Elucidation of key odorants and sensory propertiesof five different extra virgin olive oils from Turkey by GC-MS-Olfactometry

2.1. Sampling

EVOOs were used in this work. The EVOOs from the Ayvalık (AY), Memecik (ME), Gemlik (GE), Sarı Ulak (SU) and Beylik (BE) cultivars were collected from the South Aegean, North Aegean, South Marmara, West and East Mediterranean Regions in Turkey, respectively. All olives were harvested during the yellowish-purplish period, which we call the ideal harvest time. All the EVOOs provided by the producers were obtained under a three-phase extraction system during the 2014/15 and 2015/16 crop seasons. The olives were crushed with a hammer crusher after leaf separation and washing. They were then subjected to malaxation at 30-35 °C for 30-45 minutes. Then, olive oil, pomace and olive oil mill wastewater were separated from the olive paste with the help of a decanter, and the olive oil obtained was purified from remaining impurities by passing it through a separator with 200 L of water per hour. 500 mL were taken from each sample and then stored in bottles at 4 °C. Quality parameters (QP), volatile compounds and GC-MS-O, aroma extract dilution, and sensory analyses were performed for both crop seasons.

2.2. Quality parameters of samples

The QP detected were free fatty acidity (FFA) (represented as an oleic acid percentage) (IOC, 2017aIOC (International Olive Council). 2017a. Determination of free fatty acids, cold method, COI/T.20/Doc. No 34/Rev.1. https://www.internationaloliveoil.org/wp-content/uploads/2019/11/COI-T.20-Doc.-No-34-Rev.-1-2017.pdf. (Accessed: 04-October-2021).), peroxide value (PV) (represented as meqO₂/kg of oil) (IOC, 2017bIOC (International Olive Council). 2017b. Determination of peroxide value, COI/T.20/Doc. No 35/Rev. 1. https://www.internationaloliveoil.org/wp-content/uploads/2019/11/Method-COI-T.20-Doc.-No-35-Rev.-1-2017.pdf (Accessed 19-October-2021).) and characteristics of ultraviolet absorption at 232 and 270 wavelengths (K₂₃₂ and K₂₇₀) (IOC, 2019IOC (International Olive Council). 2019. Spectrophotometric investigation in the ultraviolet, COI/T.20/Doc. No 19/Rev. 5. https://www.internationaloliveoil.org/wp-content/uploads/2019/11/Method-COI-T.20-Doc.-No-19-Rev.-5-2019-2.pdf (Accessed 30-October-2021).). The samples were analyzed in triplicate.

2.3. Sensory assessment of samples

The sensorial evaluation of the EVOO was performed by an IOC (International Olive Council)-approved panel of fully qualified judges. The samples were sensory analyzed according to the parameters outlined in the IOC approved technique COI/T.20/Doc. no 15 (IOC, 2018IOC (International Olive Council). 2018. Sensory analysis of olive oil - method for the organoleptic assessment of virgin olive oil COI/T.20/Doc. No 15/Rev.10. https://www.internationaloliveoil.org/wp-content/uploads/2019/11/COI-T20-Doc.-15-REV-10-2018-Eng.pdf (Accessed 30-October-2021).). A 15-ml sample was placed in a blue tasting glass. The temperature of the sample was maintained at 28±2 °C. The sensory assessment of the sample was defined using the median of panelists’ scores obtained via sensory analysis.

2.4. Analysis of volatile compounds and GC-MS- Olfactometry conditions

The aroma substances in the EVOO samples were analyzed using the purge and trap extraction technique. Representative tests were performed on the aromatic extract to determine the extraction method’s reliability. The aroma compounds of the EVOOs were extracted according to the method of Amanpour et al. (2016)Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 . The extract was passed through sodium sulfate and concentrated to 0.5 mL at 40 °C in a “Vigreux” distillation column. The concentrated extract was directly injected into GC-FID (Flame Ionization Detector), GC-MS and GC-MS-O systems and the AACs were determined. The extractions were performed in three replicates (Kesen et al., 2013Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 ). The GC with a flame ionization and a mass selective detector (Agilent 5973, USA), and a sniffing port (Gerstel ODP-2, USA) were used in the GC system at 250 ºC. A capillary column (DB-WAX 0.25 mm x 0.4 m x 60 m) was used for the separation of the aroma components. Chemical standards, retention index, and a mass spectral database were used to identify aroma compounds (NIST 98, Wiley 11). The injector and temperature were set to the same parameters as the GC. The substances were quantified in scan mode using a mass range of 29-350 amu and mass spectra were acquired in electron impact mode (energy voltage: 70 eV). Peaks were identified using standard solutions. The internal standard ((4-nonanol) method was used to calculate the volatile concentrations.

2.5. Aroma active compounds of samples

Three qualified sniffers used GC-MS-O to evaluate the AACs. The extract sniffing process was completed in three stages (25 min each). Based on previous research, the AEDA technique was used to determine the FD factors of the AAC (Ozkara et al., 2019Ozkara KT, Amanpour A, Guclu G, Kelebek H, Selli S. 2019. GC-MS-Olfactometric Differentiation of aroma-active compounds in Turkish heat-treated sausages by application of aroma extract dilution analysis. Food Anal. Methods. 12, 729-741. https://doi.org/10.1007/s12161-018-1403-y ). CH₂Cl₂ was used to dilute the EVOOs: 1:1, 1:2, 1:4, … 1:4096. Sniffing continued until no odorant was detected.

