CHEMOMETRIC CHARACTERIZATION OF FIVE TUNISIAN VARIETALS OF OLEA EUROPAEA L. OLIVE FRUIT ACCORDING TO DIFFERENT MATURATION INDICES ABSTRACT

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1 CHEMOMETRIC CHARACTERIZATION OF FIVE TUNISIAN VARIETALS OF OLEA EUROPAEA L. OLIVE FRUIT ACCORDING TO DIFFERENT MATURATION INDICES MANEL ISSAOUI 1, BELIGH MECHRI 1,2, ADEL ECHBILI 1, SAMIA DABBOU 1, ABDELMAJID YANGUI 3, HASSEN BELGUITH 3, AHMED TRIGUI 3 and MOHAMED HAMMAMI 1,4 1 Laboratory of Biochemistry UR: Human Nutrition and Metabolic Disorder Faculty of Medicine Monastir 5019, Tunisia 2 Institute of Olivier, Sousse, Tunisia 3 Institute of Olivier, Sfax, Tunisia Submitted for Publication December 12, 2007 Revised Received and Accepted February 21, 2008 ABSTRACT The usefulness of some chemical parameters as a tool to discriminate five varietals of olives was evaluated. Fruits were collected from the same orchard in order to eliminate geographical and climatic effects. Biochemical and quality characteristics in the fruits at complete ripeness were also investigated. Results showed that pomological characteristics and fatty acid composition may differ, depending on the variety and ripeness state, which is confirmed by an analysis of variance. A linear trend between mannitol and oil content was observed (r = 0.665; P < 0.001). Jeddaria Chaal olive oil had both the highest triolein (39.33%) and oleic acid value (74.43%). However, Chemlali Chouamekh olive oil showed the highest pigment (59.29 and mg/kg of chlorophylls and carotenes, respectively) and a-tocopherol content ( mg/kg). Thus, the cultivars were clearly differentiated in their metabolic behavior and the biochemical characteristics of their oils. Ripening state is used mainly as a guideline to establish the olive harvest time. The present data provide information about the maturation patterns of different cultivars and how it can influence the quality of the final product. 4 Corresponding author. TEL: ; FAX: ; mohamed. hammami@fmm.rnu.tn Journal of Food Lipids 15 (2008) All Rights Reserved. 2008, The Author(s) Journal compilation 2008, Wiley Periodicals, Inc. 277

2 278 M. ISSAOUI ET AL. PRACTICAL APPLICATIONS This research is strongly oriented to the agronomic application. The obtained results can orient an agronomist to the optimum ripening date of olive fruits with a good amount of oil and a balanced fatty acid composition. INTRODUCTION The evergreen olive cultivar (Olea europea L., Oleaceae) is an important Mediterranean tree (Kiple and Ornelas 2000; Grati-Kamoun et al. 2006). Tunisian oleiculture constitutes one of the principal economical and agricultural strategic sectors that are known for their richness of varieties (Abaza et al. 2001). Now, the olive plantation, occupying about 1.6 million ha, is dominated by two main cultivars. The Chétoui is omnipresent in the north, while the Chemlali is ubiquitous to the rest of the country (Ben Temime et al. 2006). The olive-growing areas spread from the northern to the southern regions, where a wide range of edaphic-climatic conditions are prevailing. These two cultivars cover 50% of different olive tree plantations; several minor varieties are maintained in restricted areas in different plantations (Khlif et al. 2002; Grati-Kamoun et al. 2006; Baccouri et al. 2007; Hannachi et al. 2007). Maturity is one of the most important factors associated with the quality evaluation of fruits and vegetables (Bianchi 2003; Beltrán et al. 2004a,b; Matos et al. 2007). The variation of the color of olives is marked by a series of transformation that occurs during the ripening process (Criado et al. 2005; Matos et al. 2007). As ripening progresses, photosynthetic activity decreases (Criado et al. 2005), whereas the amount of oil increases (Nergiz and Engez 2000). Sugars are the main soluble components in olive tissues (Donaire et al. 1977) and play important roles, providing energy and acting as precursors for olive oil biosynthesis (Jimenez et al. 1995; Nergiz and Engez 2000). The important precursor for fatty acid (FA) biosynthesis is acetyl Co-A. Acetyl Co-A is the product of the degradation of six carbon sugars via glycolysis in the plastid (Sanchez and Harwood 2002). In addition, harvest timing can have a significant effect on oil quality. Garcia et al. (1996) suggests that in order to obtain a characteristically fragrant and delicately flavored olive oil, it is therefore imperative that it is properly extracted from the best degree of ripeness. This illustrates the need to determine the quality of olive oil from a range of harvest times and cultivars to establish an optimum harvest time. In fact, there are different cultivars of O. europaea, each one with specific physical and biochemical characteristics, providing fruit and oil with typical composition and performance. The aim of the present study was to focus on the changes of pomological characteristics, oil content, FA composition and sugars occurring during rip-

