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Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations
FRIN-04645; No of Pages 6 Food Research International xxx (2013) xxx–xxx
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Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations Raquel Garcia ⁎, Nuno Martins, Maria João Cabrita ICAAM — Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 8 April 2013 Accepted 4 May 2013 Available online xxxx Keywords: Olive oil Adulterations Refined olive oil Extra virgin olive oil
a b s t r a c t Authentication is becoming an issue of increasing relevance in olive oil and is generally motivated by economic benefits. Blending of extra virgin olive oil (EVOO) with refined olive oil (ROO) constitutes one of the most common types of adulteration of this top grade product. Concerning this particular topic, the most recent attempts of several research groups on the finding of some target compounds as markers of adulteration of EVOO with ROO will be reviewed. All efforts developed until now to find markers of adulteration in blends of EVOO with ROO have contributed to increase the knowledge about this topic although it is mandatory to exploit new compounds that could be assigned as reliable adulteration markers able to detect with high selectivity, sensitivity and accuracy this fraudulent practice. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays, olive oil authentication has emerged as an issue of the most relevance world-wide (Angerosa, Campestre, & Giansante, 2006; Frankel, 2010). In the last years, much attention has been given into fraudulent practices associated with olive oil traceability focused with special emphasis on the botanical origin due to the recent introduction in the market of high-quality monovarietal olive oil. Aiming to find traceability markers several studies have been performed allowing the discrimination of compositional and genetical markers (Montealegre, Alegre, & García-Ruiz, 2010). The guarantee of olive oil geographical origin is another matter of concern for the olive oil industry and recently some researchers have proposed new methodologies based on Near Infrared (NIR) Spectroscopy (Woodcock, Downey, & O'Donnell, 2008), Nuclear Magnetic Resonance (NMR) Spectroscopy (Agiomyrgianaki, Petrakis, & Dais, 2012; Mannina & Sobolev, 2011) and Synchronous Fluorescence Spectroscopy (Kunz, Ottaway, Kalivas, Georgiou, & Mousdis, 2011). Since extra virgin olive oil (EVOO) is considered the top grade of olive oil, it could be more susceptible of economic fraud, being the most common practice the addition of seed oils, such as sunflower, soybean and hazelnut oil. Recent work by Sánchez-Hernández, Marina, and Crego (2011) studied the role of non-protein amino acids as novel markers for the detection of EVOO adulterated with seed oils using a new analytical methodology based on capillary electrophoresis– mass spectrometry enabling the identification and quantification of ornithine and alloisoleucine in EVOO adulterated with soybean
⁎ Corresponding author. Tel.: +351 266 760 869; fax: +351 266 760 828. E-mail addresses: [email protected], [email protected] (R. Garcia).
oil. This methodology seems to be promising allowing the quality evaluation of EVOO as well as its authentication. Additionally, Chen et al. (2011) have performed preliminary screening studies related with the usefulness of δ-tocopherol as marker of EVOO/sunflower, EVOO/hazelnut and EVOO/peanut blends. More recently, studies developed by Calvano, De Ceglie, D'Accolti, and Zambonin (2012) have proposed phospholipids as eventual markers of EVOO adulteration with hazelnut oil based on a methodology comprising a previous selective extraction and enrichment of phospholipids from EVOO and hazelnut oil (HO) followed by the analyses using matrix- assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). The results seem to indicate that this methodology allows the detection of low percentages of hazelnut oil in blends of EVOO and HO. Another possible fraudulent economic practice is the forbidden addition of cheaper olive oil, namely refined olive oil (ROO) to EVOO. The detection of adulterations requires the use of analytical methodologies which can easily, rapidly and accurately detect those fraudulent practices. Recently, Torrecilla, García, García, and Rodríguez (2011) have proposed a methodology based on thermophysical properties to quantify adulterations of EVOO with ROO at relatively low concentrations allowing also the quantification of impurities during olive oil production to control on-line the quality of EVOO before bottling. These studies were also extended to quantify adulterations of EVOO with refined olive pomace, sunflower or corn oils. Indubitably, the detection of those illegal practices is crucial either for consumer's health and wealth protection (García-González & Aparicio, 2010), as well as for quality assurance. European Union and global food policies continually demand even stricter monitoring and control of food quality. Since adulterations have become more sophisticated, new methodologies will be required with suitable sensitivity, selectivity and accuracy to detect those fraudulent practices.
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Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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In the last years, in order to assure the authenticity of EVOO several research groups have been focused on the development of analytical methodologies comprising mainly the use of chromatographic techniques, namely Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) and more recently on spectroscopic techniques, such as Nuclear Magnetic Resonance (NMR) (Dais & Hatzakis, 2013; Fragaki, Spyros, Siragakis, Salivaras, & Dais, 2005; Ogrinc, Košir, Spangenberg, & Kidrič, 2003). Some approaches relying with electronic nose (Oliveros et al., 2002), near-infrared spectroscopy (Christy, Kasemsumran, Du, & Ozaki, 2004), fluorescence spectroscopy (Sikorska, Khmelinskii, & Sikorski, 2011), differential scanning calorimetry (Chiavaro et al., 2008), weak chemiluminescence (Papadopoulos, Triantis, Tzikis, Nikokavoura, & Dimotikali, 2002), Raman Spectroscopy (Dong, Zhang, Zhang, & Wang, 2012; Zou et al., 2009) and gas chromatograph–ion mobility spectrometer with an ultraviolet source (Garrido-Delgado, Arce, & Valcárcel, 2012; Garrido-Delgado, Mercader-Trejo, Arce, & Valcárcel, 2011; Garrido-Delgado et al., 2011) have been also introduced as analytical methodologies to discriminate olive oil commercial categories. Generally, detection of olive oil adulterations with other vegetable oils has been focused on the major constituents of olive oil, namely fatty acids (FA) and triacylglycerols (TAG) (Angerosa et al., 2006; Frankel, 2010). Since authentication of EVOO is of mandatory relevance many methodologies based on the identification of eventual markers have been explored. Triacylglycerols have been explored as a tool in the authentication and genuineness of EVOO and the results proved that those compounds have potential for the detection of changes in the EVOO composition due to forbidden blending (Bosque-Sendra, Cuadros-Rodríguez, Ruiz-Samblás, & de la Mata, 2012). More recently, Lukic, Lukic, Krapac, Sladonja, and Pilizota (2013) have proved that sterols and triterpene diols can be used as reliable indicators of variety and ripening degree among olive oils. Since the detection of EVOO adulteration based on fatty acid composition analysis combined with traditional current analytical techniques constitute a very challenging task, there is a growing interest on exploring an alternative methodology based on the application of DNA-based detection methods (Agrimonti, Vietina, Pafundo, & Marmiroli, 2011) in order to assess the role of DNA as a tool to detect adulterations (Costa, Mafra, & Oliveira, 2012; He et al., 2013; Kumar, Kahlon, & Chaudhary, 2011; Zhang et al., 2012). Particularly, to detect bleeding of EVOO with ROO several attempts have been proposed mainly based on some target compounds which belong to olive oil's bioactive compounds, namely chlorophylls, diacylglycerols (DGs), ester derivatives of FA, straight chain wax esters, sterol components and TAGs. Due to sample complexity, the detection of olive oil adulteration constitutes a really difficult and challenging analytical hindrance (Maggio, Cerretani, Chiavaro, Kaufman, & Bendini, 2010). Moreover, blending of EVOO with ROO does not produce an easily detectable modification on the chemical composition of the final blend due to the mild conditions used in the deodorization process and, consequently, the identification of eventual adulteration is a demanding analytical task (Saba, Mazzini, Raffaelli, Mattei, & Salvadori, 2005). The finding of chemical compounds which could be assigned as adulteration markers in EVOO and ROO blends is considered of crucial significance and indubitably will contribute to detect more accurately those fraudulent practices. Aiming to contribute to the current knowledge about the usefulness of some target compounds as potential candidates of adulteration markers of EVOO and ROO blends, the most significant attempts introduced in the last years on the detection of this kind of adulteration will be focused in this paper. 2. Olive oil commercial categories Olive oil has been reported as a primary consumption product and an essential component of healthy diet for majority of people living in