2.6. Statistical analysis

Analysis of variance (ANOVA) and Principal component analysis (PCA) were carried out on the Minitab® 17 program (Minitab Inc., State College, PA, USA) to reveal the discrimination pattern of the EVOO samples.

3.1. Quality parameters of samples

The general QP of the samples are shown in Table 1. As seen, the FFA, PV, K₂₃₂ and K₂₇₀ values of the VOOs did not exceed the limit defined for EVOO by IOC (IOC, 2021IOC (International Olive Council). 2021. Trade standard applying to olive oils and olive pomace oils. COI/T.15/NC No 3/Rev. 17. https://www.internationaloliveoil.org/wp-content/uploads/2021/11/COI-T15-NC3-REV-17_ENK.pdf. (Accessed 30-October-2021).). The percentage of FFA in the oils ranged from 0.30 to 0.59 for the 2014 harvest year and from 0.22 and 0.77 for the 2015 harvest year. FFA is known to be the main criterion for classifying VOO. All the samples were determined to contain less than the maximum legal limit of 0.8 (oleic acid %) for EVOO (IOC, 2021IOC (International Olive Council). 2021. Trade standard applying to olive oils and olive pomace oils. COI/T.15/NC No 3/Rev. 17. https://www.internationaloliveoil.org/wp-content/uploads/2021/11/COI-T15-NC3-REV-17_ENK.pdf. (Accessed 30-October-2021).). The PV of the oils ranged from 5.07 to 9.73 meqO₂/kg oil and from 4.59 to 12.33 meqO₂/kg oil, for 2014 and 2015 harvest years, respectively. The results showed that the PV of the samples were below the limit of 20 meqO₂/kg oil as established by the IOC (IOC, 2021IOC (International Olive Council). 2021. Trade standard applying to olive oils and olive pomace oils. COI/T.15/NC No 3/Rev. 17. https://www.internationaloliveoil.org/wp-content/uploads/2021/11/COI-T15-NC3-REV-17_ENK.pdf. (Accessed 30-October-2021).) for classifying EVOO. According to the IOC limit, K₂₃₂ and K₂₇₀ values must be less than 2.50 and 0.22 for EVOO, respectively. The K₂₃₂ and K₂₇₀ values of the oils were determined to be below this legal limit. The results agree with previous studies carried out with cv. AY, MY and GE (Kesen et al., 2013Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 ), AY and ME (Karagoz et al., 2017Karagoz SG, Yilmazer M, Ozkan G, Carbonell-Barrachina AA, Kiralan M, Ramadan M F. 2017. Effect of cultivar and harvest time on C6 and C5 volatile compounds of Turkish olive oils. Eur. Food Res. Technol. 243, 1193-1200. https://doi.org/10.1007/s00217-016-2833-7 ), ME and GE (Köseoğlu et al., 2016Köseoğlu O, Sevim D, Kadiroğlu P. 2016. Quality characteristics and antioxidant properties of Turkish monovarietal olive oils regarding stages of olive ripening. Food Chem. 212, 628-634. https://doi.org/10.1016/j.foodchem.2016.06.027 ). ANOVA analysis was performed to determine the significance of difference according to the varieties of olive oil samples. The difference among the means of the quality parameter results was not found significant at the 95% confidence level.

3.2. Sensory assessment of samples

Sensory analysis is a quality criterion in VOO standards, and evaluating the sensory quality of VOOs comprises assessing positive and negative properties. The results of the sensory assessment of the samples are shown in Figure 1. According to the IOC standard (IOC, 2021IOC (International Olive Council). 2021. Trade standard applying to olive oils and olive pomace oils. COI/T.15/NC No 3/Rev. 17. https://www.internationaloliveoil.org/wp-content/uploads/2021/11/COI-T15-NC3-REV-17_ENK.pdf. (Accessed 30-October-2021).), samples are classified as EVOOs if the median of defects is “0” and the median of fruity is greater than “0”. Sensory assessment showed that the studied oils had no defects and therefore considered as EVOOs. The highest fruity medians were found at 5.3 and 4.95 for the SU EVOO, from 2014 and 2015, respectively. With regards to the bitterness value, the highest medians were detected in the BE EVOO at 4 and 4.4 from 2014 and 2015, respectively. The pungent values were determined at the highest values in the AY (4.0), ME (4.0) and BE (4.0) EVOO from 2014 and in the BE (4.6) EVOO from 2015. According to the IOC standard (IOC, 2018IOC (International Olive Council). 2018. Sensory analysis of olive oil - method for the organoleptic assessment of virgin olive oil COI/T.20/Doc. No 15/Rev.10. https://www.internationaloliveoil.org/wp-content/uploads/2019/11/COI-T20-Doc.-15-REV-10-2018-Eng.pdf (Accessed 30-October-2021).) the term, “robust” can be used when the median of the positive attribute is more than “6.0”, “medium” can be used when the median of the attribute is between “3.0” and “6.0” and “delicate” can be used when the median of attribute is less than “3.0”. In this research, all samples were characterized as medium for fruity properties from both harvest years. Regarding the the bitterness value, the BE EVOO was classified as medium from 2014 and the AY, ME and BE EVOOs were classified as medium from 2015. The other samples were characterized as delicate. When we look at the results of pungent values, we can characterize the samples as medium for both years, except for the SU EVOO. SU EVOO was classified as delicate.