3 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 279 ening in some Tunisian olive varieties studied for the first time in an attempt to establish an optimum harvest period. Biochemical and quality characteristics in the fruits at complete ripeness were also investigated. The studied cultivars were from the same orchard and, consequently, were planted under the same pedoclimatic conditions and agricultural practices. The results were subjected to statistical analysis in order to evaluate the influence of cultivar and ripening stage on those parameters and to check if principal components analysis (PCA) and cluster hierarchical analysis of the evaluated parameters could be used as a tool for cultivar discrimination. MATERIALS AND METHODS Plant Material This study included the olive fruit (O. europaea L.) of five Tunisian cultivars: Zarrazi Zarzis (L1 L5), Jeddaria Chaal (L6 L10), Chemchali Chouamekh (L11 L15), Chemlali Zarzis (L16 L20) and Chemchali Gafsa (L21 L25). The cultivars were planted under the same pedoclimatic conditions located in the olive-growing area of the National Collection Boughrara in Sfax, southwest of Tunisia (25 km in the northeast of Sfax). The orchard was founded in 1993 and grouped about 159 different cultivars with 43% introduced cultivars and 57% autochtonous cultivars. Trees were not irrigated (grown under natural rainfall). This olive grove was kept under a biological agricultural system. The climate of this region is typical Mediterranean semiarid to arid (with an average annual temperature of 31.02C and an average rainfall of 190 mm/year). The trees were identified and carefully marked, and three trees of each cultivar were sampled. The olive fruits were handpicked. In this way, we can be sure that the only factors affecting the differences among samples are the cultivar and the ripening stage the only influences that are considered in this study. The collected samples from each variety were transported to the laboratory on the same day and stored at 20C until analyzed. Olive Fruit Analysis Maturity Indices. The maturity index was determined as described by Boskou (1996) on 100 randomly selected olives from each sample. Olives were cut in half to expose the internal flesh and were sorted into categories. Oil Content and Pomological Study. Oil content was determined by extracting dry material at 40 60C with petroleum ether using a Soxhlet apparatus for 4 h (Bettach et al. 1996; Wajda-Dubos et al. 1996). The extract was

4 280 M. ISSAOUI ET AL. dried at 70C and weighed. Mean fresh weights of 100 fruits were measured. Some fruits were destoned and the flesh (pulp) was separated. Fruit and flesh were crushed, and 60 g of olive paste were dried in a forced-air oven at 105C for 24 h. The dried paste was weighed and the moisture content recorded (Beltrán et al. 2004a). FA Composition. Extraction of the lipophilic fraction was carried out with the procedure described by Allen and Good (1971) and Hannachi et al. (2007). The FA methyl esters (FAMEs) were prepared by dissolving 0.1 g of oil in 2 mlof heptanes and a solution of KOH (0.2 N) in methanol in which FAMEs were obtained. Individual FAMEs were separated and quantified by gas chromatography (GC). The temperature was programmed to increase from 170 to 270C at a rate of 5C/min. Nitrogen was used as a carrier gas. Injection volume was 1 ml. The results were expressed as relative area percent of total FAMEs. Sugar Composition. The soluble carbohydrates were extracted according to the method described by Bartolozzi et al. (1997). Briefly, the soluble carbohydrates from composite olive samples were extracted twice with 80% ethanol at 70C. Extracts were dried and converted into trimethylsilyl ethers with a silylation mixture made up of pyridine, hexamethyldisilazane and trimethylchlorosilane. Identification of individual carbohydrates was achieved by the use of the relative retention times, i.e., in comparison with that of the standards. These were compared with those identified earlier by GC-mass spectrometry and expressed as relative area percent of total sugars (Mechri et al. 2008). Ripened Olives Analysis Olive Processing. Oil extraction was carried out by using an Abencor analyzer (MC2 Ingenierias y sistemas, Sevilla, Spain). Olives ( kg) were crushed with a hammer mill and were then slowly mixed for 30 min at 25C. The paste obtained was centrifuged at 2,000 g for over 3 min. The oil was separated by decanting. All samples were filtered with anhydrous Na 2 SO 4 and stored at 4C in darkness using amber glass bottles. Triacylglycerol (TAG) Composition. TAG molecular species of olive oil were separated by high-performance liquid chromatography (HPLC) equipped with a reverse phase C18 column (5 mm; mm, Waters Associates, Milford, MA). The eluent was monitored by refractive index detector. The mobile phase was acetone/acetonitrile (60:40, v/v) with a flow rate of 1.50 ml/min. All solvents were of HPLC grade. Samples (5 ml) were prepared by dissolving the oil in acetone (9:91, v/v). Peak assignment was

