From a quantitative point of view, the main modification that occurs during oil extraction is the considerable reduction in the ratio of total chlorophylls to total carotenoids as the pigments pass from the fruit to the oil. A mass balance of the extraction process reveals that despite the lipophilic nature of these pigments, their transfer to the oil phase is only partial; a part of these pigments is retained in the alperujo while another part is oxidized to colorless products (**Figure 7**). A higher percentage of carotenoids are transferred to the oil phase than chlorophyllic pigments; this explains why the ratio between the two fractions is lower in the oil than in the fruit. These differences mainly lie in the lower retention of carotenoids in the alperujo, with around 25% of total carotenoids retained, whereas the retention of chlorophylls can reach a level up to 60%. The percentage of pigments that are oxidized to colorless products has been estimated to be between 20 and 25% for both pigment fractions [64, 65].

Each portion of pigments that is transferred to the oil, remains in the alperujo, or is degraded to colorless compounds, is different and this variability depends on the technological innovations introduced at each stage of the extraction process. Some techniques permit the processing of less mature olive fruits, such as thermal treatments with warm air [73], or a water bath [74– 76], and their use allows the production campaign to start earlier in the season. These techniques can also decrease the stability to some extent, modulate the intensity of bitterness and lead to an increase in pigmentation of the VOO. Pitted olives produce oils with optimal qualitative characteristics, with increased content of volatile compounds and phenols; yet pitting has a negative effect on the transfer of chlorophylls and carotenoids, leading to the production of oil with a less intense color [77, 78]. During malaxation, it has been observed that as the mixing time and temperature are increased in an optimal range between 20 and 30°C, the pigment concentration in the oil is greater [79–81] due to a larger release from the plant tissue [79]. For temperatures higher than 30°C, further increase in the pigment concentration is not observed, due to the thermal destruction of the pigments [69, 79]. The use of extraction co-adjuvants that generally improve the yield of oil, like plant enzymes with pectinolytic, cellulolytic, and hemicellulolytic activities [69], micronized talc [67], or common salt (NaCl) [82], also produce oils that are richer in pigments, although the only additives authorized for VOO extraction by the EU are those of physical action such as micronized natural talc and kaolinite clay [83].

#### **4.2. Thermal degradation kinetics**

**Figure 8.** Pigment profile in fresh virgin olive oil and changes associated to aging process, thermal treatment, or colo-

From a quantitative point of view, the main modification that occurs during oil extraction is the considerable reduction in the ratio of total chlorophylls to total carotenoids as the pigments pass from the fruit to the oil. A mass balance of the extraction process reveals that despite the lipophilic nature of these pigments, their transfer to the oil phase is only partial; a part of these pigments is retained in the alperujo while another part is oxidized to colorless products (**Figure 7**). A higher percentage of carotenoids are transferred to the oil phase than chlorophyllic pigments; this explains why the ratio between the two fractions is lower in the oil than in the fruit. These differences mainly lie in the lower retention of carotenoids in the alperujo, with around 25% of total carotenoids retained, whereas the retention of chlorophylls can reach a level up to 60%. The percentage of pigments that are oxidized to colorless products has been

Each portion of pigments that is transferred to the oil, remains in the alperujo, or is degraded to colorless compounds, is different and this variability depends on the technological innovations introduced at each stage of the extraction process. Some techniques permit the processing of less mature olive fruits, such as thermal treatments with warm air [73], or a water bath [74– 76], and their use allows the production campaign to start earlier in the season. These techniques can also decrease the stability to some extent, modulate the intensity of bitterness and lead to an increase in pigmentation of the VOO. Pitted olives produce oils with optimal qualitative characteristics, with increased content of volatile compounds and phenols; yet pitting has a negative effect on the transfer of chlorophylls and carotenoids, leading to the production of oil with a less intense color [77, 78]. During malaxation, it has been observed

Minor compounds. \*Exclusive pigments for certain olive varieties as Arbequina.

estimated to be between 20 and 25% for both pigment fractions [64, 65].

rant addition. 1

80 Products from Olive Tree

Olive oil will deteriorate over time even if suitable conditions for storage are used, protected from light and heat [84]. This loss of freshness or aging in VOO can be monitored by measuring any parameter that is sensitive to some degradation that inevitably occurs during storage. The values of parameters and their kinetic variations will depend on both the operative conditions, essentially the temperature, and on the compositional characteristics of the oil matrix. Some studies on actual modification of these parameters have been reported, which have led to develop some empirical models able to perform predictions under specific conditions. One more advanced step in this area is the development of kinetic models, capable of predicting the evolution of the selected parameter, not only under specific conditions, but in terms of the different variables that affect storage.

