisia is a very important country in the olive oil producing world, the largest African exporter and fourth worldwide after Spain, Italy and Greece.The olive tree (Olea europaea L.) is present in practically every region of the country, up to the border of the southern dessert.The conventional method of olive tree propagation in Tunisia is based on vegetative multiplication using cuttings, grafting, or suckers.These methods have been frequently used for the propagation of some highly valued cultivars, particularly the Chemlali cultivar.The Chemlali cultivar is ubiquitous in the Tunisian arable land and contributes to up to 80% of the national olive oil production.Chemlali olive oil is characterized by the relatively low levels of oleic acid (53-55%) and high levels of palmitic and linoleic acids (Manaï et al., 2007).Recently, a major effort has been made to improve the quality of olive oil produced in Tunisia.Perhaps, production improvement can be made once the main drawbacks are known (cultivar type and ⁄ or the origin of olives used for oil extraction).This is the first evaluation of the chemical composition of Chemlali virgin olive oils in relation to the method used for the olive tree propagation (cuttings, suckers).Because of the importance of this cultivar for Tunisian oil production, the aim of this work was to characterize virgin olive oils of

SUMMARY Chemical composition of virgin olive oils from the
Chemlali cultivar with regard to the method of the olive tree propagation.
This paper reports for the first time a discrimination study based on the antioxidant compounds, oxidative stability and volatile compounds of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivar (Chemlali) using two methods of olive tree propagation (suckers and cuttings).There were significant differences between the oils from the two methods.Olive oil samples obtained from the fruits of trees from suckers had a higher content of oleic acid (63.8%), higher contents of chlorophyll and carotenoids (3.01 mg/ kg and 1.9 mg/kg respectively), a higher content of (E)-2 hexenal (66.1%) and a higher content in total phenols (890 mg/kg).Interestingly, more stable oil was obtained from the olives CHEMICAL COMPOSITIOn OF VIRGIn OLIVE OILS FROM THE CHEMLALI CULTIVAR wITH REGARD TO THE METHOD… Chemlali trees from cuttings and suckers based on the study of major (fatty acids) and minor compounds (phenols, chlorophylls, carotenoids and volatiles) as well as on the oxidative stability.

Oil Sample Extraction
Olive oil samples were obtained from fruits of the main Tunisian olive cultivar, Chemlali, which were picked by hand at the same stage of maturity from three trees during the crop season 2009/2010 (October) in a 4 ha olive orchard located in the Souassi center of Tunisia (35°, 49' n, 10°, 30' E).The olive trees were planted in 1988 and were subjected to an identical fertilization regime and to all common olive cultivation practices.The same laboratory mill was used to prepare the olive oil samples.Only healthy fruits, without any kind of infection or physical damage, were processed in triplicate from trees from cuttings and from suckers.After harvesting, fresh olives (1.5-2.0 kg) from each fruit sample were washed and the leaves were removed.They were then crushed with a hammer crusher, and the paste was mixed at 25 °C for 30 min, centrifuged without the addition of warm water (oil produced from each extraction was 200-250 mL/kg), transferred to dark glass bottles, and stored for one week in the dark at 4 °C until analysis.

Determination of Oil Quality Parameters
Free acidity, expressed as percent of oleic acid (%18:1); peroxide value, given as milliequivalents of active oxygen per kilogram of oil (meqO2/kg); and UV absorption characteristics (K 232 and K 270 ) were determined according to the analytical methods described in the European Union Commission Regulations EEC/2568/91 and EEC/1429/92.

Fatty Acid Composition
The fatty acids were converted to fatty acid methyl esters before analysis by shaking a solution of 0.2 g oil and 3 mL of hexane with 0.4 mL of 2-n methanolic potassium hydroxide, and analyzed using a Hewlett-Packard (HP 4890D; Hewlett-Packard Company, wilmington, DE) chromatograph equipped with a capillary column (Supelcowax: 30 m 3 0.53 mm; 0.25 mm), a split/splitless injector and a flame ionization (FID) detector.The carrier gas was nitrogen at a flow rate of 1 mL/min.The temperatures of the injector, the detector and the oven were held at 220, 250 and 210 °C, respectively.The injection volume was 1 mL.

