**1. Introduction**

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

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Olive oil is the oil extracted exclusively from the fruit of *Olea europea* L. only by means of mechanical methods or other physical procedures that do not cause any alteration of the glyceric structure of the oil thus preserving its characteristic properties. Olive oil is a highly appreciated edible oil, which is an important component of the Mediterranean diet, and is recognized for its potential health benefits.

The International Olive Council (IOC) establishes the definitions and classes of olive oils, based on methods of production and the free acidity of the oil, as well as the trade standard for their commercialization (International Olive Council, 2011). Much analytical work has been done on the authentication and quality assessment of this high added value agricultural product, as well as on the detection of its adulteration for both economic and health considerations (Frankel, 2010; Guillen & Ruiz, 2001). However, these issues continue to be major analytical challenges. In this context, the European Commission launched the TRACE project (http://www.trace.eu.org/) through the Sixth Framework Program under the Food Quality and Safety Priority with the aim of providing reliable analytical strategies to address this kind of problem.

This chapter reports research work on the use of 1H-NMR fingerprinting, combined with pattern recognition techniques, for the quality assessment of olive oil. Two major issues have been studied, the geographical origin of virgin olive oil (VOO) and the stability of VOO at room temperature.

<sup>\*</sup> Corresponding Author

The information contained in this chapter reflects the authors' views; the European Commission is not liable for any use of the information contained herein.

Quality Assessment of Olive Oil by <sup>1</sup>

origin at the national, regional and/or PDO level.

**1.2 Stability of virgin olive oil** 

Ruiz, 2001).

H-NMR Fingerprinting 187

statistical analysis have been used to distinguish olive oils from different types, botanical, geographical origins and pedoclimatic conditions. For this purpose, fatty acids (Matos et al., 2007; Ollivier et al., 2006), triglycerides (Aranda et al., 2004), sterols (Alves et al., 2005; Matos et al., 2007; Temime et al., 2008), phenolic compounds (Lopez Ortiz et al., 2006; Vinha et al., 2005), and pigments (Cichelli & Pertesana, 2004) have been analyzed by conventional methods that usually require time-consuming pre-treatment methods (solvent extraction, isolation and/or derivatization) followed by chromatographic techniques (Aparicio & Aparicio-Ruiz, 2000) such as GC-MS and/or GC-FID (Bechir Baccouri et al., 2007; Haddada et al., 2007; Temime et al., 2006; Vichi et al., 2005) and HPLC-MS (Canabate-Diaz et al., 2007; Lopez Ortiz et al., 2006). PDO olive oils were distinguished using physicochemical parameters of the oils and chemometric class-modeling tools (Federico Marini et al., 2006), sensory parameters and fatty acid profiles of the oils (Ollivier et al., 2006), or the oil sterol composition (Alves et al., 2005). IRMS measurements of the alcohol and sterol fractions of olive oil also proved to be useful for its geographical characterization (Angerosa et al., 1999). Fingerprinting techniques such as NMR (Mannina & Segre, 2002), NIR (Mignani et al., 2011), MIR (Reid et al., 2006), fluorescence (Kunz et al., 2011), FT-IR, FT-MIR and FT-Raman (Baeten et al., 2005; Lopez-Diez et al., 2003; Yang et al., 2005) spectroscopies, MS (Vaclavik et al., 2009), GC (Pizarro et al., 2011; Vaz-Freire et al., 2009) and DNA fingerprinting (Martins-Lopes et al., 2008; Ranalli et al., 2008) have been used for the determination of food authenticity (Reid et al., 2006). These types of techniques are particularly attractive since they are non selective, require little or no sample pre-treatment; use small amounts of organic solvents or reagents; and the analysis takes only a few minutes per sample. Chemometric analysis of NIR spectra of virgin olive oils allow us to determine its composition and geographical origin (Galtier et al., 2007). 1H, 13C and/or 31P-NMR analysis of the bulk oil (Rosa M. Alonso-Salces et al., 2010b; Rosa M. Alonso-Salces et al., 2011b; Mannina et al., 2010; Petrakis et al., 2008; Rezzi et al., 2005) or the unsaponifiable fraction of olive oil (R. M. Alonso-Salces et al., 2010), in combination with multivariate techniques, have been used to distinguish VOOs from certain geographical origins. In section 3.2., the achievements of 1H-NMR fingerprinting and chemometrics for the geographical characterization of VOO is reported. 1H-NMR fingerprints of a statistically significant number of authentic VOOs from seven countries, namely Italy, Spain, Greece, France, Turkey, Cyprus and Syria and from three different harvests (2004/05, 2005/06 and 2006/07) were analyzed by pattern recognition and classification techniques, such as principal component analysis (PCA), linear discriminant analysis (LDA) and partial least square discriminant analysis (PLS-DA), to evaluate the best approach to identify the geographical

