Quality Properties, Fatty Acid and Sterol Compositions of East Mediterranean Region Olive Oils.

In this study, important physicochemical properties, fatty acid and sterol compositions of olive oils from the olives which harvested from Mersin (Buyuk Topak Ulak, Gemlik, Sari Ulak), Adana (Gemlik), Osmaniye (Gemlik) and Hatay (Gemlik, Kargaburun, Hasebi, Halhali) in the Eastern Mediterranean region of Turkey have been investigated. Ripening index and oil yield analysis of the olives and free fatty acids, peroxide value, UV absorbance (K232, K270), fatty acid composition, sterol composition, erythrodiol+uvaol content, and total sterol analysis of the olive oil samples were carried out. The levels of free acidity in the olive oil samples ranged from 0.39% (Hatay Gemlik: HG3) to 2.23% (Mersin Gemlik: MG). Peroxide value ranged from 8.87 to 18.87 meq O2/kg. As K232 values in the oils fluctuated between 1.4370 and 2.3970, K270 values varied between 0.1270 and 0.1990. The results showed that all ΔK values were lower than the maximum legal limit of 0.01. The main fatty acid in all oil samples was oleic acid, ranging from 58.72% (Hatay Hasebi: HHs) to 74.54% (Hatay Gemlik: HG2). Palmitic acid values were within the percentage of 12.83% (Hatay Kargaburun: HK) to 18.50% (HHs). Total sterol content varied from 720.41 mg/kg (Hatay Kargaburun: HK) to 4519.17 mg/kg (Buyuk Topak Ulak: BTU). The β-sitosterol percentage of olive oils ranged from 76.12% (Adana Gemlik: AG) to 94.23% (Buyuk Topak Ulak: BTU). The results of this study indicated that variety significantly affect the quality indices, fatty acid and sterol compositions of olive oils significantly varied among varieties.


Olive sampling
This study was carried out during the crop season of 2015 -2016. Olive samples of common Turkish olive varieties; Gemlik, Halhalı, Hasebi, Kargaburnu, Büyük Topak Ulak, Sarı Ulak were picked by hand from their growing area in the East Mediterranean region of Turkey. The olive samples were harvested from four trees as replicates of the different orchard for each cultivar from Hatay, Adana, Mersin and Osmaniye provinces. A representative 3 kg sample of olives was collected to represent the whole tree and coded with respective growing regions, varieties and harvesting dates of olive samples are illustrated in Table 1.
Moreover, geographical characteristics about the locations from which olives were collected and given are listed in Table 2.

Ripening index RI
The RI was determined by using 100 olive fruits randomly selected from each olive fruit batch. It is reported earlier that the RI values ranged from 0 100 intensely green skin to 7 100 purple flesh and black skin 14 .

Olive oil extraction
Three kg of olive samples selected from each variety were eliminated from damaged and decay fruits. Olive oil extraction was performed using a laboratory scale mechanical extractor Hakkı Usta, Turkey with a crusher, a vertical malaxer and a two-phase centrifuge. Malaxation and centrifuge processes was carried out at 25 for 30 min and at 3000 rpm, respectively. The oil was separated by decanting and put into dark glass bottles. Oil samples were kept at 4 until chemical analysis which were duplicated.

Oil content
Oil content was performed according to the method described in American Oil Chemists Society AOCS Official Methods Am 2-93 34 by Soxhlet extraction method using nhexane at 80 for 6 h.

Fatty acid compositions
The fatty acid composition of the oils was performed according to the method proposed by the International Olive Oil Council, COI/T.20/Doc.No.24 35 . Fatty acid methyl esters FAME were prepared by vigorous shaking of a solution of oil in n-heptane 0.1 g in 2 mL with 0.2 mL of 2 N methanolic potassium hydroxide. The analysis of FAME was performed by Thermo Focus gas chromatography system ThermoFisher Scientific, Milan, Italy, using a hydrogen flame ionization detector FID and a capillary column SGE BPX 90 100 m length, 0.25 mm i.d. and 0.25 μm film thickness . The temperatures of detector and injector were set at 290 and 250 , respectively. Helium was employed as carrier gas with a flow rate of 1 mL/min and the split ratio was 1:10. The injection volume was 1 μL. The results were expressed as a relative area percentage of total. Fatty acids were determined by comparing their retention times with those of reference compounds.

