Trends in Food Science & Technology 26 (2012) 43e55

Review

Advances in vegetable oil authentication by DNA-based markers

Joana Costa, Isabel Mafra* and M. Beatriz P.P. Oliveira
rio de Bromatologia REQUIMTE, Laborato e Hidrologia, Faculdade de Farm acia, Universidade do Porto, Rua An bal Cunha, 164, Porto 4099-030, Portugal (Tel.: D351 222078902; fax: D351 222003977; e-mail: isabel.mafra@ff.up.pt)
The suitability of DNA markers in providing unequivocal identiers for authentication and traceability of food has been a subject of an increasing number of reports. Even in complex food matrices such as vegetable oils, the use of molecular markers as diagnostic tools has been exploited. Considering the wide variety of vegetable oils available for consumers and the differences in prices, especially among premium olive oil and other oils, species adulteration leading to economic losses and loss of consumer condence can arise. In this review, the advances of DNA extraction protocols are emphasised as a crucial step to overcome. Specic identication of several plant oils as potential adulterants of olive oil has been a subject of very recent progresses. When the oilseed crops are the source for vegetable oil production, additional concerns due to the presence of genetically modied organisms have prompted to further improvements in DNA analysis. In the specic case of olive oil, the use of genetic markers has provided analytical tools to assess authenticity regarding cultivar identication as independent markers from environmental uctuations.

Introduction In the recent years, a great interest has been devoted to the use of vegetable fats in human diet, especially regarding
* Corresponding author.
0924-2244/$ - see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2012.01.009

olive oil and other vegetable edible oils. Since these oils have a vast connotation for human health, mainly due to their nutritional properties, they have been a source of several studies. Over the last decade, a number of studies have established that most olive oils and some vegetable oils, which are naturally rich in monounsaturated fatty acids, antioxidants (vitamin E) and phytosterols, may contribute to health benets such as prevention of coronary diseases and possibly some forms of cancer and other diseases (Giugliano & Esposito, 2005). Vegetable oils are of utmost signicance for human consumption, not only from the nutritional point of view, but also for their use as technical components in chemical, pharmaceutical and cosmetic industries. Recently, they have also been used as raw material for renewable energy. The importance of vegetable oils to the global economy becomes clear when considering the amount of vegetable oils produced and consumed worldwide. The increased attention to food safety has stimulated the interest in food authentication (Consolandi et al., 2008). Certication of the origin of food, feed and ingredients has become of primary importance for the protection of consumers, in particular for fraud prevention (Woolfe & Primrose, 2004). In the global economy, traceability can be dened as the ability to track any food, feed, foodproducing animal or substance that will be used for consumption, along all steps of production, processing and distribution (http://ec.europa.eu/food/food/foodlaw/trace ability/index_en.htm). Various vegetable oils have been reported as adulterants of olive oil, namely hazelnut, almond, maize, palm and sunower oils (Arvanitoyannis & Vlachos, 2007; Krankel, 2010). Although in most cases, the adulteration of vegetable oils does not pose a threat to the consumers health, in the specic cases of potentially allergenic foods such as hazelnut, its use might represent a risk for sensitised individuals (Arlorio et al., 2010). The use of adulterants also implies an economical fraud, a disloyal competition among producers and it violates the consumers right to make informed choices regarding the products they acquire. In the special case of olive oil, as a food commodity that can reach premium prices, two main issues should be considered, namely the protable adulteration by blending it with lower value vegetable oils, and the fraudulent mislabelling regarding the information about the geographical origin, the cultivars and/or the production methodology (Bell & Gillat, 2010).

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Table 1. Summary of DNA extraction protocols used for vegetable oil samples. DNA extraction protocol CTAB-based method Oil matrix Monovarietal virgin olive oil Monovarietal extra virgin olive oil Starting amount Not declared 50e100 mL Pre-concentration Centrifugation (1 g pellet) Double centrifugation. Pellet (0.5 g) frozen in liquid nitrogen and immersed in 65  C bath. No No No No DNA yield Not declared Very low yields DNA markers and/or PCR method RAPD AFLP References Muzzalupo and Perri (2002) Busconi et al. (2003)

Filtered and unltered monovarietal olive oil Monovarietal and commercial olive oil Commercial monovarietal olive oil Monovarietal olive oil

2e40 g 6 mL 1 mL 100 mL

w10 ng/mL 7.75 ng/mL Not declared 40 ng/mL

SSR-CE, nested PCR RAPD, ISSR and SSR Real-time PCR SNP markers, multiplex PCR with LDR universal array Species-specic PCR with thin-lmbiosensor chips Real-time PCR Real-time PCR SNP markers, multiplex PCR with LDR universal array RAPD, ISSR and SSR Real-time PCR Species-specic PCR Species-specic PCR and real-time PCR AFLP SCAR from AFLP RAPD, ISSR and SSR AFLP SNP markers, multiplex PCR with DR universal array RAPD, ISSR and SSR Species and event-specic RR soybean PCR and

Testolin and Lain (2005) Martins-Lopes et al. (2008) J. Costa et al. / Trends in Food Science & Technology 26 (2012) 43e55 Gim enez et al. (2010) Consolandi et al. (2008)

CTAB-Hexane method CTAB-Hexane-Chloroform method Hexane method

Canola, cotton, maize, soybean, peanut, sunower, sesame, palm commercial oils Commercial monovarietal olive oil Commercial monovarietal olive oil Monovarietal olive oil

16 mL

No

0.1 fmol

Bai et al. (2011) Gim enez et al. (2010) Gim enez et al. (2010) Consolandi et al. (2008)

1 mL 500 mL 2 mL

No No No

Not declared Not declared 27 ng/mL 35.2 ng/mL Not declared 1.86 mg/g of crude oil Not declared Not declared Not declared 13.2 ng/mL Not declared 24 ng/mL 23 ng/mL 28.3 ng/mL crude 3.90e32.5 ng/mL

Hexane based method using guanidine thiocyanate Based on DNA extraction from parafn with modications Nucleospin plant kit (MachereyeNagel) Nucleospin food kit (MachereyeNagel)