Another matter of major concern regarding the quality of edible oils is their oxidation, not only from the technological and economic point of view but also for safety reasons, due to the undesirable properties of some compounds produced during this process (Guillen &

The high resistance to oxidative deterioration of VOO is due to both its triglyceride composition, which is low in polyunsaturated fatty acids, and its antioxidant constituents, i.e. polyphenols and tocopherols. The oxidative stability of VOO has been evaluated by

#### **1.1 Geographical origin of virgin olive oil**

At present, 75% of the global production of olive oil takes place in the Mediterranean basin, mainly in Spain, Italy and Greece (International Olive Council, 2011). VOO is permitted to be marketed under a Protected Designation of Origin (PDO), Protected Geographical Indication (PGI), or Traditional Specialty Guaranteed (TSG) label, on the basis of its area and method of production [Council Regulations (EC) No 510/2006 and No 509/2006]. In this context, the characterization of the geographical origin of VOO is becoming increasingly important. According to the EU definition, PDO products are most closely linked to the concept of *terroir* — a sense of place discernible in the flavor of the food. PDO products must be produced, processed and prepared in a specific region using traditional production methods. The raw materials must also be from the defined area whose name the product bears. The quality or characteristics of the product must be due essentially or exclusively to its place of origin, i.e., climate, the nature of the soil and local know-how. Food products with a PGI status must have a geographical link in at least one of the stages of production, processing or preparation. The European Commission has already registered in the "Register of protected designations of origin and protected geographical indications" 86 PDO and 15 PGI olive oils, produced in Italy, Greece, Spain, France, Portugal and Slovenia. As can be expected, given the financial benefits associated with these prestigious labels, it is very likely that economic fraud occurs (e.g. labeling a non-PDO product as a PDO one or adulteration with olive oils that do not fulfill the PDO requirements).

Another fraudulent practice is the mislabeling of the geographical origin of olive oils. The EU established new labeling rules that make origin labeling compulsory for virgin and extra virgin labeled olive oil [Commission Regulation (EC) No 182/2009]. So, oil produced from olives from just one EU Member State or third country has to be labeled with the name of the country of origin. VOO produced from olives from more than one EU Member State has to be labeled as a 'blend of Community olive oils', while oil produced using olives from outside the EU would be labeled as a 'blend of non-Community olive oils' or 'blend of Community and non-Community olive oils', or a reference to the EU and/or third countries of origin. Therefore, analytical methods are urgently needed to guarantee the authenticity and traceability of PDO and PGI olive oils, as well as the country of provenance, to help prevent illicit practices in this sector, and to support the antifraud authorities dealing with these issues.

