Fruit and Oil Characteristics of Olive Candidate Cultivars from Turkey

This is the first study in Turkey that evaluated olive fruit and oil characteristics of 23 cultivar candidates selected among 393 olive genotypes, on the bases of agronomical characteristics; the candidate cultivars were obtained by cross breeding between standard cultivars (‘Gemlik’, ‘Edinciksu’, ‘Uslu’, ‘Tavşan Yüreği’, ‘Karamürselsu’) and foreign cultivars (‘Lucques’, ‘Belle d’Espagne’ and ‘Manzanilla’). Fruit traits such as fruit weight, flesh to seed ratio, fruit and seed size, as well as oil characteristics such as moisture, oil content, specific absorption at ultraviolet light, free acid content, peroxide value, chlorophyll and fatty acid composition were determined. Fruits and oils of intensely cultivated ‘Gemlik’ and ‘Ayvalık’ olive cultivars were also analyzed to compare them with the new candidate cultivars. According to the oil content, ‘GE067’ (23.30%), ‘GE181’ (23.97%), ‘GE366’ (22.52%), ‘GE417’ (22.16%), ‘GT009’ (24.14%), ‘GT014’ (21.02%), ‘GU247’ (22.48%), ‘GU320’ (27.15%) and ‘GU434’ (21.89%) gave better results, thus having good potential for registration as new cultivars for oil production. ‘GE366’ and ‘GU434’ were determined as the best cultivar candidates for producing dual purpose olives (table olives and oil). ‘GE363’ had the best fatty acid composition, which is an important feature in terms of oxidative stability and disease prevention.


Introduction
The olive tree (Olea europaea L.) is one of the most important fruit trees in the Mediterranean countries.Olive oil and table olives are important components of the Mediterranean diet and are greatly consumed worldwide.The Mediterranean region is the major international olive-growing area, accounting for almost 95% of the world's olive tree plantations.The biggest olive-producing countries in the Mediterranean region are Spain, Italy, Greece and Turkey (FAO, 2012).
Olive cultivars are generally identified by morphological traits regarding habitus and fruit or by oil characteristics (Kaya and Tekintas, 2006).Most of the olive breeding programs have focused to improve the quality of olive oil, but few were initiated in order to obtain quality table olives (León et al., 2011; Ozdemir and Kurultay, 2015).Fruit size is a critical character for selection of olive cultivar candidates (Padula et al., 2008).Selection steps of olive breeding programs lead to achieving outstanding cultivars and eliminate others which do not have the potential to increase quality of produced olive oil or table olives (Sánchez de Medina et al., 2015).Classic breeding programs based on crossing and selection within the progenies were conducted in

Material
In this study, 23 olive candidate cultivars with determined genitors were evaluated (Table 1).These trees were planted at a 1.5 × 3 m distance in olive genotype observation orchard from Ataturk Central Horticultural Research Institute (Yalova/Turkey).The investigated genotypes were chosen on the basis of their high productivity and agronomic characteristics, among 492 olive genotypes.

Fruit analysis
Maturation index of olives was determined according calculation method of Hermoso et al. (1991) by using the color of olive skin and flesh.Fruit weight and flesh to seed ratio were established according to official method TS 774 (1992).Fruit weight was determined by weighting 100 olive fruits.Flesh to seed ratio was calculated by using the ratio of flesh and seed weight of 100 olive fruits.Fruit and seed size were measured by using digital caliper (Mitutoyo, Japan).
Before the oil analysis, olive seeds were removed, after which olives were crushed and dried.Oil of the dried olive paste was extracted by soxhlet apparatus, for at least 8 hours, with petroleum ether extraction at 50 °C (Cemeroglu, 2007).

Obtaining olive oil
Olives were washed without delay; diseased and damaged olives were removed.Then olives were turned into paste by laboratory scale hammer (100 rev/min) and kneader (45 minutes) after which 0.5 kg batches of olive paste was put into press cloth and pressed (250-300 kg/cm 2 ) with a hydraulic press.Liquid phase from press was separated into water and oil phase by using separatory funnel.The obtained oil was centrifuged (8,000 rev/min) and filtered through a coarse filter (20 µm).Finally, oil was filled into dark glass bottles without any air space and stored at 4 °C until analyze.