5 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 281 carried out by comparison with chromato grams reported in the literature and with the retention times of some pure standards. Total Phenol Content Determination. Total phenolic compounds were quantified colorimetrically (Gutfinger 1981). They were isolated by extraction of a solution of oil in hexane with water/methanol (60:40, v/v). The Folin- Ciocalteau reagent was added to a suitable aliquot of the combined extracts, and the absorption of the solution at 725 nm was measured. Pigment Content. Carotenoids and chlorophylls (mg/kg oil) were determined at 470 and 670 nm, respectively, in cyclohexane using the specific extinction values according to the method of Minguez-Mosquera et al. (1991). Tocopherol Content Determination. Tocopherol was measured by the method of Gimeno et al. (2000). The oil sample was diluted in hexane (1:10, v/v). Therefore, 200 ml was transferred to another test tube, where 600 ml of methanol and internal standard solution (300 mg/ml of a-tocopherol acetate in ethanol) was added. The samples were filtered through a 0.45-mm pore size filter, and the aliquot was directly injected into the chromatograph. Analyses were performed on an Agilent (1100) Series HPLC system chromatograph (Hewlett-Packard, Waldbronn, Germany). Merceological Parameters. The determination of free acidity, peroxide value and specific ultraviolet (UV) absorption characteristics was carried out following the analytical methods described in Regulation ECC/2568/91 of the European Union Commission (1991). Statistical Analysis Analysis of variance (ANOVA) was applied in order to evaluate the influence of cultivar and season on fruit parameters. The software SPSS (SPSS Inc., Chicago, IL) was utilized. Tukey s test (P < 0.05) was used to determine significant differences among means. Correlation analysis was performed by using Pearson s test. PCA was performed to detect structure in the relationships between variables, allowing its classification and the separation of each cultivar. PCA and cluster analysis were performed with XLSTAT 2006 version (Addinsoft, New York, NY). RESULTS AND DISCUSSION Maturity Indices Table 1 reports the list of samples and maturation indices (MI) for each variety. The ripening indices showed a different pattern for each olive cultivar

6 282 M. ISSAOUI ET AL. TABLE 1. SAMPLE CODE, RIPENING INDICES, OIL AMOUNT AND FRUIT WEIGHT OF SOME TUNISIAN OLIVE CULTIVARS ACCORDING TO HARVESTING STATE Time period Zarrazi Zarzis (L1 L5) Jeddaria Chaal (L6 L10) Chemlali Chouamekh (L11 L15) Chemlali Zarzis (L16 L20) Chemchali Gafsa (L21 L25) Ripening indices September 29, * e, d, d, d, d,2 October 13, d, c, d, c, c,2 October 27, c, c, c, b, b,2 November 9, b, b, b, a, a,2 November 23, a, a, a, a, a,2 December 7, December 20, January 5, January 20, February 2, P value <0.001 <0.001 <0.001 <0.001 <0.001 Oil amount September 29, d, e, c, c, d,1 October 13, c, d, c, b, d,1 October 27, b, c, b, b, c,1 November 9, a, b, b, a, b,1 November 23, a, a, a, a, a,1 P value <0.001 <0.001 <0.001 <0.001 <0.001 Fruit weight September 29, c, b, c, c, c,2 October 13, b, a, c, c, b,2 October 27, b, a, b, b, b,2 November 9, a, a, b, b, a,2 November 23, a, a, a, a, a,2 P value <0.001 <0.001 <0.001 <0.001 <0.001 Contents with letters within each maturation scale are statistically different at P 0.05; contents with numbers within each cultivar are statistically different at P * Mean and SD for a confidence coefficient of 95% (n-3).

7 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 283 (Cerretani et al. 2004). Studied varieties have a significant effect for maturation state (P < 0.001) and cultivar kind (P < 0.001). From the beginning of harvest date, we can detect that Zarrazi Zarzis is a cultivar with an early scale of maturation. At the third period of sampling, the MI of Zarrazi Zarzis markedly increases at 5.67, being the first to change color of the skin and throughout the fruit. Cultivar Jeddaria Chaal, being the latest to ripen, did not exceed the value 5 until the last season. Pomological Study Table 1 shows the mean values of olive fruit parameters of the cultivars for the ripening period studied. In fact, the fresh weight of fruits differed for each cultivar, and was thus genetically dictated (Beltrán et al. 2004a). There are significant differences in fresh fruit weight between cultivars (P < 0.001). Oil Content The oil accumulation behavior observed in the olive fruit of Tunisian cultivars studied is illustrated in Table 1. The amount of oil on a dry weight basis varied with cultivar (Beltrán et al. 2004a). In fact, at the initial stage of ripening, some varieties showed a good amount of oil, such as Chemchali Gafsa and Chemlali Zarzis, whereas Zarrazi Zarzis proved a lower amount of oil accumulation. In addition, the amount of oil increased in some varieties during the progression of the ripening state: Zarrazi Zarzis from to 29.06%; Chemlali Chouamekh from to 42.18%; Chemchali Gafsa from to 46.81%; and Chemlali Zarzis from to 46.03%. Statistical analysis shows that Zarrazi Zarzis had the lowest means of oil amount (mean value of ); however, the cultivar Chemchali Gafsa had the highest oil amount (mean value of ), followed by the cultivar Chemlali Zarzis (mean value of ). Sugar Content A linear trend between mannitol and oil content was observed (r = 0.665, P < for all cultivars) for the cultivar Chemlali Chouamekh (r = 0.958, P < 0.001) (Table 2). However, other cultivars showed a significant correlation coefficient (r = 0.77 for Chemchali Gafsa and r = for Jeddaria Chaal; P < 0.001). Thus, the mannitol in the olive, as well as other polyols in many other higher plants, might be of specific importance in the metabolic transformation and synthesis of the fruit storage material (Fernandez Diez 1975). In all cultivars, we showed that glucose, the principal free sugar, decreased gradually as maturation progresses, probably because it was metabolized via acetyl-coa to oil (Stumpf 1983; Salas et al. 2000). This behavior has already been found

