J Am Oil Chem Soc (2016) 93:1499–1508 DOI 10.1007/s11746-016-2904-8

ORIGINAL PAPER

Effects of Variety, Maturation and Growing Region on Chemical Properties, Fatty Acid and Sterol Compositions of Virgin Oils

Dilsat Bozdogan Konuskan1 · Burcu Mungan1

Received: 11 July 2016 / Revised: 21 July 2016 / Accepted: 15 September 2016 / Published online: 26 September 2016 © AOCS 2016

Abstract Chemical properties, fatty acid and sterol com- Keywords Olive cultivar · Maturity · Growing region · positions of olive oils extracted from Gemlik and Halhalı Fatty acid · Sterol varieties grown in Hatay and Mardin provinces in Turkey were investigated during four maturation stages. The samples were analyzed for their chemical properties Introduction such as free acidity, peroxide value, total carotenoid, total chlorophyll, total phenolic contents, antioxidant activity, Virgin olive oil (VOO) has nutritional, sensorial and func- fatty acid and sterol compositions. Chemical properties, tional characteristics that make it unique among other veg- fatty acids and sterol profiles of olive oil samples gener- etable oils and a basic component of the Mediterranean ally showed statistically significant differences depending diet [1, 2]. VOO is a juice obtained by solely mechanical on the varieties, maturation and growing areas (p < 0.05). or other physical means from the fruit of the olive tree, As free fatty acid contents and total phenolic contents without the use of chemicals [3]. Thus generally lipophilic increased, total carotenoid and chlorophyll contents components of the drupe are transferred to the oil, which decreased throughout the maturity stages. Total carote- in turn retains the organoleptic properties of [4]. The noid and chlorophyll contents of oil samples from Mardin composition of VOO is mainly constituted of triacylglycer- were higher than those of Hatay. The total phenolic com- ols and minor compounds (0.5–2 %) which is called unsa- pounds of olive oil samples ranged from 20.62 in Gemlik to ponifiable or nonglycerol fraction 5[ ]. Olive oil has high 525.22 mg GAE/kg oil in Halhalı from Hatay. In general, content of monounsaturated fatty acids (%56–84) mainly the phenolic contents and antioxidant activities of olive oil oleic acid [6, 7]. The high percentage of oleic acid is an samples were positively associated. Oleic acid content was important component of the nutritional profile of olive oils, the highest 71.53 % in H1 samples in Hatay. Total sterol particularly in relation to its effect on cardiovascular sys- contents were 1194.33 mg/kg in Halhalı and 2008.66 mg/ tem health [8]. Olive oil naturally contains main minor kg in Gemlik from Hatay. Stigmasterol contents of oils compounds such as hydrocarbons, sterols, aliphatic alco- obtained from Hatay were lower than those of Mardin. hols, polyphenols, tocopherols, pigments and flavor com- Oleic acid, palmitic acid, β-sitosterol, ∆-5-avenasterol and ponents [9, 10]. Phytosterols are main constituents of the campesterol contents fluctuated with maturation for each of unsaponifiable fraction of olive oil 11[ ]. Sterol composition variety from both growing regions. These results showed is related to the quality of the oil and are broadly used for that the variety, growing area and maturation influence the checking its genuineness and detecting adulteration, since chemical properties, fatty acid and sterol compositions. sterols can be considered as its real fingerprint 9[ , 11, 12]. Clinical studies have demonstrated that the dietary intake of phytosterols may reduce blood cholesterol levels inhib- * Dilsat Bozdogan Konuskan iting its absorption from the small intestine. Also it has [email protected] anti-inflammatory, antibacterial, antifungal, antioxidant 1 Food Engineering Department, Faculty of Agriculture, and antitumoral activities [13–15]. The main olive oil ster- Mustafa Kemal University, Hatay, Antakya, Turkey ols are β-sitosterol (%75–90), Δ-5-avenasterol (%5–20)