VOO is made up of triglycerides (more than 98%) and minor components (about 1-2%) such as squalene, -tocopherol, phytosterols, phenolic compounds, carotenoids, and aliphatic and terpenic alcohols, which constitute the unsaponifiable fraction of the oil (Bortolomeazzi et al., 2001; Harwood & Aparicio, 2000). The chemical composition of this fraction may vary both qualitatively and quantitatively depending on vegetal species, climatic conditions, extraction and refining procedures and storage conditions (Canabate-Diaz et al., 2007; Harwood & Aparicio, 2000), which also greatly influence the organoleptic quality and stability of the oil. The diversity and interdependence between all these factors makes it highly unlikely that these influences would be the same in different regions. Hence, the geographical characterization of VOO addresses all these agronomic, pedoclimatic and botanical aspects which are unique to the oil of each origin (Aparicio et al., 1994).

A considerable number of sensorial (Pardo et al., 2007), physical (F. Marini et al., 2004; Federico Marini et al., 2006) and chemical (Aguilera et al., 2005; Boggia et al., 2002; Lanteri et al., 2002; Federico Marini et al., 2006; Federico Marini et al., 2007) approaches combined with

At present, 75% of the global production of olive oil takes place in the Mediterranean basin, mainly in Spain, Italy and Greece (International Olive Council, 2011). VOO is permitted to be marketed under a Protected Designation of Origin (PDO), Protected Geographical Indication (PGI), or Traditional Specialty Guaranteed (TSG) label, on the basis of its area and method of production [Council Regulations (EC) No 510/2006 and No 509/2006]. In this context, the characterization of the geographical origin of VOO is becoming increasingly important. According to the EU definition, PDO products are most closely linked to the concept of *terroir* — a sense of place discernible in the flavor of the food. PDO products must be produced, processed and prepared in a specific region using traditional production methods. The raw materials must also be from the defined area whose name the product bears. The quality or characteristics of the product must be due essentially or exclusively to its place of origin, i.e., climate, the nature of the soil and local know-how. Food products with a PGI status must have a geographical link in at least one of the stages of production, processing or preparation. The European Commission has already registered in the "Register of protected designations of origin and protected geographical indications" 86 PDO and 15 PGI olive oils, produced in Italy, Greece, Spain, France, Portugal and Slovenia. As can be expected, given the financial benefits associated with these prestigious labels, it is very likely that economic fraud occurs (e.g. labeling a non-PDO product as a PDO one or

Another fraudulent practice is the mislabeling of the geographical origin of olive oils. The EU established new labeling rules that make origin labeling compulsory for virgin and extra virgin labeled olive oil [Commission Regulation (EC) No 182/2009]. So, oil produced from olives from just one EU Member State or third country has to be labeled with the name of the country of origin. VOO produced from olives from more than one EU Member State has to be labeled as a 'blend of Community olive oils', while oil produced using olives from outside the EU would be labeled as a 'blend of non-Community olive oils' or 'blend of Community and non-Community olive oils', or a reference to the EU and/or third countries of origin. Therefore, analytical methods are urgently needed to guarantee the authenticity and traceability of PDO and PGI olive oils, as well as the country of provenance, to help prevent illicit practices in this sector, and to support the antifraud authorities dealing with these issues. VOO is made up of triglycerides (more than 98%) and minor components (about 1-2%) such as squalene, -tocopherol, phytosterols, phenolic compounds, carotenoids, and aliphatic and terpenic alcohols, which constitute the unsaponifiable fraction of the oil (Bortolomeazzi et al., 2001; Harwood & Aparicio, 2000). The chemical composition of this fraction may vary both qualitatively and quantitatively depending on vegetal species, climatic conditions, extraction and refining procedures and storage conditions (Canabate-Diaz et al., 2007; Harwood & Aparicio, 2000), which also greatly influence the organoleptic quality and stability of the oil. The diversity and interdependence between all these factors makes it highly unlikely that these influences would be the same in different regions. Hence, the geographical characterization of VOO addresses all these agronomic, pedoclimatic and

botanical aspects which are unique to the oil of each origin (Aparicio et al., 1994).

A considerable number of sensorial (Pardo et al., 2007), physical (F. Marini et al., 2004; Federico Marini et al., 2006) and chemical (Aguilera et al., 2005; Boggia et al., 2002; Lanteri et al., 2002; Federico Marini et al., 2006; Federico Marini et al., 2007) approaches combined with

adulteration with olive oils that do not fulfill the PDO requirements).