Mediterranean countries due to its rich nutritional value, namely high monounsaturated fatty-acid and antioxidant properties (Bendini et al., 2007; Frankel, 2011; Visioli, Bogani, & Galli, 2006). According to the European Union legislation (EC. Off. J. Eur. Communities, 2003), olive oil is classified into some categories reflecting its quality and organoleptic properties, namely extra virgin olive oil (EVOO), virgin olive oil (VOO), lampante virgin olive oil (LVOO) and also refined olive oil (ROO) among others. In particular, EVOO is considered to be the oil of the highest quality since it is obtained from olive fruits solely by mechanical or other physical means that do not lead to alterations of the oil, and also has not undergone treatment other than washing, decantation, centrifugation and filtration. Its free acidity expressed as oleic acid must be b0.8% and other characteristics are fixed for this category by the International Olive Oil Council (IOOC) and European Community (EC) Regulations (IOOC, 2011; Official Journal of European Community, 2003). The high cost of EVOO makes it prone to adulteration with olive oils of lower categories in order to increase economic benefits. However, this practice deteriorates its quality and nutritional value leading to major economic losses for the consumers and the loss of consumer confidence can also arise (Fragaki et al., 2005; Gurdeniz & Ozen, 2009; Mignani et al., 2011). One of the most common adulteration practices consists of blending EVOO with ROO (Fragaki et al., 2005; Frankel, 2010) which is obtained usually from virgin olive oil mechanically extracted from damaged olive fruits or from olives stored in unsuitable conditions and using refining methods that does not lead to alterations in the initial glyceridic structure. It has a free acidity, expressed as oleic acid, of not more than 0.3 g per 100 g (IOOC, 2011). Particularly, the refining process can be accomplished in two ways: alkali or physical refining. In the alkali refining, the oil is treated with dilute acid (the two most common degumming agents are citric and phosphoric acid) to precipitate the phosphatides and proteinaceous material, which are separated by settling or centrifugation. After degumming, the oil is neutralized either in a continuous or batched system. Next, the oil obtained is bleached under vacuum with mixtures of various adsorbents and filtered by filter presses equipped with a solvent system for recovering oil which is then entrained in the bleaching earth, followed by deodorization of the bleached oil. Finally, the refined oil is mixed with virgin oil in order to improve the organoleptic attributes as well as the chemical properties of the final oil. In the process of physical refining, the oil is initially degummed and bleached and after that, in order to remove the volatiles and free fatty acids (92–95%) the oil is deodorized (continuous distillation). Usually, deodorization is finished before removal of all of the free fatty acids, and the oil is alkali refined to remove the remainder of the free fatty acids. The advantage of this procedure is the improvement of the oil stability by hampering the formation of oxidation products (Firestone, 2005; Petrakis, 2006). In the current context, it is crucial to ensure EVOO authenticity in order to guarantee the safety and consumer protection and to avoid the image of a hypothetical uncontrolled distribution of adulterated olive oil into the market.
3. Putative markers of EVOO adulterations with ROO During the last years, several researchers have been devoted to the study of reasonable marker candidates of adulteration of EVOO with ROO. It should be taken into account that most of those compounds belong to the major constituents of olive oil and are chosen based on some quality indicators, such as TAG composition and sterols. More recently, new approaches have been explored based on minor components of olive oil. In this section will be discussed in detail the more significant compounds belonging to those class which were proposed as putative markers of EVOO adulteration with ROO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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of DG (1,3-DG) is ascribed to enzymatic or chemical hydrolysis of TAGs which are formed before or during olive oil extraction process (Pérez-Camino, Moreda, & Cert, 2001). Then, olive oils obtained with poor-quality olive fruits show a significant raise of 1,3-DG while olive oils originating from healthy olive fruits contain almost exclusively 1,2-DG. Mild refining process of virgin oil which involves some steps, such as neutralization, washing and deodorization of the oil at low temperatures under vacuum leads to a decrease of total contents of DGs and subsequent raise of 1,3-DG/1,2-DG ratio (Pérez-Camino et al., 2001). During the storage, 1,2-DG is gradually transformed into 1,3-DG and hence 1,3-DG/1,2-DG ratio has been considered as an valuable marker of olive oil's freshness and quality (Mannina & Sobolev, 2011). For the past 20 years, the role of DGs as possible markers of adulteration has been investigated and within this purpose several approaches have been explored. One of the first studies developed by Pérez-Camino et al. (2001), aimed to assess the evolution of 1,3- and 1,2-DG isomers in olive oils obtained from healthy olives during storage and to detect deodorized oils in virgin olive oils (VOO) using an analytical procedure comprising a solid phase extraction (SPE) followed by GC analysis on polar columns. This approach proved to have scarce utility on the detection of blends of mild refined oils in VOO due to the lower DG contents. However, particularly for VOO with low acidities the usefulness of 1,3-DG/1,2-DG ratio to evaluate their genuineness and the storage conditions as well as on the determination of VOO aging has been claimed by the authors. Furthermore, Mannina, Sobolev, and Segre (2003) have applied 1H NMR spectroscopy to evaluate 1,2-DG and 1,3-DG contents in EVOO and ROO. Results obtained in this study also corroborated the hypothesis that 1,2-DGs usually present in olive oils are transformed progressively during the storage into 1,3-DGs. Thus, these compounds
3.1. Natural chlorophyll Natural chlorophylls comprising chlorophyll A and B are the pigments responsible for the characteristic green color of the olive drupe. During the ripening of the olives some chemical and physical changes occur leading to changes in the chlorophyll fraction profile which affects the color of the olives as well as the olive oil extracted from them (Giuliani, Cerretani, & Cichelli, 2011). Those compounds are irreversibly converted into more stable pigments called as pheophytins, in which the central Mg 2+ ion of the porphyrin ring is replaced by two hydrogen atoms. Pyropheophytins are another class of natural chlorophylls which are also formed resulting from the removal of the carboxy-methyl group from the pheophytin structure (Giuliani et al., 2011). Chemical structures of pheophytin A and pyropheophytin A are depicted in Fig. 1. Earlier studies of Serani and Piacenti (1992) were related with the study of pheophytin A photodecomposition in EVOO aiming to assess the influence of temperature and light intensity on the kinetic constant of this reaction. More recently, Gertz and Fiebig (2006) have developed a procedure to determine the degradation products of chlorophyll A, namely pheophytin A and pyropheophytin A, in olive oil. However, since pyropheophytin A content increases along EVOO shelf life only scarcely this compound can be considered a marker of adulteration. 3.2. Diacylglycerols Diacylglycerols (DGs) are present in EVOO in two isomeric forms 1,2- and 1,3-isomers, as depicted in Fig. 2, ranging from 1 to 3% (Mannina & Sobolev, 2011). While the occurrence of 1,2 isomers of DG (1,2-DG) is attributed to the incomplete biosynthesis of TAGs, the presence of 1,3 isomers
H3C
H
3
CH3
CH3
O
H3C N H
H
O N
NH
CH3
O
N
CH3
H CH3
CH3
O CH2
CH3
H
CH3
CH3
O Pheophytin A
NH
N
H
N
HN
H
O
O O
O Pyropheophytin A Fig. 1. Natural chlorophyll present in EVOO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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CH2OOCR
CH2OOCR R'COO
C
H
CH2OH sn-1,2- diacylglycerol
HO
C
H
CH2OOCR' sn-1,3- diacylglycerol
Fig. 2. Diacylglycerol isomeric forms in olive oil.