3.3. Volatile compounds of samples

Table 2 shows the identified and classified (µg/kg) volatile components in the samples. As shown in Table 2, a total of 52, 57, 51, 57 and 54 compounds, including aldehydes, alcohols, terpenes, acids, volatile phenols, ketones, esters, lactones, hydrocarbons and furans, were qualitatively and quantitatively identified in AY, ME, GE, SU and BE EVOO, respectively. GC-MS chromatograms of EVOOs are shown in Figure 2. As seen from these chromatograms, the most volatile components were found in BE EVOO. The highest amount of total volatile compounds (45364 and 31990 µg/kg) was determined in the BE EVOO, for 2014 and 2015, respectively. It was followed by the AY EVOO (34890.1 µg/kg) from 2014 and the SU EVOO (15282 µg/kg) from 2015. The lowest volatile compounds were found in the GE EVOO (20947.8 and 8677.8 µg/kg) from 2014 and 2015, respectively. The majority of the volatile components found in this research had been previously identified in the VOOs of the same and different olive cultivars (Aparicio and Morales, 1998Aparicio R, Morales M T. 1998. Characterization of olive ripeness by green aroma compounds of virgin olive oil. J. Agric. Food Chem. 46, 1116-1122. https://doi.org/10.1021/jf970540o ; Vichi et al., 2007Vichi S, Guadayol JM, Caixach J, Tamames EL, Buxaderas S. 2007. Comparative study of different extraction techniques for the analysis of virgin olive oil aroma. Food Chem. 105, 1171−1178. https://doi.org/10.1016/j.foodchem.2007.02.018 ; Baccouri et al., 2008Baccouri O, Bendini A, Cerretani L, Guerfel M, Baccouri B, Lercker G, Zarrouk M, Ben Miled D D. 2008. Comparative study on volatile compounds from Tunisian and Sicilian monovarietal virgin olive oils. Food Chem. 111, 322−328. https://doi.org/10.1016/j.foodchem.2008.03.066.; Giuffrè et al., 2019Giuffrè A M, Capocasale M, Macrì R, Caracciolo M M, Zappia C, Poiana M. 2019. Volatile profiles of extra virgin olive oil, olive pomace oil, soybean oil and palm oil in different heating conditions. LWT Food Sci. Technol. 17, 108631. https://doi.org/10.1016/j.lwt.2019.108631 ; Žanetić et al., 2021Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 ). Among the volatile compounds detected in different EVOOs, aldehydes were identified and quantified as the major components with regard to the volatile part, followed by alcohols. The lipoxygenase pathway is activated during the olive oil extraction process, resulting in the release of enzymes. Aldehyde compounds are produced by the hydroperoxide lyase enzyme, which is then reduced into alcohols by the alcohol dehydrogenase enzyme in VOOs (Amanpour et al., 2016Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 ). C5 and C6 aldehydes and alcohols are the four most common chemical groups of the 18 components which contribute positively to the aroma composition of VOOs from the positive sensory properties (Procida et al., 2016Procida G, Cichelli A, Lagazio C, Conte L S. 2016. Relationships between volatile compounds and sensory characteristics in virgin olive oil by analytical and chemometric approaches. J. Sci. Food Agric. 96, 311-318. https://doi.org/10.1002/jsfa.7096 ). As previously stated by Issaoui et al. (2015)Issaoui M, Gharbi I, Flamini G, Cioni P L, Bendini A, Gallina Toschi T, Hammami M. 2015. Aroma compounds and sensory characteristics as biomarkers of quality of differently processed Tunisian virgin olive oils. Int. J. Food Sci. 50, 1764-1770. http://dx.doi.org/10.1111/ijfs.12830 , Caporaso et al. (2016)Caporaso N. 2016. Virgin olive oils: environmental conditions, agronomical factors and processing technology affecting the chemistry of flavor profile. J. Food Chem. Nanotech. 2, 21-31. https://doi.org/10.17756/jfcn.2016-007 and Žanetić et al. (2021) Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 the volatile component profile of VOOs is affected by the region where it is grown, the geographical origin, the pedoclimatic conditions, the variety, the extraction systems and VOO storage conditions. It was also seen in the study that the volatile component profile changed according to year, variety and region.

