*2.1. Geographical Distribution and Selection of Olive Oil Samples*

A total of seventy-one (71) olive oil samples were obtained in one successive harvesting year (2014–2015), cultivated in the geographical Messinia region, southwest of Peloponnese, in Greece (see Figure 1). All samples were produced from olive trees representing the typical Koroneiki cultivar of Messinia. Sampling was made from different points in the prefecture of Messinia so as to have the utmost homogeneity. As mentioned earlier, the European Commission recently approved the extension of "Kalamata PDO olive oil" throughout the whole Messinia region, enlarging the area covered by the PDO. On this basis, Messinian extra virgin olive oils may be classified as PDO, if they meet the corresponding parameters [21]. The whole region is characterized by the same climatic conditions as described in the relevant EC Commission Regulation for Kalamata PDO olive oil [19–21].

**Figure 1.** Map of Greece, focusing on the Messinia region (in red), southwest of the Prefecture of Peloponnese (in orange). Adapted from Wikipedia [22].

#### *2.2. Sampling and Sample Maintenance*

– – Sampling was carried out during the 2014–2015 olive fruit-harvesting period. Provision was made to harvest olive fruits at the optimal stage of maturity. Samples were transferred to local oil mills in solid, vented, food-grade harvest bins or in suitable waist harvest bags. Olive mills were equipped with two or three-phase centrifugal systems (decanters), as olive mills in Messinia operate with both extraction methods (the ratio of two- and three-phase olive mills in Messinia is approximately 50:50). Olive fruits were processed within 24 h, according to the relevant EC Regulation for Kalamata PDO olive oil and the same post-harvest conditions were maintained in all cases. In detail, the leaves were removed from the olive fruits, washed and then sent to the crusher. Malaxation was carried out at low temperatures (27–28 ◦C) for 30 min according to the above-mentioned regulation. The obtained olive paste was horizontally centrifuged (decanted) (three- or two-phase system) and the resulting olive oil was finally centrifuged. Olive oil samples were stored directly in 1 L air-tight dark-green glass bottles at 4 ◦C until further analysis. Quality parameters were analyzed in triplicate, while all the other examined chemical parameters were determined in duplicate.

#### *2.3. Determination of the Physicochemical Quality Parameters*

Free acidity, peroxide value and spectroscopic indices (K<sup>232</sup> and K268) were carried out, following the analytical methods described in Regulation EEC/2568/91 of the European Commission and later amendments [23]. Free acidity was expressed as the percentage of oleic acid and peroxide value was given as milliequivalents of active oxygen per kilogram of oil (meq O<sup>2</sup> kg −1 ). K<sup>232</sup> and K<sup>268</sup> extinction coefficients were calculated from absorption at 232 and 268 nm respectively. Spectrophotometric examination in the ultraviolet provides information on the olive oil quality, its state of preservation and changes brought about by technological processes (due to the presence of conjugated diene and triene systems resulting mainly from oxidation processes). These absorptions are expressed as specific extinctions E (the extinction of 1% *w*/*v* solution of the oil in isooctane, in a 10 mm cell) conventionally indicated by K 'extinction coefficient'. Free acidity (FA), peroxide value (PV), K<sup>232</sup> and K<sup>268</sup> were immediately determined for each sample in order to avoid any kind of olive oil deterioration. Solvents used were purchased from Sigma (St. Louis, MO, USA).

## *2.4. Determination of Sterols and Triterpene Dialcohols*

The individual sterols, total sterols and triterpene dialcohols were determined according to the method adopted by EEC/2568/91 regulation, Annexes V with later amendments [23]. The oil sample, with added α-cholestanol (Sigma, St. Louis, MO, USA), as an internal standard, was saponified with

'extinction coefficient'. Free acidity (FA), peroxide value (PV)

potassium hydroxide in ethanolic solution and the unsaponifiable matter was extracted with diethyl ether. The sterol and triterpene dialcohol fractions were separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate (Fluka, Buchs, Switzerland). The fractions recovered from the silica gel were transformed into trimethylsilyl ethers (TMSE) by the addition of pyridine-hexamethyldisilizane-tri-methylchlorosilane (9:3:1, *v*/*v*/*v*) (Supelco, Bellefonte, PA, USA). Sterols (%) and triterpene dialcohol contents were determined with a Shimadzu (GC-2010) gas chromatograph equipped with a flame ionization detector (FID), a DB-5 (30 m × 0.32 mm × 0.25 µm) capillary column and an autosampler injector. The operating conditions were as follows: injection temperature 280 ◦C, column temperature 265 ◦C, detector temperature 310 ◦C, splitting ratio (1:50), flow rate 1.4 mL/min and amount of substance injected 1 µL of TMSE solution. The sterols and triterpene dialcohols were eluted in the following order: cholesterol, 24-methylen-cholesterol, campesterol, campestanol, stigmasterol, ∆7-campesterol, ∆5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆5-avenasterol, ∆5,24-stigmastadienol, ∆7-stigmastenol, ∆7-avenasterol, erythrodiol and uvaol (calculated as total erythrodiol). Individual peaks were identified on the basis of their relative retention times with respect to the internal standard. The sum of ∆5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆5-avenasterol, and ∆5,24-stigmastadienol represents apparent b-sitosterol. Mean values of duplicate experiments in each sample were used for further statistical analysis.

## *2.5. Determination of Fatty Acid Composition*

The fatty acid profile was determined according to the official method of the Regulation EEC/2568/91, Annex IV with amendments [23]. The fatty acid methyl esters (FAME) were obtained by cold alkaline transesterification with methanolic potassium hydroxide solution and extracted with n-heptane. FAME were analyzed on a model GC-2010 Shimadzu chromatograph, equipped with an BPX-70, (60 m × 0.25 mm × 0.25 µm), capillary column and a flame ionization detector (FID). The carrier gas was helium, with a flow of 1.5 mL/min. The temperatures of the injector and detector were set at 250 and 260 ◦C respectively and the oven temperature was increased gradually from 165 to 225 ◦C in 35 min. The injection volume was 1 µL. Quantification was achieved using a FAME standard mixture purchased from Sigma (St. Louis, MO, USA). The results were expressed as a percentage of individual fatty acids. Analytical-grade methanol, heptane, and potassium hydroxide were purchased from Sigma (St. Louis, MO, USA).

#### *2.6. Determination of Wax Content*

The wax content of olive oil samples was determined according to the Regulation EEC/2568/91, Annex IV with later amendments [23]. A suitable amount of internal standard (lauryl arachidate) was added to 0.5 g of olive oil sample and then fractionized by chromatography on a hydrated silica gel column. The chromatographic elution was carried out with a mixture of n-hexane/diethyl ether, keeping a rate of flow of approximately 15 drops every 10 s. The subsequent fraction was completely dried and finally resolved in 2 mL of n-hexane. Waxes were analyzed on a model GC-2010 Shimadzu chromatograph equipped with an on-column injector, a flame ionization detector and a MEGA-5 HD (10 m × 0.32 × 0.10 mm) capillary column. The operating conditions were as follows: detector temperature 370 ◦C; the column temperature was increased from 80 to 160 ◦C at 40 ◦C/min and up to 340 ◦C at 5 ◦C/min for 7 min; the amount of substance injected was 1 µL of the n-hexane solution. The identification of the peaks was based on retention time by comparison with wax mixtures of known retention times analyzed under the same conditions.
