*3.2. Analysis of Sterolic Profile and Triterpenic Dialcohol Content of Messinian Olive Oils, Greece*

Phytosterols are important components of the unsaponifiable fraction of olive oil beneficial for the human health and nutrition. Sterol composition and content are broadly used for the control of olive oil authenticity and adulteration. Sterol content varies between 1000 and 3000 mg/kg depending the botanical variety, olive ripening, storage conditions and geographical origin [26–31]. Numerous studies have shown that each variety has a characteristic sterol "fingerprint". Therefore, those minor components can be considered as an important and useful tool for detecting oil adulteration and/or classifying virgin olive oils in accordance with their variety [32–35]. The influence of geographical origin on the sterol composition of virgin olive oil has been evaluated by various authors, pointing out the great potential of different analytical techniques followed by chemometric tools for this purpose [36–38].

Although several studies have been conducted for cv Koroneiki in other regions of Greece, mainly in Crete [39–43], very little information is available in the literature regarding the sterolic profile of cv Koroneiki in Peloponnese generally and more precisely in the Messinia region.

In the present study, we evaluated the sterolic composition of the examined Messinian olive oils. Table 2 lists the mean values expressed as percentages of the total sterols and their standard deviations of the main sterols present in the olive oil sampled. The main sterols detected were β–sitosterol, ∆5-avenasterol and campesterol, with mean values of 80.73%, 12.28% and 3.71%, respectively. The first two represent over 90% of the total sterol content, with β-sitosterol being the most abundant phytosterol (over 80% of the total sterol content). The calculated parameter, the apparent β-sitosterol, falls within the established regulatory limits, with a mean value of 94.63%. Finally, the cholesterol and ∆7-stigmastenol values were low and quite below the limits set by EU regulation (0.5%), with a mean value of 0.11% and 0.19% of total sterols, respectively (Table 2).

In contrast, several major deviations were observed in the case of the sterolic profile for the Messinian olive oils. Most importantly, 43.5% of the examined olive oil samples did not surpass the required limit of 1000 mg/kg in total sterol concentration according to the EEC Regulation 2568/91. In addition, as illustrated in Figure 3, the regulated limit for Kalamata PDO olive oil is established at 1100 mg/kg. As a result, a really high percentage (66.7%) of the examined samples was below the established PDO limit. The mean total sterols content was 1033 mg/kg and ranged from 744 to

1283 mg/kg. A similar case was observed in campesterol, where a total of 21.7% of the examined samples exceeded the legal maximum of 4%, with a mean value of 3.71% and ranged from 2.78 to 4.70%. A trend of higher campesterol has also been reported for cv Koroneiki, as well as for other cultivars cultivated in different countries [44,45], whereas the total sterol concentration of the most studied Spanish and Italian cultivars is always within the minimum limit of 1000 mg/kg [46–50].


**Table 2.** Sterolic profile and triterpene diols determined in Messinian olive oil, Greece.

Results are expressed as the means ± standard deviation (SD). *N* = 69.

**Figure 3.** (**a**) Scatter plots visualizing the chemical parameters: (**a**) total sterols (43.5% of the examined olive oil samples did not surpass the EEC limit of 1000 mg/kg and 66.3% of the examined olive oil samples did not surpass the PDO limit of 1100 mg/kg in total sterols); (**b**) campesterol (21.7% of the examined olive oil samples exceeded the legal maximum of 4%); (**c**) total erythrodiol (8.06% of the examined olive oil samples exceeded the upper set limit of 4.5%). Note. Dotted line: limits according to EEC/2568/91 for the EVOO category; straight line: limits according to Council Regulation (EC) 510/2006 for Kalamata PDO olive oil [20,21,23].

Although no information exists in the literature regarding Kalamata PDO olive oils, results show that cv Koroneiki in the Messinian region shows a clear tendency of low concentrations of total sterols and high concentrations of campesterol. Low mean values on total sterol concentration for cv Koroneiki were reported earlier in Crete, in 2001, by Stefanoudaki et al., who studied the effect of drought stress on olive oil characteristics, without giving emphasis on the mentioned tendency [41].

In general, such problems (fluctuations from EU regulations) could inevitably raise questions regarding the authenticity of Kalamata PDO extra virgin olive oils in the olive oil sector, and so they certainly require further investigation.

It is known that total erythrodiol levels are high in solvent-extracted oils, indicating adulteration with olive-pomace oil [51]. The mean total erythrodiol content was 2.85% (Table 2). However, a small but noteworthy percentage of 8.06% of the examined samples exceeded the upper set limit of 4.5% as shown in Figure 3. A possible assumption may be the inappropriate higher degree of olive crushing during the extraction process, leading to an increase in erythordiol levels from the olive's exocarp.

Finally, almost no significant differences were observed in the sterol composition and triterpene dialcohols using the two industrial decanters (*p* > 0.05) (please see Supplementary data). The amount of water added during oil extraction does not affect their levels due to their lipophilic nature and because they are sparingly soluble in water. This is in agreement with previous reported data for cv Koroneiki among other cultivars [40,52].