Oil analysis
Free acid content and peroxide value were assessed by titrimetric methods according to the official methodologies of Turkish Food Codex -Communiqué of Analysis Methods of Olive Oil and Pomace Oil (Anonymous, 2015).For determination of specific absorbance value, 0.5 g of oil were weighted (with 0,0001 accuracy) and dissolved into 50 mL cyclohexane 50 mL.Mixture was put into 1 cm quartz cuvette and its absorbance was measured at 232 and 270 nm with spectrophotometer (Hitachi, Japan).
Chlorophyll content was determined by spectrophotometric method according to Kiritsakis (1998).Olive oil samples were put into quartz cuvette and absorbance values were taken at 630, 670 and 710 nm against carbon tetrachloride.The chlorophyll content was calculated by the following formula: Chlorophyll content (mg /kg) = = [A 670 -(A 630 + A 710 )/2] / 0.901 L where: A = the absorbance value of the oil sample; L = 1 cm length tub.
Fatty acid methyl ester composition was determined by gas chromatography according to Anonymous (2015).0.2 g oil and 10 ml of hexane were put into a vial and shaken.After that, 0.5 ml of a methanolic KOH solution (2N) was added and stirred.0.5 µl were taken from the upper phase and injected into the gas chromatography (Hewlett-Packard, USA).

Statistical analysis
Research plan was established according to completely randomized design and trial was performed in 3 replications.The data obtained throughout 3 years were determined whether there was a significant difference among the characteristics of samples with the analysis of variance.If significant differences were found between the samples' characteristics, they were further evaluated by testing with Fischer LSD test of multiple comparison procedure.The significance value was taken as 0.05.Analyses were performed using the GLM procedure of SAS statistical software package program.Principal component analysis was applied on the obtained data.The average of analysis result of the 3 years data was used in this analysis.Kinship tree was created using NTSYSPC 2.0 software package program, to show the degree of similarity.Genetic similarities were determined by using SAHN cluster analysis of UPGMA (Unweighted Pair-Group Method Algorithm).

Results and Discussion
High oil content and oil quality characteristics, with high fruit weight and flesh to seed ratio are reported as desired traits for new olive cultivar candidates.Fruit and seed dimensional characteristics (width and length), fruit weight and flesh to seed ratio are being used as descriptive fruit characters and thus are required for the new cultivar registration procedure for olive cultivar candidates in breeding studies.Measurements for these fruit characters of candidate cultivars tested within the current experiment are given in Table 2.
Olive maturity is one of the important factors that significantly affects all characters of the fruits and oils (Owen et al., 2000).Therefore, maturity index was determined along with fruit and oil characters.Maturity index and oil and dry matter contents of genotypes are given in Table 3.
In order to define the oil characters of candidate cultivars, free fatty acid ratio, peroxide value, specific absorption value at ultraviolet light and chlorophyll content of oil samples were determined and results are given in Table 4. Free fatty acid ratio, peroxide value and chlorophyll value were between 0.47 ('LE003') and 0.98 ('ME015'); 3.70 ('LT019') and 9.01 meqO2/kg ('ME015'); 0.11 ('GU320') and 0.63 mg/kg ('GE363') (Table 4).There were significant statistical differences among genotypes in terms of peroxide value and chlorophyll content (p<0.05).Olive oil is characterized by high oleic acid content (Ollivier et al., 2003).Free acidity of extra virgin and virgin olive oil expressed as oleic acid, should not be more than 0.8% and 2.0%, respectively (Anonymous, 2015).According to free fatty acid content, 'GU320', GU439, GT005, 'GU434' and 'ME015' fit to virgin olive oil, while the rest of the oil samples fit to extra virgin olive oil category (Anonymous, 2015).Diraman ( 2007) and Kiralan et al. (2009) reported peroxide value of olive oils of different cultivars  Fatty acid composition is one of the key parameters used to characterize olive oils.The unique fatty acid composition of olive oil is distinguished from other vegetable oil with this feature (Lee et al., 1998).Furthermore, distribution is also effective on oxidative stability and nutritional physiology (Owen et al., 2000).Sánchez de Medina et al. (2015) reported that oleic and palmitic acid content in olive oil is critical for the quality and fatty acid composition, thus it has been reported to be an important parameter used to decide the selection of olive genotypes in olive breeding programs.
Oleic acid/linoleic acid and MUFA/PUFA parameter is seen as an important indicator for oxidative stability of olive oil (Kiritsakis et al., 1998).Most studies referring to nutrients needed for a healthy life recommend the consumption of fatty acids in the form of omega-6/omega-3 fatty acid ratio which is desired to be approximately 1 (Stoll, 2001;Simopoulos, 2002), whereas the ratio of dietary habits in Western countries was reported as 15/1 to 16.7/1 (Simopoulos, 2002;Allport, 2007).For olive oils, omega-6/omega-3 fatty acid ratio is depending on LA/LNA ratio (Simopoulos, 2008).In this research, GE363 had a remarkable balance on LA and LNA results, with a ratio of 5.67, which represent an important feature in terms of disease prevention.
Specific absorbance of olive at 232 and 270 nm is reported as a common character used for quality determination (Boskou, 2006).Bıyıklı (2009), Ilyasoglu and Ozcelik (2011) reported specific absorbance of olive oils at 232 and 270 nm to be between 1.95-2.67 and 0.123-0.420respectively.Results of the current research were similar with these literature data.Beltran et al. (2005) determined the chlorophyll content of olive oil to be between 0.50-49.8mg/kg.Ranalli et al. (2000) reported the chlorophyll content of olive oils of an olive genotype (I-77) from three different regions as 8.8-12.7 mg/kg.In this research, chlorophyll content of the olive oil was found to be between 0.11 to 0.63 mg/kg, thus lower than the mentioned literature data.
Major and minor fatty acids compositions of olive oils are given in Tables 5 and 6.The most distinctive character of olive oil is fatty acid composition, which is used for identification and grouping of olive oils (Lee et al., 1998).Many different characteristics related with oxidative stability or nutritional physiology can be calculated by using distribution of fatty acids of olive oil (Kyriakidis and Katsiloulis, 2000).High MUFA and MUFA/PUFA correlated with low LA/LNA values are important for stability of oil against oxidation and for the beneficial effect on consumers (Kiritsakis et al., 1998).Thus SFA, MUFA, PUFA, MUFA/PUFA, LA/LNA and IN values of candidate cultivars were calculated.The obtained values are given in Table 7.
The first component was positively correlated to fruit width, weight and length and negatively to dry matter.The second component was mainly positively associated with chlorophyll, stearic acid and oleic acid and negatively with absorbance value 152 As a result of principal component analysis of determined fruit and oil characteristics, tree diagram representation of component analysis were obtained (Fig. 1); plot of vector loadings on principal components 1 and 2 for fruit and oil characteristics are given in Fig. 2.