8 284 M. ISSAOUI ET AL. TABLE 2. SUGAR COMPOSITION (%) OF TUNISIAN OLIVE CULTIVARS WITH DIFFERENT MATURATION INDICES HP Y Cultivars Glucose Fructose Mannitol Galactose Inositol Sorbitol Sucrose MI1 L a, d, d, b, a, c, b,3 MI2 L b, c, d, a, b, c, a,3 MI3 L c, b, c, d, c, d, c,4 MI4 L d, b, b, c, c, b, c,5 MI5 L e, a, a, d, c, a, c,5 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 MI1 L a, d, d, c, a, a, b,1 MI2 L c, b, c, d, b, b, b,2 MI3 L b, c, c, a, c, c, d,3 MI4 L c, b, b, b, b, c, c,2 MI5 L d, a, a, c, b, c, a,1 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 MI1 L a, d, d, d, b, a, d,2 MI2 L b, d, c, b, a, b, a,1 MI3 L b, c, b, c, a, c, b,1 MI4 L d, b, a, a, c, c, c,1 MI5 L c, a, a, e, c, c, d,2 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 MI1 L c, c, b, a, a, b, a,1 MI2 L b, a, c, b, a, b, a,1 MI3 L c, a, b, b, a, b, a,1 MI4 L a, b, c, a, c, c, b,3 MI5 L c, a, a, b, b, a, b,3 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 MI1 L ab, a, c, a, a, b, c,3 MI2 L a, b, b, c, a, a, a,2 MI3 L bc, a, a, d, c, d, b,2 MI4 L abc, a, a, c, c, d, d,4 MI5 L d, a, a, b, b, c, d,4 P value = = <0.001 <0.001 <0.001 <0.001 <0.001 Contents with letters within each maturation scale are statistically different at P 0.05; contents with numbers within each cultivar are statistically different at P HP, harvesting period.

9 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 285 for other cultivars (Nergiz and Engez 2000; Marsilio et al. 2001). Cultivar effect showed a greater influence on these sugars (37.33%) than harvest timing (27.12%). Fructose, the second sugar in olive pulp, was detected at variable concentration and depends mainly on the MI (24.35%) and the appropriate cultivars (15.55%). Generally, the percentage of fructose increased considerably during the ripening process. This result agreed with those of Nergiz and Engez (2000) and Marsilio et al. (2001). The percentage of galactose varied, especially with the appropriate cultivars (33.18%, P < 0.001). Sucrose was detected at lower percentage and diminished markedly when the fruit color surface changed from green to cherry. Harvesting time (33.17%) had greater influence on sucrose than the effect of cultivars (27.35%). FA Composition The effect of harvesting time on the FA composition of the five cultivars is shown in Table 3. The variability for oleic acid can be explained mainly by cultivar kind (38.14%) and ripening time (38.52%), and then by the interaction between cultivars and maturation state (21.50%). Five cultivars analyzed show higher oleic acid content that oscillated between and 74.43%. At the first period of harvesting, the oleic acid for the majority of cultivars was higher (67.30 and 70.84% for Chemlali Chouamekh and Zarrazi Zarzis, respectively) until the end of the period of harvest timing, and the percentage of the FA increased (74.13 and 72.54%, respectively). As the ripening process goes on, the oleic acid content rises throughout the maturation period. Olive fruit from Jeddaria Chaal and Chemlali Chouamekh showed a faster increase in oleic acid percentage as maturation progresses. Results showed that the C16:0 decreased with the progression of the maturation process (Beltrán et al. 2004a; Ayton et al. 2007; Mana et al. 2007). The decreases in the level of palmitic acid are variety dependent (55.92% of variability), and there was a significant cultivar kind (P < 0.001) and harvest timing (42.65% of variability; P < 0.001) effect in all varieties studied. Hence, Zarrazi Zarzis show a decline of about one-half at the last part of maturation (from to 7.74%); in contrast, a slight decrease was shown for the cultivars Chemlali Chouamekh and Chemlali Zarzis (from to 13.81% and from to 13.61%, respectively). Stearic acid has been observed in Tunisian cultivars at lower values ( %). Maturation state did not show significance as a variability source (17.33%); however, the main factor was cultivar kind, which explains 65.82% of its variation. During olive ripening, its content increased; this trend is similar to other studies on different olive varieties (Beltrán et al. 2004b; Matos et al. 2007). Like the cultivar Jeddaria Chaal, the cultivars Chemlali Zarzis and Chemlali Chouamekh showed lower levels of linoleic acid among the cultivars studied. For this FA, the most important source of variability was cultivar kind