1 3 1500 J Am Oil Chem Soc (2016) 93:1499–1508 and campesterol (%1–4). Total sterol content of olive oil (Germany) and Sigma-Aldrich (Germany). The fatty acid is affected by the cultivar, stage of maturity of the olives, methyl ester (FAME) mix was obtained from Supelco extraction method, environmental conditions and storage (Bellefonte, USA). time prior to oil extraction [16, 17]. Phenolic compounds which are important minor compounds and main antioxi- Olive Sampling dants in VOO are used in the characterization and authen- tication with respect to geographical origin and cultivars This study was performed during the growing seasons [15, 18, 19]. They have potent antioxidant activity and between 2014 and 2015. Olive samples of two common contribute significantly to the remarkable stability of VOO Turkish olive varieties; Gemlik and Halhalı, were picked by against oxidation [10, 20]. The benefits of phenolic on hand in their growing area in Hatay (Mediterranean region human health are correlated with their antioxidant activity of Turkey) and Mardin (south eastern Anatolia region of [7, 21]. They are also responsible for the nutritional and Turkey) provinces. The olive samples were collected from organoleptic properties such as bitter and pungent taste of three trees (as replicates) of the same orchard for each VOO [4, 22]. Olive oil color is correlated with its pigment individual cultivar and at four different harvesting times, composition, which is considered quality criterion. Chloro- 20 day intervals, beginning from the 5th of September to phyll and carotenoid pigments are mainly responsible for the 5th of November and coded accordingly from 1 to 4 as the color of VOO, ranging from yellow–green to greenish H1-H4 for Halhalı and G1–G4 for Gemlik cultivars. Only gold [23, 24]. Chlorophyll pigments act as photosensitiz- healthy olive drupes, without any infection or physical ers, promoting oxidation, while antioxidant activity was damage were selected. reported in the dark [25, 26]. Carotenoids, as singlet oxy- gen quenchers, protect oils from photo-oxidation [23, 27]. Therefore, these minor compounds show great importance since their antioxidant activity, nutritional and organoleptic Oil extraction was carried out within 24 h from the har- properties effects on VOO. Antioxidant content of the oil is vested olives. A representative 3 kg olive sample was not constant; it depends on the cultivar, maturation stage, extracted to obtain the oil by a laboratory scale mechanical agroclimatic conditions and olive growing techniques [23, mill (Hakkı Usta, Turkey) with a crusher, a vertical malaxer 28]. Turkey is one of the most important Mediterranean and a two-phase centrifuge. Malaxation and centrifuge pro- countries, such as Spain, Italy, Greece because of olive and cesses was performed at 25 °C for 30 min and at 5000 rpm, olive oil production [29]. Turkey has adequate climate and respectively. The oil was separated by decanting and put soil conditions for olive production [30]. Despite the great into dark glass bottles. Oil samples were kept at 4 °C until production and economic importance of olive oil, relatively chemical analysis which were duplicated. little information is available in the literature about the characteristics particularly sterol profiles of Turkish olive Chemical Properties oil. The aim of this work was to determine the effect of the cultivar, maturation and growing area on the chemical Free acidity (% oleic acid) and peroxide value (mequiv O2/ compositions (free fatty acids, peroxide value, total chloro- kg) were performed according to the AOCS official meth- phyll, total carotenoid, total phenolic contents, antioxidant ods Ca 5a-40 and Cd 8–53, respectively [31].Chlorophyll activity, fatty acids) and sterol profiles of olive oils. and carotenoid compounds were determined at 670 and 470 nm, respectively, in cyclohexane using the specific extinction values, by the method of Minguez-Mosquera Materials and Methods et al. [32]. The chlorophyll and carotenoid contents are expressed as mg of pheophytin “a” and lutein per kg of oil, Reagents and Standards respectively.

All reagents used in the experiments were of analytical Extraction of Phenolics grade. Folin-Ciocalteu’s reagent, gallic acid, 2,2′-diphenyl- 1picryhydrazyl (DPPH), tri-methyl chlorosilane, hexa- The phenolic extraction procedure was performed accord- methyl chlorosilane, pyridine, 2,7 dichlorofluorescein, 5 ing to the method described by Pirisi et al. [33]. Firstly, a α-cholestan-3beta ol, sodium carbonate, beta sitosterol, 2-ml oil sample was weighed into a centrifuge tube and campesterol, stigmasterol, cholesterol, methanol, n-hexane, 1 ml hexane and 2 ml methanol:water (60:40, v/v) were cyclohexane, ethyl ether, ethanol, diethyl ether, acetone, added. This mixture was stirred for 2 min in a vortex and toluene, formic acid, chloroform, acetic acid, sodium sul- the tube was then centrifuged at 3000 rpm for 5 min. The fate and potassium iodine were purchased from Merck methanol:water phase was separated and the extraction was

1 3 J Am Oil Chem Soc (2016) 93:1499–1508 1501 repeated twice. Finally, phenolic extracts were recovered percentage of the total. The injection volume was 1 μL. with a syringe and then filtered through a 0.45 µm mem- Fatty acids were identified by comparing their retention brane filter (Millipore, USA) before analysis. times with those of reference compounds.

Total Phenolic Content Sterol and Triterpene Dialcohols

THe total phenolic content was colorimetrically determined Sterol composition were carried out according to Interna- according to the Folin-Ciocalteu procedure as reported by tional Olive Council method COI/T.20/Doc.No30/Rev. 1 Montedoro et al. [34], with some modifications. Firstly, [37]. Identification and quantification of sterols and diols 0.2 ml phenolic extract was mixed with 0.5 ml Folin-Cio- as trimethylsilyl ethers was performed by gas chromatogra- calteu reagent. Then, 1 ml saturated sodium carbonate solu- phy (GC 2010, Shimadzu, Japan) equipped with a Supelco tion was added and the mixture shaken for 0.5 min. Finally, (SPBTM-5 24034, Bellefonte, USA) capillary column the solution was brought up to a volume of 10 ml with dis- (30 m 0.25 mm i.d 0.25 mm film thickness) and flame × × tilled water. After 45 min of reaction at ambient tempera- ionization detector (FID). Injector, column and detec- ture in the dark, the absorbance was measured at 765 nm tor temperatures were 280, 260 and 290 °C, respectively. in a UV–Vis spectrophotometer (Hitachi U1900, Japan). Helium was used as the carrier gas with a flow rate of 1 ml/ The quantitative results were calculated using an analytical min and the split ratio of 50:1. Individual sterols and two curve of gallic acid equivalents per kg of VOO (mg GAE/ triterpendiols (erythrodiol and uvaol) in oils were identified kg). based on their relative retention times with respect to the internal standard, cholestanol, according to the standard- Antioxidant Activity ized reference method.