Aldehydes. A total of 7, 8, 4, 11 and 12 aldehydes and 7, 8, 3, 10 and 12 aldehydes were identified in AY, ME, GE, SU and BE EVOO from 2014 and 2015, respectively. The primary aldehyde compounds in EVOO were (E)-2-hexenal and hexanal (Table 2). Total concentrations of aldehydes were found in the AY EVOO at 14055.7 and 5440.1 µg/kg, in the ME EVOO at 14722.2 and 6277.5 µg/kg, in the GE EVOO at 3465.8 and 993 µg/kg, in the SU EVOO at 20894.8 and 8194.4 µg/kg and in the BE EVOO at 27598.9 and 17346 µg/kg, for the 2014 and 2015 seasons, respectively. According to the results, the aldehyde compounds were higher in 2014 than in 2015. In the SU EVOO (E)-2-hexenal was determined to be the highest aldehyde compound with 14280 and 5265 µg/kg, and it was followed by the ME EVOO with 12363 and 5145 µg/kg, for 2014 and 2015, respectively. In the BE EVOO hexanal was found to be the highest aldehyde compound with 8753 and 6312 µg/kg, and it was followed by the AY EVOO with 4790 and 1091 µg/kg, and ME EVOO 886 and 468 µg/kg, for 2014 and 2015, respectively. It was reported by other authors (Kesen et al., 2014Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 ; Sacchi et al., 2015Sacchi R, Caporaso N, Paduano A, Genovese A. 2014. Industrial-scale filtration affects volatile compounds in extra virgin olive oil cv. Ravece. Eur. J. Lipid Sci. Technol. 117, 2007-2014. https://doi.org/10.1002/ejlt.201400456 ; Ben Brahim et al., 2018Ben Brahim S, Amanpour A, Chtourou F, Kelebek H, Selli S, Bouaziz M. 2018. Gas chromatography-mass spectrometry-olfactometry to control the aroma fingerprint of extra virgin olive oil from three Tunisian cultivars at three harvest times. J. Agric. Food Chem. 66, 2851-2861. https://doi.org/10.1021/acs.jafc.7b06090 ; Giuffrè et al., 2019Giuffrè A M, Capocasale M, Macrì R, Caracciolo M M, Zappia C, Poiana M. 2019. Volatile profiles of extra virgin olive oil, olive pomace oil, soybean oil and palm oil in different heating conditions. LWT Food Sci. Technol. 17, 108631. https://doi.org/10.1016/j.lwt.2019.108631 ; Žanetić et al., 2021Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 ) that (E)-2-hexenal and hexanal are common aldehydes in many VOOs, including AY, GE, ME, Halhalı, Nizip Yağlık, and Kilis Yağlık from Turkey, Mari from Iran, Jemri, Touffehi and Fakhari from Tunisian, Arbequina, Cornicabra, Morisca, Picolimon, Picudo and Picual from Spain, and Ravece from Italy. According to studies, the percentage of C6 aldehydes, particularly (E)-2-hexenal, increased during olive ripening, which was primarily detected when the olive fruit skin color changed from yellow-green to purple (Ben Brahim et al., 2018Ben Brahim S, Amanpour A, Chtourou F, Kelebek H, Selli S, Bouaziz M. 2018. Gas chromatography-mass spectrometry-olfactometry to control the aroma fingerprint of extra virgin olive oil from three Tunisian cultivars at three harvest times. J. Agric. Food Chem. 66, 2851-2861. https://doi.org/10.1021/acs.jafc.7b06090 ). The amount of hexanal. which was the second major aldehyde in the samples, mostly decreased with maturation. In this study, the results are in agreement with these reports, that our samples were harvested during the yellowish-purplish period, which we consider the ideal harvest time. Among the aldehydes, (E)-2-hexenal and hexanal are responsible for the positive green sensory attributes in EVOO. The results showed that our samples’ positive sensory attributes are in accordance with this criterion.