Pigment Content
Chlorophyll and carotenoid contents were determined colorimetrically as previously described (Mínguez-Mosquera et al., 1991).The maximum absorption at 670 nm is related to the chlorophyll fraction, while the maximum absorption at 470 nm is related to the carotenoid fraction.The values of the coefficients of specific extinction applied were E0 5 613 for pheophytin, a major component in the chlorophyll fraction, and E0 5 2,000 for lutein, a major component in the carotenoid fraction.Thus, the pigment contents were calculated as follows: Chlorophyll (mg/ kg) 5 (A 670 3 10 6 ) (613 3 100 3 d) Carotenoid (mg/ kg) 5 (A 470 3 10 6 ) (2,000 3 100 3 d) where A is the absorbance and d is the spectrophotometer cell thickness (1 cm).

Total phenolic content
Total phenol contents were quantified colorimetrically (Ranalli et al., 1999).Phenolic compounds were isolated by the triple extraction of a solution of oil (10 g) in hexane (20 mL) with 30 mL of a methanol and water mixture (60:40, v/v).The Folin-Ciocalteau reagent (Merck Schuchardt OHG,Hohenbrunn, Germany) was added to a suitable aliquot of the combined extracts, and the absorption of the solution at 725 nm was measured.Values are given as milligrams of caffeic acid per kilogram of oil (Gutfinger, 1981).

Volatile compound analyses
Solid phase micro extraction was used as a technique for headspace sampling of virgin olive oils.SPME devices coated with polydimethylsiloxane (PDMS, 100 mm) were used to sample the headspace of 2 mL of olive oil inserted into a 5 mL glass septum vial and allowed to equilibrate for 30 min.After the equilibration time, the fiber was exposed to the headspace for 50 min at room temperature (25 °C).Once sampling was finished, the fiber was withdrawn into a needle and transferred to the injection port of the GC-FID and GC-MS system.GC-EIMS separations were performed with a Varian CP 3800 gas chromatograph equipped with a DB-5 Capillary column (30 m 3 0.25 mm; coating thickness 5 0.25 mm) and a Varian Saturn 2000 ion trap mass detector.
Analytical conditions were as follows: injector and transfer line temperature at 250 and 240 °C, respectively; oven temperature was programmed from 60 to 240 °C at 3 °C min -1 ; carrier gas, helium at 1 mL min -1 ; splitless injection.Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial (nIST 98 and ADAMS) and homemade library mass spectra built from pure substances and components of known oils and MS literature data (Jennings and Shibanoto1980; Davies, 1990;Adam, 1995).Moreover, the molecular weights of all the identification M. GUERFEL, M.B.MAnSOUR, Y. OUnI, F. GUIDO, D. BOUJnAH AnD M. ZARROUK substances were confirmed by GC-CIMS, using MeOH as the CI ionizing gas.

Oil Stability
Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989).Stability was expressed as the oxidation induction time (h), measured with the Rancimat 743 apparatus (Metrohm, Herisau Switzerland), using an oil sample of 3.6 g.The oil temperature was 101.6 °C and the air flow was 10 L/h.

Statistical Analysis
Significant differences between means were determined by an analysis of variancewhich applied a Duncan's test.Differences were considered statistically significant when the probability was greater than 99% (P , 0.01).The statistical analysis was performed using SPSS 13.0 for windows (SPSS Inc., 2004).

Fatty Acid Composition
Compared to the olive oil samples obtained from cuttings, the olive oil samples obtained from suckers had a higher content of oleic acid (63.8%) (Table 1).The palmitic acid content varied between 14.3% and 17.5%.The olive oil samples also contained low amounts of linolenic acid (C18:3), arachidic acid (C20:0) and palmitoleic acid (C16:1).The olive samples obtained from trees cuttings were found to be rich in total saturated fatty acids (21.1%), essentially due to their high content of palmitic acid.The olive oil samples obtained from suckers were found to show a higher content in total monounsaturated fatty acids (64%), due to their high percentage of oleic acid.The olive oil samples obtained from trees from cuttings were found to have a higher percentage of polyunsaturated fatty acids (20%) due to their high content in linoleic acid.

Oil Quality Parameters and Pigment Contents
The olives from both methods of propagation yielded extra virgin olive oils, but the profile of the analytical parameters (e.g., free fatty acid content, peroxide value and extinction coefficients at 232 and 270 nm) showed some slight differences (Table 2).The oils from suckers had higher contents of chlorophyll and carotenoids.

Changes in Oxidative Stability
The olive oil samples obtained from the fruits of the trees from suckers were found to have higher contents in total phenols (890 mg/kg) (Table 2).Therefore, significant differences between the two propagation methods were observed with regard to the total phenol contents, with more stable oil being obtained from the trees from suckers (50.2 h).

