*3.4. Analysis of Wax Content of Messinian Olive Oil, Greece*

Waxes are important constituents of olive oil used in order to distinguish olive oil obtained by pressing and that obtained by extraction (olive-residue oil) [56,57]. Waxes are present on the external fruit wax cuticle in olives so as to protect the fruit from transpiration and insect damage [57]. In dry hot weather, plants produce more waxes in order to control the rate of transpiration so that reduction of water loss is achieved. As a result, high temperature increases the wax production as a mechanism of fruit defense from environmental factors (climate) [58]. Generally, it has been found that wax compositions are influenced mainly by cultivar, harvest year and malaxation conditions [59–61].

In the present study, six wax esters were detected in the GC chromatogram: C36, C38, C40, C42, C44 and C46. Due to the latest amendment of the EEC regulation [23], the sum of wax esters was classified in two groups. Since only C42, C44 and C46 are now included in the most recent EU regulation for extra virgin olive oil, they were grouped together as Wax Esters (WEs 42–46). Since the sum of C40, C42, C44 and C46 was previously calculated, they were also grouped together as Total Wax Esters (TWEs 40–46). As shown in Table 4, the mean value of WEs 42–46 of the examined olive oil samples was 28.38 mg/kg and ranged from 16.89 to 58.33 mg/kg, well short of the upper legal limit of 150 mg/kg. Respectively, the TWEs 40–46 had a mean value of 67.20 mg/kg and ranged between 42.84 to 140.31 mg/kg. Although there is no previous study on wax determination for Kalamata PDO olive oils, according to our knowledge, the obtained results were similar to the wax content of olive oils extracted in other hot climates such as those in southern Italy for cv Carolea and in Australia for cv Koroneiki [44.46]. Finally, we found no differences in wax content caused by extraction (*p* > 0.05) (please see Supplementary data).


**Table 4.** Wax esters of Kalamata PDO olive oils produced in Messinia (southwest of Peloponesse).

Results are expressed as the means ± standard deviation (SD).

Using chemometric analysis, we verified that the extraction method (two- or three-phase decanter) causes non or minor changes on the examined chemical characteristics.

The score plot of Principal Component Analysis (PCA) is used to assess the data structure and detect clusters, outliers, and trends. As shown in Figure 4 groupings of data on the plot based on two-phase and three-phase decanters in Messinian olive oil samples showed that the points are randomly distributed around zero. As a result, no correlations for both two-phase and three-phase samples were presented. Therefore, the extraction method (two- or three-phase decanter) has verified no changes to the chemical parameters examined.

**Figure 4.** Score plot of Principal Component Analysis (PCA) for two-phase and three-phase decanters in Messinian olive oil.

#### **4. Conclusions**

The evaluation of extra virgin olive oils produced in the Messinia region, southwest of Peloponnese, denoted some challengeable characteristics. On the one hand, the results depict the high qualitative profile of Messinian olive oils, which is in agreement with similar studies examining cv Koroneiki from different geographical regions of Greece. On the other hand, major fluctuations were observed from the established EU regulatory limits. Most importantly, results show that Messinian extra virgin olive oils show low concentration of total sterols, with 66.7% of the examined samples being below the regulated set limits for Kalamata PDO status. Although no information exists in the literature regarding Kalamata PDO olive oils, as mentioned previously, analysis of VOOs from cv Koroneiki in completely different geographical regions such as Crete and Australia, has also shown a tendency of low total sterol concentration. Thus, low mean value in total sterols may clearly depict a "special characteristics" for Koroneiki cultivar, yet completely opposed to the existing standard limits of Kalamata PDO status. In contrast, total sterol concentration of the most studied Spanish and Italian cultivars is always quite above the limit of 1000 mg/kg.

a "special characteristics" for Koroneiki cultivar, yet complete In addition, our results show that olive oil samples of cv Koroneiki in the Messinia region present a high concentration of campesterol, with a total of 21.7%, exceeding the legal maximum of 4.0% and a light tendency of high total erythrodiol content. A trend of higher campesterol has been reported for cv Koroneiki, cv Barnea and cv Cornicabra cultivated in other geographical regions such as in Australia and Spain. Furthermore, although the fatty acid composition of the examined samples was within the range for the EVOO category, the extremely narrow established PDO limits in specific fatty acid composition may result in further fluctuations excluding Messinian olive oils from PDO certification. As far as wax content is concerned, although no information exists in the literature for Messinian olive oils for comparison, the obtained results are within the regulatory EU limits. Finally, in accordance with previous reported data for cv Koroneiki in Greece, the extraction method (two- or three-phase decanter) caused non or minor changes on the examined chemical characteristic.

In general, as PDO-certified products are a crucial strategic tool to enhance rural economy and development, through the added value of the PDO trademark, in terms of the higher price such products can enjoy, the above-mentioned deviations could inevitably lead to a controversy regarding the authenticity of Kalamata PDO extra virgin olive oils in the olive oil sector and consequently result in diminishing its reputation.

As this work is the first systematic attempt focusing on the evaluation of "Kalamata PDO olive oil" characteristics, further in depth research, with a higher number of samples and more crop years, is under way. The continued study of Messinian olive oils with the addition of more examined parameters (e.g., sensory analysis) will provide adequate datasets and allow supporting the improvement of the current EU regulation through the update and the re-adjustment of the established limits for Kalamata PDO status.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/8/12/610/s1, Table S1: Influence of the extraction method on the sterolic profile, Table S2: Influence of the extraction method on wax esters content.

**Author Contributions:** V.S. designed and performed the experiments; P.T. performed statistical analysis; T.V. and V.S. wrote, edited and reviewed the paper.

**Funding:** This research project was funded under the Project 'Research and Technology Development Innovation Projects'-AgroETAK, MIS 453350, in the framework of the Operational Program 'Human Resources Development'. It is co-funded by the European Social Fund through the National Strategic Reference Framework (Research Funding Program 2007–2013) coordinated by the Hellenic Agricultural Organization–DEMETER.

**Acknowledgments:** V.S. would like to thank A.S. Vekiari and the Dept. of Olive and Horticultural crops, Kalamata, Greece, for the support provided. All the experiments were conducted in the accredited laboratory of the Dept. of Olive and Horticultural crops, Kalamata, Greece, according to ISO 17025:2008.

**Conflicts of Interest:** The authors declare no conflict of interest.