Monovarietal and commercial olive oil Monovarietal olive oil Crude soybean oil Crude and degummed soybean oil Monovarietal olive oil Monovarietal olive oil Monovarietal and commercial olive oil Monovarietal olive oil at different times of storage Monovarietal olive oil

2 mL 1 mL 2 g, 5 g 75 g crude 365 g degu. 2 mL 2 mL 1 mL Not declared 2 mL

No No No No No No No Not declared No

Martins-Lopes et al. (2008) nez et al. (2010) Gime Gryson et al. (2002) Gryson et al. (2004) Pafundo et al. (2005) Pafundo et al. (2007) Martins-Lopes et al. (2008) Pafundo et al. (2010) Consolandi et al. (2008)

Monovarietal and commercial olive oil Crude and rened (neutralised, washed, bleached and

4.2 mL 50 g crude 200 g steps

No Centrifugation

Martins-Lopes et al. (2008) Costa et al. (2010b)

deodorised) soybean oil Blended and rened oils QIAamp DNA Stool kit (Qiagen) Monovarietal olive oil Filtered and unltered monovarietal olive oil Destoned oil and oil with pits olive oil Filtered olive oil spiked with lDNA Plant oils

of rening 200 g 200 mL 2e40 g 100, 200, 300 mL 50 mL 50 mL

Centrifugation No No No Centrifugation Centrifugation

steps of rening 3.6 ng/mL 5e10 ng/mL w10 ng/mL 3.67e15.0 ng/mL 10 mg lDNA Very low yields

quantitative real-time PCR Species-specic PCR and real-time PCR SSR-CE SSR-CE, nested PCR SSR lDNA specic PCR PCR of chloroplast trnL intron polymorphisms with CE SSR-CE, nested PCR RAPD, ISSR and SSR SSR

Costa et al. (2010a) Ayed et al. (2009) Testolin and Lain (2005) Muzzalupo et al. (2007) Spaniolas, Bazakos, Nturou et al. (2008) Spaniolas, Bazakos, Awad et al. (2008) J. Costa et al. / Trends in Food Science & Technology 26 (2012) 43e55 Testolin and Lain (2005) Martins-Lopes et al. (2008) Pasqualone et al. (2007)

DNeasy Plant mini kit (Qiagen)

Gene Elute plant kit (Sigma)

Filtered and unltered monovarietal olive oil Monovarietal and commercial olive oil Filtered and unltered monovarietal and binary varietal olive oils Monovarietal olive oil Monovarietal olive oil

2e40 g 1 mL 50 mL unltered oil 200 mL ltered oil 200 mL 250 mL 400 mL 80 mL 40 mL 2e40 g 120 mL

No No Centrifugation (50 mL pellet)

w10 ng/mL 1.75 ng/mL Not declared

Hydroxyapatite Biogel (Sigma) Silica kit (Sigma) Wizard Magnetic purication system for food (Promega)

Commercial olive oils (PDO, PGI) Commercial olive oils (PDO, PGI) Commercial olive oils (PDO, PGI) Filtered and unltered monovarietal olive oil Monovarietal olive oil

Centrifugation (pellet of cellular residues) Centrifugation (pellet of cellular residues) No No No No No

Not declared 5 ng/mL Not declared Not declared Not declared w10 ng/mL 34 ng/mL

AFLP SSR-CE SSR SSR SSR SSR-CE, nested PCR SNP markers, multiplex PCR with LDR universal array Species-specic real-time PCR Species-specic PCR PCR-CE-SSCP

Montemurro et al. (2008) Alba et al. (2009) Breton et al. (2004) Breton et al. (2004) Breton et al. (2004) Testolin and Lain (2005) Consolandi et al. (2008)

Commercial LB Link-Bioteck ExtMan Ofcial Swiss method for lecithin and oil DNA extraction

Sunower and maize commercial oils Crude and degummed soybean oil Olive, maize, rapeseed, sesame, soybean, peanut and sunower oils Filtered and unltered monovarietal olive oil Monovarietal olive oil

Not declared 5 mL 160 mL

No No No

Not declared 3.2 mg crude 1.7 mg, degummed 0.1 pg/uL olive oil

Doveri and Lee (2007) Bogani et al. (2009) Wu et al. (2011)

2e40 g 2.5 mL

No No

w10 ng/mL Low yield

SSR-CE, nested PCR SSR

Testolin and Lain (2005) Doveri et al. (2006)

(continued on next page) 45

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Considering the importance of authentication and traceability for food processors, regulatory authorities and consumers, different analytical methodologies have been proposed over the last years. Instrumental techniques based on chromatographic analysis of different families of compounds are among the most suggested and used approaches for monitoring the quality and authenticity of vegetable oils. Other methodologies mainly based on spectroscopic techniques, such as near-infrared spectroscopy and nuclear magnetic resonance (NMR) spectroscopy have also been proposed to assess vegetable oil identity (Casale, Casolino, Ferrari, & Forina, 2008; Cunha, Amaral, & Oliveira, 2011; Luykx & van Ruth, 2008). Compared to chromatographic, spectroscopic techniques are considered faster, simpler and less expensive. Regarding olive oil, since its chemical composition may differ among seasons and growing area, depending on the environmental conditions, the use of chemical markers for authenticity assessment of olive cultivar is not effective in this case (Gim enez, Pist on, Mart n, & Atienza, 2010). In the last years, there has been a growing interest towards the application of methodologies based on the analysis of DNA regarding food authentication (Mafra, Ferreira, & Oliveira, 2008). DNA analysis presents several advantages such as, a high durability of DNA molecules compared to other compounds such as proteins, associated to their ubiquity in cells. These advantages make the use of DNA markers as effective targets independent from geographical, climatic or agronomical factors. Most DNA-based methods rely on the high specic amplication of one or more DNA fragments by means of polymerase chain reaction (PCR). A number of papers is available reporting the application of different DNA ngerprinting methods to olive oil traceability and cultivar identication (Alba, Sabetta, Blanco, Pasqualone, & Montemurro, 2009; Breton, Claux, Metton, Skorski, & Berville, 2004; Martins-Lopes, Gomes, Santos, & GuedesPinto, 2008; Montemurro, Pasqualone, Simeone, Sabetta, & Blanco, 2008; Pafundo, Agrimonti, & Marmiroli, 2005). While olive oils have been essentially a subject of authenticity and traceability studies due to their high economic value, other vegetable oils are further focus on genetically modied organism (GMO) detection, beyond species identication. With the increasing commercial use of GM oilseed crops, DNA has also been considered as a preferable target for the traceability of transgenic material in vegetable oils. In this context, the aim of the present work is to provide an updated overview of the DNA-based methods applied to vegetable oil matrices. DNA extraction from vegetable oil matrices For the effective application of PCR techniques, a critical step to overcome in the case of complex and highly processed food matrices is the DNA extraction and purication. Adequate strategies are required to ensure efcient recovery of nucleic acids and removal of PCR inhibitors. Substances such as polysaccharides, phenolics and others are not entirely