Alcohols. Alcohols are associated with positive sensory properties such as green, bitter, fruity aromatic, and they present weaker sensory attributes than aldehydes (Žanetić et al., 2021Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 ). Alcohols produced by the ADH enzyme, are found in plants and are responsible for the production of volatile alcohols which contribute to the aroma of VOO (Kesen et al., 2014Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 ). A total of 12, 16, 14, 16 and 15 alcohols and 16, 16, 11, 14 and 15 alcohols were determined in the AY, ME, GE, SU and BE EVOOs in the 2014 and 2015 seasons, respectively. In all EVOO samples (Table 2) alcohols were determined to be the second main group of volatile compounds, as confirmed in previous studies (Kesen et al., 2014Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 ; Karagoz et al., 2017Karagoz SG, Yilmazer M, Ozkan G, Carbonell-Barrachina AA, Kiralan M, Ramadan M F. 2017. Effect of cultivar and harvest time on C6 and C5 volatile compounds of Turkish olive oils. Eur. Food Res. Technol. 243, 1193-1200. https://doi.org/10.1007/s00217-016-2833-7 ; Žanetić et al. 2021Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 ). The highest amount of alcohols was found in the first year, which was likely due to increased ADH enzyme activity, and determined in the ME EVOO at 6767.1 µg/kg, followed by the SU EVOO at 5820.9 µg/kg, and the GE EVOO at 5310.5 µg/kg, the BE EVOO at 5193 µg/kg and the AY EVOO at 5094.0 µg/kg. The second year, the highest total amount of alcohols was found in the BE EVOO at 4108 µg/kg, followed by SU EVOO at 3678 µg/kg, the ME EVOO at 3294.8 µg/kg, the GE EVOO at 3038 µg/kg and the AY EVOO at 2587.5 µg/kg. The results showed that (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol and 1-hexenol were the dominant C6 alcohols in all the analyzed samples. These results are in agreement with other researchers (Baccouri et al., 2008Baccouri O, Bendini A, Cerretani L, Guerfel M, Baccouri B, Lercker G, Zarrouk M, Ben Miled D D. 2008. Comparative study on volatile compounds from Tunisian and Sicilian monovarietal virgin olive oils. Food Chem. 111, 322−328. https://doi.org/10.1016/j.foodchem.2008.03.066.; Karagoz et al., 2017Karagoz SG, Yilmazer M, Ozkan G, Carbonell-Barrachina AA, Kiralan M, Ramadan M F. 2017. Effect of cultivar and harvest time on C6 and C5 volatile compounds of Turkish olive oils. Eur. Food Res. Technol. 243, 1193-1200. https://doi.org/10.1007/s00217-016-2833-7 ). The contents of (Z)-3-hexen-1-ol main alcohols in the ME EVOO were 1625 µg/kg and 1115 µg/kg in the BE EVOO from 2014 and 2015, respectively. (Z)-3-hexen-1-ol is the most prominent green note, and in our study this chemical appears to have a green-leaf characteristic similar to freshly cut grass. (E)-2-hexen-1-ol were determined to be the predominant alcohols in the ME EVOO (2318 and 1199 µg/kg); while 1-hexenol was found to be the most abundant alcohol in the GE EVOO (1618 and 1033 µg/kg) for both years.

Terpenes. Terpenes (dl-Limonene, styrene, β-ocimene, α-copaene, (E)-α-bergamotene, (Z,E)-α-farnesene, β-sesquiphellandrene, α-muurolene and α-farnesene) were found to be the third most abundant group of volatile compounds in the samples, with total amounts of 6136 µg/kg, 2593.2 µg/kg, 5029.4 µg/kg, 3857.2 µg/kg and 7711.7 µg/kg in 2014, and 2551.8 µg/kg, 1438.5 µg/kg, 1711.3 µg/kg, 686.7 µg/kg and 4297.3 µg/kg in the AY, ME, GE, SU and BE EVOO from 2015, respectively. A total of 7, 7, 8, 8 and 6 terpenes and 8, 6, 4, 6 and 5 terpenes were found in the AY, ME, GE, SU and BE EVOO from 2014 and 2015, respectively. The highest amounts of terpenes were determined in the BE EVOO from both years. In the AY (4525 and 1675 µg/kg) and GE (4200 and 1240 µg/kg) EVOO α-farnesene was determined to be the prominent terpene from both years. α-Copaene was found as the highest terpene for the ME (894 and 744 µg/kg) and for the BE (3022 and 3259 µg/kg) EVOO from 2014 and 2015. β-ocimene was identified as the highest terpene for the SU EVOO (2654 and 239 µg/kg) from both years. These terpenes were also detected in Turkish VOOs (Kaftan and Elmaci, 2011Kaftan A, Elmaci Y. 2011. Aroma Characterization of virgin olive oil from two Turkish olive varieties by SPME/GC/MS. Int. J. Food Prop. 14, 1160-1169. https://doi.org/10.1080/10942910903453371 ; Kesen et al., 2013Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 ; Guclu et al., 2016Guclu G, Sevindik O, Kelebek H, Selli S. 2016. Determination of volatiles by odor activity value and phenolics of cv. Ayvalik early-harvest olive oil. Foods. 5 (3), 46. https://doi.org/10.3390/foods5030046 ), Greek VOOs (Issaoui et al., 2015Issaoui M, Gharbi I, Flamini G, Cioni P L, Bendini A, Gallina Toschi T, Hammami M. 2015. Aroma compounds and sensory characteristics as biomarkers of quality of differently processed Tunisian virgin olive oils. Int. J. Food Sci. 50, 1764-1770. http://dx.doi.org/10.1111/ijfs.12830 ), Tunisian VOOs (Ben Brahim et al., 2018Ben Brahim S, Amanpour A, Chtourou F, Kelebek H, Selli S, Bouaziz M. 2018. Gas chromatography-mass spectrometry-olfactometry to control the aroma fingerprint of extra virgin olive oil from three Tunisian cultivars at three harvest times. J. Agric. Food Chem. 66, 2851-2861. https://doi.org/10.1021/acs.jafc.7b06090 ) and Iranian VOO (Amanpour et al., 2016Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 ). Kelebek et al. (2015)Kelebek A, Kesen S, Selli S. 2015. Comparative study of bioactive constituents in Turkish olive oils by LC-ESI/MS/MS. Int. J. Food Prop. 18, 10, 2231-2245. https://doi.org/10.1080/10942912.2014.968788 reported that terpenes mostly affected the varieties of VOOs. The results of our study support this assertion.

