## 3 Materials and Methods
### Hop Cone Analysis  
#### 3.3.1 Hop Oil GC-MS Analysis  
Essential oils were first extracted in triplicate by steam distillation with a Clevenger apparatus, using 25.0 g of dried hops left for 4 h in a 2.0 L round-bottom flask with 1250 mL of pure water. Anhydroous sodium sulfate (Sigma-Aldrich, Milan, Italy) was then added to the extracted essential oils, which were subsequently stored at 4 \({}^{\circ}\)C. Before analysis, the essential oils were diluted in CH\({}_{2}\)Cl\({}_{2}\) (1:200 \(v/v\)), to which 400 ppm of Toluene had been added as the internal standard ([PERSON], Milan, Italy).  
All samples were analysed with a Thermo Scientific (San Jose, CA, USA) TRACE 1300 gas-chromatograph coupled to a Thermo Scientific ISQ\({}^{\mathrm{TM}}\) Single Quadrupole mass spectrometer. The gas-chromatograph was equipped with Supelcowax 10 (30 m \(\times\) 0.25 mm, f.t. 0.25 \(\upmu\)m) (Supelco, Bellefonte, PA, USA) capillary columns and helium was used as a carrier gas (1 mL min\({}^{-1}\)). The gas chromatography-mass spectrometry (GC-MS) oven temperature gradient started from 50 \({}^{\circ}\)C, a condition that was maintained for 3 min, after which the temperature was raised to 200 \({}^{\circ}\)C (5 \({}^{\circ}\)C min\({}^{-1}\)). This final temperature was maintained for 18 min. The injector was maintained at 230 \({}^{\circ}\)C, operating in split modality with a ratio of 1:20. The mass spectrometer was equipped with an electron impact (EI) source (70 eV) and the acquisition mode was full scan (from 40 to 500 \(m/z\)). A solvent delay time of 4 min was applied. The main terpenic volatile compounds were identified on the basis of their mass spectra compared with the reference mass spectra libraries (NIST, 2005 software, Mass Spectral Search Program V.2.0d, Washington, DC, USA version 2.2 June 2014) and on the basis of their calculated Retention Indexes (RIs) through the application of the Kovats' index (KI) formula compared with those reported in the literature [25; 35; 36]. When it was not possible to find the KI in the literature, a tentative identification was obtained by matching the value obtained with mass spectra library data: a minimum match quality of 98% was used as a criterion. In order to determine the RI of the components, a mixture of alkanes (C8-C20) was injected in the GC-MS equipment and analysed under the same conditions described earlier. After the identification of target volatile compounds  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameter** & **Value** & **Unit** \\ \hline pH & 7.33 & \\ EC & 264 & \(\upmu\)S/cm \\ CEC & 30.86 & \(\mathrm{cmol_{c}/Kg}\) \\ \hline
**Element** & **Value** & **Unit** \\ \hline (N) tot & 2 & g/Kg \\ Organic matter & 3.1 & \% \(w/w\) \\ (P) available & 17.8 & mg/kg \\ (P\({}_{2}\)O\({}_{5}\)) available & 40.9 & mg/kg \\ Ca & 13.65 & mmol/L \\ Mg & 1.1 & mmol/L \\ K & 1.23 & mmol/L \\ Na & 0.22 & mmol/L \\ Zn & 41 & mg/kg \\ Fe & 17.4 & g/kg \\ B & 11 & mg/kg \\ \hline \hline \end{tabular} \(\mathrm{EC}\) = Electric Conductivity; CEC = Cation Exchange Capacity.  
\end{table}
Table 6: Chemical properties of the soil and elements.  
(VOC), peak areas of target volatile compounds (AVOCs) were obtained on the basis of the major ion then normalised with the internal standard peak area (AIS). Data were expressed as a percentage based on the equation below:  
\[\left(\text{A}_{\text{voc}}/\text{A}_{\text{SI}}/\sum\text{A}_{\text{voc}}/ \text{A}_{\text{SI}}\right)*100\]  
\(\text{A}_{\text{voc}}\) = integrated peak area  
\(\text{A}_{\text{SI}}\) = area of the internal standard  
All samples were analysed in triplicate.