**1.1 Geographical origin of virgin olive oil** 

statistical analysis have been used to distinguish olive oils from different types, botanical, geographical origins and pedoclimatic conditions. For this purpose, fatty acids (Matos et al., 2007; Ollivier et al., 2006), triglycerides (Aranda et al., 2004), sterols (Alves et al., 2005; Matos et al., 2007; Temime et al., 2008), phenolic compounds (Lopez Ortiz et al., 2006; Vinha et al., 2005), and pigments (Cichelli & Pertesana, 2004) have been analyzed by conventional methods that usually require time-consuming pre-treatment methods (solvent extraction, isolation and/or derivatization) followed by chromatographic techniques (Aparicio & Aparicio-Ruiz, 2000) such as GC-MS and/or GC-FID (Bechir Baccouri et al., 2007; Haddada et al., 2007; Temime et al., 2006; Vichi et al., 2005) and HPLC-MS (Canabate-Diaz et al., 2007; Lopez Ortiz et al., 2006). PDO olive oils were distinguished using physicochemical parameters of the oils and chemometric class-modeling tools (Federico Marini et al., 2006), sensory parameters and fatty acid profiles of the oils (Ollivier et al., 2006), or the oil sterol composition (Alves et al., 2005). IRMS measurements of the alcohol and sterol fractions of olive oil also proved to be useful for its geographical characterization (Angerosa et al., 1999).

Fingerprinting techniques such as NMR (Mannina & Segre, 2002), NIR (Mignani et al., 2011), MIR (Reid et al., 2006), fluorescence (Kunz et al., 2011), FT-IR, FT-MIR and FT-Raman (Baeten et al., 2005; Lopez-Diez et al., 2003; Yang et al., 2005) spectroscopies, MS (Vaclavik et al., 2009), GC (Pizarro et al., 2011; Vaz-Freire et al., 2009) and DNA fingerprinting (Martins-Lopes et al., 2008; Ranalli et al., 2008) have been used for the determination of food authenticity (Reid et al., 2006). These types of techniques are particularly attractive since they are non selective, require little or no sample pre-treatment; use small amounts of organic solvents or reagents; and the analysis takes only a few minutes per sample. Chemometric analysis of NIR spectra of virgin olive oils allow us to determine its composition and geographical origin (Galtier et al., 2007). 1H, 13C and/or 31P-NMR analysis of the bulk oil (Rosa M. Alonso-Salces et al., 2010b; Rosa M. Alonso-Salces et al., 2011b; Mannina et al., 2010; Petrakis et al., 2008; Rezzi et al., 2005) or the unsaponifiable fraction of olive oil (R. M. Alonso-Salces et al., 2010), in combination with multivariate techniques, have been used to distinguish VOOs from certain geographical origins. In section 3.2., the achievements of 1H-NMR fingerprinting and chemometrics for the geographical characterization of VOO is reported. 1H-NMR fingerprints of a statistically significant number of authentic VOOs from seven countries, namely Italy, Spain, Greece, France, Turkey, Cyprus and Syria and from three different harvests (2004/05, 2005/06 and 2006/07) were analyzed by pattern recognition and classification techniques, such as principal component analysis (PCA), linear discriminant analysis (LDA) and partial least square discriminant analysis (PLS-DA), to evaluate the best approach to identify the geographical origin at the national, regional and/or PDO level.

#### **1.2 Stability of virgin olive oil**

Another matter of major concern regarding the quality of edible oils is their oxidation, not only from the technological and economic point of view but also for safety reasons, due to the undesirable properties of some compounds produced during this process (Guillen & Ruiz, 2001).