The chlorophyll and carotenoid pigments can be good tracers of storage conditions and preservation of VOO, as they are quite sensitive to operational factors such as temperature, light, and oxygen. The kinetic and thermodynamic parameters related to the oxidation of the main chlorophyll and carotenoid pigments have been characterized by thermodegradation studies of VOO at different temperatures and in the absence of light and oxygen. First-order kinetics of an irreversible reaction mechanism was determined appropriate to describe the thermal degradation of pheophytin *a* [85], lutein, β-carotene, and β-cryptoxanthin [86], as well as the epoxidized xanthophylls, neoxanthin, violaxanthin, and mutatoxanthin [87]. Details have been reviewed by Gandul-Rojas et al. [5].

A complex kinetic mechanism of parallel and consecutive reactions has been proposed for pheophytin *a* (phy a) degradation (**Figure 9**) [85]. Of these competitive reactions, the formation of pyropheophytin *a* (pyphy *a*) shows significantly higher kinetic constants in dark conditions and in the absence of air. This result confirms that pyphy *a* could be a good chemical marker for monitoring the aging of VOO during storage in dark conditions. Oxygen availability is a critical factor in the allomerization reactions that give rise to 132 -OH-pheophytin *a* and 151- OHlactone-pheophytin *a*. Allomerization takes place via mechanisms involving free radicals [88] and are, therefore, favored when VOO is exposed to the air [89]. The final reactions that produce colorless compounds are prevalent when VOO is exposed to the light and the chlorophyllic structure is photo-oxygenated by a singlet oxygen [90] due to its photosensitizing capability [88] through a self-destruction mechanism of porphyrins.

**Figure 9.** Kinetic mechanisms for thermodegradation of pheophytin *a* in virgin olive oil and expression of a first-order kinetic model for prediction of the percentage of pyropheophytin *a* [85, 102]. Abbreviations as in **Figure 3**.

An isokinetic study compared the kinetic and thermodynamic parameters of three VOO matrices obtained from olive fruits of distinct levels of maturity and with a different pigment content (high, medium, or low); it demonstrated that the oil matrix does not significantly affect the reaction mechanisms that predominate in dark conditions and in conditions of limited oxygen availability [85]. Consequently, the kinetic parameters obtained as a function of the temperature (according to the Arrhenius equation) could be used to develop mathematical models to predict the formation of pyphy *a* (**Figure 9**), the *Z* isomers of lutein, and 5,8-furanoid xanthophylls, in addition to the global degradation of carotenoids to colorless products [86, 87, 91]. In contrast, the mechanism for the formation of 132 -OH-pheophytin is affected by the composition of VOO, which is logical due to the presence of compounds that act as radical scavengers in VOO that can partially inhibit the mechanism of degradation by free radicals. A clear relationship between the content of radical scavengers and the formation of OH-phy in VOO stored in darkness has not been found [92]. A marked effect of temperature has been discovered for the thermodegradation reactions of pigments in VOO, and perhaps contrary to what one might expect, the kinetic constants for the degradation of the carotenoid fraction were about 3.6-fold higher than for the chlorophyll fraction, demonstrating a structure that is more stable against discoloration. Likewise, higher activation energy in chlorophylls indicates that a lower increase of temperature is needed in order to increase the kinetic constants for the carotenoid fraction [91].