Pasqualone et al. (2001)

de la Torre et al. (2004)

Hellebrand et al. (1998)

Doveri and Lee (2007)

References

DNA markers and/or PCR method

SCAR makers from RAPD

Species-specic PCR, nested PCR

Species-specic PCR, nested PCR Species-specic real-time PCR Not declared Not declared 300 mL No Cold-and warm-pressed soybean oil prior to ltration Sunower and maize rened oils 1 mL

Pauli et al. (1998)

0.2e2 ng/g oil sediment 0.1e0.5 pg/g oil Not declared

ISSR and SSR

Not declared

DNA yield

Centrifugation and concentration by mini-concentrators (pellet 200e300 mL) No

Pre-concentration

Centrifugation (pellet 1 g)

Centrifugation

Starting amount

25e500 g

250 mL

200 mL

Sediments, unltered and ltered monovarietal olive oil

Olive drupes with no amplication for olive oil

Cold-pressed and rened rapeseed oil

Oil matrix

TEA based buffer (Tris, EDTA and ascorbic acid) method TNE based buffer (Tris, NaCl, EDTA, SDS) method with Wizard DNA Clean-up resin (Promega) TNE based buffer (Tris, NaCl, EDTA, SDS) method

Table 1 (continued)

DNA extraction protocol

Wizard DNA Extraction method (Promega) NIAB Protocol B and Gene Clean III kit, Bio 101 (combined kits) Petroleum ether/PBS extraction

Crude palm oil

30 mL

No

LOD 10 pg

Species-specic real-time PCR

Zhang et al. (2009)

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removed during classical extraction protocols, remaining as contaminants in the nal DNA preparations. These inhibitors can interfere with the reaction at some levels, causing a decrease or even a complete inhibition of DNA polymerase activity. In the specic case of vegetable oils, besides the problem of being a lipidic matrix containing minor amounts of DNA, another difculty to overcome is the low integrity of DNA as a consequence of rening treatment, needed in most vegetable oils (Costa, Mafra, Amaral, & Oliveira, 2010b). Regarding the extraction of DNA from olive oil, it has been considered a hard task due to its low amount and integrity caused by DNA nucleases present in the olive oil (Muzzalupo & Perri, 2002). Numerous methods have been attempted for DNA extraction from vegetable oils, mainly olive oils, which are summarised in Table 1. It can be noted that the classical CTAB-based protocol with or without modications (cetyltrimethylammonium bromide) is one of the most reported methods for DNA extraction from olive oil, although its application on rened vegetable oils has not been described as successful (Costa, Mafra, Amaral, & Oliveira, 2010a). From the other reported methods, emphasis is given to the kit Wizard Magnetic purication system for food that was successfully applied to several types of vegetable oils. Nucleospin kits were also effective in the extraction of DNA from monovarietal olive oils and rened soybean oil, while QIAamp DNA Stool kit was successful in ltered olive oils and other plant oils. When the extraction regards olive oil, a wide range of starting amounts, from low (100 mL) to relatively high amounts (500 g), with or without pre-concentration step, has been reported. This is possible because olive oil is the oily juice mechanically separated from the other components of fruit pulp, being unique among common vegetable oils since it can be consumed in the crude form. The extraction of olive oil involves a set of steps that correspond mainly to fruit cleaning (removal of leaves and washing), crushing (laceration of cells to access the fat content), malaxation (enhancement of the effect of crushing to make the paste uniform), pressing and centrifugation (to separate different liquid densities) (Petrakis, 2006). Still regarding DNA extraction from olive oil, one important issue to consider is the storage period after milling. According to Pafundo, Busconi, Agrimonti, Fogher, and Marmiroli (2010), for a good traceability of olive oil, the sample should be as fresh as possible to avoid oxidation damages to DNA and to obtain good repeatability and reliability of results. Their study showed that AFLP proles of some olive oil varieties remained similar until a maximum period of one month. To monitor the DNA fragmentation in olive oil during storage, Spaniolas, Bazakos, Ntourou et al. (2008) used lDNA as a marker. The amplication of 415 and 691 bp amplicons was not successful for samples stored longer than 20 and 10 days, respectively, while the 107 bp amplicon was obtained for all the samples regardless of both concentration of spiking lDNA and storage period.