The antioxidant activities (free radical scavenging capacity) Statistical Analysis of phenolic extracts, was measured using 2-2′-diphenyl- 1-picrylhydrazyl radical (DPPH) according to the proce- Statistical analysis was performed using SPSS 15 statistical dure of Brand-Williams [35] with some modifications. software (SPSS Inc., Chicago, USA).Data were analyzed Briefly, 1 ml of extracts were diluted with 1.9 ml DPPH according to Analysis of Variance (one way ANOVA). Sig- methanolic solution. Samples were incubated at room tem- nificant differences between samples were determined by perature in the dark for 60 min and then the absorbance Duncan’s multiple range test at the 5 % confidence level. was measured at 515 nm against a blank (MeOH). The Interaction analysis was performed by using the Student t percentage of inhibition was calculated from the following test [38]. equation: Ac − As DPPH inhibition (%): × 100. Results and Discussion Ac Ac and As refer to the absorbances at 515 nm of DPPH Chemical Properties in the control and sample solutions, respectively. All meas- urements were carried out in triplicate. The chemical properties of olive oil such as free fatty acid- ity, peroxide value, total carotenoid, total chlorophyll, total Fatty Acid Composition phenol contents and antioxidant activities are shown in Table 1. As can be seen in Table 1, free fatty acids of olive Determination of the fatty acid composition was made oils ranged between %0.34 (H2 from Mardin)– %2.49 (G4 according to the International Olive Oil Council, COI/T.20/ from Hatay). Free fatty acids of oils were within the limits Doc.No.24 [36]. Methyl-esters were prepared by vigorous ( 2) established by European regulations for virgin olive ≤ shaking of a solution of oil in n-heptane (0.1 g in 2 mL) oil except for G1 from Hatay. The free fatty acid contents with 0.2 ml of 2 N methanolic potassium hydroxide and of oils from Mardin was lower than those of from Hatay anajyzed by Agilent gas chromatography system (Agi- and it was observed that this content increased during rip- lent 6850, USA) equipped with a hydrogen flame ioniza- ening slightly. The free acidity of oils showed statistically tion detector (FID) and a capillary column DB-23 of 60 m significant differences depending on the varieties, ripen- length 0.25 mm i.d. and 0.25 μm of film thickness. The ing and growing area (p < 0.05). Peroxide values varied × carrier gas was helium at 1.0 ml/min ratio. Injector, oven between 7.10 (H1 from Hatay) and 18.36 (G1 from Mar- and detector temperatures were 250, 230 and 280 °C, din) mequiv O2/kg oil and peroxide values of all samples respectively. The results were expressed as the relative area were below the legal limit (<20) for virgin olive oil. There

1 3 1502 J Am Oil Chem Soc (2016) 93:1499–1508 D;E B;F D;Y;E D;X A;Y;F B;X;E A;F A;E A;X C;Y;E C;X;E D;Y;E 92.16 ± 12.49 34.82 ± 8.60 36.20 ± 8.62 1.60 ± 0.17 1.65 ± 0.13 18.6 ± 0.16 13.97 ± 1.34 3.08 ± 0.78 4.74 ± 0.15 5.46 ± 0.22 11.04 ± 0.50 120.77 ± 13.90 H4 B;Y;F B;X;E B;X;E C;Y;F B;X B;X;E B;X;F B;Y;F B;Y B;Y;E B;X;E C;Y;E H3 188.82 ± 37.81 48.42 ± 7.56 42.53 ± 7.65 0.74 ± 0.09 0.70 ± 0.05 13.06 ± 0.38 8.56 ± 2.25 4.24 ± 0.15 5.28 ± 0.24 7.69 ± 0.33 11.57 ± 0.34 415.38 ± 53.80 A;X;F C;Y;E C;E B; A;X B;Y;E B;X;E B;X;F C;Y C;Y;F B;Y;E B;X;E H2 7.72 ± 0.71 10.36 ± 0.51 12.15 ± 0.51 232.09 ± 24.62 45.48 ± 5.20 45.81 ± 9.40 0.56 ± 0.06 0.34 ± 0.07 13.68 ± 0.67 4.88 ± 0.10 5.26 ± 0.12 322.13 ± 38.14 A;Y;F A;X;E A;X;E A;Y;E A;X;F A;E A;E A;X;F B;F B A;Y;E C;Y 59.39 ± 8.42 53.73 ± 6.27 Halhalı 0.63 ± 0.09 0.54 ± 0.09 7.1 ± 0.87 11.29 ± 0.44 5.66 ± 0.15 6.40 ± 0.26 12.29 ± 0.34 13.59 ± 1.00 525.22 ± 65.59 225.81 ± 27.91 H1 C;X;E B;X C;Y;F A B;E B;Y;F A;X;E C;Y;F D;X;F C;F D;F A;Y;F G4 37.29 ± 9.23 18.12 ± 2.74 17.45 ± 0.90 2.32 ± 0.20 3.57 ± 0.66 3.69 ± 0.30 188.48 ± 31.7 31.73 ± 4.30 2.49 ± 0.17 0.93 ± 0.1 1.56 ± 0.06 43.31 ± 9.44 A;X;E A;X;E BC;Y;F B;X D;Y BC;Y;F B;X;E C;Y;F C;X;F C;F A;Y;E C;F 10.52 34.73 ± 6.21 45.93 ± 1.84 ± 0.07 0.94 ± 0.0 12.70 ± 0.42 10.64 ± 0.71 1.68 ± 0.15 3.48 ± 0.31 4.5 ± 0.48 5.27 ± 0.80 34.23 ± 8.31 465.20 ± 71.9 G3 C;Y;F AB;X;E C;Y;F A;X B;Y;E C;X C;X;E B;Y;F B;X;F B;F B;F B;Y 23.73 ± 4.50 46.15 ± 8.14 1.65 ± 0.07 0.47 ± 0.1 12.17 ± 0.65 14.31 ± 0.21 2.08 ± 0.25 4.42 ± 0.10 7.00 ± 0.16 7.36 ± 0.50 20.62 ± 16.85 421.23 ± 19.4 G2 A;Y;F B;X;E A;X;F B;Y;F A;X;E C;X;E C;Y A;Y;F A;F F B;Y A;X;F 1.56 ± 0.05 51.19 ± 5.21 3.18 ± 0.26 8.03 ± 0.65 74.57 ± 13.09 8.05 ± 0.67 37.63 ± 6.21 0.56 ± 0.1 18.36 ± 0.52 5.04 ± 05 9.25 ± 0.51 355.28 ± 41.4 G1 Hatay Hatay Hatay Hatay Hatay Hatay Mardin Gemlik Mardin Mardin Mardin Mardin Mardin Grow. Grow. region / 2 Chemical properties of olive oil samples Chemical properties of olive For each parameter different letters for the same cultivar and maturity indicate statistically significant differences ( p < 0.05) between region and maturity indicate statistically significant differences letters for the same cultivar each parameter different For For each parameter different letters for the same cultivar and region indicate statistically significant differences ( p < 0.05) between maturation indicate statistically significant differences and region letters for the same cultivar each parameter different For For each parameter different letters for the same maturity and region indicate statistically significant differences ( p < 0.05) between cultivar indicate statistically significant differences letters for the same maturity and region each parameter different For