The high resistance to oxidative deterioration of VOO is due to both its triglyceride composition, which is low in polyunsaturated fatty acids, and its antioxidant constituents, i.e. polyphenols and tocopherols. The oxidative stability of VOO has been evaluated by

Quality Assessment of Olive Oil by <sup>1</sup>

**2.1 Chemicals and plant material** 

Chemie (Steinheim, Germany).

**2. Experimental** 

**2.2 NMR analysis** 

H-NMR Fingerprinting 189

Deuterated chloroform for NMR analysis (99.8 atom %D) was provided by Sigma-Aldrich

Virgin olive oils (963 samples) from seven countries of the Mediterranean basin, namely Italy (661 VOOs), Spain (144 VOOs), Greece (97 VOOs), France (39 VOOs), Turkey (14 VOOs), Cyprus (6 VOOs) and Syria (2 VOOs), were collected directly from the producers (olive oil mills) from most of the main producing regions of these countries during three harvests (2004/05, 2005/06 and 2006/2007). The sample collection was carried out with the collaboration of Dipartimento di Chimica e Technologie Farmaceutiche ed Alimentari - Università degli Studi di Genova (Italia), Laboratorio Arbitral Agroalimentario (Ministry of Agriculture and Fishery, Spain), General Chemical State Laboratory D'xy Athinon (Greece), General State Laboratory (Ministry of Health, Cyprus), Departamento de Química Orgánica - Universidad de Córdoba (Spain), Istituto di Metodologie Chimiche - Laboratorio di Risonanza Magnetica Annalaura Segre – CNR (Italy), Fondazione Edmund Mach - Istituto San Michele all'Adige (Italy), and Eurofins Scientific Analytics (France), in the framework of the EU TRACE project. The true type (virgin or extra virgin) and origin of the olive oils at the national, regional and PDO level were assured. The Italian samples were representative of the olive oil producing areas, which are markedly influenced by

For the study of VOO stability, about a liter of VOO was divided into aliquots contained in dark glass 40mL-vials completely filled and kept at -20ºC in a freezer. Each month, over a period of more than 3 and half years (samples for the months 20th, 32nd, 38th and 42nd are missing), one vial was taken from the freezer and stored at room temperature (r.t.) in a closed box. A preliminary supposition was made; this considered that the degradation of VOO at -20ºC is not significant and thus the last aliquot taken out of the freezer was time 0. All aliquots were analyzed by 1H-NMR once the last sample was taken from the freezer.

Aliquots of 40 µL of each VOO were dissolved in 200 µL of deuterated chloroform, shaken in a vortex, and placed in a 2 mm NMR capillary. The 1H-NMR experiments were performed at 300K on a Bruker (Rheinstetten, Germany) Avance 500 (nominal frequency 500.13 MHz) equipped with a 2.5 mm broadband inverse probe. The spectra of the samples used for the study of the geographical origin of VOOs were recorded using a 7.5 µs pulse (90°), an acquisition time of 3.0 s (32k data points) and a total recycling time of 4.0 s, a spectral width of 5500 Hz (11 ppm), 64 scans (+ 4 dummy scans), with no sample rotation. The spectra of the samples used for the VOO stability study were recorded using a 6.7 µs pulse (90°), an acquisition time of 3.5 s (50k data points) and a total recycling time of 7.0 s, a spectral width of 7100 Hz (14 ppm), 32 scans (+ 4 dummy scans), with no sample rotation. Prior to Fourier transformation, the free induction decays (FIDs) were zero-filled to 64k and a 0.3 Hz linebroadening factor was applied. The chemical shifts are expressed in scale (ppm), referenced to the residual signal of chloroform (7.26 ppm) (Hoffman, 2006). The spectra were phase- and baseline-corrected manually, binned with 0.02 ppm-wide buckets, and normalized to total intensity over the region 4.10-4.26 ppm (glycerol signal). TopSpin 1.3 (2005) and Amix-Viewer 3.7.7 (2006) from Bruker BioSpin GMBH (Rheinstetten, Germany)

pedoclimatic factors from the North to the South of the country.

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.

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 principal component analysis (PCA).