Sterol and triterpene diols erythrodiol uvaol com-
position Sterol compositions were determined depending on the official method IOC/T.20/No10 36 . Identification and quantification of sterols and diols as trimethylsilyl ethers was performed by gas chromatography GC 2010, Shimadzu, Japan equipped with a SGE BPX5 capillary column 25 m, 0.32 mm i.d. and 0.25 μm film thickness and a flame ionization detector FID . Injector, column and detector temperatures were 280, 265 and 290 , respectively. Helium was used as the carrier gas with a flow rate of 1 mL/min and the split ratio of 10:1. Individual sterols and two triterpendiols erythrodiol and uvaol in oils were identified based on their relative retention times with respect to the internal standard, cholestanol, according to the standardized reference method.

Statistical analysis
Statistical analysis was carried out using the SPSS 10.0 statistical software SPSS Inc., Chicago, USA . Data were analyzed by a one-way analysis of variance ANOVA . Significant differences between samples were determined by Duncan s multiple range test at the 5 confidence level 37 .

Quality indices
Ripening index, oil content of olives and quality indices FFA, peroxide value and K 232 , K 270 , ΔK values of olive oils  23 ; Noorali et al. 38 . All the studied olive oils exhibited the values of some quality indices peroxide value ≤ 20 meq O 2 /kg; K 232 ≤ 2.5; K 270 ≤ 0.22 and ΔK ≤ 0.01 below the limits established by EU regulation for extra virgin olive oil. However, free fatty acidity values of the samples were over the limit of 0.8 fixed for extra virgin olive oil category except for HG1 and HG3. Our results were higher than those reported by Bouarroudj et al. 39 whereas in they were in agreement with those by Yorulmaz and Bozdogan Konuskan 3 . Free fatty acid which is a quality criteria for olive oils can show variations according to variety, location, olive fly and unsuitable storage conditions 8, 40, 41 .

Fatty acid compositions
The fatty acid compositions of olive oils samples are presented in Table 4. The major fatty acids were palmitic C16:0 , oleic C18:1 and linoleic C18:2 acids while palmitoleic C16:1 , linolenic C18:3 , stearic C18:0 , arachidic C20:0 , behenic C22:0 acids occurred in minor amounts. According to the results, the fatty acid content of olive oil samples was within the legal limits proposed by EU regulation for extra virgin olive oil. The fatty acid compositions were significantly influenced by variety in the oil samples p 0.05 . The major fatty acid in all oil samples was oleic acid, varying between 58.72 Hatay Hasebi and 74.54 Hatay Gemlik2 . Xiang et al. 15 reported that the oleic acid content of four olive oil varieties from China was in the range of 60.94-74.03 . Our results showed similarity with those of the results obtained by Xiang et al. 15 . In another study by Manai-Djebali et al. 2 oleic acid content of five Tunisian olive oil varieties varied between 67.2 Aloui and 78. 9 Hor Kesra , indicating that they obtained higher values of oleic acid content when compared with our results. Palmitic acid, the main saturated fatty acid of olive oils, ranged from 12.83 HK to 18.50 HHs . Lopez-Cortes et al. 42 stated that palmitic acid content from eight Spanish varieties ranged from 9.84 to 18.44 as in accordance with our findings. However, Arbequina olive oil during 2011 crop season contained higher palmitic acid than our varieties according to the work by Abenoza et al. 41 . Linoleic acid content varied between 4.88 HG2 and 17. 18 BTU . These results were in agreement with those obtained by Bouarroudj et al. 39 Laincer et al. 43 and Xiang et al. 15 , although the linoleic acid content of Brazilian olive oil samples in the study carried out by Ballus et al. 44 was lower than our values. Stearic, palmitoleic, linolenic, arachidic, and behenic acids were present in the range between 2.49 and 3.77, 0.46 and 1.58, 0.46 and 0.87, 0.39 and 0.58 and 0.10 and 0.16 , respectively. The fatty acid composition of olive oil is significantly influenced by the cultivar, ripeness stage of the fruit, climatic conditions, latitude, irrigation management and zone of production 38, 45, 46 .