(%C18:1) (mequiv O (mequiv (mg/kg) (mg/kg) content (mg/ kg) kgoil) Free fatty acids Free fatty %Inhibition Chemical prop - Chemical erties Peroxide value Carotenoid Chlorophyll phenolic Total 1 Table A–C X–Y E–F

1 3 J Am Oil Chem Soc (2016) 93:1499–1508 1503 were statistically significant differences between the perox- with minor exceptions (p < 0.05). The main fatty acid in all ide values of the cultivars in growing area and ripening. The oil samples was oleic acid, ranging from 62.34 % (G2 from carotenoid and chlorophyll contents decrease significantly Hatay) to 71.53 % (H1 from Hatay). Oleic acid contents of throughout ripening in both olive oils from two regions. Halhalı olive oils were higher than Gemlik and these con- These results were in accordance with those of Abenoza et tent showed fluctuation for olive oils from both locations. al. [28]. Carotenoid and chlorophyll contents ranged from Similarly, stearic acid contents also showed apparent vari- 1.56 to 6.40 mg/kg and from 3.57 to 13.59 mg/kg, respec- ation in olive oils. Palmitic acid, the main saturated fatty tively. Carotenoid and chlorophyl contents were found to acid of olive oils, ranging from 13.87 % (G1 from Mar- be the highest in Halhalı from Mardin while their contents din) to 16.70 % (H1 from Mardin). Palmitic acid content were the lowest in Gemlik from Hatay. increased slightly during ripening in Gemlik oils while this content decreased in Halhalı oils from both of two Total Phenolic Contents locations. Linoleic acid content ranged between 5.02 % (H1 from Hatay) and 11.93 % (G2 from Hatay). Linoleic Total phenolic contents (Table 1) decreased generally acid content of Gemlik olive oils was higher than Halhalı during ripening. The highest (525 mg/kg) and the low- olive oils. Linoleic acid content increased during matura- est (20.62 mg/kg) total phenol contents were determined tion, confirming the results reported by Yorulmazet al. [40] in Halhalı and Gemlik from Hatay, respectively. There which is probably because of the results of oleate desatu- were significant differences among the cultivars, ripening rase enzyme activity transforming oleic into linoleic acid. and location the in total phenolic contents of the olive oil The fatty acid compositions of oils from both olive varie- samples (p < 0.05). Kesen et al. [19] reported that the total ties were within the legal limits established by the EU regu- phenolic contents of two olive oil varieties from different lations, with a minor exception that could have been due regions in Turkey ranged from 74.71 to 96.97 mg of GA/ to genetic factors and climatic conditions [41]. Palmitoleic, kg of oil. Also, Del Monaco et al. [7] studied 12 olive oils linolenic, arachidic and behenic acids were between 0.75– from different regions and cultivars in Italy to characteri- 1.67, 0.56–1.06, 0.50–0.83, 0.08–0.18 %, respectively. The zation of olive oils with respect to varieties, geographical fatty acid composition of olive oil is significantly influ- region and harvest date. They reported that the phenolic enced by the cultivar, ripeness stage of the fruit, climatic content of olive oils showed large variation based on varie- conditions, latitude, irrigation management and zone of ties, ripening and geographical region, ranging from 290 to production [42, 43]. 2180 mg/kg. Sterol and Triterpene Dialcohol Compositions Antioxidant Activity The sterol and triterpene diols (erythrodiols uvaol) com- + Radical scavenger capacity of oil samples was performed positions of olive oil were presented in Table 3. While by using a spectrophotometric method based on the color β-sitosterol, ∆-5-avenasterol and campesterol were deter- change of the radical DPPH. Results were expressed mined as the main sterols in the oil samples, cholesterol, as %Inhibition. The highest antioxidant activity was brassicasterol, 24-methylenecholesterol, campestanol, determined as 59.39 % (H1 from Hatay) while the low- stigmasterol, ∆-7-campesterol, clerosterol, sitostanol, est 23.73 % (G2 from Hatay). Antioxidant activities were ∆-5,24-stigmastadienol, ∆-7-stigmastenol, ∆-7-ave- in accordance with total phenolic content in all oil sam- nasterol and two triterpene dialcohols (erythrodiol and ples with minor exceptions. The differences in antioxidant uvaol) were found as minor sterols. These results were activities may depend on the composition and profile of in good agreement with data reported elsewhere [1, 16, phenolic compounds rather than total phenol contents as in 44, 45]. As shown in Table 3., campesterol, stigmasterol, the previous works [39]. ∆-5-avenasterol, β-sitosterol, ∆-5,24-stigmastadional, erythrodiol uvaol and total sterols contents were sig- + Fatty Acid Compositions nificantly affected by the variety, maturity and growing region (p < 0.05) with minor exceptions. The total sterol The fatty acid compositions of olive oils samples are compositions of olive oil samples were higher than the shown in Table 2. The major fatty acids were palmitic minimum limits (1000 mg/kg) established by EU regula- (C16:0), oleic (C18:1) and linoleic (C18:2) acids. Palmit- tion, ranging from 1194 (H2 from Hatay) to 2008 mg/kg oleic (C16:1), linolenic (C18:3), stearic (C18:0), arachidic (G1 from Hatay) and the apparent β-sitosterol contents sig- (C20:0), behenic (C22:0) acids were detected in minor nified as the sum of the contents of β-sitosterol and other amounts. The fatty acid compositions were significantly sterols (sitostanol, Δ-5,24-stigmastadienol, clerosterol, affected by variety, maturation and location in oil samples and Δ-5-avenasterol) were higher than the legal threshold