10 286 M. ISSAOUI ET AL. TABLE 3. FATTY ACID COMPOSITION (%) OF TUNISIAN OLIVE CULTIVARS WITH DIFFERENT MATURATION INDICES HP Cultivars C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 MI1 L a, a, a1,2 * ab,1ns a, c, c, a, ab, ab,1 * c,1ns MI2 L b, a, b,1 * b,1ns a, c, b, b, a, b, c,2 MI3 L c, ab, b, a, c,1, b, a, c,1, b1, a, b,1,2ns MI4 L cd, a, b,1ns a, c,1, b, a, c, a, b, a,1 MI5 L d, b, ab,3 nd ac2ns b, a, a, a,1ns ab,1ns ab,2,3 nd d,1 value P <0.001 = = = 0.03 <0.001 <0.001 <0.001 <0.001 = = 0.08 <0.001 MI1 L a, a, c2,3 * b,1ns a, d,2, c,3, ab, b, b,2,3 * c,1,2ns MI2 L b, b, c,1,2 * b,1ns b, c, a, b, b, b, b,1ns MI3 L c, c, ab, a, c, b, a, b, b, b, d,2 MI4 L d, c, b,1 nd c, d,1, a, b, ab, b, b, a,3 MI5 L e, c, a1,2 nd c,2ns d, a, d, a,1ns a,1ns a,1 nd e,1 P value <0.001 <0.001 <0.001 = <0.001 <0.001 <0.001 = = <0.001 <0.001 MI1 L b1, ab, a,3 * a,1ns a, b3, b, a, ab, bc,3 * b,2ns MI2 L a, a, a,1 * a1ns b, b, c, ab, bc, d, b,1,2ns MI3 L c, b, a, a, c, b, a, ab, c,2, cd, a,1 MI4 L d, c, a,1ns nd b, bc, a, c, a, b, b, b,2 MI5 L d, c, a,3 nd b,2ns a, a,1, c, b,1ns a,1ns a,2 nd b1 P value <0.001 <0.001 = = <0.001 <0.001 <0.001 = = <0.001 = MI1 value L a, a, b,2,3 * a,1ns a, c, b, a, b2, a,1,2 * a,1,2ns MI2 L a, a, b,2 * a,1ns b, b, c, b, a, c, b,3ns MI3 L b, c, b, a, b, a, b, b, a, bc, ab,2 MI4 L b, b, b,1ns nd b, b, a, b, b, ab, b, b,3 MI5 L c, b, a,1 nd b,2ns b, a,2, a, b,1ns ab,1ns b,3 nd c,1 P <0.001 <0.001 <0.001 <0.001 = <0.001 <0.001 <0.001 = <0.001 <0.001 MI1 value L b1, b, a,1 * a,1ns a, ab, c, b, a, c,2,3 * c,1,2ns MI2 L a, a, b,2 * a,1ns a, b, c, b, a, c, c,2,3ns MI3 L c, b, b, a, b, a, b, b, a,1, a, b,1,2 MI4 L d, b, b,1ns a, c, a, b, a, a, b, a,1 MI5 L e, c, a,2, a,1ns d, a, a, b,1ns a,1ns a,2,3 nd d,1 P <0.001 = = = <0.001 = <0.001 <0.001 = <0.001 <0.001 Contents with letters within each maturation scale are statistically different at P 0.05; contents with numbers within each cultivar are statistically different at P 0.05). * Mean and SD for a confidence coefficient of 95% (n-3). L1 L5, Zarrazi Zarzis at maturation state 1 5; L6 L10, Jeddaria Chaal at maturation state 1 5; L11 L15, Chemlali Chouamekh at maturation state 1 5; L16 L20, Chemlali Zarzis at maturation state 1 5; L21 L25, Chemchali Gafsa at maturation state 1 5. MI1 MI5, maturation indices 1 5. ns, not significant; nd, not detected.

