## 2 Materials and Methods
### Extraction of EOs
#### 2.1.3 Experimental Procedure  
Two different objectives were considered: (a) PEF impact as a pre-treatment for EO extraction; (b) optimization of PEF parameters. PEF Impact as a Pre-Treatment for EO Extraction in Eucalyptus and Rosemary  
Eucalyptus and rosemary were used as plant materials. Both plant materials were used within 15 days of harvesting, and thus, the results obtained were limited to the quantity harvested, as the plant's quality changes over time. The eucalyptus leaves were reduced in size, using the shredder mode of a Bimby\({}^{\circledR}\). Two different protocols were followed: (_i_) the control (i.e., without PEF), and (_ii_) the one considering the PEF application. It is important to mention that for each condition considered (i.e., plant material, distillation time, \(t_{dist}\), and specific energy, \(W_{s}\), applied), the experiments were done in duplicate. For the control, 50 g of plant material was weighed and introduced into a 1 L volumetric flask alongside with 500 mL of tap water, which was then connected to the Clevenger apparatus, seen in Figure 1c. The mixture was heated up to the boiling point and the distillation would go on for 30 or 60 min. The volume of the EO extracted was measured in the Clevenger apparatus, and then recovered into a dark flask that was covered with Al foil and stored in the fridge, at 4 \({}^{\circ}\)C, for a few days. The EO's density was determined, calculated the recovered mass and, finally, the extraction yield, \(\eta\), was determined by (8). \[\eta=\frac{Mass\ of\ EO\ extracted}{Mass\ os\ plant\ material}.100 \tag{8}\]  
For the PEF experiments, 50 g of plant material was weighed and mixed with 250 mL of tap water in the batch treatment chamber, in order to have a uniform mixture. PEF was applied to achieve a \(W_{s}\) of approximately 10 kJ/kg (i.e., less than 2.5 \({}^{\circ}\)C temperature increase expected), with a voltage of 10 kV, frequency of 5 Hz, pulse width of 10 \(\upmu\)s, and field strength of 2 kV/cm. The mixture is then transferred to a 1 L volumetric flask, where an additional 250 mL of tap water was introduced. The volumetric flask was then connected to the Clevenger apparatus, and the same procedure as described for the control was followed. For clearness, an example of PEF parameters calculation is given in Table 1, for one eucalyptus processing experiment. These calculations were made for all the PEF experiments, using the equations presented in Section 2.1.2. It is important to note that the conductivity, \(\rho\), was calculated, using Equation (4), after applying the first pulse and measuring the pulse current amplitude for a given pulse protocol and batch condition. After this, the number of pulses, n, was calculated in order that the specific energy is approximately 10 kJ/kg. Optimization of PEF Parameters for Thyme EO Extraction  
For this experiment, two different methods were used: (i) the control method; and (ii) the one considering the PEF application. It is important to mention that for each condition considered (i.e., plant material, distillation time, \(t_{dist}\), and specific energy, \(W_{s}\), applied), the experiments were done only once. For the control, a sample of 10 g of plant material was weighed and introduced into a 1 L volumetric flask, alongside with 220 mL of tap water, which was then connected to the Clevenger apparatus. The mixture was heated up to the boiling point and the distillation would go on for 30 or 60 min. The volume of EO extracted was measured in the Clevenger apparatus, then recovered into a dark flask, and the mass of EO extracted was weighed. Finally, the flask was covered with aluminum foil and stored in the fridge, at 4 \({}^{\circ}\)C, for a few days. Finally, the extraction yield, \(\eta\), was determined by (8). For the PEF experiments, a sample of 10 g of plant material was weighed and introduced into a treatment chamber, alongside with 90 mL of tap water.