#### **4.3. Role of the chlorophylls in the photo-oxidation of virgin olive oil**

VOO is one of the vegetable oils most resistant to oxidation due to its composition of triacylglycerols low in polyunsaturated fatty acids and the presence of natural antioxidants [93]. However, it also contains other minor compounds such as chlorophyllic pigments, which can have a catalytic effect on the oxidation of VOO in the presence of light [94]. The possible participation of the chlorophylls and their derivatives in the photo-oxidation of VOO has been studied by several groups. Initial investigations were carried out with bleached olive oil, to which compounds including chlorophyll *a*, pheophytin *a* and *b*, and others were added, demonstrating the capacity of these pigments to act as photosensitizers and promote the oxidation of the oil in the presence of light [95]. In a later study, Gutiérrez et al. [96] did not observe any pro-oxidant effects when chlorophylls *a* and *b* were added to a real VOO system that was maintained for three months under artificial light. The authors concluded that the effect of the light itself caused a greater oxidation of the oil than the added chlorophylls, with the chlorophyllic pigments totally destroyed after one week in these study conditions. Nevertheless, the authors did find a slight antioxidant effect when the same VOO system was maintained in dark conditions, with a more pronounced effect for chlorophyll *a* than for chlorophyll *b*. Other later studies carried out with VOOs have found a positive influence of the chlorophyllic pigments on the photo-oxidation of oils subjected to different conditions of light and storage times [90, 97, 98]. In relation to this result, Psomiadou and Tsimidou [90] suggested a concentration-dependent photosensitizing activity for pheophytin *a* that is favored by the availability of oxygen.

#### **4.4. Pigment profiles as authenticity and freshness indices of virgin olive oil**

**Figure 9.** Kinetic mechanisms for thermodegradation of pheophytin *a* in virgin olive oil and expression of a first-order

An isokinetic study compared the kinetic and thermodynamic parameters of three VOO matrices obtained from olive fruits of distinct levels of maturity and with a different pigment content (high, medium, or low); it demonstrated that the oil matrix does not significantly affect the reaction mechanisms that predominate in dark conditions and in conditions of limited oxygen availability [85]. Consequently, the kinetic parameters obtained as a function of the temperature (according to the Arrhenius equation) could be used to develop mathematical models to predict the formation of pyphy *a* (**Figure 9**), the *Z* isomers of lutein, and 5,8-furanoid xanthophylls, in addition to the global degradation of carotenoids to colorless products [86,

composition of VOO, which is logical due to the presence of compounds that act as radical scavengers in VOO that can partially inhibit the mechanism of degradation by free radicals. A clear relationship between the content of radical scavengers and the formation of OH-phy in VOO stored in darkness has not been found [92]. A marked effect of temperature has been discovered for the thermodegradation reactions of pigments in VOO, and perhaps contrary to

kinetic model for prediction of the percentage of pyropheophytin *a* [85, 102]. Abbreviations as in **Figure 3**.

87, 91]. In contrast, the mechanism for the formation of 132

82 Products from Olive Tree

The qualitative changes in the profiles of chlorophyllic and carotenoid pigments, namely their structural transformations to other detectable colored compounds during the oil extraction process, were described in Section 4.1. Such modifications leave a signature specific for a product and they can be used as an index of its authenticity and quality (**Figure 8**) [28, 99]. Beside the basic profile of pigments common to all VOOs, the presence of specific pigments, such as de-esterified chlorophyllic derivatives, α-carotene, or esterified xanthophylls, is a good marker of the varietal origin of the oil [28, 59]. Moreover, three ratios have been proposed to determine the authenticity and correct processing of Spanish VOOs [28, 99]. The genuine pigment profile of the fresh VOO is modified also during its storage, and it is therefore a good indicator of the suitability of the conditions employed in the preservation of the product [100].

For commercial olive oil products (a mix of VOO and refined olive oil), different intensities of the sensory characteristics can be obtained depending on the proportion of VOO. As such, we can find olive oils labeled as intense flavor or mild flavor, which should correspond to the percentage of VOO used in the mix, although this concept is not yet regulated. The color of the VOO can range from dark green to pale yellow depending on the composition of the pigments of the olive fruit used as the raw material. As described in Section 4.1, this content is subject to wide variability—for chlorophylls between 1 and 40 mg/kg, and for carotenoids between 2 and 20 mg/kg—depending on the olive variety (of high or low pigmentation) and the stage of fruit maturity [28, 64]. Therefore, the color of bottled oil is indicative but it cannot be used to identify exactly the quantity of VOO present in the mixture. Some methods exist to determine a global quality index of olive oil color via the measurement of absorption at the wavelengths of maximum absorption of the oil against an air blank. The use of such methods allows for a measurement of the oil's color to be made, but in no instance are valid to scientifically discriminate taste or the proportion of VOO in the mix, nor can they substitute the analysis of individual pigments as a method for the detection of the adulteration of color. In this case, only the detection of pigments outside the common profile as defined for the VOO, or changes in the aforementioned quantitative ratios of certain pigments, would indicate the color adulteration of commercialized olive oil [55,84,101].