To extract other rened plant oils, it becomes evident the need of higher starting amounts to overcome the low DNA integrity, beyond the low yield. Thus, reports stating the need for relatively high oil amounts that were subjected to a pre-concentration step prior to DNA extraction are a more frequent practice in the case of rened oils. The heat treatments, the use of activated clays and charcoal, and the pH variations during rening may affect the quantity and quality of the DNA that remains in the fully rened oil (Gryson et al., 2002). In spite of the difculties, some studies have evidenced positive results for the DNA detection in crude oils, although in rened oils the number of studies is still limited, especially when compared to olive oil (Table 1). Hellebrand, Nagy, and M orsel (1998) reported the extraction of DNA from cold-pressed and rened rapeseed oil from initial start samples of 200 mL. After extracting the oil with water, the aqueous phase was separated and concentrated to a volume of 4 mL, which was further concentrated by centrifugation using mini-concentrators. The residue of about 200e300 mL was then incubated at 37  C overnight using TNE buffer, containing sodium dodecyl sulphate and proteinase K. After digestion, the sample was extracted using phenol and trichloromethane based solvents and the DNA puried by cold ethanol precipitation overnight. Although this protocol enabled the isolation of detectable amounts of DNA, it was laborious needing at least two working days. In another work concerning the DNA extraction from soybean oil (Pauli, Liniger, & Zimmermann, 1998), the Wizard DNA extraction kit produced ampliable DNA from a subsample of 300 mL (starting sample of 5 mL) of crude soybean oil achieved by cold-pressing. However, when applied to commercial rened oils no ampliable DNA was obtained, concluding that the rst step of the rening process could have removed the DNA to an extent below the limit of detection. Gryson et al. (2002) and Gryson, Messens, and Dewettinck (2004) reported the possibility of getting DNA fragments from crude soybean oil samples based on the use of hexane and guanidine thiocyanate as the extraction buffer. Like Pauli et al. (1998), those authors were not able to produce ampliable DNA from samples submitted to chemical rening process after the degumming step, indicating that almost all DNA was transferred to lecithin water solution. Even, in samples obtained from physical rening process, they failed to produce ampliable DNA after the referred step (Gryson et al., 2002). However, increasing the amount of degummed oil to 365 g, Gryson et al. (2004) veried that the degumming step does not remove DNA completely from the crude oil and higher amounts of test sample could lead to positive amplications. Spaniolas, Bazakos, Awad, and Kalaitzis (2008) compared three different methods to extract DNA from sunower and olive oil: the DNAExtractor Fat kit, the Wizard Magnetic DNA Purication System for Food kit and the QIAamp DNA Stool mini kit. The results showed that the QIAamp DNA Stool kit gave the stronger PCR

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amplication signal compared to the DNAExtractor Fat kit, whereas the Wizard kit gave only one signal for diluted DNA. The successful results with QIAamp DNA Stool kit were obtained extracting the pellet after centrifugation of 50 mL of rened sunower oil. Doveri and Lee (2007) referred that using the kit Wizard Magnetic DNA Purication System for Food and a combination between two different methods (NIAB Protocol B and Gene Clean III kit, Bio 101) it was possible to extract ampliable DNA from pure sunower and maize oils. Although they did not state the initial oil amount used for the rst method, 1 mL of oil sample was extracted with the combination of the two referred methods. Bogani et al. (2009) obtained ampliable DNA from crude and degummed soybean oils when they increased the sample amount from 500 mL to 5 mL using the Wizard Magnetic DNA Purication System for Food, with a total of DNA extracted of 3.20 and 1.70 mg, respectively. To obtain ampliable DNA from commercial samples of fully rened edible oils, Costa et al. (2010a) tested the performance of four DNA extraction methods: two commercial kits (Nucleospin food and Wizard Magnetic DNA Purication System for Food) and two in-house based methods (CTAB and Wizard). The results showed that only the Nucleospin food kit was able to extract ampliable soybean DNA from all rened oil samples. The same authors performed another study where oil samples were collected at an industrial rening unit comprising crude, degummed/neutralised, washed, bleached and deodorised soybean oil, as nal product (Costa et al., 2010b). They demonstrated that the detection of ampliable DNA in all stages of the soybean oil rening using the selected Nucleospin food kit from 50 g of crude oil and 200 g of neutralised, washed, bleached and rened (deodorised) oil was possible. The successful DNA amplication was attributed to the combination of the pre-concentration step of a relatively high amount of oil samples (200 g), the extraction protocol based on the use of DNA adsorption to silica columns and guanidine reagent, and the amplication of small DNA fragments (103e106 bp). Identifying species of origin in vegetable oils The main sources of vegetable oil production in the world are oilseeds, totalising more than 85% in 2010, from which soybean was the major contributor (58%), followed by rapeseed, cottonseed and sunower (SoyStats, 2011). According to the same source of information, soybean oil was the second most consumed vegetable oil, accounting for 29%, after palm oil with 33%, while olive oil accounted with only 2% of the world vegetable oil consumption in 2010. Olive oil is undoubtedly one of the oils more prone to fraudulent practices as it commands a higher price than other vegetable oils. The peculiar organoleptic characteristics of olive oil associated to its proved benecial health effects have increased its popularity and demand in the last years.

Most vegetable oils cannot be used as crude oils since they contain several substances that may contribute to undesirable colour, taste and aroma, limiting their application and shelf-life. To remove those substances, the oils must be submitted to a rening process that can be performed either physically or chemically, depending on the oil characteristics. Physical rening is essentially done in oils with low content of free fatty acids and phospholipids (palm and coconut), which includes a distillation step that is enough to remove these components followed by washing and deodorisation. The chemical rening comprehends an alkali treatment with NaOH in order to remove the free fatty acids and the phospholipids. This rening process also includes degumming, washing, bleaching and deodorisation stages (Johnson, 2002). By the end of the oil production, the nal product is considered suitable for daily dietary. Authentication of vegetable oils can be carried out by a variety of methods, from the classical physic-chemical techniques to more recent chromatographic, spectroscopic and molecular-based methodologies, among others. Instrumental techniques based on chromatographic analysis of different families of compounds are among the most used approaches suggested for monitoring the quality and authenticity of vegetable oils. High performance liquid chromatography (HPLC) or gas chromatography (GC) have been applied for obtaining qualitative and quantitative data regarding different compounds such as fatty acids, triacylglycerols, phytosterols, tocopherols and tocotrienols, hydrocarbons, phenolic compounds, pigments and volatile compounds. In general, chemical pre-treatment of the sample prior to the chromatographic analysis is required, making some of these methodologies time consuming and labour intensive. Several other alternative approaches mainly based on spectroscopic techniques, such as near-infrared (NIR), mid-infrared (MIR), Fourier transform infrared (FTIR), front face uorescence (FFF) and nuclear magnetic resonance (NMR) spectroscopy, are also being increasingly used for evaluating vegetable oils identity (Cunha et al., 2011). The rising interest towards the use of DNA for food authentication is also patented in the case of vegetable oils. Table 1 presents the resumed DNA-based methods to authenticate vegetable oils. To overcome the problems of high degradation and minute amounts of DNA present in vegetable oils, the amplication of small DNA fragments has been a recommended practice. Thus, taking advantage of the favoured real-time PCR kinetics for the amplication of very small fragments, recent papers have proposed this technique for vegetable oil authentication. The 5S spacer of the DNA was successfully used to detect the presence of DNA in rened vegetable oils by multiplex PCR and real-time PCR with SYBR Green coupled to melting curve analysis. Pure sunower and maize oils produced fragments of 108 bp and 75 bp size, respectively, reinforcing the specicity and sensitivity of the DNA analysis and suggesting its usefulness for the identication of vegetable oil adulteration (Doveri & Lee, 2007).