Acids. Fourteen acid components were found in the studied samples. A total of 7, 11, 13, 10 and 11 acids and 8, 10, 6, 11 and 12 acids were detected in the AY, ME, GE, SU and BE EVOO from 2014 and 2015, respectively. Kesen et al. (2013)Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 determined acetic acid, nonanoic acid, and decanoic acid as the major acids in AY, GE, ME VOOs, respectively. Acetic acid was found in the highest concentration in Mari VOO by Amanpour et al. (2016)Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 , and acetic acid was identified as a major acid in Tunisian and Sicilian VOOs by Baccouri et al. (2008)Baccouri O, Bendini A, Cerretani L, Guerfel M, Baccouri B, Lercker G, Zarrouk M, Ben Miled D D. 2008. Comparative study on volatile compounds from Tunisian and Sicilian monovarietal virgin olive oils. Food Chem. 111, 322−328. https://doi.org/10.1016/j.foodchem.2008.03.066.. In our study, the most representative acid was hexadecanoic acid with 1104 and 234 µg/kg, 145 and 143 µg/kg, 153 and 248 µg/kg and 309 and 734 µg/kg, in the AY, ME, GE and SU EVOO from 2014 and 2015, respectively. Hexadecanoic acid (375 µg/kg) and hexanoic acid (2522 µg/kg) were the highest acids in the BE EVOO from 2014 and 2015, respectively.

Volatile Phenols. Five volatile phenols, namely guaiacol, phenol, p-cresol, 4-ethyl-phenol and 2,4-di-tert-butylphenol, were identified in the studied EVOOs. They are generally responsible for the bitter and pungent attributes of VOOs (Amanpour et al., 2016Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 ). A total of 4, 4, 4, 4 and 3 volatile phenols and 4, 4, 0, 0 and 3 volatile phenols were determined in the AY, ME, GE, SU and BE EVOO from 2014 and 2015, respectively. The highest total volatile phenols were found in the AY EVOO (192.9 and 124.8 µg/kg), followed by the BE EVOO (162.7 and 114.8 µg/kg), from 2014 and 2015, respectively. Guaiacol, phenol and 4-ethyl-phenol were detected in Turkish VOOs (Kesen et al., 2013Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 ) and Iranian VOO (Amanpour et al., 2016Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 ).

Ketones. A total of 4, 3, 3, 3 and 1 ketones and 4, 2, 2, 3 and 1 ketones were identified in the AY (1415 and 571 µg/kg), ME (800 and 479 µg/kg), GE (2347 and 1288 µg/kg), SU (1236 and 553 µg/kg) and BE (1484 and 1090 µg/kg) EVOO from 2014 and 2015, respectively. 3-methyl-3-buten-2-one was detected in the AY, ME, GE and SU EVOO from both years. 2-pentanone was found only in the GE EVOO from both years. 1-penten-3-one was determined in the AY, ME, SU and BE EVOO from both years. 2-methyl-3-buten-2-one was identified only in the AY EVOO from both years. Acetophenone were detected in the AY and SU EVOO from both years and in the ME and GE EVOO from 2014. It was reported by Kalua et al. (2007)Kalua C M. Allen M S, Bedgood D R, Bishop A G, Prenzler PD, Robards K. 2007. Olive oil volatile compounds, flavour development and quality: A critical review. Food Chem. 100, 273-286. https://doi.org/10.1016/j.foodchem.2005.09.059 that especially the short ketones are responsible for the positive sensory attributes in VOOs.

Esters. A total of 3, 3, 2, 2 and 1 esters were determined in the AY, ME, GE, SU and BE EVOOs from both years. Hexyl acetate, (Z)-3-hexenyl acetate and methyl salicylate esters were determined in the studied EVOOs. The highest total esters were found in the AY EVOO with 4042.1 and 1500.7 µg/kg, followed by the GE EVOO with 3646.7 and 1019.1 µg/kg and the ME EVOO with 2850.7 and 844.3 µg/kg, from 2014 and 2015, respectively. Esters are accountable for the pleasant fruity and flowery odor of the olive fruits (Kelebek et al., 2015Kelebek A, Kesen S, Selli S. 2015. Comparative study of bioactive constituents in Turkish olive oils by LC-ESI/MS/MS. Int. J. Food Prop. 18, 10, 2231-2245. https://doi.org/10.1080/10942912.2014.968788 ). These compounds were also detected in the AY, ME, GE VOOs in previous studies by Kesen et al. (2013)Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 , Karagoz et al. (2017)Karagoz SG, Yilmazer M, Ozkan G, Carbonell-Barrachina AA, Kiralan M, Ramadan M F. 2017. Effect of cultivar and harvest time on C6 and C5 volatile compounds of Turkish olive oils. Eur. Food Res. Technol. 243, 1193-1200. https://doi.org/10.1007/s00217-016-2833-7 , Guclu et al. (2016)Guclu G, Sevindik O, Kelebek H, Selli S. 2016. Determination of volatiles by odor activity value and phenolics of cv. Ayvalik early-harvest olive oil. Foods. 5 (3), 46. https://doi.org/10.3390/foods5030046 and in Jemri, Touffehi and Fakhari OOs by Ben Brahim et al. (2018)Ben Brahim S, Amanpour A, Chtourou F, Kelebek H, Selli S, Bouaziz M. 2018. Gas chromatography-mass spectrometry-olfactometry to control the aroma fingerprint of extra virgin olive oil from three Tunisian cultivars at three harvest times. J. Agric. Food Chem. 66, 2851-2861. https://doi.org/10.1021/acs.jafc.7b06090 . Our results are in agreement with these studies.