1 3 1504 J Am Oil Chem Soc (2016) 93:1499–1508 C;Y;F D;X;E A;F F C;Y;F B;X;E C;E A;E A;X;F A;Y;F B;Y C;X;E C;Y A;X;E A A;F 1.25 ± 0.22 1.22 ± 0.10 2.99 ± 0.27 3.51 ± 0.21 7.88 ± 0.31 6.84 ± 0.16 0.77 ± 0.26 0.84 ± 0.11 0.54 ± 0.14 0.65 ± 0.34 0.34 ± 0.01 0.34 ± 0.01 14.30 ± 0.28 15.75 ± 0.44 68.28 ± 0.30 69.08 ± 0.38 H4 B;Y;F C;X B;X;E B;Y;E C;Y;F X;F B;Y;E A;X;E B;X;F A;Y;F C;Y;F B;X B;Y;E A;X;E A;X;F C;Y;F 0.82 ± 0.15 1.23 ± 0.14 3.50 ± 0.17 3.67 ± 0.39 7.48 ± 0.23 6.82 ± 0.35 0.68 ± 0.26 0.87 ± 0.22 0.60 ± 0.29 0.62 ± 0.28 0.32 ± 0.01 0.31 ± 0.01 14.37 ± 0.18 15.92 ± 0.42 70.21 ± 0.14 68.75 ± 0.27 H3 A;Y;F B;X;E C;Y;F ;X;E B;Y;E C;X;E D;Y;E C;X;F C;Y;F C;X;F B;Y;F BC;X;F A;X;E B;Y;F B;X;E C;Y;F 0.83 ± 0.32 1.22 ± 0.23 3.53 ± 0.21 3.44 ± 0.23 5.23 ± 0.12 6.33 ± 0.24 0.74 ± 0.20 0.84 ± 0.29 0.59 ± 0.32 0.57 ± 0.30 0.37 ± 0.00 0.31 ± 0.00 14.46 ± 0.14 16.40 ± 0.48 67.20 ± 0.22 67.45 ± 0.13 H2 A;Y;F B;E A;X D;Y;E A;X;E D;Y;E D;Y;F B;X;F A;Y;E A;X;F B;Y;E A;X;E E A;Y;F B;X;F B;X;F 1.11 ± 0.12 1.26 ± 0.14 3.60 ± 0.15 2.96 ± 0.21 5.02 ± 0.32 6.64 ± 0.21 0.87 ± 0.14 0.97 ± 0.18 0.51 ± 0.21 0.56 ± 0.19 0.36 ± 0.01 0.33 ± 0.01 14.47 ± 0.34 16.70 ± 1.19 71.53 ± 0.32 66.32 ± 0.18 Halhalı H1 C;X;E B;X;E B;X;E B;Y;F B;F D;F C;Y;E A;X;E B;X D;Y;F A;X;E A;Y;F A;Y;E BC;X D;Y;F C;Y 1.56 ± 0.20 3.57 ± 0.26 3.27 ± 0.20 8.86 ± 0.33 0.76 ± 0.34 0.67 ± 0.20 0.36 ± 0.02 1.34 ± 0.11 0.54 ± 0.29 0.44 ± 0.20 0.34 ± 0.02 15.13 ± 0.41 67.07 ± 0.32 66.33 ± 0.22 11.13 ± 0.12 14.74 ± 0.15 G4 B;E B A;X;E A;Y;E C;Y;F A;X;F C;Y;F C;X;F B;Y;E B;X;E A; E C C;Y;F C;X;F B;X;E B;Y;E 1.67 ± 0.20 3.07 ± 0.06 3.44 ± 0.32 0.86 ± 0.22 0.87 ± 0.42 0.34 ± 0.01 1.35 ± 0.10 0.50 ± 0.29 0.58 ± 0.11 0.35 ± 0.01 15.62 ± 0.54 65.50 ± 0.27 65.98 ± 0.30 10.25 ± 0.13 10.55 ± 0.30 14.57 ± 0.31 G3 A;X;E C;Y;F A;X;E B;Y;F D;Y;F C;X;F D;Y;F A;X;E A;X;E C;Y;E A;Y;E B;X;E A;F A;E A;X,E A;Y,E 1.63 ± 0.28 1.05 ± 0.20 2.58 ± 0.20 3.11 ± 0.30 8.29 ± 0.42 0.88 ± 0.31 0.95 ± 0.21 0.69 ± 0.22 0.63 ± 0.30 0.38 ± 0.02 0.32 ± 0.02 16.23 ± 0.96 13.96 ± 0.20 62.34 ± 0.23 67.90 ± 0.42 11.93 ± 0.14 G2 D;X;E C;Y;F C;Y;F C;X;F A;X D;Y;F A;X;F D;Y;F D;Y;E D;X;E C;Y;F A;X;E B;Y;E B;X;E C,;X;F C,;Y;F 0.75 ± 0.29 3.61 ± 0.32 7.68 ± 0.26 0.56 ± 0.27 0.58 ± 0.19 0.32 ± 0.02 0.85 ± 0.20 2.85 ± 0.31 7.54 ± 0.20 1.01 ± 0.02 0.65 ± 0.02 0.30 ± 0.02 68.29 ± 0.19 13.87 ± 0.30 64.14 ± 0.22 15.33 ± 0.22 G1 Grow. Grow. region Hatay Hatay Hatay Hatay Hatay Hatay Hatay Gemlik Mardin Mardin Mardin Mardin Mardin Mardin Mardin Mardin Hatay Fatty acid compositions of olive oil samples(%) acid compositions of olive Fatty For each parameter different letters for the same cultivar and maturity indicate statistically significant differences ( p < 0.05) between region and maturity indicate statistically significant differences letters for the same cultivar each parameter different For For each parameter, different letters for the same cultivar and region indicate statistically significant differences ( p < 0.05) between maturation indicate statistically significant differences and region letters for the same cultivar different each parameter, For For each parameter different letters for the same maturity and region indicate statistically significant differences ( p < 0.05) between cultivar indicate statistically significant differences letters for the same maturity and region each parameter different For

