*3.1. Physico-Chemical Parameter of the Two Major Olive Cultivars of Southern Peloponnese*

It is well established that the quality parameters of olive oil are mainly altered by factors causing injuries to the olive fruits such as olivefly attacks, improper methods during olive harvesting as well as poor post-extraction conditions (e.g., inappropriate storage and packaging) [19]. Free fatty acid, peroxide value and spectrophotometric absorption were examined in the studied olive oils. Mean values for each analytical parameter, as well as minimum and maximum values of the measured parameters are reported in Table 1. It is clear that all analysed samples obtained from the two examined cultivars in the southern region of Peloponnese (cv. Koroneiki and cv. Mastoides), are classified in the highest quality category as "extra virgin olive oil (EVOO)" as they fullfil the demands of the current EU Regulation 2568/91 [18]. In particular, olive oils of both cultivars exhibited low mean values on their qualitative parameters. The mean free fatty acid was 0.34% for olive oils of cv Koroneiki and 0.39% for olive oils of cv. Mastoides. Respectively, the mean peroxide value for cv. Koroneiki was 7.24 meqO<sup>2</sup> kg−<sup>1</sup> and 6.96 meqO<sup>2</sup> kg−<sup>1</sup> for olive oils of cv. Mastoides. Likewise, K<sup>232</sup> and K<sup>268</sup> mean values were quite below the limit set by the EU Regulation 2568/91. The results depict the overall high quality of south Peloponesse olive oil production, one of the most important olive-growing regions in Greece.


Results are expressed as means ± standard deviation (SD). N = 112.

*3.2. Evaluation and Discrimination of the Two Examined Cultivars of Southern Peloponnese According to Their Fatty Acid Composition*

Fatty acid composition is a crucial parameter for the quality and characterization of an olive oil [20]. Because of the fact that the fatty acid content is a fundamental parameter for the determination of the nutritional properties of olive oil, the description of a specific cultivar on the basis of their fatty acid composition is of utmost importance. As a result, many researchers have used fatty acid composition in order to group olive oils according to the origin of the cultivar [21–23].

In the present study, the GC-FID analysis of the 112 olive oil samples from Koroneiki and Mastoides cultivars showed their complete fatty acid composition. As shown in Table 2, all values of the thirteen fatty acids identified, were in conformity to the normal range expected for olive oil category for both cultivars. Generally, olive oils of Koroneiki cultivar had a mean value of 76.70% for the mono-unsaturated oleic acid (C18:1) compared to olive oils of Mastoides cultivar which had a mean value of 75.93% (*p* < 0.05). Moreover, olive oils of Koroneiki presented a higher concentration with respect to the poly-unsaturated linolenic acid (C18:3) with a mean value of 0.68% compared to cv. Mastoides (0.55%). On the other hand, olive oils of cv. Mastoides were characterized by a clearly higher concentration in heptadecanoic acid (C17:0) with a mean value at 0.14% and in heptadecenoic

acid (C17:1) with a mean value at 0.25% compared to the olive oils of cv. Koroneiki which had almost a three-fold lower concentration, with mean values 0.05% and 0.08%, respectively. No differences were observed for the following fatty acids: myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and linoleic acids (C18:2) as shown in Table 2.


**Table 2.** Fatty acid profile of cv. Koroneiki and cv. Mastoides cultivated in southern Peloponnese.

Results are expressed as means ± standard deviation (SD). n.s = not-significant. The statistical significance level was set at *p* < 0.05.

As mentioned in the introduction, there is one relevant publication by Stefanoudaki et al. [17] where the authors examined the same cultivars in the island of Crete. They reported that olive oils of Koroneiki cultivar were characterized by lower concentrations of oleic (C18:1) and heptadecanoic acids (C17:0) and higher concentrations of linoleic (C18:2) and palmitic acids (C16:0). Those differences can be explained by the fact that apart from the olive cultivar other secondary factors, mainly environmental, (e.g., different climatic conditions such as temperature, rainfall, humidity at each growing site), have a significant effect on the composition of the fatty acid profile [24,25].

As shown in Table 2, the fatty acid composition data of the 112 olive oil samples were subjected to analysis of variance. It was revealed that, apart from C14:0, C16:0, C16:1, and C18:2, substantial differences were observed between Koroneiki and Mastoides cultivars in all the rest analyzed fatty acids (*p* < 0.05). Additionally, principal component analysis (PCA) on fatty acid composition data was performed to confirm and enhance the classification according to the cultivar. PCA can be used to decrease the initial variables into a limited number of new variables (principal components) describing most of the variation in the originals. The main purpose of the key factor analysis, taken together, is to define related variables. The first two principal components are significant and explain approximately the 84% of the variation in the data. Thus, based on PCA in Figure 1 we showed the score plot of PCA for cv. Koroneiki and cv. Mastoides according to their fatty acid composition. In this case we found that most of the points for K are pointed to the left of PC1, meaning that K has large negative loadings on component 2. On the other hand, points for M are presented on the right of PC1, meaning that M has large positive loadings on component 1. The K and M regions are therefore independent of each other and the chemical properties studied are also independent and the regions are affected by them. Hence, the application of the PCA algorithm to the fatty acid data revealed a discrete separation between the two cultivars, by creating two distinctive clusters. The results are in agreement with studies by Stefanoudaki et al. where they concluded that fatty acid compositional data of Koroneiki and Mastoides cultivar showed significant potential for olive oil classification [17].

**Figure 1.** Score plot of principal component analysis (PCA) for cv. Koroneiki and cv. Mastoides obtained from olive trees in southern Peloponnese according to their fatty acid composition. K corresponds to Koroneiki olive oils (blue dots) and M to Mastoides olive oils (red dots).

There are other studies focusing on olive oil compounds with the capability to differentiate among cultivars, highlighting that fatty acid composition data can be used as a traceability marker of the botanical origin [21,26–28]. For example, D' Imperio et al. by analyzing Sicilian extra virgin olive oils from 22 cultivars found out that oleic, linoleic and palmitic fatty acids were crucial in the characterization of the olive oil cultivars [21]. Likewise, Krichene et al. determined the content of fatty acids and phenolic compounds, as well as other olive oil minor components in Tunisian olive cultivars; observing clear differences between them [28].