could be considered as a good indicator of freshness and quality of an olive oil (Mannina et al., 2003), but seem to be not suitable as markers of EVOO adulteration with ROO. Recently, studies performed by Fragaki and co-workers have allowed the determination in a single experiment of 1,2-DG, 1,3-DG and total DGs' (TDGs) contents and the ratios of 1,2-DGs over TDGs using 31P NMR spectroscopic experiments on different grades of olive oil, namely EVOO, ROO and blended olive oil composed of ROO and EVOO (BOO) (Fragaki et al., 2005). To achieve this purpose an analytical methodology has been employed comprising a derivatization step involving the replacement of the labile hydrogens of hydroxyl and carboxyl groups with 2-chloro-4,4,5,5-tetramethyl dioxaphospholane leading to the production of the correspondent phosphitylated compounds, followed by the subsequent 31P NMR analysis (Spyros & Dais, 2000; Vigli, Philippidis, Spyros, & Dais, 2003). The results have shown the usefulness of this methodology to detect olive oil adulteration applying a 31P NMR method and a multivariate statistical analysis allowing the estimation of BOO sample composition. According to these results, EVOO samples possess 1,3-DG contents much lower than the corresponding values in the ROO samples which corroborate the fact that isomerization of 1,2-DGs to 1,3-DGs, which frequently occurs during prolonged olive oil storage, also happens through olive oil refinement (Fragaki et al., 2005). This finding also corroborates that the use of diacylglycerols isomers as markers of adulterations must be done carefully since it represents some limitations. 3.3. Fatty acids and wax esters Olive oil includes on its composition a class of natural neutral lipids called fatty acid alkyl esters (FAAEs) which are formed by esterification of free fatty acids (FA). These compounds were detected firstly by Mariani and Fedeli (1986) which isolated the “nonpolar fraction” of olive oil by means of column liquid chromatography and analyzed it by GC. Formation of FAAEs is promoted by unsuitable practices related with olive oil extraction and scarce quality of olive fruits (Pérez-Camino, Moreda, Mateos, & Cert, 2002). Recent studies developed by Pérez-Camino and co-workers are devoted to the evaluation of soft deodorization on the concentration of FAAEs and the potential utility of these compounds as adulteration markers on the detection of blends of mildly ROO with EVOO (Pérez-Camino, Cert, Romero-Segura, Cert-Trujillo, & Moreda, 2008). Within this purpose, the authors have quantified FAAE contents using a previous sample preparation method based on the isolation of those compounds with a silica gel solid phase extraction cartridge followed by GC analysis. The study has been applied to around one hundred of Spanish olive oils of several categories, varieties and geographical origin allowing differentiating of EVOO from other categories of olive oils that has been subjected to a mild refining process. The results shown that blending process of EVOO with refined low quality olive oils can be assessed by the measurement of their alkyl ester concentrations (Pérez-Camino et al., 2008). The profiles of fatty acids and fatty alcohol esters are related with olive oil categories thus straight chain wax esters were indicated as useful quality and purity indicators of olive oils. Hence, the distinction of VOO from ROO based on wax esters could be achieved since VOO
posses a higher content of C36 and C38 waxes than of C40, C42, C44 and C46 while ROO exhibits an inverse relation (Aparicio & Aparicio-Ruíz, 2000). Since straight chain wax esters were indicated as potentially useful quality/purity markers in olive oils some attention were given to those compounds in order to evaluate their relevance on the detection of EVOO and ROO blends (Biedermann, Hase-Aschoff, & Grob, 2008). Those compounds are located in the waxy surface layer of the olive but are poorly extracted from the olive fruit by physical pressing operation. According to the literature, olive oils of low quality possess these wax esters at higher concentrations. Biedermann and co-workers have optimized an LC–GC–FID method to measure methyl and ethyl oleate contents as well as wax esters' fraction of the C26 and C28 alcohol with unsaturated C18 fatty acids in order to clarify their significance for the evaluation of olive oil quality (Biedermann et al., 2008). Results have shown that the presence of high wax ester content is a consequence of degrading olives and their formation also occurs during the storage of olive oils. Therefore, assignment of straight chain wax esters as quality markers is more controversial since those compounds are continuously formed during storage constituting a limitation for their use as adulteration markers (Aparicio & Aparicio-Ruíz, 2000; Biedermann et al., 2008). 3.4. Sterols Sterols are present on the unsaponifiable matter of almost all fats and oils. The analysis of the unsaponifiable fraction is being considered as a powerful tool to detect adulteration allowing the differentiation between olive oils of different quality (Lerma-Garcia, 2012). These compounds have been ascribed the genuineness of some vegetable oils because this fraction is more characteristic than the fatty acid profile, which explains why this family of compounds has been widely used in olive oil authentication (Martínez-Vidal, Garrido-Frenich, Escobar-García, & Romero-González, 2007). Further investigations on variations of the ratio of free to total sterols in different categories of olive oil and the usefulness of this ratio as a parameter for olive oil quality have been evaluated by Pasqualone and Catalano (2000). In particular, VOO possesses high levels of β-sitosterol and Δ5-avenasterol (Fig. 3) constituting a potential differentiation from other oils (Aparicio & Aparicio-Ruíz, 2000). In general, refining process leads to a decrease in the total content of sterols and increase esterified sterols in the oil (Jiménez de Blas & dell Valle González, 1996; Phillips, Ruggio, Toivo, Swank, & Simpkins, 2002). Concretely, during refining process of edible oils and fats a dehydration of β-sitosterol occurs yielding
HO
HO
Δ5- Avenasterol
β-Sitosterol
H H H H
H
Stigmasta 3,5- diene
Fig. 3. Some sterols present in VOO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
R. Garcia et al. / Food Research International xxx (2013) xxx–xxx