2 Table acids Fatty C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 A–C X–Y E–F C16:0

1 3 J Am Oil Chem Soc (2016) 93:1499–1508 1505 B;Y;E C;X;E C;F C A;E E A;X Y;F D;Y B;X;E C A;Y;E A;X;E C A;X B;Y BC;X;F Y C;X;E D;Y;F A;X;F B;Y;F C;X;F B;Y C;X;F B;Y;F 0.24 ± 0.01 0.21 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 0.14 ± 0.01 0.08 ± 0.01 2.27 ± 0.01 0.08 ± 0.01 3.62 ± 0.03 0.09 ± 0.02 1.96 ± 0.02 2.72 ± 0.01 0.02 ± 0.00 0.02 ± 0.01 1.06 ± 0.01 0.96 ± 0.02 0.72 ± 0.01 0.31 ± 0.02 7.06 ± 0.02 2.74 ± 0.01 0.75 ± 0.03 0.44 ± 0.03 0.40 ± 0.01 0.26 ± 0.02 0.66 ± 0.03 0.31 ± 0.01 H4 84.45 ± 0.03 86.95 ± 0.02 C;E C;E B;Y;F X B;E F C;Y;E A;X;E B;C;F B;Y;F B;X;E B;X Y BA;X B;Y C;Y;F A;X;E C;X Y;F A;X;E C;Y;F A;X;F AB;Y;F B;X;E A;Y;F B;X;E B;Y;F 0.23 ± 0.01 0.23 ± 0.01 0.01 ± 0.00 0.03 ± 0.55 0.09 ± 0.02 0.09 ± 0.02 2.91 ± 0.01 3.66 ± 0.02 0.10 ± 0.02 0.09 ± 0.02 0.93 ± 0.02 1.35 ± 0.01 0.05 ± 0.01 0.02 ± 0.01 1.02 ± 0.02 0.98 ± 0.02 0.52 ± 0.39 0.46 ± 0.02 7.87 ± 0.02 3.22 ± 0.02 0.75 ± 0.02 0.49 ± 0.02 0.49 ± 0.02 0.31 ± 0.03 0.97 ± 0.01 0.31 ± 0.01 83.70 ± 0.02 88.69 ± 0.03 H3 A;X;E A;Y;E B E C F A;Y;E B;X;E A;X;F Y C;Y;F D;X;F B E B;E A;F A;X;E B;Y;E A;X;E Y;F D;Y;F A;X;F B;F AB;F B;X;E AB;Y D;Y;F A;X;F 0.01 0.03 ± 0.01 0.39 ± 0.01 0.31 ± 0.01 0.02 ± 0.01 0.06 ± 0.01 0.08 ± 0.01 3.49 ± 0.01 3.60 ± 0.01 0.17 ± 0.01 0.08 ± 0.02 0.78 ± 0.01 1.15 ± 0.05 ± 0.01 0.03 ± 0.01 0.99 ± 0.01 1.03 ± 0.02 1.39 ± 0.02 0.50 ± 0.02 5.37 ± 0.02 3.73 ± 0.01 0.47 ± 0.02 0.46 ± 0.02 0.47 ± 0.01 0.27 ± 0.01 0.43 ± 0.01 0.47 ± 0.01 85.87 ± 0.03 88.19 ± 0.02 H2 B;X;E B;Y;E B;F C;Y X;F B;Y;E A;X;E B;X;E Y D;Y;F C;X;E A;X Y B;Y;E A;X;F D;Y;F B;X;E AB;X;E Y;F B;X;E B;Y;F A;X;F A;Y;F A;X;E AB;Y;E A;X;E A;Y;F 0.09 ± 0.01 0.11 ± 0.01 0.13 ± 0.01 0.31 ± 0.01 0.27 ± 0.02 0.02 ± 0.01 0.01 ± 0.00 0.06 ± 0.01 0.09 ± 0.01 3.11 ± 0.01 3.68 ± 0.01 0.66 ± 0.02 1.27 ± 0.02 0.04 ± 0.01 0.99 ± 0.02 1.06 ± 0.01 1.06 ± 0.02 0.53 ± 0.01 7.41 ± 0.01 3.42 ± 0.02 0.75 ± 0.02 0.50 ± 0.02 0.55 ± 0.02 0.30 ± 0.01 1.02 ± 0.01 0.49 ± 0.02 83.21 ± 0.02 88.21 ± 0.03 H1 Halhalı B;Y;E X B;F B;F Y X;E C;X C;Y;F A;X;F B;Y;F A A;X C;X;F A;X;E A;Y;F B;X;E X;E A;Y;E D;Y C;Y;F C;Y B;X;E B;Y;E B;Y D;X;E 0.07 ± 0.00 0.02 ± 0.01 0.17 ± 0.01 0.22 ± 0.00 0.02 ± 0.00 0.01 ± 0.00 0.08 ± 0.01 0.15 ± 0.00 1.87 ± 0.01 1.52 ± 0.20 0.07 ± 0.02 1.84 ± 0.02 1.14 ± 0.01 0.09 ± 0.01 1.07 ± 0.02 0.97 ± 0.0 1.03 ± 0.01 0.59 ± 0.0 6.88 ± 0.02 0.87 ± 0.02 0.76 ± 0.0 0.45 ± 0.01 0.28 ± 0.0 0.92 ± 0.01 1.08 ± 0.0 84.73 ± 0.02 72.10 ± 0.1 20.93 ± 0.1 G4 B;F F B;X;E B;Y Y;F X;E B;X;F C;Y;F E B;X;E C;Y;F B B B;X;E B B;X;F B;E X;F D;Y;F C D;Y;F B;E A;Y;E A;E A;Y;E A;X;E 0.18 ± 0.01 0.19 ± 0.00 0.06 ± 0.02 0.01 ± 0.00 0.06 ± 0.01 0.16 ± 0.00 2.27 ± 0.02 1.53 ± 0.01 0.06 ± 0.01 0.07 ± 0.00 1.69 ± 0.05 0.96 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 0.98 ± 0.03 1.02 ± 0.0 0.69 ± 0.01 0.65 ± 0.0 4.83 ± 0.06 0.90 ± 0.02 0.85 ± 0.0 0.43 ± 0.02 0.38 ± 0.0 0.62 ± 0.02 1.69 ± 0.0 87.24 ± 0.09 71.21 ± 0.1 21.30 ± 0.1 G3 B;F F B B;F Y X;E A;X;F A;Y;F Y;E X C;Y;E A;X;E X B;Y;F C;Y;F A;X;F C;Y;F C;Y;E A;X;E X;F C;Y;E A;X;E A;Y;F AB;X;E D;X;E A;Y;E BC;Y C;X;E 0.17 ± 0.02 0.22 ± 0.00 0.02 ± 0.00 0.01 ± 0.00 0.06 ± 0.02 0.16 ± 0.00 2.96 ± 0.02 2.01 ± 0.00 0.05 ± 0.00 0.08 ± 0.00 0.92 ± 0.02 1.27 ± 0.00 0.01 ± 0.00 0.86 ± 0.01 1.16 ± 0.0 0.33 ± 0.08 0.70 ± 0.0 4.16 ± 0.05 1.43 ± 0.09 0.91 ± 0.0 0.44 ± 0.01 0.23 ± 0.0 0.68 ± 0.02 1.17 ± 0.0 0.06 ± 0.01 87.96 ± 0.16 77.13 ± 0.1 15.05 ± 0.0 G2 A;F F A;X;E A;Y Y X;E A;X;F B;Y;F Y;F X C;Y;E B;X;F B C;Y;F A;X;E D;Y;F C;Y;F A;X;E X;F B;Y;F B;X;E B;Y;F A;X;E C;X;E A;Y;E C;Y;F B;X;E 0.21 ± 0.01 0.20 ± 0.00 0.02 ± 0.00 0.17 ± 0.00 1.93 ± 0.00 0.12 ± 0.00 1.16 ± 0.00 0.03 ± 0.01 1.10 ± 0.0 0.90 ± 0.01 0.75 ± 0.0 0.85 ± 0.0 0.22 ± 0.0 0.83 ± 0.01 1.28 ± 0.0 0.23 ± 0.05 0.08 ± 0.01 2.98 ± 0.05 0.06 ± 0.01 0.28 ± 0.06 0.23 ± 0.02 4.07 ± 0.08 1.51 ± 0.02 0.43 ± 0.03 0.77 ± 0.02 76.60 ± 0.1 15.53 ± 0.0 88.03 ± 0.07 G1 Hatay Grow Grow region Gemlik Mardin Mardin Mardin Mardin Mardin Mardin Mardin Mardin Mardin Hatay Mardin Mardin Mardin Mardin Hatay Mardin Hatay Hatay Hatay Hatay Hatay Hatay Hatay Hatay Hatay Hatay Hatay Sterol compositions of olive oil samples (%) Sterol compositions of olive lesterol Stigmastadional Cholesterol 3 Table Stigmasterol Sterol Clerosterol Brassicasterol - 24-Methylene-cho Campesterol Campestanol Δ -7-Campesterol β -Sitosterol Sitostanol Δ -5-Avenasterol Δ -5,24- Δ -7-Stigmastenol Δ -7-Avenasterol