Conclusion
High fruit weight and flesh to seed ratio for table olives were preferred from consumers' perspective.So that, according to flesh to seed ratio, 'GU358', 'GE110', 'GE061' proved good potential for registration as table olive cultivar competitors, while according to fruit weight and flesh to seed ratio, 'ME015' and 'BK013' were suitable for table olive registration.Never the less, based on the oil percentage in dry matter, 'GE061' and 'ME015' were not appropriate to register for oil production, whereas 'GU358', 'GE110' and 'BK013' had high oil percentage in dry matter and thus they can be registered for dual purpose or for oil production.According to the overall evaluation of fruit and seed size, oil and free fatty acid content, peroxide value, specific absorption at ultraviolet light and fatty acid composition, 'GE366' and 'GU434' were determined as the best candidate cultivars, their fruits having the ability to be used as raw material for both table olive and olive oil industry.Olive oil of 'GE363' had the highest MUFA (79.30%) and MUFA/PUFA (14.69%) and the lowest LA/LNA (5.67%) values, so that it comes forward for its oxidative stability and consumer nutrition physiology.

Fig. 1 .
Fig. 1.Tree diagram representation of principal component analysis result of fruit and oil characteristics

Table 1 .
Evaluated olive genotypes and their genitors

Table 2 .
Fruit and seed characteristics of tested olive genotypes and standard cultivars Different letters in the same column refers to the statistical difference (p<0.05).

Table 3 .
Maturity index, dry matter and oil content of tested olive genotypes and standard cultivars

Table 4 .
Free fatty acid content, peroxide value, specific absorption at ultraviolet light and amount of chlorophyll in oil of tested olive genotypes and standard cultivars Different letters in the same column refers to the statistical difference (p<0.05).
.51%, respectively.Olive oil of 'GE363' had highest MUFA (79.30%) and MUFA/PUFA (14.69%) and the lowest LA/LN (5.67%) values.Highest PUFA and lowest IN results were detected in olive oils of 'GE067' and 'GU434'.Olive oils of all candidate cultivars tested in the current study had lower IN than cv.'Ayvalık'.from different regions of Turkey between 4.21-20.60and 4.30-8.81meqO2/kg.According to Trade Standard Applying to Olive Oils and Olive-Pomace Oil, peroxide value of extra virgin olive oil

Table 5 .
Major fatty acids in oil (%) of tested olive genotypes and standard cultivars

Table 6 .
Minor fatty acids in oil (%) of tested olive genotypes and standard cultivars Different letters in the same column refers to the statistical difference (p<0.05).