11 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 287 (74.07%), whereas harvesting time represented 11.60%. During the ripening process, the relative level of linoleic acid increases, as described in general, for several olive varieties (Gutierrez et al. 1999; Beltrán et al. 2004b; Matos et al. 2007). In olive oil, the highest degree of unsaturation is shown by linolenic acid, which achieves a mean value of 0.35% in the cultivar Chemchali Gafsa, followed by 0.49% in the cultivar Chemlali Chouamekh, less than that reported for the cultivars Chemlali Zarzis (0.72%) and Jeddaria Chaal (0.89%). The ripening state showed a greater influence on this FA (24.80%). During maturation, linolenic content dropped; similar results were observed in the finding of Beltrán et al. (2004b) and Matos et al. (2007). The evaluation of the influence of olive ripening degree suggested that the composition and the variation of FA depends more on the ability of different cultivars to metabolize substrates needed to synthesize the appropriate FA with the appropriate levels and times. The factors that influence saturated FA highlight the maturation state, which explains 52.02% of the variability found, whereas cultivar kind explains only 45.81%. The monounsaturated FA comprises the largest group in virgin olive oils. The variability observed for these FA may be explained by the ANOVA, where the main variation source is the cultivar kind (48.43%), above the effect of the harvesting time (30.88%). Similar results were observed for polyunsaturated FA; for these acids, the cultivar kind had a great importance of 74.92%. The ratios of oleic to linoleic acid were statistically very different among cultivars (75.22% of variability), and also varied along ripening (6.59%). In fact, the increase in oleic acid content is a result of the active biosynthesis of TAGs, which takes place through fruit ripening, involving a fall in the relative percentage of the oil s palmitic acid content (Gomez-Rico et al. 2005). At the same time, the increase in linoleic acid content is a result of the transformation of oleic acid into linoleic acid by the oleate desaturase activity (Sanchez and Harwood 2002). The oleic to linoleic acid ratio is frequently used as a stability parameter (Velasco and Dobarganes 2002; Beltrán et al. 2004b; Matos et al. 2007). A matrix containing all the five cultivars at the five sampling times, each with the above-mentioned parameters, was used in order to observe discrimination between studied varieties based on their metabolic behavior. As can be seen in the 68.38% of confidence in Fig. 1A, the discrimination of cultivars was accomplished, although some elements were distant from the formed groups. In the cluster analysis, we also observed the formation of groups based on the similarities between the samples. From this point of view, the formation of clusters and its linkage could be compared and related with groups formed in PCA I. For instance, the cluster composed by cultivar Zarrazi Zarzis according to different maturation states, L1, L2, L3, L4 and L5, could be plotted in Fig. 1B, with samples cultivars L3 and L4 having a percentage of similarity superior to 90%, being very similar in what concerns the chemical parameters

12 288 M. ISSAOUI ET AL. A 2 Biplot (PC1 and PC2 : %) 1,5 L10 -- PC2 (17.66 %) --> 1 0,5 0-0,5-1 L17 L19 L14 L18 MUFA/PUFA L8 O/L Galactose L6 C16:0 L16 SFA L12 L11 L15 L7 L9 L21 L22 L20 Oil Glucose L13 C18:1 M annitol L1 Fructose RI FW C18:2 PUFA L2 L23 L24 L25 L3L4 L5-1, ,5-1 -0,5 0 0,5 1 1,5 2 --PC1 (50.71%) --> B Dendrogram 0,700 0,600 0,500 Similarity 0,400 0,300 0,200 0,100 0 L7 L6 L11 L12 L16 L17 L9 L10 L8 L14 L15 L18 L19 L20 L1 L2 L3 L4 L5 L21 L22 L13 L23 L24 L25 FIG. 1. PRINCIPAL COMPONENTS ANALYSIS I (A) AND DENDOGRAM ANALYSIS (B) BASED ON SOME FATTY ACID, SUGARS, OIL AMOUNT, FRUIT WEIGHT AND RIPENING INDICES OF OLIVE FRUIT CULTIVARS ZARRAZI ZARZIS, JEDDARIA CHAAL, CHEMLALI CHOUAMEKH, CHEMLALI ZARZIS AND CHEMCHALI GAFSA WITH DIFFERENT MATURATION INDICES L1 L5, Zarrazi Zarzis; L6 L10, Jeddaria Chaal; L11 L15, Chemlali Chouamekh; L16 L20, Chemlali Zarzis; L21 L 25, Chemchali Gafsa.

