# Understanding Olive Oil Stability Using Filtration and High Hydrostatic Pressure
## 4 Materials and Methods
### Analyses  
The acidity (% oleic acid), peroxide value (meq O\({}_{2}\) kg\({}^{-1}\)) and UV spectroscopic indices (K\({}_{232}\), K\({}_{270}\) and AX) were measured according to the official EU method and subsequent amendments [45]. The panel test was carried out according to the official IOC method [46]. The panel was made up of five men and three women, aged from 28 to 57; all of the panellists were non-smokers and had been trained following the official IOC procedure. The panellists worked for the Taste Commission of the Ministero delle Politiche Agricole Alimentari, Forestali e del Turismo (MIPAAAFT - Italian Ministry of Agri-Food and Forestry Policy and Tourism). The degree of turbidity was measured in nephelometric turbidity units (NTU) using a Hach Model 2100 turbidimeter (Hach, Loveland, CO, USA). About 25 g of the oil samples were put in the standard glass vessel, which was inserted in the closed vessel chamber of the turbidimeter; the degree of turbidity was measured at equilibrium after approx. 1 h.  
Water content (% _w/w_) was analysed using a Karl Fischer Kit for visual water determination without a titrator (37858 HYDRANAL--Moisture Test Kit, Honeywell FlukaTM, Bucharest, Romania). The oil sample (1 mL) was dissolved in previously neutralized HYDRANAL--Solvent E, and the titrating reagent (HYDRANAL--Titrant 5E) was added until the equivalence point was reached. Water activity (A\({}_{w}\)) was measured using a Rotronic Hyroskop DT hygrometer (Michell Italia SrI, Milan, Italy). The samples (approx. 6.5 mL) were placed in the standard sample cups and the water activity was measured at equilibrium after approx. 12 h.  
The solid particle content was measured using the method described by the literature [15]. A 5 g aliquot of filtered oil was vacuum-filtered to saturate Whatman grade 1 filter paper (Merck KGaA, Darmstadt, Germany). The same filter paper was used to filter approx. 30 g of the oil samples and then it was weighed using an analytical balance. The solid particle content was calculated by weighing the difference and quantified in % _w/w_. The microorganisms were enumerated according to the method reported by the literature [47], with some modifications: an aliquot of each sample (i.e., approx. 20 mL) was taken from each bottle in sterile conditions and filtered through a 0.45 \(\upmu\)m sterile nitrocellulose membrane. Then, the membrane was transferred into a 50 mL sterile Falcon tube containing 20 mL of sterile physiological solution (NaCl 0.85%), and homogenized using an UltraTurrax (mod. T25 homogenizer, IKA, Milan, Italy). Of each homogenized sample, 200 \(\upmu\)L serial dilutions were plated onto a YPD agar medium. After 48-72 h of incubation at 28 \({}^{\circ}\)C, the colonies with different morphologies were counted and, for each kind, the cell morphology was observed through a light microscope. The extraction, identification and determination of the phenolic compounds were performed by RP-HPLC using the official IOC method [48]. Briefly the HPLC apparatus consisted an Agilent 1200 series system (Agilent technologies, Santa Clara, CA, USA) composed by a quaternary pump equipped with a diode-array detector and autosampler. The analytical conditions were: HPLC column: LiChroCART@250-4.6 Purospher@STAR RP-18E, 5 \(\upmu\)m (250 \(\times\) 4.6 mm id, Merck KGaA) equipped with a: LiChroCART@4-4 Purospher@STAR RP-18E, 5 \(\upmu\)m pre-column (4 \(\times\) 4 mm); elution condition: water 0.2% H\({}_{3}\)PO\({}_{4}\) (_v/v_), methanol, acetonitrile gradient following the official IOC method [48]; injection volume: 20 \(\upmu\)L; wavelength: 280 nm. Syringic acid was used as the internal standard; syrigenic acid and tyrosol were chosen as the external calibration standards to evaluate the relative response factor (i.e., RRF = 4.87). Phenolic compounds were quantified in mg\({}_{\text{tyrosol}}\) kg\({}_{\text{oi}}\)\({}^{-1}\).