**2. Experimental**

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

several methods: Rancimat test (Di Lecce et al., 2009; Esquivel et al., 2009; Kamvissis et al., 2008; Mateos et al., 2006; Platero-López & García-Mesa, 2007), oxygen stability index (OSI) (Carrasco-Pancorbo et al., 2007; Ceci & Carelli, 2010; Cercaci et al., 2007; Gómez-Caravaca et al., 2007; Márquez-Ruiz et al., 2008), peroxide value (Carrasco-Pancorbo et al., 2007; Di Lecce et al., 2009; Márquez-Ruiz et al., 2008), AOCS method (Diraman, 2008), conjugated dienes (Deiana et al., 2002) and conjugated trienes (Hrncirik & Fritsche, 2005) analyses, K232 and K270 UV indexes (Antolin & Meneses, 2000; Cañizares-Macías et al., 2004a; Márquez-Ruiz et al., 2008; Platero-López & García-Mesa, 2007), thermogravimetry analyses (Coni et al., 2004; García Mesa et al., 1993; Gennaro et al., 1998; Santos et al., 2002; Vecchio et al., 2009), differential scanning calorimetry analyses (Vecchio et al., 2009), 13C NMR on chromatographically enriched oil fractions (Hidalgo et al., 2002), high-resolution chromatographic techniques coupled with UV spectrometry or mass spectrometry (B. Baccouri et al., 2008; Gallina-Toschi et al., 2005; Tena et al., 2009), capillary electrophoresis (Carrasco-Pancorbo et al., 2007; Gallina-Toschi et al., 2005), ultrasound-assisted method (Cañizares-Macías et al., 2004b; Platero-López & García-Mesa, 2007), microwave-assisted method (Cañizares-Macías et al., 2004a), chemiluminescence (Navas & Jiménez, 2007), electron paramagnetic resonance (Papadimitriou et al., 2006), and ORAC assay (Ninfali et al., 2002), among others. Fingerprinting techniques such as NMR (Alonso-Salces et al., 2011a; Guillen & Ruiz, 2001, 2006), FTIR (Guillen & Cabo, 2000), and fluorescence (Guimet et al., 2005; Tena et al., 2009) spectroscopies, DNA fingerprinting (Spaniolas et al., 2008), electronic nose (Lerma-García et al., 2009), and Oxitest method (Kamvissis et al., 2008; Mora

et al., 2009) have been also used successfully to study edible oil stability.

principal component analysis (PCA).

Most of the studies on the oxidative stability of olive oil employed questionably hightemperatures which, unfortunately, cannot be considered reliable to predict the stability of olive oils at room temperature (Frankel, 2010), i.e. under normal storage conditions. This is due to the fact that the mechanism of lipid oxidation changes at the elevated temperatures at which these experiments were run. In this sense, the rate of lipid oxidation is independent of O2 pressure at ambient temperatures; whereas it does become dependent on O2 pressures at elevated temperatures due to the decrease in solubility of O2. This causes the O2 concentration to become a significant limiting factor that increases with the degree of oxidation. For this reason, in oxidative stability studies the use of several temperatures, in a range as low as practical, preferably at or below 60 ºC, is an important consideration. Moreover, polymerization and cyclization of PUFA, which mainly occurs at elevated temperatures, are not significant at room temperature. Furthermore, volatile acids that are measured by the Rancimat and OSI methods are produced only at elevated temperatures (Frankel, 2010). For all these reasons, the results of the studies on the oxidative stability of olive oil at high temperatures are neither relevant nor can be extrapolated to normal storage conditions at room temperature. Olive oil stability at room temperature is of great interest, for instance, to know its storage shelf-life. Because VOO is relatively stable to oxidation due to its particular chemical composition, there has been apparently little or no control of its stability under normal storage conditions in the past. To provide some knowledge on this issue, the stability of olive oil at room temperature while protected from light by 1H-NMR fingerprinting was studied (Alonso-Salces et al., 2011a). The 1H-NMR spectra of the VOO aliquots kept under these conditions, over a certain period of time, were analyzed by