stigmasta-3,5-diene (Fig. 3) (León-Camacho, Serrano, & Constante, 2004). Experimentally, its quantification is performed by means of an analytical methodology comprising the previous purification of stigmasta-3,5-diene by preparative column chromatography followed by its analysis using gas chromatography. However, this procedure is considered a time-consuming and laborious work. Kinetic studies performed by León-Camacho et al. (2004) using different independent variables namely time, temperature, flow of stripping gas and the thickness of oil layer have allowed the measurement of apparent thermodynamic parameters related with the formation of stigmasta-3,5-diene through the deodorization refining process. According to these studies, the formation of stigmasta-3,5-diene from β-sitosterol and therefore stigmasta-3,5-diene contents are strongly dependent of the temperature used during the physical refining (León-Camacho et al., 2004). Then, stigmasta-3,5-diene has been considered potentially remarkable on the detection of ROO in VOO (León-Camacho et al., 2004). However, stigmastadienes could be only seen as reliable indicators of olive oil adulteration when their concentrations ranged between 0.01 and 4 mg/kg (Al-Ismail, Alsaed, Ahmad, & Al-Dabbas, 2010) limiting their use on the detection of ROO and olive oil blends. More recently, Martínez-Vidal et al. (2007) have introduced an analytical methodology based on liquid chromatography coupled to mass spectrometry (LC–MS) with atmospheric-pressure chemical ionization in positive ion mode which enables the determination of the sterol composition of different types of olive oils, namely EVOO and ROO. This methodology seems to be most advantageous since it allows the reduction of sample handling and analysis time. According to these results, sterol contents can be used to distinguish between different types of oil, in particular authentic olive oil and seed oils (Martínez-Vidal et al., 2007). 3.5. TAG oligopolymers Recently, studies of Caponio and co-workers are committed to the discrimination between VOO and ROO based on the quantification of some polar compounds (PC), namely triacylglycerol oligopolymers (TAGP), oxidized triacylglycerols (ox-TAG) and diacylglycerols (DAG) by means of High Performance Size-Exclusion Chromatography (HPSEC) involving a previous step comprising a purification of olive oil samples by silica gel column chromatography. In particular, TAGP is produced during deodorization in the refining process due to the higher temperatures achieved, being considered as a relevant indicator of the secondary oxidative degradation of olive oil since their presence is independent of processing conditions. The other compounds — ox-TAG and DAG, are linked with primary oxidation level of the olive oil and the hydrolytic degradation of ROO, respectively (Caponio et al., 2011). Ox-TAG could undertake polymerization reactions promoting the formation of TAGP. It's assumed that TAGP were absent or present at very low concentrations in EVOO although present in relatively high amounts in ROO and the same trend is observed for ox-TAG. Therefore, especially TAGP could be a valuable marker to discriminate olive oils complemented with HPSEC (Caponio et al., 2011). Since the formation of TAGP could be ascribe to the bleaching and especially deodorization steps during the refining process, this compound displays a potential role on the discrimination among EVOO and ROO. However, the analytical methodology implemented by Caponio et al. (2011) has not been applied to more complex experimental designs including several blends of oils thus the usefulness of that methodology for the detection of EVOO adulterated with ROO requires more detailed studies. 4. Conclusions Adulteration of olive oil is an issue of crucial significance because of its impact in quality, nutritional value and safety of consumers.
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Due to the inherent EVOO high-cost, the adulteration of this kind of product with low-quality olive oils seems to be actually one of the most common types of fraud. The development of analytical methodologies which enable detection of adulterations is warranted since the addition of ROO to EVOO at low percentages could be a very challenging task. In spite of all attempts developed until now, it seems to be relatively consensual that none of the compounds described above can be considered as a reliable adulteration marker of EVOO and ROO blends, since their appearance is not exclusively related with the refining process used on the ROO production. Thus, the assignment of some components intimately ascribed to the refining step could be the key to overcome the limitations of the methodologies proposed until now to detect this kind of fraudulent practice. Nevertheless, the discovery of putative markers that could identify possible adulteration of EVOO with ROO could be considered of mandatory importance and will open new avenues in the field of “Food Authenticity”. Briefly, more efforts are needed to exploit new compounds that could be assigned as reliable adulteration markers able to detect with high selectivity, sensitivity and accuracy blends of EVOO with ROO. Acknowledgments This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors — COMPETE and national funds through FCT — Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011. References Agiomyrgianaki, A., Petrakis, P. V., & Dais, P. (2012). Influence of harvest year, cultivar and geographical origin on Greek extra virgin olive oils composition: A study by NMR spectroscopy and biometric analysis. Food Chemistry, 135, 2561–2568. Agrimonti, C., Vietina, M., Pafundo, S., & Marmiroli, N. (2011). The use of food genomics to ensure the traceability of olive oil. Trends in Food Science and Technology, 22, 237–244. Al-Ismail, K. M., Alsaed, A. K., Ahmad, R., & Al-Dabbas, M. (2010). Detection of olive oil adulteration with some plant oils by GLC analysis of sterols using polar column. Food Chemistry, 121, 1255–1259. Angerosa, F., Campestre, C., & Giansante, L. (2006). Analysis and authentication. In D. Boskou (Ed.), Olive oil: Chemistry and technology (pp. 113–172). American Oil Chemists' Society Press. Aparicio, R., & Aparicio-Ruíz, R. (2000). Authentication of vegetable oils by chromatographic techniques. Journal of Chromatography. A, 881, 93–104. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A. M., Segura-Carretero, A., Fernández-Gutiérrez, A., et al. (2007). Phenolic molecules in virgin olive oils: A survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules, 12, 1679–1719. Biedermann, M., Hase-Aschoff, P., & Grob, K. (2008). Wax ester fraction of edible oils: Analysis by on-line LC–GC–MS and GC × GC-FID. European Journal of Lipid Science and Technology, 110, 1084–1094. Bosque-Sendra, J. M., Cuadros-Rodríguez, L., Ruiz-Samblás, C., & de la Mata, A. P. (2012). Combining chromatography and chemometrics for the characterization and authentication of fats and oils from triacylglycerol compositional data — A review. Analytica Chimica Acta, 724, 1–11. Calvano, C. D., De Ceglie, C., D'Accolti, L., & Zambonin, C. G. (2012). MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent. Food Chemistry, 134, 1192–1198. Caponio, F., Summo, C., Bilancia, M. T., Paradiso, V. M., Sikorska, E., & Gomes, T. (2011). High performance size-exclusion chromatography analysis of polar compounds applied to refined, mild deodorized, extra virgin olive oils and their blends: An approach to their differentiation. LWT — Food Science and Technology, 44, 1726–1730. Chen, H., Angiuli, M., Ferrari, C., Tombari, E., Salvetti, G., & Bramanti, E. (2011). Tocopherol speciation as first screening for the assessment of extra virgin olive oil quality by reversed-phase high-performance liquid chromatography/fluorescence detector. Food Chemistry, 125, 1423–1429. Chiavaro, E., Rodriguez-Estrada, M. T., Barnaba, C., Vittadini, E., Cerretani, L., & Bendini, A. (2008). Differential scanning calorimetry: A potential tool for discrimination of olive oil commercial categories. Analytica Chimica Acta, 625, 215–226. Christy, A. A., Kasemsumran, S., Du, Y., & Ozaki, Y. (2004). The detection and quantification of adulteration in olive oil by near-infrared spectroscopy and chemometrics. Analytical Sciences, 20, 935–940. Costa, J., Mafra, I., & Oliveira, M. B. P. P. (2012). Advances in vegetable oil authentication by DNA-based markers. Trends in Food Science and Technology, 26, 43–55.