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Wu et al. (2008) reported the successful application of a real-time PCR assay to distinguish olive oil from other plant oils. By the use of olive-specic primers targeting the cDNA encoding the Olea europaea plasma intrinsic protein and a novel uorescent dye (EvaGreen) for real-time PCR, they were able to establish a sensitive method to detect the adulteration of olive oil with other plant oil species. Gim enez et al. (2010) determined the relative quantity of target molecules by real-time PCR using the universal SYBR Green dye of nuclear and chloroplastidial DNA. Independently of the DNA isolation protocol, they veried that the amount of amplicons around 80 bp was much higher than those of 200 bp, suggesting that the determination of optimal amplicon size should be considered for olive oil authentication. Moreover, regarding the DNA type, the quantity of chloroplastic DNA was higher than the nuclear DNA for all the test extraction protocols, with the exception for hexane method. Thus, considering the results presented, more attention should be paid when choosing the DNA target for olive oil authentication. Beyond differences in quantity, cytoplasmatic DNA is maternal inherited, offering several advantages over nuclear DNA. According to Spaniolas, Bazakos, Spano, Zoghby, and Kalaitzis (2010), the use of DNA makers to authenticate plant oils requires polymorphic and high copy analytical targets such as the plastid region of the trnL (UAA) intron that has been used for discriminating several plant species. The variability in length of the chloroplast trnL intron among plant species was exploited to identify ten oil producing species with extra emphasis on olive oil (Spaniolas, Bazakos, Awad et al., 2008). DNA templates from olive, sunower, soybean, sesame, hazelnut, maize, cotton, walnut, almond and avocado were mixed prior to PCR amplication, resulting in the detection of 6 peaks, based on the combinatorial use of a PCR assay with a lab-on-a-chip capillary electrophoresis system. However, olive, sesame and avocado produced amplicons with similar electrophoretic mobility making their discrimination not feasible. To further improve the resolution of PCR products from trnL (UAA) intron and develop a reliable analytical method with emphasis on the detection of sesame in olive oil, the same researchers used a capillary DNA sequencer system (Spaniolas et al., 2010). Eleven plant species could be discriminated on the basis of differential length amplicons, except olive and avocado. In the same study, a complementary approach based on SNP detection technology enabled the discrimination of the two referred species. Although the approaches of Spaniolas, Bazakos, Awad et al. (2008) and Spaniolas et al. (2010) seem very promising for plant oil authentication, their applicability was mainly tested on leafs and seeds, with the drawback of using high length PCR amplicons (300e400 bp). To identify several vegetable oils blended in olive oil, a PCR assay coupled to capillary electrophoresis and single-strand conformation polymorphism (CE-SSCP) method targeting the rbcL gene of chloroplast genome was proposed (Wu et al., 2011). The method presented an

absolute LOD of 0.1 pg/mL of olive DNA, a relative LOD of less than 10% DNA from other plant oils and a practical LOD of 30e50% soybean oil. Applicability to commercial oils was demonstrated, but further improvements are required to increase sensitivity to vegetable oils for its effective use on olive oil authentication. Because palm oil is readily available and sold at low prices, adulteration of higher valued oils such as peanut oil and soybean oil with palm oil is currently a widespread practice (Zhang et al., 2009). Based on the MT3-B sequence, conventional and real-time PCR assays were established to detect palm oil contamination by amplifying an amplicon of 109 bp. The methods were able to detect ve haploid copies of palm DNA (10 pg) and were effectively applied to commercial edible vegetable oils, indicating the presence of unlabelled palm oil. Bai et al. (2011) developed a thin-lm biosensor chipbased analytical device to rapidly authenticate eight vegetable oils. The method used primers and probes to specically detect canola, cotton, maize, soybean, peanut, sunower, sesame and palm, relying on the hybridization of biotinylated PCR fragments with covalently attached probes to a thin-lm silicon biosensor chip in a specic array. The method could specically detect trace levels of oil DNA down to 0.1 fmol. However, information about the sources of vegetable oils is missing, as well as the DNA extraction method, which after contacting the corresponding author we were informed it was based on CTAB method (Table 1). Tracing GMO in vegetable oils The International Service for the Acquisition of Agribiotech Applications estimates that in 2010, 15 million farmers cultivated genetically modied (GM) crops over 148 Mha spread across 29 countries (James, 2010). The major GM crop species are soybean, maize, cotton and canola, whose cultivation is concentrated in the developed countries, dominating global trade of these commodities. Soybean is the main genetically modied crop, corresponding to 81% of total planted soybean and to 50% of global biotech area (James, 2010). Consequently, the use of crops for oil production has been rapidly increasing. Recent data have revealed that in 2010, 29% of the worlds vegetable oil consumption was from soybean, with a major contribution arising from GM seeds since the main exporting countries (USA, Argentina and Brazil) adopted mainly this kind of seeds (SoyStats, 2011). The increase of novel food production and the lack of information and condence within society regarding GMO have led to the establishment of specic traceability guidelines and compulsory labelling requirements by some regulatory agencies. The EU regulations, based on precautionary principles, established both the legal basis for the approval procedure of GMO and the post market traceability and labelling requirements for GMO and GMO-derived food and feeds (Regulations (EC) No. 1829/2003, 1830/ 2003). Accordingly, any food containing more than 0.9% GM content should be labelled as such.