Lactones, hydrocarbons, furans. The other minor volatile compounds in the samples were lactones, hydrocarbons and furans. Lactone (γ-caprolactone) was identified in the ME, GE and BE EVOO; hydrocarbon (3-ethyl-1,5-octadiene) was detected in the all EVOO samples and furan (2-pentylfuran) was determined only in the BE EVOO. Kesen et al. (2014)Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 reported that lactones contribute to the characteristic fruity odors of VOOs.

3.4. Aroma active compounds of samples

Table 3 shows the results of AACs detected using AEDA, as well as their FD values and odor descriptions. AAC odor intensities were measured as FD factors and ranged from 4 to 2048. Aromatic extracts of the samples revealed a total of 29 AACs. Aromatic extracts of AY, ME, GE, SU, and BE EVOOs contained a total of 22, 21, 18, 22 and 21 AACs, respectively.

One of the most important AACs which affects the overall composition of VOO is aldehydes. Ten odorants were defined as aroma active aldehydes (Table 3). The most dominant was (E)-2-hexenal, which had a cut grass-green odor and an FD factor ranging from 128 to 2048. The first year, FD factor was determined at the highest level with 2048 in ME, SU and BE EVOOs. AY EVOO followed it with an FD factor of 1024. The second year, it was determined in AY EVOO with the highest 1024 FD factor. The FD factor was determined as 512 in ME, SU and BE EVOO s. (E)-2-hexenal was followed by hexanal with a green-cut grass odor and an FD factor ranging from 128 to 2048. In 2014 and 2015, the FD factor was determined at the highest level with 2048 in BE EVOO. Guth et al. (1991)Guth G, Grosch W A. 1991. Comparative-study of the potent odorants of different virgin olive oils. Eur J Lipid Sci. Tech. 93, 335−339. https://doi.org/10.1002/lipi.19910930903 states that (E)-2-hexenal contributes to the aroma of VOOs with its strong odor. Solinas et al. (1988)Solinas MF, Angerosa F, Marsili V. 1988. Research of some flavor components of virgin olive oil in relation to olive varieties. Riv. Ital. delle Sostanze Grasse. 65, 361−368. also suggests that (E)-2-hexenal can be used for distinguishing a monovariatel VOO. Other aldehydes are (E)-2-pentenal, (Z)-3-hexenal, 3-hexenal, heptanal, octanal, nonanal. (E,E)-2.4-hexadienal and (E,E)-2.4-heptadienal were determined to impart fresh-plant, fresh-cut grass, pleasant-cut grass, green-oily, oily-floral, oily-citrusy, oily and oily odors, respectively. The detection threshold of aldehydes is low. It is known that aldehydes have a significant effect which can change the general properties of VOOs, even at low detection thresholds and low concentrations (Kesen et al., 2013Kesen S, Kelebek H, Sen K, Ulas M, Selli S. 2013. GC-MS-Olfactometric characterisation of the key aroma compounds in Turkish olive oils by application of the aroma extract dilution analysis. Int. Food Res. J. 54, 1987-1994. https://doi.org/10.1016/j.foodres.2013.09.005 ). It can be seen from previous studies that the detected aldehydes are commonly found in many VOOs (Guth et al., 1991Guth G, Grosch W A. 1991. Comparative-study of the potent odorants of different virgin olive oils. Eur J Lipid Sci. Tech. 93, 335−339. https://doi.org/10.1002/lipi.19910930903 ). The results are consistent with the studies performed.

Alcohols are the second most important aroma active compounds which influence the VOO’s overall composition. Aldehydes have a higher sensory value than alcohols. The FD factors of the samples varied from 8 to 1024. As aroma active alcohols, 10 odorants were detected in the samples (Table 3). Among them, (E)-2-hexen-1-ol, (Z)-3-hexen-1-ol and 1-hexanol were the most dominant with grassy-cool, herbal-cut grass and floral-herbal odors and an FD factor ranging from 32 to 1024. In 2014, (E)-2-hexen-1-ol was determined at the highest level in ME and GE EVOOs with an FD factor of 1024. It was followed by SU EVOO with an FD factor of 512. In 2015, the highest FD value was determined for GE and SU EVOOs with an FD factor of 512. The (Z)-3-hexen-1-ol aroma-active compound was determined at the highest level with 512 FD factor in AY, ME and SU EVOOs from 2014. In 2015, it was determined at the highest level in BE EVOO with an FD factor of 1024. The results are in accordance with other studies (Kesen et al., 2014Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 ; Amanpour et al., 2016Amanpour A, Kelebek H, Kesen S, Selli S. 2016. Characterization of aroma-active compounds in Iranian cv. Mari olive oil by aroma extract dilution analysis and GC-MS-olfactometry. J. Am. Oil Chem. Soc. 93 (12), 1595-1603. https://doi.org/10.1007/s11746-016-2906-6 ).