±0.000
Results are signified as mean±SD of three sample replicates. Different small letters express significant statistical differences (Duncan' s Test p＜0.05) among varieties.
terol and two triterpene dialcohols erythrodiol and uvaol were identified in small amounts. There was a significant differentaition between cultivars in relation to the sterol composition of olive oil samples p 0.05 . The total sterol content of the studied olive oils which ranged from 720.41 HK to 4519.17 BTU mg/kg was found to be above the allowable limits 1000 mg/kg for extra virgin olive oils specified by EU regulations except for 720.41 mg/kg with HK, 788.80 mg/kg with HH, 880.18 mg/kg with HG2 and 980.65 mg/kg with HG3. Additionally, Gemlik oil samples from different locations of Hatay showed significant changes in terms of total sterol content. β-sitosterol was the predominant phytosterol in all oil samples, varying between 76.12 AG and 94. 23 BTU . The studied Turkish oils exhibited high contents of β-sitosterol when compared with those from other olive oils, namely the Tunisian ones worked by Baccouri et al. 17 , the Iranian ones studied by Noorali et al. 38 and the Spanish ones studied by Fernandez-Cuesta et al. 23 . Apparent β-sitosterol content of the all samples, calculated as the sum of the contents of β-sitosterol and other sterols sitostanol, Δ-5,24-stigmastadienol, clerosterol, and Δ-5-avenasterol , were higher than the legal minimum of 93 established by EU regulations. These results were in good harmony with those of Baccouri et al. 17 who found the values of apparent β-sitosterol content of the oils from different Tunisian varieties ranging from 92.63 to 95.33 .
Δ-5-avenasterol, which is the second most plentiful sterol in olive oils, fluctuated between 1.82 BTU and 18.91 AG . These results were in consistent with those of Lukic et al. 14 Hannachi et al. 47 and Damirchi et al. 20 even though Fernandez-Cuesta et al. 23 obtained higher values of the Δ-5-avenasterol content of Spanish olive oils. Campesterol content of the oil samples was below the maximum limit of 4 required by EU regulations, varying between 0.75 OG and 2.90 HHs . Manai-Djebali et al. 2 obtained higher campesterol content of Tunisian olive oils while our values were in agreement with those of the results found by Temime et al. 27 . The content of campesterol in olive oils can show variations according to olive ripening and variety 3 . Stigmasterol is the main sterol associated to the low quality of virgin olive oil 38 . The contents of stigmasterol of oils were lower than those of campesterol required by EU regulations, ranging from 0.58 MG to 1.35 HK . Moreover, Δ-7 stigmastenol and cholesterol contents of all oils were within the legal maximum of 0.5 . The triterpenic dialcohols erythrodiol and uvaol , which are the part of unsaponifiable fraction of olive oil, are analyzed together with the sterol fraction 2 . The percentage of triterpenic dialcohols erythrodiol and uvaol was below the maximum limit of 4.5 required by EU regulations, varying between 0.07 MS and 4. 27 AG . Gorassini et al. 48 determined the erythrodiol and uvaol contents of natural olive oils between 1.6-3.8 using different methods. These findings are similar to our study.

Conclusion
This study showed that olive oils from different varieties Halhalı, Gemlik, Kargaburun, Büyük Topak Ulak, Hasebi, Sarı Ulak in different growing regions of East Mediterranean have significant variations based on chemical properties, fatty acid and sterol compositions, and these results were in accordance with the internationally accepted limits. Gemlik oils obtained from five different locations had significant variations especially in terms of oleic acid, β-sitosterol content, total sterol content. The varieties growing in East Mediterranean should be intensely cultivated and certified with Protected Designation of Origin due to their higher quality properties.