13 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 289 evaluated and used in these analyses. Chemchali Gafsa is grouped in the first cluster with the cultivar Zarrazi Zarzis. Similarity between the metabolic behaviors of the two cultivars may be explained by the near origin location and pedoclimatic conditions (Gafsa and Zarzis located in southern Tunisia). At the same time, results given with samples in different maturation states allow further conclusions. As can be observed, the cultivar Zarrazi Zarzis appears as a very homogeneous group where ripening time has generally no influence. Zarrazi Zarzis has been studied for many years, under different maturation states, and different origin locations; results elaborated by those studies proved that there is no effect on their metabolic behavior. However, the majority of the studied samples, especially cultivars Chemlali Chouamekh and Chemlali Zarzis, present an evident dispersion of values considered to be very heterogeneous cultivars. The maximum amount of oil on the tree and the best FA profiles were found since late in November to December. Therefore, results suggest that this period led the balance between the highest quantity and the best quality. Chemical Composition and Analytical Parameters in Ripened Olives Concerning four (free acidity, peroxide value, K232 and K270) of the numerous parameters considered by regulation EC 2568/91 for the merceological classification of olive oils, the values reported in Table 4 showed that all the monovarietal oil samples analyzed were labeled as extra-virgin merceological class. TAG Composition of Oil. As reported by several authors (Cerretani et al. 2006), the triglyceride profile helps classify and characterize monovarietal oils. Results for TAG content, expressed in percentage of total TAGs of oil samples, are shown in Table 4. The main TAGs were 1,2,3-trioleylglycerol (OOO), 2,3-dioleyl-1 palmitoylglycerol (POO) and 2,3-dioleyl-1-linoleylglycerol (LOO). Other minor TAGs were 2,3-dioleyl-1-stearoylglycerol (SOO), 2-oleyl-3-palmitoyl-1-stearoylglycerol (SOP), 1,2-dilinoleyl-3-palmitoylglycerol (LLP), 1-linolenoyl-2-linoleyl-3-oleylglycerol (LnLO) and 1,2,3- trilinoleylglycerol (LLL). The level of triolein (OOO), the main TAG in all olive oil varieties, was remarkably high for Jeddaria Chaal (39.33%), followed by Chemlali Chouamekh (37.86%) and Zarrazi Zarzis (36.37%). The second peak in order of quantitative importance in the studied virgin olive oils was POO. Both Jeddaria Chaal and Chemlali Chouamekh had the highest level (27.27 and 27.97%, respectively). The presence of a high triolein (OOO) level in inverse proportion to trilinolein (LLL), constitutes a favorable authenticity indicator (Baccouri et al. 2007). Oil from Chemlali Chouamekh had the lowest level of LLL (0.25%) in comparison with other samples.

14 290 M. ISSAOUI ET AL. TABLE 4. ANALYTICAL PARAMETERS, TRIACYLGLYCEROL COMPOSITION, OXIDATIVE SUSCEPTIBILITY AND ANTIOXIDANT COMPOUNDS OF SOME TUNISIAN VIRGIN OLIVE OIL Cultivars Zarrazi Zarzis Jeddaria Chaal Chemlali Chouamekh Chemlali Zarzis Chemchali Gafsa Quality parameters Fatty acid (%) b * b b b a PV (mequiv O2/kg) a a a a a K b b a a b K a a a a a IV a b c b c OS a d e c b Triacylglycerol composition (%) LLL a a b a a OLLn b d d a c LLO a d d c b OOLn c d d a b PLnO b a a c c PPL c b b a a PLL c b b a b OOL a d d b c POL c d c a b POP c b b b a OOO ab a a c c POO + SLO d a a c b SOO a b b c b SPO b a a c a AOO a c b d a Antioxidant compounds (mg/kg) Chlorophylls d b a c c Carotenes d b a c c Total phenols a b c c a,b O-diphenols a b c c b a-tocopherol e c a b d Contents with letters within each cultivar are statistically different at P PV, peroxide value; P, palmitic; S, stearic; O, oleic; L, linoleic.

15 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 291 Total Phenolic and O-diphenolic Content. The phenolic compounds present in the virgin olive oils are one of the basis of the nutritional importance of this oil. In studied oils, the phenol content varied between and mg/kg. Zarrazi Zarzis olive oil, which had the highest level of phenol ( mg/kg), also showed the highest O-diphenolic content ( mg/kg). In contrast, Chemlali Chouamekh olive oil had both the lowest phenol ( mg/kg) and O-diphenol content (42.84 mg/kg). Furthermore, significant negative correlations were found to exist between phenol content and merceological parameters: (r = between phenol and peroxide value, r = between phenol and K232, and r = between phenol and K270; P < 0.05). Our results are similar to those of Lavelli (2002). a-tocopherol Content. Vitamin E is a general term used to refer to a group of minor but important lipid-soluble compounds that are believed to be involved in a diversity of physiological and biochemical functions. However, other tocopherol isomers (b-, g- and d-tocopherols) are presented, but at lower amounts, in olive oils (Matos et al. 2007). The Tunisian olive oil samples, which are the subject matter of the present work, have various a-tocopherol amounts ranging from to mg/kg (Table 4). In the studied olive oil samples Chemlali Choumekh had the highest level ( mg/kg). In contrast, Zarrazi Zarzis olive oil had the lowest level (97.68 mg/kg). These results are in agreement with previously studies, suggesting that tocopherol content was highly variety dependent (Baldioli et al. 1996). Tukey s test showed significant (P < 0.05) differences in tocopherol level between the studied cultivars. Pigment Content. Chlorophylls and carotenoids are the main pigments in virgin olive oils (Gandul-Rojas and Minguez-Mosquera 1996). These pigments, responsible for the characteristic color of the oil, are well known to act as photosensitizers during light exposure (Psomiadou and Tsimidou 2002; Skevin et al. 2003). In the studied oils, chlorophyllic pigments can be found at concentrations between and mg/kg (Table 4). Chemlali Chouamekh olive oil had the highest amount, while Zarrazi Zarzis showed the lowest value. Carotenoid pigment contents varied between 7.71 and mg/kg according to the appropriate cultivar (Table 4); high amounts were observed in the oils of Chemlali Chouamekh and Jeddaria Chaal, while low ones were observed in Zarrazi Zarzis. ANOVA test show significant differences between cultivars (P < 0.001). In order to study discrimination between cultivars for their quality of oils from ripened fruits, another PCA was carried. After an explorative analysis of