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Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations Raquel Garcia ⁎, Nuno Martins, Maria João Cabrita ICAAM — Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 8 April 2013 Accepted 4 May 2013 Available online xxxx Keywords: Olive oil Adulterations Refined olive oil Extra virgin olive oil
a b s t r a c t Authentication is becoming an issue of increasing relevance in olive oil and is generally motivated by economic benefits. Blending of extra virgin olive oil (EVOO) with refined olive oil (ROO) constitutes one of the most common types of adulteration of this top grade product. Concerning this particular topic, the most recent attempts of several research groups on the finding of some target compounds as markers of adulteration of EVOO with ROO will be reviewed. All efforts developed until now to find markers of adulteration in blends of EVOO with ROO have contributed to increase the knowledge about this topic although it is mandatory to exploit new compounds that could be assigned as reliable adulteration markers able to detect with high selectivity, sensitivity and accuracy this fraudulent practice. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays, olive oil authentication has emerged as an issue of the most relevance world-wide (Angerosa, Campestre, & Giansante, 2006; Frankel, 2010). In the last years, much attention has been given into fraudulent practices associated with olive oil traceability focused with special emphasis on the botanical origin due to the recent introduction in the market of high-quality monovarietal olive oil. Aiming to find traceability markers several studies have been performed allowing the discrimination of compositional and genetical markers (Montealegre, Alegre, & García-Ruiz, 2010). The guarantee of olive oil geographical origin is another matter of concern for the olive oil industry and recently some researchers have proposed new methodologies based on Near Infrared (NIR) Spectroscopy (Woodcock, Downey, & O'Donnell, 2008), Nuclear Magnetic Resonance (NMR) Spectroscopy (Agiomyrgianaki, Petrakis, & Dais, 2012; Mannina & Sobolev, 2011) and Synchronous Fluorescence Spectroscopy (Kunz, Ottaway, Kalivas, Georgiou, & Mousdis, 2011). Since extra virgin olive oil (EVOO) is considered the top grade of olive oil, it could be more susceptible of economic fraud, being the most common practice the addition of seed oils, such as sunflower, soybean and hazelnut oil. Recent work by Sánchez-Hernández, Marina, and Crego (2011) studied the role of non-protein amino acids as novel markers for the detection of EVOO adulterated with seed oils using a new analytical methodology based on capillary electrophoresis– mass spectrometry enabling the identification and quantification of ornithine and alloisoleucine in EVOO adulterated with soybean
⁎ Corresponding author. Tel.: +351 266 760 869; fax: +351 266 760 828. E-mail addresses: [email protected], [email protected] (R. Garcia).
oil. This methodology seems to be promising allowing the quality evaluation of EVOO as well as its authentication. Additionally, Chen et al. (2011) have performed preliminary screening studies related with the usefulness of δ-tocopherol as marker of EVOO/sunflower, EVOO/hazelnut and EVOO/peanut blends. More recently, studies developed by Calvano, De Ceglie, D'Accolti, and Zambonin (2012) have proposed phospholipids as eventual markers of EVOO adulteration with hazelnut oil based on a methodology comprising a previous selective extraction and enrichment of phospholipids from EVOO and hazelnut oil (HO) followed by the analyses using matrix- assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). The results seem to indicate that this methodology allows the detection of low percentages of hazelnut oil in blends of EVOO and HO. Another possible fraudulent economic practice is the forbidden addition of cheaper olive oil, namely refined olive oil (ROO) to EVOO. The detection of adulterations requires the use of analytical methodologies which can easily, rapidly and accurately detect those fraudulent practices. Recently, Torrecilla, García, García, and Rodríguez (2011) have proposed a methodology based on thermophysical properties to quantify adulterations of EVOO with ROO at relatively low concentrations allowing also the quantification of impurities during olive oil production to control on-line the quality of EVOO before bottling. These studies were also extended to quantify adulterations of EVOO with refined olive pomace, sunflower or corn oils. Indubitably, the detection of those illegal practices is crucial either for consumer's health and wealth protection (García-González & Aparicio, 2010), as well as for quality assurance. European Union and global food policies continually demand even stricter monitoring and control of food quality. Since adulterations have become more sophisticated, new methodologies will be required with suitable sensitivity, selectivity and accuracy to detect those fraudulent practices.
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Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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In the last years, in order to assure the authenticity of EVOO several research groups have been focused on the development of analytical methodologies comprising mainly the use of chromatographic techniques, namely Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) and more recently on spectroscopic techniques, such as Nuclear Magnetic Resonance (NMR) (Dais & Hatzakis, 2013; Fragaki, Spyros, Siragakis, Salivaras, & Dais, 2005; Ogrinc, Košir, Spangenberg, & Kidrič, 2003). Some approaches relying with electronic nose (Oliveros et al., 2002), near-infrared spectroscopy (Christy, Kasemsumran, Du, & Ozaki, 2004), fluorescence spectroscopy (Sikorska, Khmelinskii, & Sikorski, 2011), differential scanning calorimetry (Chiavaro et al., 2008), weak chemiluminescence (Papadopoulos, Triantis, Tzikis, Nikokavoura, & Dimotikali, 2002), Raman Spectroscopy (Dong, Zhang, Zhang, & Wang, 2012; Zou et al., 2009) and gas chromatograph–ion mobility spectrometer with an ultraviolet source (Garrido-Delgado, Arce, & Valcárcel, 2012; Garrido-Delgado, Mercader-Trejo, Arce, & Valcárcel, 2011; Garrido-Delgado et al., 2011) have been also introduced as analytical methodologies to discriminate olive oil commercial categories. Generally, detection of olive oil adulterations with other vegetable oils has been focused on the major constituents of olive oil, namely fatty acids (FA) and triacylglycerols (TAG) (Angerosa et al., 2006; Frankel, 2010). Since authentication of EVOO is of mandatory relevance many methodologies based on the identification of eventual markers have been explored. Triacylglycerols have been explored as a tool in the authentication and genuineness of EVOO and the results proved that those compounds have potential for the detection of changes in the EVOO composition due to forbidden blending (Bosque-Sendra, Cuadros-Rodríguez, Ruiz-Samblás, & de la Mata, 2012). More recently, Lukic, Lukic, Krapac, Sladonja, and Pilizota (2013) have proved that sterols and triterpene diols can be used as reliable indicators of variety and ripening degree among olive oils. Since the detection of EVOO adulteration based on fatty acid composition analysis combined with traditional current analytical techniques constitute a very challenging task, there is a growing interest on exploring an alternative methodology based on the application of DNA-based detection methods (Agrimonti, Vietina, Pafundo, & Marmiroli, 2011) in order to assess the role of DNA as a tool to detect adulterations (Costa, Mafra, & Oliveira, 2012; He et al., 2013; Kumar, Kahlon, & Chaudhary, 2011; Zhang et al., 2012). Particularly, to detect bleeding of EVOO with ROO several attempts have been proposed mainly based on some target compounds which belong to olive oil's bioactive compounds, namely chlorophylls, diacylglycerols (DGs), ester derivatives of FA, straight chain wax esters, sterol components and TAGs. Due to sample complexity, the detection of olive oil adulteration constitutes a really difficult and challenging analytical hindrance (Maggio, Cerretani, Chiavaro, Kaufman, & Bendini, 2010). Moreover, blending of EVOO with ROO does not produce an easily detectable modification on the chemical composition of the final blend due to the mild conditions used in the deodorization process and, consequently, the identification of eventual adulteration is a demanding analytical task (Saba, Mazzini, Raffaelli, Mattei, & Salvadori, 2005). The finding of chemical compounds which could be assigned as adulteration markers in EVOO and ROO blends is considered of crucial significance and indubitably will contribute to detect more accurately those fraudulent practices. Aiming to contribute to the current knowledge about the usefulness of some target compounds as potential candidates of adulteration markers of EVOO and ROO blends, the most significant attempts introduced in the last years on the detection of this kind of adulteration will be focused in this paper. 2. Olive oil commercial categories Olive oil has been reported as a primary consumption product and an essential component of healthy diet for majority of people living in
