*2.1. Olive Fruits, Storage Treatments and Virgin Olive Oil Production*

Healthy olive fruits from Istarska bjelica (IB) and Rosinjola (RO), two Croatian autochthonous olive cultivars (*Olea europaea* L.), were manually harvested at the end of October 2016 and the beginning of November 2016, respectively. Both cultivars were grown in the same experimental olive orchard of the Institute of Agriculture and Tourism (Poreˇc, Croatia). The ripening index of olive fruits was determined by the protocol described by Beltran et al. [30].

Olive fruits were divided in twelve batches of 3 kg per cultivar. Three batches per cultivar were processed into oil immediately after harvest (control oil), and the rest of the olive fruits was stored for seven days at three different temperatures prior to olive oil production: at room temperature of 22 ± 4 ◦C (RT), at +4 ◦C in a refrigerator and at −20 ◦C in a freezer. The olive fruits stored at 4 ◦C and −20 ◦C were allowed to reach room temperature before milling, which lasted about 2 and 6 h, respectively. Fruits were crushed by a hammer crusher and olive paste was malaxed for 45 min at 25 ◦C using vertical thermostated olive paste mixers. Olive oil extraction was done by a laboratory centrifuge (Abencor, MC2 Ingeneria y Sistemas, Seville, Spain). In order to obtain enough oil for analysis, fruits representing one batch of 3 kg, were processed in triplicates and obtained oils were mixed in one oil sample. Obtained oil samples (*n* = 12 per cultivar, 3 control oils and 3 samples per each storage time/temperature) were left to sediment naturally for ten days and were then decanted. The analyses of oil samples started immediately after decantation. Samples were stored in non-transparent bottles at 16–18 ◦C during the time of analysis.

## *2.2. Oil Content, Oil Yield and Extractability Index*

Theoretical oil content in the fruit (expressed on fresh and on dry weight based on the gravimetric determination of water in fruit) was determined from the olive paste obtained after crushing using Soxtec Avanti 2055 apparatus (Foss Tecator, Höganäs, Sweden) according to the method described by Brki´c et al. [31].

Oil yield (%) was calculated from three parallel processing repetitions, multiplying by 100 the mass ratio of mechanically extracted oil (g) and centrifuged olive paste (g) [32].

Olive oil extractability index (EI) was calculated according to Beltran et al. [30] using the formula: EI = V × d/W × F × 100, where V (mL) is a volume of olive oil extracted, d (0.915 g/mL) is the average olive oil density, W (g) is olive paste weight and F (%) is the oil content of the fruit (on fresh weight).