Aldehydes and alcohols are affected according to the region where the olive is grown, especially cis-3-hexenal, cis-3-hexenol, hexanal, hexanol, trans-2-hexenal, trans-3-hexenol and trans-2-hexenol. (Vicchi et al., 2003Vichi S, Guadayol JM, Caixach J, Tamames EL, Buxaderas S. 2007. Comparative study of different extraction techniques for the analysis of virgin olive oil aroma. Food Chem. 105, 1171−1178. https://doi.org/10.1016/j.foodchem.2007.02.018 ; Žanetić et al., 2021Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 ).

Five terpenes were determined in the study: α-farnesene, β-ocimene, α-copaene, dl-Limonene and α-pinene (Table 3). α-farnesene (floral, green-plant odor) was detected as the highest aroma-active terpene with an FD factor of 1024. The first year, it was found to be the highest in AY and GE EVOOs; the second year it was found at its highest in GE EVOO. β-ocimene was determined to have a fruity-leafy odor with an FD factor of ≤ 512. In 2014, all the samples were determined to have an FD factor which ranged from 32 to 512. In 2015, the FD factor decreased, and ranged from 16 to 64. α-Farnesene aroma-active compound was previously determined in Kilis Yağlık Turkish VOO by Kesen et al. (2014)Kesen S, Kelebek H, Selli S. 2014. Characterization of the key aroma compounds in turkish olive oils from different geographic origins by application of aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 62, 391-401. https://doi.org/10.1021/jf4045167 , and has also been determined as a key odorant in Moroccan green olives (Iraqi et al., 2005Iraqi R, Vermeulen C, Benzekri A, Bouseta A, Collin S. 2005. Screening for key odorants in Moroccan green olives by gas chromatography-olfactometry/aroma extract dilution analysis. J. Agric. Food Chem. 53, 1179−1184. https://doi.org/10.1021/jf040349w ).

Esters are associated with sweet and fruity sensory properties. Two esters, hexyl acetate with fruity-plant odor and (Z)-3-hexenyl acetate with fruity-green odor, were identified. (Z)-3-hexenyl acetate was detected only in ME and GE EVOOs. The FD factor was ≤ 1024 in 2014, and ≤ 512 in 2015. Hexyl acetate was detected in AY, ME and GE EVOOs (Table 3). Žanetić et al. (2021)Žanetić M, Jukić Špika M, Ožić MM, Brkić Bubola K. 2021. Comparative study of volatile compounds and sensory characteristics of Dalmatian monovarietal virgin olive oils. Plants 10, 1995. https://doi.org/10.3390/plants10101995 stated that hexyl acetate caused significant differences in the differentiation of Dalmatian monovariatel EVOO.

Hexanoic acid was detected as a butter-cheesy odor in AY and BE EVOOs with an FD factor of ≤ 64 and only in 2015 (Table 3).

The most powerful AACs in the extracts were identified using the FD factor for AY EVOO hexanal, (E)-2-hexenal and α-farnesene (FD:1024) in 2014 and (E)-2-hexenal in 2015, for ME OO (E)-2-hexenal (FD:2048) in 2014 and (E)-2-hexenal (FD: 512) in 2015, for GE EVOO (Z)-3-hexenyl acetate, (E)-2-hexen-1-ol and α-farnesene (FD:1024) in 2014 and (Z)-3-hexenyl acetate, (E)-2-hexen-1-ol and 1-hexanol (FD: 512) in 2015, for SU EVOO (E)-2-hexenal (FD:2048) in 2014 and hexanal, (E)-2-hexenal, (Z)-3-hexen-1-ol and (E)-2- hexen-1-ol (FD:512) in 2015, for BE EVOO hexanal and (E)-2-hexenal (FD:2048) in 2014 and hexanal in 2015.

3.5. Principal component analysis

Total volatile compounds were used to construct the PCA models for the EVOOs of from different varieties of olives. The PCA model was formed with 4 components. PCA score plot and biplot are illustrated in Figure 3 based on 2 main components. The first major component explains 46.7% of the total variance, and the second major component explains 21.9% of the total variance. When the classification pattern of EVOO samples was examined, it was seen that EVOOs obtained in the first crop season were grouped and separated from the 2nd crop season except for the BE variety of EVOOs. The PCA biplot was used to establish the relationship between the varieties and total volatiles in the EVOOs.

Total ES is negatively correlated on PC1, while ALC, TER, PHE, KET, ES and HYD are positively correlated on PC2. SU1, ME1, GE1 and AY1 were characterized by T-ALC, T-ES, T-TER, T-PHE and T-KET groups of volatile compounds. T-ALD, T-TER, T-LAC and T-HYD characterized BE1 EVOOs, while T-AC and T-FU characterized BE2 EVOOs. Total volatile compounds were not found to be effective for the characterization of SU2, GE2 or AY2 EVOOs.