Mediterranean countries due to its rich nutritional value, namely high monounsaturated fatty-acid and antioxidant properties (Bendini et al., 2007; Frankel, 2011; Visioli, Bogani, & Galli, 2006). According to the European Union legislation (EC. Off. J. Eur. Communities, 2003), olive oil is classified into some categories reflecting its quality and organoleptic properties, namely extra virgin olive oil (EVOO), virgin olive oil (VOO), lampante virgin olive oil (LVOO) and also refined olive oil (ROO) among others. In particular, EVOO is considered to be the oil of the highest quality since it is obtained from olive fruits solely by mechanical or other physical means that do not lead to alterations of the oil, and also has not undergone treatment other than washing, decantation, centrifugation and filtration. Its free acidity expressed as oleic acid must be b0.8% and other characteristics are fixed for this category by the International Olive Oil Council (IOOC) and European Community (EC) Regulations (IOOC, 2011; Official Journal of European Community, 2003). The high cost of EVOO makes it prone to adulteration with olive oils of lower categories in order to increase economic benefits. However, this practice deteriorates its quality and nutritional value leading to major economic losses for the consumers and the loss of consumer confidence can also arise (Fragaki et al., 2005; Gurdeniz & Ozen, 2009; Mignani et al., 2011). One of the most common adulteration practices consists of blending EVOO with ROO (Fragaki et al., 2005; Frankel, 2010) which is obtained usually from virgin olive oil mechanically extracted from damaged olive fruits or from olives stored in unsuitable conditions and using refining methods that does not lead to alterations in the initial glyceridic structure. It has a free acidity, expressed as oleic acid, of not more than 0.3 g per 100 g (IOOC, 2011). Particularly, the refining process can be accomplished in two ways: alkali or physical refining. In the alkali refining, the oil is treated with dilute acid (the two most common degumming agents are citric and phosphoric acid) to precipitate the phosphatides and proteinaceous material, which are separated by settling or centrifugation. After degumming, the oil is neutralized either in a continuous or batched system. Next, the oil obtained is bleached under vacuum with mixtures of various adsorbents and filtered by filter presses equipped with a solvent system for recovering oil which is then entrained in the bleaching earth, followed by deodorization of the bleached oil. Finally, the refined oil is mixed with virgin oil in order to improve the organoleptic attributes as well as the chemical properties of the final oil. In the process of physical refining, the oil is initially degummed and bleached and after that, in order to remove the volatiles and free fatty acids (92–95%) the oil is deodorized (continuous distillation). Usually, deodorization is finished before removal of all of the free fatty acids, and the oil is alkali refined to remove the remainder of the free fatty acids. The advantage of this procedure is the improvement of the oil stability by hampering the formation of oxidation products (Firestone, 2005; Petrakis, 2006). In the current context, it is crucial to ensure EVOO authenticity in order to guarantee the safety and consumer protection and to avoid the image of a hypothetical uncontrolled distribution of adulterated olive oil into the market.
3. Putative markers of EVOO adulterations with ROO During the last years, several researchers have been devoted to the study of reasonable marker candidates of adulteration of EVOO with ROO. It should be taken into account that most of those compounds belong to the major constituents of olive oil and are chosen based on some quality indicators, such as TAG composition and sterols. More recently, new approaches have been explored based on minor components of olive oil. In this section will be discussed in detail the more significant compounds belonging to those class which were proposed as putative markers of EVOO adulteration with ROO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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of DG (1,3-DG) is ascribed to enzymatic or chemical hydrolysis of TAGs which are formed before or during olive oil extraction process (Pérez-Camino, Moreda, & Cert, 2001). Then, olive oils obtained with poor-quality olive fruits show a significant raise of 1,3-DG while olive oils originating from healthy olive fruits contain almost exclusively 1,2-DG. Mild refining process of virgin oil which involves some steps, such as neutralization, washing and deodorization of the oil at low temperatures under vacuum leads to a decrease of total contents of DGs and subsequent raise of 1,3-DG/1,2-DG ratio (Pérez-Camino et al., 2001). During the storage, 1,2-DG is gradually transformed into 1,3-DG and hence 1,3-DG/1,2-DG ratio has been considered as an valuable marker of olive oil's freshness and quality (Mannina & Sobolev, 2011). For the past 20 years, the role of DGs as possible markers of adulteration has been investigated and within this purpose several approaches have been explored. One of the first studies developed by Pérez-Camino et al. (2001), aimed to assess the evolution of 1,3- and 1,2-DG isomers in olive oils obtained from healthy olives during storage and to detect deodorized oils in virgin olive oils (VOO) using an analytical procedure comprising a solid phase extraction (SPE) followed by GC analysis on polar columns. This approach proved to have scarce utility on the detection of blends of mild refined oils in VOO due to the lower DG contents. However, particularly for VOO with low acidities the usefulness of 1,3-DG/1,2-DG ratio to evaluate their genuineness and the storage conditions as well as on the determination of VOO aging has been claimed by the authors. Furthermore, Mannina, Sobolev, and Segre (2003) have applied 1H NMR spectroscopy to evaluate 1,2-DG and 1,3-DG contents in EVOO and ROO. Results obtained in this study also corroborated the hypothesis that 1,2-DGs usually present in olive oils are transformed progressively during the storage into 1,3-DGs. Thus, these compounds
3.1. Natural chlorophyll Natural chlorophylls comprising chlorophyll A and B are the pigments responsible for the characteristic green color of the olive drupe. During the ripening of the olives some chemical and physical changes occur leading to changes in the chlorophyll fraction profile which affects the color of the olives as well as the olive oil extracted from them (Giuliani, Cerretani, & Cichelli, 2011). Those compounds are irreversibly converted into more stable pigments called as pheophytins, in which the central Mg 2+ ion of the porphyrin ring is replaced by two hydrogen atoms. Pyropheophytins are another class of natural chlorophylls which are also formed resulting from the removal of the carboxy-methyl group from the pheophytin structure (Giuliani et al., 2011). Chemical structures of pheophytin A and pyropheophytin A are depicted in Fig. 1. Earlier studies of Serani and Piacenti (1992) were related with the study of pheophytin A photodecomposition in EVOO aiming to assess the influence of temperature and light intensity on the kinetic constant of this reaction. More recently, Gertz and Fiebig (2006) have developed a procedure to determine the degradation products of chlorophyll A, namely pheophytin A and pyropheophytin A, in olive oil. However, since pyropheophytin A content increases along EVOO shelf life only scarcely this compound can be considered a marker of adulteration. 3.2. Diacylglycerols Diacylglycerols (DGs) are present in EVOO in two isomeric forms 1,2- and 1,3-isomers, as depicted in Fig. 2, ranging from 1 to 3% (Mannina & Sobolev, 2011). While the occurrence of 1,2 isomers of DG (1,2-DG) is attributed to the incomplete biosynthesis of TAGs, the presence of 1,3 isomers
H3C
H
3
CH3
CH3
O
H3C N H
H
O N
NH
CH3
O
N
CH3
H CH3
CH3
O CH2
CH3
H
CH3
CH3
O Pheophytin A
NH
N
H
N
HN
H
O
O O
O Pyropheophytin A Fig. 1. Natural chlorophyll present in EVOO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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R. Garcia et al. / Food Research International xxx (2013) xxx–xxx
CH2OOCR
CH2OOCR R'COO
C
H
CH2OH sn-1,2- diacylglycerol
HO
C
H
CH2OOCR' sn-1,3- diacylglycerol
Fig. 2. Diacylglycerol isomeric forms in olive oil.
