**2.2.2 Total phenols analysis**

4 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

The olive oils investigated (60 Italian and 60 Tunisian) were single cultivar virgin olive oils (SCVOOs) produced in different regions of Tunisia (Chamlali Cv.) and Italy (Coratina Cv.). Olives were handpicked at the optimal olive ripening degree. Immediately after harvest, olive fruits were transported and cleaned, each fruit sample was divided into three portions of 20 Kg. One portion was extracted using pressure system (see paragraph 2.1.1), the second and the third were extracted by centrifugation systems, three and two phases, respectively (see paragraph 2.1.2 and 2.1.3). The oils obtained were stored in three types of packaging (opaque glass, transparent glass and polyethylene terephtalate PET) and monitored for six months.

Olives are ground into an olive paste using large millstones. In general, the olive paste stays under the stones for 45–50 minutes. After grinding, the olive paste is spread onto fibre disks, that are easier to clean and maintain, stacked on top of each other and then placed into the press. Afterwards, this pile of disks are put on a hydraulic piston where a pressure of about 400 atm is applied. By the action of this pressure, a olive paste and a liquid phase is produced. Finally, the liquid phase containing oil and vegetation water is separated by a standard

This system does not need water addition and produces a liquid phase (oil) and a solid waste-water-dampened phase (pomace). The olive paste is kneaded for 60 minutes at 27°C and the oil is extracted with a horizontal centrifugation decanter and separated by means of

This system allows the crushing of olives into a fine paste. This paste is then malaxed for 60 minutes in order to achieve the coalescence of small oil droplets. The aromas are created during these two steps through the action of enzymes. Then, the paste is pumped into an industrial decanter where the phases are separated. Water (500 liters per ton) is added to facilitate the extraction process with the paste. The high centrifugal force created into the decanter separates the phases readily according to their different densities (solid phase pomace, vegetation water, oil). The solid materials is pushed out of the system by the action of a conical drum that rotates with a lower speed. The separated oil and vegetation water are then rerun through a vertical centrifuge, which separates the small quantity of

The physic-chemical and organoleptic analysis of VOO were carried out according to the

methods described by the European Union Regulations (UE 61/2011).

**2. Materials and methods** 

**2.1.1 Pressure system (PS)** 

process of decantation.

**2.1.2 Two-phase centrifugation (2P)** 

an automated discharge vertical centrifuge.

**2.1.3 Three-phase centrifugation (3P)** 

vegetation water still contained in the oil.

**2.2 Analytical methods** 

**2.1 Extraction of olive oil and storage** 

Total phenols content was determined according to the method developed by Gutfinger (1981). Briefly, an amount of olive oil (2.5 g) was dissolved with hexane (5 mL) and extracted with a solution of methanol and water (5 mL; 60/40). The mixture was then vigorously agitated for 2 minutes. Folin-Ciocalteu reagent (0.5 mL) and bi-distilled water (4.8 mL) were added to the phenolic fraction. The absorbance of the mixture was measured at 725 nm and results were given as mg of caffeic acid per Kg of oil.

#### **2.2.3 Free fatty acids, peroxides, ultra-violet light absorption**

Acidity value, peroxide value (PV) and ultra-violet light absorption, conjugated diene (K232) and conjugated trienes (K270), were determined according to the Regulation EEC 2568 91 of the European Union Commission (EEC, 1991).

#### **2.2.4 Sensory analysis**

Olive oils were evaluated by a panel according to the official method for the Organoleptic assessment of virgin olive oil referenced COI/T.20/Doc. No 15/Rev. 2.