16 292 M. ISSAOUI ET AL. Biplot (PC1 and PC2 : %) 5 4 Chemlali Zarzis 3 -- PC2 (33.92 %) --> POL Chemchali Gafsa OOLn OLLn PLL PPL LLO LLL OOL k270 Fee fatty acidity POP k232 OS IV Total phenols Tocopherols PV I EeOO Carotenes SPO diphenols POO+SLO SOO Chlorophylls PLnO OOO Zarrazi Zarzis -2-3 Chemlali Chouamekh Jeddaria Chaal PC1 (46.14 %) --> FIG. 2. PRINCIPAL COMPONENTS ANALYSIS II BASED ON TRIACYLGLYCEROLS, a-tocopherol, PHENOLS, O-DIPHENOLS, PIGMENTS AND ANALYTICAL PARAMETERS OF STUDIED VIRGIN OLIVE OIL SAMPLES FROM CULTIVARS OF RIPENED OLIVES data, with regard to the higher explained variance, the a-tocopherol content, minor components, analytical parameters and TAG were selected to carry out a second PCA (80.06% of confidence) (Fig. 2), which showed that the selected variables may be able to distinguish oils obtained from different cultivars. The application of the PCA II algorithm to the data showed three distinctive groups (Fig. 2). The first group is composed of Chemlali Chouamekh and Jeddaria Chaal olive oil. The second group is composed of the cultivars Zarrazi Zarzis and Chemchali Gafsa. Moreover, we observed the presence of a third group composed of a single sample (Chemlali Zarzis) located on the center of the plot. Figure 2 gives some explanations for understanding this classification. Indeed, the first group is correlated with high levels of carotenes, chlorophylls, tocopherols and triolein. The second group had the highest levels of phenols, O-diphenols, LLL and oxidative susceptibility. This PCA of olive oils showed the oil composition similarity of Zarrazi Zarzis and Chemchali Gafsa.

17 CHEMOMETRIC CHARACTERIZATION OF OLIVE VARIETIES 293 CONCLUSIONS It was possible to conclude that the results obtained for such parameters allow the differentiation of the cultivars. Hence, it had been demonstrated by the ANOVA that cultivar has more influence than maturation state on the studied olive varieties. Moreover, it has been found that there are significant differences in metabolic behavior according to ripening indices in the olive fruits studied. Therefore, the variables chosen to differentiate the cultivars also permitted, in some cases, to appreciate the influence of the MI. These findings are also valuable in determining the acceptable times of harvest to ensure that FAs are within the limits for extra-virgin olive oil. The appropriate harvest timing based on the scientific criteria is a key factor in the determination of the balance between oil quality and quantity. For each studied sample, it can be also said that the olive oils that were the subject of this study show good nutritional characteristics. ACKNOWLEDGMENTS This study was supported by a grant from the Ministère de l Enseignement Supérieur, de la Recherche Scientifique et de la Technologie UR/03-ES08 (Nutrition Humaine et Désordres Métaboliques) and l Institut de l olivier de Sfax. We are grateful to Mr I. Cheraief for his expert technical support. We also thank the reviewers for their constructive criticisms of the manuscript. REFERENCES ABAZA, L., DAOUD, D., MSALLEM, M. and ZARROUK, M Evaluation biochimique des huiles de sept variétés d Olivier cultivées en Tunisie. Oléa. Corps Gr. Lip. 9, ALLEN, C. and GOOD, P Acyl lipids in photosynthetic systems. In Methods in Enzymology, Vol. 23 (S.P. Clowic and N.O. Kaplan, eds.) pp , Academic Press, New York, NY. AYTON, J., MAILER, R.J., HAIGH, A., TRONSON, D. and CONLAN, D Quality and oxidative stability of Australian olive oil according to harvest date and irrigation. J. Food Lipids 14, BACCOURI, B., BEN TEMIME, S., TAAMALI, W., DAOUD, D., MSALLEM, M. and ZARROUK, M Analytical characteristics of virgin olive oils from two new varieties obtained by controlled crossing on Meski variety. J. Food Lipids 14,

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