DISCUSSION
The fatty acid composition of Chemlali from trees from suckers and cuttings was characterized by relatively high levels of oleic and linoleic acids.These results are similar to those reported by several authors for other olive oil varieties cultivated in Tunisia (Ben Temime et al., 2006;Krichene et al., 2007).The differences in the method of olive tree propagation led to differences in the fatty acid composition of virgin olive oil.Morello et al. (2004) reported that several agronomic parameters could modify the fatty acid composition of olive oil.The most studied aspects include cultivar and origin, fruit ripening, harvest period, climatic conditions and soil characteristics.However, our results show that the origin of the tree had little influence on the analytical parameters (free fatty acid content, peroxide value and extinction coefficients at 232 and 270 nm) (Table 2), which are affected by factors causing damage to the fruits (e.g.olive fly attacks or improper methods of harvesting, transport and storage of olives) (Kiritsakis et al., 1998).In addition, olive oil samples obtained from the fruits of trees from both propagation methods were found to have varied chlorophyll and carotenoids contents.The total pigment content in olive oil is an important parameter for evaluating olive oil quality.Furthermore, pigments are involved in auto-oxidation and photo-oxidation mechanisms (Gutiérrez, 1989).
Stability to oxidation is an important property of olive oil, which is improved by synergistic interactions between the various antioxidants present in the oil itself, and also depends on the lipid composition.Oxidative stability of the Chemlali olive oils varied according to the method adopted for olive tree propagation (Table 2).Moreover, the two studied olive oils presented a good correlation between total phenols (r 5 0.80), and oxidative stability measured by Rancimat.A good direct correlation between oxidative stability and total phenolic content has been previously reported by other authors (Gutiérrez et al., 2001).Significant differences among the two propagation methods were observed with regard to the total phenol contents.Olive oil is the only vegetable oil which contains appreciable amounts of phenolic compounds (which were represented basically by o-diphenols) acting as antioxidant substances and conferring to it a greater stability against oxidation during storage (Bendini et al., 2007).
(E)-2-hexenal was the principal volatile identified in the oils from the two propagation methods.(E)-2hexenal was also the main volatile reported among the constituents of olive oil aroma (Ben Temime et al., 2006;Vichi et al., 2003).From the levels of the esters in the olive oils samples it can be hypothesized that levels of alcohol acetyl transferase (AAT) are not dependent on the origin of the olive tree.However, some differences in the levels of some terpenes hydrocarbons were observed in our olive oils samples.The hydrocarbons of olive oil have been studied by different authors as possible markers to distinguish virgin olive oil from different olive varieties or geographical origins (Vichi et al., 2003;Aparicio and Luna, 2002;Bortolomeazzi et al., 2001;Guinda et al., 1996).

CONCLUSION
In conclusion, the olive oils samples from both methods of olive tree propagation were within the limits established in the European Regulation, allowing them to be classified as extra virgin olive oils.However, our results showed that olive oil quality was different when the olive tree is from suckers or from cuttings.The oils obtained from the fruits of trees from suckers and compared to oils from trees from cuttings showed high levels of antioxidants along with an increased oxidative stability and high level of oleic acid.

Table 1 Fatty acid composition of virgin olive oil samples from the two ways of olive tree propagation Olives from cuttings Olives from suckers
a, b Mean 6 S.D. (n 5 6).Significant differences within the same row are shown by different letters (P , 0.01).CHEMICAL COMPOSITIOn OF VIRGIn OLIVE OILS FROM THE CHEMLALI CULTIVAR wITH REGARD TO THE METHOD…

Table 2 Quality parameters of Chemlali olive oil Samples from the two ways of olive tree propagation Olives from cuttings Olives from suckers
Mean 6 S.D.(n 5 6).Significant differences within the same row are shown by different letters (P , 0.001).PV, peroxide value; K 232 and K 270 , values of specific extinction given as absorbance at 232 and 270 nm, respectively.

Table 3 Composition of the volatile fraction obtained from Chemlali virgin olive oils extracted by HS-SPME LRI Olives from cuttings Olives from suckers
: linear retention indices; Data values expressed in mg/kg.a, b Mean 6 S.D.(n 5 3).Significant differences within the same row are shown by different letters (P , 0.005).-compound not detected.M. GUERFEL, M.B.MAnSOUR, Y. OUnI, F. GUIDO, D. BOUJnAH AnD M. ZARROUK LRI