**3.3. Molecular markers for the traceability of olive oil**

The food crisis situation seen in last years and the controversy about genetically modified organisms (GMO), with a sharp increase in basic food prices, highlights the extreme susceptibility of the current agricultural and food model and the need for more strict food quality control, which should include determination of the origin of the product and the raw

materials used in it. That's why a well documented traceability system has become a requirement for quality control in the food chain.

Olive Oil Traceability 277

(SNPs) markers have been recently developed in olive and utilized to study the genetic diversity of olive trees (Reale *et al.*, 2006). SNP detection can be delivered in a number of ways, but the simultaneous detection of multiple SNPs from a single DNA sample is of particular interest (Consolandi *et al*., 2008). PDO oils are typically not monovarietal, so a method for quantifying the components of the mixture is essential if conformity with certification depends on a prescribed proportion of varietal types. So far, application of Real-Time as a tool for olive oil authentication has been explored by Giménez *et al*., (2010). The authors evidenced that Real-Time PCR is useful to quantify DNA extracted from oil, and thus to assess the yields of different methods of extraction. But the size of amplicon, is critical for the success of analysis. A possibility of utilising qRT-PCR to quantify varieties in PDO oils rests on the use of taqMan probes designed on SNPs specific of varieties entering in the oil composition (Marmiroli *et al*., 2009). Several sequences from noncoding spacer region between *psbA-trnH* and partial coding region of *matK* of plastid genome provided a good discrimination of pure *olive oil* and its admixture by other vegetable oils such as canola and sunflower. The plastid based molecular DNA technology has a great potential to be used for rapid detection of adulteration easily up to 5% in olive oil (Kumar *et al*., 2011).

The knowledge of genome nucleotide sequences also could be useful to identify new sequence polymorphisms, which will be very useful in the development of many new variety-specific molecular markers and in the implementation of more efficient protocols for

A recent publication reported by Papadia *et al.*, 2011 a systematic effort to obtain genetic characterization by SSR amplification, soil analyses, and 1H-NMR spectra, is carried out in order to make a direct connection between the olive tree variety (genetic information) and the NMR spectra (chemical information) of the extra virgin olive oil produced. The results reported show that a multidisciplinary approach, through the application of multivariate statistical analysis, could be used to set up a method for variety and/or geographic origin certification, based on the construction of a suitable database. Further research will be directed to the growth of an organic genetic/NMR/soil database, in order to improve the prediction ability of the LDA, and furthermore to develop a way to correlate 1H-NMR spectra of commercial extra virgin olive oils with their geographical and genetic origin.

Electronic noses (e-noses) and electronic tongues (e-tongues) for liquid analysis, based on the organizational principles of biological sensory systems, developed rapidly during the last decade. E-noses and e-tongues crudely mimic the human smell and taste sensors (gas and liquid sensors) and their communication with the human brain. The human olfactory system is by far the more complex and contains thousands of receptors that bind odor molecules and can detect some odors at parts per trillion levels (Breer, 1997) and include between 10 and 100 million receptors (Deisingh *et al*., 2004). Apparently some of the receptors in the olfactory mucus can bind more than one odor molecule and in some cases one odor molecule can bind more than one receptor. This results in a mind-boggling amount

**3.4. Electrochemical sensor analysis for the traceability of olive oil** 

tracking and protect olive oil origin.

Almost 84% from the total olive oil production derives from the European Union, especially from Spain, Italy and Greece. The olive oil is a main constituent of the Mediterranean diet. However there has recently been an increase in olive oil consumption internationally, due to greater availability and the current consideration of high nutritive and health benefits, including a qualified health claim from Food and Drug Administration (FDA, USA).

Molecular markers allow the detection of DNA polymorphisms and enable to effectively distinguish different varieties in an effective way, without any environmental influence.

Olive oil extraction is the process of extracting the oil present in the olive drupes for food use. Olive oil extraction is the process of separating the oil from the other fruit contents. It is possible to attain this separation by physical means alone, *i.e.* oil and water do not mix, so they are relatively easy to separate. The modern method of olive oil extraction uses an industrial decanter to separate all the phases by centrifugation. In this method the olives are crushed to a fine paste. This can be done by a hammer crusher, disc crusher, depicting machine or knife crusher. This paste is then malaxed for about 30 minutes in order to allow the small olive droplets to agglomerate.

When we blend olive oils of the same category, but from different provenances, most chemical analyses are of limited significance. Due to their high variability according to environmental conditions, neither morphological characteristics of different groups, nor the analyses of chemical composition of fatty acid and secondary metabolites can provide reliable results for oil traceability (Ben Ayed *et al.*, 2009; Papadia *et al.*, 2011). For this reason, genetic identity seems to be the most appropriate method for identifying the variety from which the olive oil under study derives.

Molecular markers allow the detection of DNA polymorphisms and enable to effectively distinguish different varieties in an effective way, without any environmental influence. In fact, DNA in oil is not affected by the environment and is identical to the mother tree DNA since the oil containing tissues are formed by diploid somatic cells of the tree (Muzzalupo *et al.*, 2007). However, depending on the molecular markers used correctly, extra alleles can be detected in the oil that do not correspond to the mother tree allele but to the pollinator alleles contained in the embryo, itself located inside the seed (Muzzalupo and Perri, 2002; Ben Ayed *et al.*, 2010).