could be considered as a good indicator of freshness and quality of an olive oil (Mannina et al., 2003), but seem to be not suitable as markers of EVOO adulteration with ROO. Recently, studies performed by Fragaki and co-workers have allowed the determination in a single experiment of 1,2-DG, 1,3-DG and total DGs' (TDGs) contents and the ratios of 1,2-DGs over TDGs using 31P NMR spectroscopic experiments on different grades of olive oil, namely EVOO, ROO and blended olive oil composed of ROO and EVOO (BOO) (Fragaki et al., 2005). To achieve this purpose an analytical methodology has been employed comprising a derivatization step involving the replacement of the labile hydrogens of hydroxyl and carboxyl groups with 2-chloro-4,4,5,5-tetramethyl dioxaphospholane leading to the production of the correspondent phosphitylated compounds, followed by the subsequent 31P NMR analysis (Spyros & Dais, 2000; Vigli, Philippidis, Spyros, & Dais, 2003). The results have shown the usefulness of this methodology to detect olive oil adulteration applying a 31P NMR method and a multivariate statistical analysis allowing the estimation of BOO sample composition. According to these results, EVOO samples possess 1,3-DG contents much lower than the corresponding values in the ROO samples which corroborate the fact that isomerization of 1,2-DGs to 1,3-DGs, which frequently occurs during prolonged olive oil storage, also happens through olive oil refinement (Fragaki et al., 2005). This finding also corroborates that the use of diacylglycerols isomers as markers of adulterations must be done carefully since it represents some limitations. 3.3. Fatty acids and wax esters Olive oil includes on its composition a class of natural neutral lipids called fatty acid alkyl esters (FAAEs) which are formed by esterification of free fatty acids (FA). These compounds were detected firstly by Mariani and Fedeli (1986) which isolated the “nonpolar fraction” of olive oil by means of column liquid chromatography and analyzed it by GC. Formation of FAAEs is promoted by unsuitable practices related with olive oil extraction and scarce quality of olive fruits (Pérez-Camino, Moreda, Mateos, & Cert, 2002). Recent studies developed by Pérez-Camino and co-workers are devoted to the evaluation of soft deodorization on the concentration of FAAEs and the potential utility of these compounds as adulteration markers on the detection of blends of mildly ROO with EVOO (Pérez-Camino, Cert, Romero-Segura, Cert-Trujillo, & Moreda, 2008). Within this purpose, the authors have quantified FAAE contents using a previous sample preparation method based on the isolation of those compounds with a silica gel solid phase extraction cartridge followed by GC analysis. The study has been applied to around one hundred of Spanish olive oils of several categories, varieties and geographical origin allowing differentiating of EVOO from other categories of olive oils that has been subjected to a mild refining process. The results shown that blending process of EVOO with refined low quality olive oils can be assessed by the measurement of their alkyl ester concentrations (Pérez-Camino et al., 2008). The profiles of fatty acids and fatty alcohol esters are related with olive oil categories thus straight chain wax esters were indicated as useful quality and purity indicators of olive oils. Hence, the distinction of VOO from ROO based on wax esters could be achieved since VOO
posses a higher content of C36 and C38 waxes than of C40, C42, C44 and C46 while ROO exhibits an inverse relation (Aparicio & Aparicio-Ruíz, 2000). Since straight chain wax esters were indicated as potentially useful quality/purity markers in olive oils some attention were given to those compounds in order to evaluate their relevance on the detection of EVOO and ROO blends (Biedermann, Hase-Aschoff, & Grob, 2008). Those compounds are located in the waxy surface layer of the olive but are poorly extracted from the olive fruit by physical pressing operation. According to the literature, olive oils of low quality possess these wax esters at higher concentrations. Biedermann and co-workers have optimized an LC–GC–FID method to measure methyl and ethyl oleate contents as well as wax esters' fraction of the C26 and C28 alcohol with unsaturated C18 fatty acids in order to clarify their significance for the evaluation of olive oil quality (Biedermann et al., 2008). Results have shown that the presence of high wax ester content is a consequence of degrading olives and their formation also occurs during the storage of olive oils. Therefore, assignment of straight chain wax esters as quality markers is more controversial since those compounds are continuously formed during storage constituting a limitation for their use as adulteration markers (Aparicio & Aparicio-Ruíz, 2000; Biedermann et al., 2008). 3.4. Sterols Sterols are present on the unsaponifiable matter of almost all fats and oils. The analysis of the unsaponifiable fraction is being considered as a powerful tool to detect adulteration allowing the differentiation between olive oils of different quality (Lerma-Garcia, 2012). These compounds have been ascribed the genuineness of some vegetable oils because this fraction is more characteristic than the fatty acid profile, which explains why this family of compounds has been widely used in olive oil authentication (Martínez-Vidal, Garrido-Frenich, Escobar-García, & Romero-González, 2007). Further investigations on variations of the ratio of free to total sterols in different categories of olive oil and the usefulness of this ratio as a parameter for olive oil quality have been evaluated by Pasqualone and Catalano (2000). In particular, VOO possesses high levels of β-sitosterol and Δ5-avenasterol (Fig. 3) constituting a potential differentiation from other oils (Aparicio & Aparicio-Ruíz, 2000). In general, refining process leads to a decrease in the total content of sterols and increase esterified sterols in the oil (Jiménez de Blas & dell Valle González, 1996; Phillips, Ruggio, Toivo, Swank, & Simpkins, 2002). Concretely, during refining process of edible oils and fats a dehydration of β-sitosterol occurs yielding
HO
HO
Δ5- Avenasterol
β-Sitosterol
H H H H
H
Stigmasta 3,5- diene
Fig. 3. Some sterols present in VOO.
Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008
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stigmasta-3,5-diene (Fig. 3) (León-Camacho, Serrano, & Constante, 2004). Experimentally, its quantification is performed by means of an analytical methodology comprising the previous purification of stigmasta-3,5-diene by preparative column chromatography followed by its analysis using gas chromatography. However, this procedure is considered a time-consuming and laborious work. Kinetic studies performed by León-Camacho et al. (2004) using different independent variables namely time, temperature, flow of stripping gas and the thickness of oil layer have allowed the measurement of apparent thermodynamic parameters related with the formation of stigmasta-3,5-diene through the deodorization refining process. According to these studies, the formation of stigmasta-3,5-diene from β-sitosterol and therefore stigmasta-3,5-diene contents are strongly dependent of the temperature used during the physical refining (León-Camacho et al., 2004). Then, stigmasta-3,5-diene has been considered potentially remarkable on the detection of ROO in VOO (León-Camacho et al., 2004). However, stigmastadienes could be only seen as reliable indicators of olive oil adulteration when their concentrations ranged between 0.01 and 4 mg/kg (Al-Ismail, Alsaed, Ahmad, & Al-Dabbas, 2010) limiting their use on the detection of ROO and olive oil blends. More recently, Martínez-Vidal et al. (2007) have introduced an analytical methodology based on liquid chromatography coupled to mass spectrometry (LC–MS) with atmospheric-pressure chemical ionization in positive ion mode which enables the determination of the sterol composition of different types of olive oils, namely EVOO and ROO. This methodology seems to be most advantageous since it allows the reduction of sample handling and analysis time. According to these results, sterol contents can be used to distinguish between different types of oil, in particular authentic olive oil and seed oils (Martínez-Vidal et al., 2007). 3.5. TAG oligopolymers Recently, studies of Caponio and co-workers are committed to the discrimination between VOO and ROO based on the quantification of some polar compounds (PC), namely triacylglycerol oligopolymers (TAGP), oxidized triacylglycerols (ox-TAG) and diacylglycerols (DAG) by means of High Performance Size-Exclusion Chromatography (HPSEC) involving a previous step comprising a purification of olive oil samples by silica gel column chromatography. In particular, TAGP is produced during deodorization in the refining process due to the higher temperatures achieved, being considered as a relevant indicator of the secondary oxidative degradation of olive oil since their presence is independent of processing conditions. The other compounds — ox-TAG and DAG, are linked with primary oxidation level of the olive oil and the hydrolytic degradation of ROO, respectively (Caponio et al., 2011). Ox-TAG could undertake polymerization reactions promoting the formation of TAGP. It's assumed that TAGP were absent or present at very low concentrations in EVOO although present in relatively high amounts in ROO and the same trend is observed for ox-TAG. Therefore, especially TAGP could be a valuable marker to discriminate olive oils complemented with HPSEC (Caponio et al., 2011). Since the formation of TAGP could be ascribe to the bleaching and especially deodorization steps during the refining process, this compound displays a potential role on the discrimination among EVOO and ROO. However, the analytical methodology implemented by Caponio et al. (2011) has not been applied to more complex experimental designs including several blends of oils thus the usefulness of that methodology for the detection of EVOO adulterated with ROO requires more detailed studies. 4. Conclusions Adulteration of olive oil is an issue of crucial significance because of its impact in quality, nutritional value and safety of consumers.
5
Due to the inherent EVOO high-cost, the adulteration of this kind of product with low-quality olive oils seems to be actually one of the most common types of fraud. The development of analytical methodologies which enable detection of adulterations is warranted since the addition of ROO to EVOO at low percentages could be a very challenging task. In spite of all attempts developed until now, it seems to be relatively consensual that none of the compounds described above can be considered as a reliable adulteration marker of EVOO and ROO blends, since their appearance is not exclusively related with the refining process used on the ROO production. Thus, the assignment of some components intimately ascribed to the refining step could be the key to overcome the limitations of the methodologies proposed until now to detect this kind of fraudulent practice. Nevertheless, the discovery of putative markers that could identify possible adulteration of EVOO with ROO could be considered of mandatory importance and will open new avenues in the field of “Food Authenticity”. Briefly, more efforts are needed to exploit new compounds that could be assigned as reliable adulteration markers able to detect with high selectivity, sensitivity and accuracy blends of EVOO with ROO. Acknowledgments This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors — COMPETE and national funds through FCT — Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011. References Agiomyrgianaki, A., Petrakis, P. V., & Dais, P. (2012). Influence of harvest year, cultivar and geographical origin on Greek extra virgin olive oils composition: A study by NMR spectroscopy and biometric analysis. Food Chemistry, 135, 2561–2568. Agrimonti, C., Vietina, M., Pafundo, S., & Marmiroli, N. (2011). The use of food genomics to ensure the traceability of olive oil. Trends in Food Science and Technology, 22, 237–244. Al-Ismail, K. M., Alsaed, A. K., Ahmad, R., & Al-Dabbas, M. (2010). Detection of olive oil adulteration with some plant oils by GLC analysis of sterols using polar column. Food Chemistry, 121, 1255–1259. Angerosa, F., Campestre, C., & Giansante, L. (2006). Analysis and authentication. In D. Boskou (Ed.), Olive oil: Chemistry and technology (pp. 113–172). American Oil Chemists' Society Press. Aparicio, R., & Aparicio-Ruíz, R. (2000). Authentication of vegetable oils by chromatographic techniques. Journal of Chromatography. A, 881, 93–104. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A. M., Segura-Carretero, A., Fernández-Gutiérrez, A., et al. (2007). Phenolic molecules in virgin olive oils: A survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules, 12, 1679–1719. Biedermann, M., Hase-Aschoff, P., & Grob, K. (2008). Wax ester fraction of edible oils: Analysis by on-line LC–GC–MS and GC × GC-FID. European Journal of Lipid Science and Technology, 110, 1084–1094. Bosque-Sendra, J. M., Cuadros-Rodríguez, L., Ruiz-Samblás, C., & de la Mata, A. P. (2012). Combining chromatography and chemometrics for the characterization and authentication of fats and oils from triacylglycerol compositional data — A review. Analytica Chimica Acta, 724, 1–11. Calvano, C. D., De Ceglie, C., D'Accolti, L., & Zambonin, C. G. (2012). MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent. Food Chemistry, 134, 1192–1198. Caponio, F., Summo, C., Bilancia, M. T., Paradiso, V. M., Sikorska, E., & Gomes, T. (2011). High performance size-exclusion chromatography analysis of polar compounds applied to refined, mild deodorized, extra virgin olive oils and their blends: An approach to their differentiation. LWT — Food Science and Technology, 44, 1726–1730. Chen, H., Angiuli, M., Ferrari, C., Tombari, E., Salvetti, G., & Bramanti, E. (2011). Tocopherol speciation as first screening for the assessment of extra virgin olive oil quality by reversed-phase high-performance liquid chromatography/fluorescence detector. Food Chemistry, 125, 1423–1429. Chiavaro, E., Rodriguez-Estrada, M. T., Barnaba, C., Vittadini, E., Cerretani, L., & Bendini, A. (2008). Differential scanning calorimetry: A potential tool for discrimination of olive oil commercial categories. Analytica Chimica Acta, 625, 215–226. Christy, A. A., Kasemsumran, S., Du, Y., & Ozaki, Y. (2004). The detection and quantification of adulteration in olive oil by near-infrared spectroscopy and chemometrics. Analytical Sciences, 20, 935–940. Costa, J., Mafra, I., & Oliveira, M. B. P. P. (2012). Advances in vegetable oil authentication by DNA-based markers. Trends in Food Science and Technology, 26, 43–55.
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Please cite this article as: Garcia, R., et al., Putative markers of adulteration of extra virgin olive oil with refined olive oil: Prospects and limitations, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.05.008