## 2 Materials and Methods
### Extraction of Polyphenols with Supercritical Fluids  
The supercritical extractions by CO\({}_{2}\) were performed on a _Spe-ed_ SFE 4 extractor (Applied Separation, Allentown, PA, USA) and following the necessary steps: loading in the 50 mL stainless steel vessel, pressurization with CO\({}_{2}\) and waiting for a transitory state of temperature, which is kept constant by an oven module. After the reaching of target temperature and pressurization, in order to guarantee the intimate contact between the CO\({}_{2}\) and the matrix, the system was kept closed for 20 min (static phase), and only after  
Figure 3: Homogenized from fresh samples of _OFI cult._ (**A**) and _OFI s.l._ (**B**).  
this time the micrometric valve was opened until a constant flow of 1 was reached. 5 L/min (dynamic phase), measured by a ball float rotameter. A graphical scheme of _Spe-ed_ SFE 4 extractor is given in Figure 4.  
The extract left the vessel through a valve, which was in advance thermostated to avoid CO2 solidification, due to the expansion. Experiments were carried out at 40 \({}^{\circ}\)C, 110 bar and 250 bar by varying the preventive sample preparation with PSE accessories, as Ottawa Sand and an Spe-edTM PSE Matrix (Hydrooscopic Samples Dispersing Agent). All the extracts were stored at \(-18\) \({}^{\circ}\)C before HPLC analysis.  
This extraction method has always attracted because it is safe and eco friendly. In addition, the supercritical fluid, in the specific case CO2, has properties similar to those of gas in the supercritical state so that it can extract in a very short time compared to the classic extraction types, returning an extract already separated from the solvent after depressurization. Supercritical fluids have a high diffusion coefficient and a low viscosity which favours their intimate contact with the matrix. On the other hand, however, this method has limits, especially in relation to the use of the supercritical solvent because CO2 has low polarity. Therefore this type of extraction technique is generally used for the extraction of oils, fats or in any case polar substances. Only the use of other solvents, such as ethanol or water, increases the solvent power of CO2 in the supercritical state.  
The different methods to prepare the samples and pressure are listed in Table 1.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **Ref.** & **Preventive Preparation** & **P (bar)** \\ \hline _OFI cult._ & CULT\_20D & 20\% Diatomee Sand & 110 \\ _OFI cult._ & CULT\_20P & 20\% Diatomee Sand & 250 \\ _OFI cult._ & CULT\_90E\_200 & Dried until 90\% + 20\% Ottawa Sand & 110 \\ _OFI cult._ & CULT\_30E\_200 & Dried until 30\% + 20\% Ottawa Sand & 110 \\ _OFI cult._ & CULT\_C\_20D & Centrifuged + 20\% Diatomee & 110 \\ _OFI s.l._ & SL\_30E\_20D & Dried until 30\% + 20\% Diatomee Sand & 110 \\ _OFI s.l._ & SL\_90E\_20O & Dried until 90\% + 20\% Ottawa Sand & 110 \\ _OFI s.l._ & SL\_90E\_20D & Dried until 90\% + 20\% Diatomee Sand & 110 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Different preparation of samples before SFE separation.  
Figure 4: Scheme of Spe-ed SFE 4 extractor unit.  
All samples were cleaned, homogenized and frozen after collection, to be subsequently analysed.  
Some samples analysed were centrifuged after defrosting at 3500 rpm for 10 min and only the precipitated part was used to extract the polyphenols while other samples were partially dried at 32 \({}^{\circ}\)C in the oven to reduce the amount of water.  
The extraction time was 1 h. The final extracts were collected in glass tubes covered with an aluminium foil and frozen until analysis. Each extraction was carried out in duplicate.