The use of DNA based technology in the field of food authenticity is gaining increasing attention. This technique makes use of molecular markers that mostly use polymerase chain reaction (PCR) and are thus easy to genotype. Even in a complex matrix such as olive oil, molecular marker techniques such as RAPDs (random amplified polymorphic DNA, Pasqualone *et al.*, 2001; Muzzalupo and Perri, 2002).), AFLPs (amplified fragment length polymorphism, Pafundo *et al.,* 2005; Montemurro *et al.,* 2007) and SSRs (simple sequence repeat, Muzzalupo *et al.*, 2007; Breton *et al.*, 2004; Bracci *et al.*, 2011; Ben Ayed *et al.*, 2009) are very useful in the study of the traceability of olive oil. Single nucleotide polymorphism (SNPs) markers have been recently developed in olive and utilized to study the genetic diversity of olive trees (Reale *et al.*, 2006). SNP detection can be delivered in a number of ways, but the simultaneous detection of multiple SNPs from a single DNA sample is of particular interest (Consolandi *et al*., 2008). PDO oils are typically not monovarietal, so a method for quantifying the components of the mixture is essential if conformity with certification depends on a prescribed proportion of varietal types. So far, application of Real-Time as a tool for olive oil authentication has been explored by Giménez *et al*., (2010). The authors evidenced that Real-Time PCR is useful to quantify DNA extracted from oil, and thus to assess the yields of different methods of extraction. But the size of amplicon, is critical for the success of analysis. A possibility of utilising qRT-PCR to quantify varieties in PDO oils rests on the use of taqMan probes designed on SNPs specific of varieties entering in the oil composition (Marmiroli *et al*., 2009). Several sequences from noncoding spacer region between *psbA-trnH* and partial coding region of *matK* of plastid genome provided a good discrimination of pure *olive oil* and its admixture by other vegetable oils such as canola and sunflower. The plastid based molecular DNA technology has a great potential to be used for rapid detection of adulteration easily up to 5% in olive oil (Kumar *et al*., 2011).

276 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

requirement for quality control in the food chain.

the small olive droplets to agglomerate.

which the olive oil under study derives.

Ben Ayed *et al.*, 2010).

materials used in it. That's why a well documented traceability system has become a

Almost 84% from the total olive oil production derives from the European Union, especially from Spain, Italy and Greece. The olive oil is a main constituent of the Mediterranean diet. However there has recently been an increase in olive oil consumption internationally, due to greater availability and the current consideration of high nutritive and health benefits,

Molecular markers allow the detection of DNA polymorphisms and enable to effectively distinguish different varieties in an effective way, without any environmental influence.

Olive oil extraction is the process of extracting the oil present in the olive drupes for food use. Olive oil extraction is the process of separating the oil from the other fruit contents. It is possible to attain this separation by physical means alone, *i.e.* oil and water do not mix, so they are relatively easy to separate. The modern method of olive oil extraction uses an industrial decanter to separate all the phases by centrifugation. In this method the olives are crushed to a fine paste. This can be done by a hammer crusher, disc crusher, depicting machine or knife crusher. This paste is then malaxed for about 30 minutes in order to allow

When we blend olive oils of the same category, but from different provenances, most chemical analyses are of limited significance. Due to their high variability according to environmental conditions, neither morphological characteristics of different groups, nor the analyses of chemical composition of fatty acid and secondary metabolites can provide reliable results for oil traceability (Ben Ayed *et al.*, 2009; Papadia *et al.*, 2011). For this reason, genetic identity seems to be the most appropriate method for identifying the variety from

Molecular markers allow the detection of DNA polymorphisms and enable to effectively distinguish different varieties in an effective way, without any environmental influence. In fact, DNA in oil is not affected by the environment and is identical to the mother tree DNA since the oil containing tissues are formed by diploid somatic cells of the tree (Muzzalupo *et al.*, 2007). However, depending on the molecular markers used correctly, extra alleles can be detected in the oil that do not correspond to the mother tree allele but to the pollinator alleles contained in the embryo, itself located inside the seed (Muzzalupo and Perri, 2002;

The use of DNA based technology in the field of food authenticity is gaining increasing attention. This technique makes use of molecular markers that mostly use polymerase chain reaction (PCR) and are thus easy to genotype. Even in a complex matrix such as olive oil, molecular marker techniques such as RAPDs (random amplified polymorphic DNA, Pasqualone *et al.*, 2001; Muzzalupo and Perri, 2002).), AFLPs (amplified fragment length polymorphism, Pafundo *et al.,* 2005; Montemurro *et al.,* 2007) and SSRs (simple sequence repeat, Muzzalupo *et al.*, 2007; Breton *et al.*, 2004; Bracci *et al.*, 2011; Ben Ayed *et al.*, 2009) are very useful in the study of the traceability of olive oil. Single nucleotide polymorphism

including a qualified health claim from Food and Drug Administration (FDA, USA).

The knowledge of genome nucleotide sequences also could be useful to identify new sequence polymorphisms, which will be very useful in the development of many new variety-specific molecular markers and in the implementation of more efficient protocols for tracking and protect olive oil origin.

A recent publication reported by Papadia *et al.*, 2011 a systematic effort to obtain genetic characterization by SSR amplification, soil analyses, and 1H-NMR spectra, is carried out in order to make a direct connection between the olive tree variety (genetic information) and the NMR spectra (chemical information) of the extra virgin olive oil produced. The results reported show that a multidisciplinary approach, through the application of multivariate statistical analysis, could be used to set up a method for variety and/or geographic origin certification, based on the construction of a suitable database. Further research will be directed to the growth of an organic genetic/NMR/soil database, in order to improve the prediction ability of the LDA, and furthermore to develop a way to correlate 1H-NMR spectra of commercial extra virgin olive oils with their geographical and genetic origin.