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To guaranty the implementation of these regulations, analytical methodologies allowing accurate determination of GMO are demanded. Specically, in blended edible oils prepared with mixtures of two or more different oils, it is important to verify the labelling statements, not only concerning their constituents, but also to assess the presence of GM material since soybean, maize or canola oils are frequently used. The most accepted analytical methods for GMO detection are based on DNA techniques, since the protein-based methods are not reliable for highly processed food analysis. However, in the specic case of rened vegetable oils, as already mentioned, it is very difcult to obtain ampliable DNA once it is present at very low amounts and, after rening, it is further reduced and degraded, affecting nal quantity and integrity (Costa et al., 2010a, b). One of the rst attempts of tracing DNA from vegetable oils for GMO detection was reported by Hellebrand et al. (1998). Those authors were able to amplify DNA from crude and rened rapeseed oil based on nested PCR to increase specicity and sensitivity with two separate set of primers in two sequentially PCR amplications. They concluded that the amplication of short fragments of DNA (rst step 350 bp and second step 248 bp) was more successful than those of longer length (1005 bp and 439 bp). These results indicate that oils might contain ampliable DNA and PCR could be used for the detection of GM oilseeds, adulteration of oils and/or identication of species. In opposition to that, Pauli et al. (1998) declared that no signal was observed applying the same technique (nested PCR) to rened soybean oils. They considered that a simple centrifugation step, which represented the degumming step of the industrial soybean oil rening, was sufcient to purify crude soybean oil at least by a factor of 10,000 with respect to DNA. Therefore, according to these authors, oil from GM soybeans did not need to be labelled as containing GMO. Later on, other researchers tried to amplify DNA from crude and degummed soybean oil (Gryson et al., 2002). The end-point PCR results conrmed that the DNA from crude oil was highly concentrated (1.86 mg/g of crude soybean oil), presenting a high quantity of fragments of 118 bp even after diluting the DNA extracts by a factor of 5000. After degumming, no ampliable DNA was observed, thus, in samples from neutralised, bleached and deodorised oil, DNA was not detected, suggesting that it remained in the water fraction (Gryson et al., 2002). Nevertheless, further studies conducted by the same researchers showed the possibility of detecting PCR fragments after the degumming step, if the DNA was extracted from a test portion with sufciently high volume (w365 g of degummed soybean oil) (Gryson et al., 2004). Bogani et al. (2009) were able to amplify PCR fragments from crude and degummed soybean oil until the size of 470 bp using construct-specic primers of Roundup Ready (RR) soybean. However, no data was presented for the other steps of rening, until the fully rened oil. Considering the difculties addressed to amplify trace amounts of degraded DNA in rened vegetable oils, Costa

et al. (2010a, b) used PCR primers to target small DNA fragments. In contrast to previous reports (Gryson et al., 2002, 2004; Pauli et al., 1998), Costa et al. (2010a) succeeded to detect DNA fragments of 103 bp targeting the soybean lectin gene in samples of rened oils, including blended vegetable oils and soybean oil. For the event-specic detection of RR soybean, primers specically designed to target the plant genome and the NOS terminator junction zone produced PCR fragments of 106 bp, which were then veried by real-time PCR with TaqMan probes. The same authors (Costa et al., 2010b), when applying the same PCR protocols, achieved for the rst time the detection soybean DNA in all the steps of industrial rening, including crude, neutralised, washed, bleached and deodorised oil samples. Moreover, the production of soybean oil with GM seeds along the total rening process was conrmed by end-point PCR. Amplication by real-time PCR with specic TaqMan probes reinforced all the results and proved that it is possible to detect and quantify GMO in the fully rened soybean oil. However, much more efforts are still needed to increase the levels of DNA detection for quantitative purposes considering the labelling requirements imposed by EU regulations. DNA markers for olive oil cultivar identication The determination of cultivar(s) of origin can be a decisive aspect regarding the authenticity of olive oil. Olive (O. europaea L.) has a considerable number of different cultivars, which present differences concerning chemical composition and sensorial characteristics. Moreover, among different countries and even in different regions of the same country, genetically identical cultivars are sometimes designated by different names (Matos et al., 2007). The chemical composition and sensorial descriptors outlining each cultivar are also affected by climatic and agronomic aspects, together with olive ripeness and the olive extraction system (Arvanitoyannis & Vlachos, 2007). To protect foods with unique characteristics, the EU has created legislation to establish and protect olive oils awarding them with certication brands PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication), ensuring both consumers rights and fair commercial trade. In this context, the determination of olive cultivar(s) used in olive oil production is of high importance for the nal product authentication as, depending on the PDO olive oil, only certain cultivars are allowed to be used. Therefore, several analytical techniques have been suggested to ensure PDO olive oil authentication regarding the cultivar. The analysis of different chemical components of olive oil coupled to chemometric techniques for data exploitation has been reported by several authors as a possible approach. Recently, DNA-based markers have been successfully applied to overcome problems associated with differences due to environmental conditions of growth and to function as a diagnostic tool for food authenticity and traceability of a variety/type composition of complex food matrices in an increasing number of worldwide projects (Consolandi

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et al., 2008; Martins-Lopes et al., 2008; Montemurro et al., 2008; Pafundo et al., 2010). After overcoming the task of successfully extracting ampliable DNA from olive oil, a number of markers such as simple sequence repeats (SSR), amplied fragment length polymorphism (AFLP), random amplied polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and single nucleotide polymorphism (SNP) have been proposed to identify olive cultivars present in olive oil samples. It is important to emphasise that the olive oil may be produced, not only from monovarietal fruits, but also from multiple cultivars, increasing even more the complexity and the usefulness of DNA-based techniques for their distinction. In Table 1 are resumed the application of DNA makers for olive oil cultivar identication.