1 3 1506 J Am Oil Chem Soc (2016) 93:1499–1508

value (93 %) except for Halhalı from Mardin (91.55 %). A;X;F C;Y;F A;X;F B;Y;F D;Y;E A;X;E β-sitosterol was found as major sterol in all oil samples, varying between 71.21 (G3) and 88.69 % (H3) in oils from Mardin. β-sitosterol contents of Gemlik oils from Hatay 2.33 ± 0.15 4.09 ± 0.03 94.46 ± 0.36 91.55 ± 0.02 H4 1477.33 ± 8.50 1455.00 ± 4.35 were higher than those of from Mardin. The second most abundant sterol was ∆-5-avenasterol, fluctuating between B;F D;F AB;X;F B;Y;F C;E D;E 2.74 (H4) and 21.30 % (G3) in oils obtained from Mar- din. Gutierrez et al. [46] and Lukic et al. [11] reported that β-sitosterol contents generally decrease during matura- 2.74 ± 0.01 1.83 ± 0.01 94.13 ± 0.02 91.55 ± 0.02 H3 1279.16 ± 6.42 1277.66 ± 6.65 tion, while ∆-5-avenasterol increase. Campesterol contents in oil samples ranged from 1.52 % (G4 from Mardin) to C;Y;F B;Y;F A;Y;F A;X;E B;X;E B;Y 3.68 % (H1 from Mardin). Cholesterol and campesterol percentages were below the limit (0.5 and 4.0 %, respec- tively) established by EU regulation. Stigmasterol is the 3.13 ± 0.01 2.30 ± 0.02 94.09 ± 0.02 93.91 ± 0.02 main sterol related to the quality of virgin olive oil and its 1194.33 ± 12.42 1568.66 ± 7.76 H2 high level contents are correlated with high acidity and low organoleptic quality [1, 47]. The mean content in the oil C;Y;F C;Y;F A;X;F B;X;E A;X;E C;F samples ranged from 0.66 to 1.96 % in Halhalı oils from Hatay. Stigmasterol percentages were lower than those of campesterol in all samples. These results are in consistent 3.50 ± 0.03 1.93 ± 0.02 93.43 ± 0.03 93.74 ± 0.03 with other research which showed the existence of differ- 1195.33 ± 13.31 1503.00 ± 6.24 H1 Halhalı ences according to variety and growing region [17, 48]. Many researchers reported that geographical region had B;X;E A;Y;E A;X;E B;F A;Y;E B;X;F significant effect on sterol amounts this may be related to the different climate conditions at each growing area, such as rainfall, temperature and humidity [1, 41, 49]. 1.71 ± 0.02 1.86 ± 0.1 94.63 ± 0.03 95.46 ± 0.10 1761.33 ± 18.50 1472.67 ± 3.1 G4 The triterpene dialcohols (erythrodiol and uvaol) were anajyzed together with the sterol fraction of the oil sam-

B;X;E ples. As can be seen in Table 3, the mean content of eryth- A;Y;E B;X;E A;Y;F C;Y;E C;X;F rodiol uvaol was below the accepted limit of 4.5 % from + the EU regulation, ranging from 1.16 (G1 from Hatay) to 1.80 ± 0.01 1.56 ± 0.1 4.09 % (H4 from Mardin). Moreover, the sum of erythro- 94.64 ± 0.01 94.95 ± 0.10 1770.66 ± 20.20 1354.67 ± 6.8 G3 diol and uvaol contents was influenced by variety, maturity and geographical region and it was determined the amounts A;X;E AB;Y;E B;X;E C;Y;F D;Y;F A;X of those in Halhalı olive oils were higher than Gemlik from both locations. These results were consistent with those of Mailer et al. [48] who pointed out erythrodiol and uvaol 1.34 ± 0.00 2.11 ± 0.2 94.59 ± 0.08 94.88 ± 0.01 contents of olive oils of ten olive cultivars from different G2 1988.00 ± 1.73 1325.33 ± 4.2 maturation stages and regions of Australia ranged from 0.4 to 4 %. B;Y;E B;X;E A;X;E D;Y;F B;Y;F A;X;E 2.08 ± 0.1 1.16 ± 0.01 Conclusions 94.53 ± 0.03 94.86 ± 0.10 1397.67 ± 7.6 2008.66 ± 1.52 G1 Analysis of olive oils obtained from two cultivars (Halhalı and Gemlik) at four maturation stages in two different growing regions (Hatay and Mardin) indicated that oil sam- Hatay Grow Grow region Gemlik Mardin Mardin Mardin Hatay Hatay ples have largely significant variations in terms of chemical properties, fatty acid and sterol compositions, and this find- ings were in accordance with the internationally accepted continued ranges. Total phenolic contents, antioxidant activity, oleic For each parameter different letters for the same cultivar and maturity indicate statistically significant differences ( p < 0.05) between region and maturity indicate statistically significant differences letters for the same cultivar each parameter different For For each parameter different letters for the same cultivar and region indicate statistically significant differences ( p < 0.05) between maturation indicate statistically significant differences and region letters for the same cultivar each parameter different For For each parameter different letters for the same maturity and region indicate statistically significant differences ( p < 0.05) between cultivar indicate statistically significant differences letters for the same maturity and region each parameter different For

acid, β-sitosterol, erythrodiol and uvaol contents of the

(%) Apparent β -sitosterol Sterol Total sterol (mg/kg) Total Erythrodiol + uvaol 3 Table A–C X–Y E–F Halhalı olive oils were higher than Gemlik oils. Halhalı

1 3 J Am Oil Chem Soc (2016) 93:1499–1508 1507 variety generally had better than Gemlik in terms of oil virgin olive oils according to their geographical origin using phe- quality properties and sterol composition. β-sitosterol and nolic compound profiles obtained by capillary electrochromatog- raphy. Food Res Int 42:1446–1452 total sterol contents of Gemlik oils from Hatay were higher 11. Lukic M, Lukic I, Krapac M, Sladonja B, Pilizota V (2013) Ster- than those of from Mardin. Therefore, the Halhalı variety ols and triterpene diols in olive oil as indicators of variety and should be intensely cultivated and certified with Protected degree of ripening. Food Chem 136:251–258 Designation of Origin especially in Hatay. The results of 12. Pardo JE, Sena E, Cuesta MA, Granell JD, Valiente J, Alvarez- Ort M (2013) Evaluation of potential and real quality of virgin this study showed that cultivar, maturation and growing olive oil from "Campos de Hellin" (Albacete, Spain). 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