Montemurro et al. (2008) demonstrated the possibility of using AFLP markers to detect the varietal origin of the olive oil. However, the AFLP proles from oil were less intense and dened than those obtained with the DNA extracted from the corresponding leaves, with a partial coincidence restricted to fragments under 350 bp, as found by Pafundo et al. (2005). RAPD Random amplied polymorphic DNA makers are widely applied to plant research such as phylogenetic studies, genome mapping, population genetic studies, as well as in cultivar identication such as in olive. The advantages of this technique rely on the simplicity of use, low cost and the need of a small amount of plant material. Muzzalupo and Perri (2002) reported the possibility of using RAPD to analyse DNA from sediments of olive oil, previously treated with proteinase K during the oil production (malaxation). Those authors veried some differences between the leaves and the oil proles, assigning this discrepancy to the crosspollination process observed in olive-trees. Busconi et al. (2003) also applied RAPD to obtain ngerprint proles from 15 olive cultivars, from which they selected two fragments that after cloning and sequencing were transformed in more reliable SCAR markers. A similar approach by the development of specic SCAR markers for olive-tree produced amplication for olive sediments, ltered and unltered olive oil (de la Torre, Bautista, C anovas, & Claros, 2004). The differences found in six SCAR patterns of three olive oil cultivars were considered as characteristic ngerprint. Nonetheless, whenever possible, the use of DNA from sediments is recommended. Martins-Lopes et al. (2008) tested eleven RAPD primers from which two produced reproducible bands in all olive oil samples under study. Among RAPD markers obtained, seven of nine bands were considered as polymorphic, reporting a mean level of polymorphism of 78%. This nding is in good agreement with the general lack of reproducibility attributed to RAPD makers (Jones et al., 1997), which in our opinion are not adequate markers for olive oil ngerprinting. ISSR Inter-simple sequence repeat markers are generated by PCR amplication of DNA regions situated between adjacent and inversely oriented 16e18 bp length simple sequence repeats. Since microsatellite loci are abundant in plant genomes, ISSR primers often produce multiple bands that may be useful for genotyping and mapping, and also for developing SSR markers. Although this technique is less used than other methods, some studies based their work in this type of DNA markers. Pasqualone, Caponio, and Blanco (2001) used ISSR markers for the distinction of drupes from different cultivars combining two sets of primers that were the most polymorphic. In spite of their unsuccessful application to olive oil, they suggested ISSR as a potential useful tool for varietal identication

AFLP AFLP allow the simultaneous screening of a large number of loci without any need of preliminary sequence knowledge. They are the most efcient markers in revealing many polymorphic bands in a single assay. When DNA extraction is difcult to achieve such as in the case of oil samples, the ability of these markers to provide many bands in a single analysis results efciently (Montemurro et al., 2008). For their advantages and high reliability, AFLP have been widely used for genotyping in a large number of crops and wild species including olive (Angiolillo, Mencuccini, & Baldoni, 1999; Sanz-Cort es et al., 2003). Busconi et al. (2003) were able to demonstrate that the DNA extracted from monovarietal olive oil could be used for AFLP analysis, whose prole revealed high correspondence to the DNA from the leaves of the same cultivar. This nding allowed cultivar identication used for olive oil production by means of the ngerprint of AFLP analysis. Pafundo et al. (2005) emphasised the DNA extraction as a critical step for the success of AFLP analysis, referring its inuence on reproducibility. They reached a maximum correspondence between AFLP proles in four cultivars and respective olive oils of 70%. Nevertheless, while AFLP proles of DNA from plants were at longer fragments, their coincidence in oils was restricted to the shorter fragments below 250 bp. To improve the traceability of olive oil, Pafundo et al. (2005) suggested that the development of sequence-characterised amplied region (SCAR) markers derived from reproducible fragments obtained through AFLP ngerprinting of monovarietal oil would be more useful. Thus, the same research group (Pafundo, Agrimonti, Maestri, & Marmiroli, 2007) converted an AFLP fragment to a robust and specic-single locus PCR-based marker to extend the use of molecular makers to complex food matrices. The amplication of the chloroplast fragment CP-rp116T was considered a SCAR marker, which allowed the classication of 56 olive oil cultivars in four groups. Though, in general, when fragments were small they could be retrieved in oil DNA, being very difcult to amplify fragments longer than 300 bp.

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purposes when milling the drupes for the production of PDO oils. Later on, ISSR markers were tested by other authors (Martins-Lopes et al., 2008) in leaves and olive oil material, obtaining a total of 18 reproducible ISSR fragments from the two most informative sets of primers. Comparing with RAPD markers, they consider the ISSR technique more informative. Microsatellites Microsatellites or simple sequence repeats are among the most reliable markers since they are characterised by a high polymorphic level due to variations of the number of repeats. Several studies have reported the successful use of the microsatellites (Table 1). Pasqualone, Montemurro, Caponio, and Blanco (2004) obtained acceptable levels of amplication from olive oil DNA with patterns identical to those of leaves and drupes of the same cultivar. Of the seven primer sets used for SSR markers, six proved to be polymorphic, yielding fragments of different lengths for each oil type under study. They concluded that DNA microsatellites were able to distinguish virgin olive oils from different cultivars, with good discriminating ability and that this technique might be applicable to mixtures of 3e4 cultivars, such as those usually adopted in PDO oils. Testolin and Lain (2005) considered the use of microsatellite polymorphism for olive oil cultivar identication a promising tool. From six SSR primer sets tested, all gave DNA amplicons of the expected sizes for unltered oil samples. In case of low DNA yield, nested PCR improved the amount of amplied DNA. Although DNA from olive oil was found degraded, they were considered to be long enough to allow making copies of fragments up to 188 bp, enabling consistent amplication of SSR from low starting amounts of oil. Doveri, OSullivan, and Lee (2006) investigated the contribution of paternal alleles to the DNA content of olive oil by the use of SSR markers, verifying that care should be taken when interpreting DNA proles from olive oil. DNA extracted from maternal tissues (leaves and olive pulp) revealed identical genetic proles by means of SSR markers. However, those authors found additional alleles in embryos (stone), also found in the paste obtained by crushing whole fruits and from oil pressed from this material. These results demonstrate that the DNA prole from olive oil is likely to represent a composite prole of the maternal alleles juxtaposed with alleles contributed by various pollen donors. In opposition to that, Muzzalupo, Pellegrino, and Perri (2007) showed that DNA puried from olive oil can be used for microsatellite analysis and that the prole of DNA puried from monovarietal oil corresponded to the prole of DNA from the leaves of the same cultivar. Ayed, Grati-Kamoun, Moreau, and Reba (2009), when assessing the potential applicability of microsatellites to trace Tunisian olive oil cultivars, compared the genetic proles from DNA extracted from oil and leaves of two cultivars. For some SSR markers, they were able to

identify alleles of the pollinators in oil samples and distinguish them from alleles of tree somatic tissues, suggesting their reliability for olive oil traceability. Alba et al. (2009) went even further reporting that the low concentration and high degradation of DNA, and the possible presence of additional paternal alleles in oils from entire drupes, should be taken in consideration when comparing SSR proles from leaves with the corresponding oils for varietal traceability purposes. Those authors amplied the SSR fragments with 85.7% rate of success and evidenced that 90% of their experiments showed identical patterns between leaves and oil DNA. The use of capillary electrophoresis by an automatic sequencer facilitated the identication of specic alleles, even for weak signals, conrming that DNA microsatellites were able to distinguish and identify olive oils from different cultivars. SNP Compared to other genetic markers, single nucleotide polymorphism are abundant in the genome and genetically stable, being effectively applied to genotype olive cultivars (Reale et al., 2006). The application of SNP to olive cultivar identication was then performed coupled to a microarray based assay by means of ligation detection reaction (LDR) in a universal array (UA) format (Consolandi et al., 2007). The same authors further improved the LDR-UA platform by introducing multiplex PCR to simultaneously amplify 13 DNA fragments containing 17 SNP loci from leaves and olive oil (Consolandi et al., 2008). The assay provided enough discriminating power to distinguish 49 olive cultivars, allowing high-throughput capacities if used as a semi-automated SNP genotyping assay to identify the origin of monovarietal olive oils. Considering the needs for authentication and the great number of olive oil cultivar, the availability of automated multiplex platforms seems to represent very promising tools to discriminate olive oil cultivars. Concluding remarks The growing interest towards the use of DNA makers for food authentication together with the increasing demands of tracing GMO have been the driving forces for the application of molecular methods in food analysis, including vegetable oil matrices. Concerning the authentication of olive oil, which is a food commodity that can reach premium prices, the efforts and progress in the application of molecular makers are patented in this review. The progress on DNA extraction protocols applied to olive oil has led to enhancements in DNA recovery and quality. Analytical parameters independent from environmental uctuations, such as DNA-based markers, have provided useful tools for cultivar discrimination, as well as plant species identication in olive oils. It is pertinent to refer the great importance of small length DNA markers to discriminate olive oil varieties, mainly due to the difculties in obtaining DNA extracts with adequate quantity and purity. Considering

J. Costa et al. / Trends in Food Science & Technology 26 (2012) 43e55 Table 2. Summary of pros and cons of the main molecular markers for vegetable oil authentication. Pros PCR                    Species identication in crude and rened vegetable oils Detection of one or more DNA fragments, including GMO, Detection of vegetable oil adulterations, Application of real-time PCR technique as a potential quantitative tool Easy performance and interpretation with the possibility of combining high-throughput technologies (arrays), Application to genotype olive cultivars, Multiple SNP detection in single DNA analysis, Ability to distinguish small differences among very similar olive oil cultivars or others oil crop species, Apparently allows high correlation between olive leaves and olive oil, Application to assess the authenticity of monovarietal olive oils. Highly polymorphic and reliable markers Easy performance and interpretation Most employed DNA markers, great discriminatory power Identication of different olive oil cultivars Application to assess the authenticity of monovarietal olive oils. Highly polymorphic and reliable markers without previous knowledge of the genome sequence One of the most used multi-locus DNA markers for the identication of olive oil cultivars Possibility of converting AFLP into SCAR markers Application to assess the authenticity of monovarietal olive oils. Cons  DNA extraction is a critical step for its application to rened vegetable oils  Unable to discriminate olive cultivars

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SNP

 Requires expensive technology (capillary electrophoresis, LDR universal array, multiplex PCR coupled with microarray technology),  More recently applied, less tested in olive oil matrices,  Lack of information concerning the comparison with other single locus markers.

SSR

 Amplication of relatively high length DNA fragments  Limited reproducibility when applied to olive oils due to the low DNA integrity

AFLP

 High complexity of the technique  Difcult to analyse the numerous bands obtained, so not suitable for varietal oil mixtures  Limited reproducibility when applied to olive oils due to the low DNA integrity

that it was demonstrated the integrity of DNA is reduced with the storage period of olive oil after milling, the use of small length DNA fragments is highly recommended if evaluation of commercial samples, with previous storage period, is required. Regarding olive oil markers, SNP seem to be the most useful markers since they allow distinguishing small differences among very similar individuals, while SSR markers have been the most widely applied because of the high discriminatory power. However, the reproducibility of SSR markers is sometimes compromised because some fragments, mainly of higher length, are not always amplied. Table 2 summarises the main advantages and disadvantages of the principal DNA markers used to identify species of origin of vegetable oils and varietal composition of olive oil. Concerning the specic identication of several plant oils as potential adulterants of olive oil, recent progresses have been done for species differentiation. Exploiting chloroplast DNA to discriminate plant oil species seem to be a powerful approach taking advantage of the high number of DNA copies, which is especially benecial for rened oils. However, amplication of short DNA fragments is recommended. When the oilseed crops, such as soybean, are the source for vegetable oil production, additional concerns due to the presence of GMO have prompted the improvements in DNA analysis for this target. It became clear and emphasised by several authors that one main constraint to overcome in the case of rened vegetable oil matrices is the isolation of acceptable quality DNA. To isolate minute

amounts of DNA present in this kind of matrices, high quantities of starting sample (w200 g) has been recommended mainly for rened vegetable oils. Considering the capacity for DNA amplication from fully rened oils or subjected to a rst step of rening (degumming), it is highly advised the use of primers targeting small fragments (w100 bp), such as highlighted in the case of olive oil. Taking in consideration the unsuccessful rst attempts in detecting DNA from rened vegetable oils, the latest developments present promising results regarding the traceability for the origin of plant oils and GMO detection. This is of major importance to verify the labelling compliance, with great potential for application in the food industries. However, future research is still required to increase the amount and quality of template DNA for quantitative analysis by real-time PCR, which is undoubtedly the technique of choice for this purpose. Acknowledgements This work has been supported by Fundac ~ ao para a Ci^ encia e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/ 2011. Joana Costa is grateful to FCT PhD grant (SFRH/BD/ 64523/2009) nanced by POPH-QREN (subsidised by FSE and MCTES). References
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