In the list of colorants permitted by the EU for use as food additives are two natural colorants named "chlorophylls" or "natural green" (E-140i) and "copper complexes of chlorophyll" (E-141i), which are obtained by solvent extraction from different edible plant sources. The colorant compounds of E-140i—chlorophylls, pheophytins, and carotenoids—undergo the same transformations as the natural pigments of VOO. The additive E-141i, produced by the addition of copper (II) salts to the pigments causing the formation of copper–chlorophyll derivatives primarily, is preferentially used for the adulteration of olive oil because of its highly stable green color. It is not a pure compound but rather a heterogeneous mixture of chlorophyllic derivatives, whose variability depends on the starting material, the process used for its synthesis, and even the particular commercial batch. In any case, analysis of different commercial samples of the colorant E-141i [101] showed that more than 76% of the compounds were copper–chlorophyll derivatives, with the major component being Cu–pyropheophytin *a*. Of the chlorophyllic pigments present in the colorant E-141i, 99.6% are not found in olive oil; therefore, a simple detection of one of these compounds in olive oil would reveal the oil's adulteration.

A maximum allowable level of copper-chlorophyll derivatives cannot be set for edible oils in general. This is because the detection of these copper–chlorophyll derivatives in VOO is indicative of adulteration by E-141i addition [101], yet this claim cannot be made for olive pomace oil. In this case, it is possible for copper–chlorophyll derivatives to form "natural" complexes via reactions between the chlorophyll pigments and copper from the olive pulp under acidic conditions during the storage and/or drying of the raw material (olive pomaces) (**Figure 7**). These fat-soluble compounds could be transferred to the crude oil during the extraction process and are not completely removed during the refining step (industrial sector private reports). At present, there is no regulation for these refined oils and the detection of copper–chlorophyll in refined olive pomace oil is necessary but not sufficient to certify that the oil has been adulterated by the addition of colorant E-141i.

The fraudulent use of yellow colorants has also been reported. The most likely additives to be used are the carotenes (E-160a) and lutein (E-161b) due to their structural similarity to the carotenoids naturally present in VOO. Cases have been reported of commercialized olive oils where minor xanthophylls were not detected and β-carotene or lutein was found exclusively (or at percentages greater than 90%). This quantitative profile of carotenoids is not within the margins defined for VOO [15, 28] and indicates adulteration by the addition of the colorants E-160a or E-161b, respectively. In other cases, esterified lutein has been detected with spectroscopic characteristics identical to those of free lutein but with different chromatographic properties due to the less polar nature. Lutein is susceptible to esterification with fatty acids on the two hydroxyl groups of its structure; this process, however, does not occur in the olive fruit (see Section 2.2)—despite its high content in fatty acids—as the fruit has a typical noncarotenogenic metabolism, and lutein and xanthophylls remain unesterified. Thus, the presence of esterified lutein in VOO is indicative of the addition of the colorant E-161b.

percentage of VOO used in the mix, although this concept is not yet regulated. The color of the VOO can range from dark green to pale yellow depending on the composition of the pigments of the olive fruit used as the raw material. As described in Section 4.1, this content is subject to wide variability—for chlorophylls between 1 and 40 mg/kg, and for carotenoids between 2 and 20 mg/kg—depending on the olive variety (of high or low pigmentation) and the stage of fruit maturity [28, 64]. Therefore, the color of bottled oil is indicative but it cannot be used to identify exactly the quantity of VOO present in the mixture. Some methods exist to determine a global quality index of olive oil color via the measurement of absorption at the wavelengths of maximum absorption of the oil against an air blank. The use of such methods allows for a measurement of the oil's color to be made, but in no instance are valid to scientifically discriminate taste or the proportion of VOO in the mix, nor can they substitute the analysis of individual pigments as a method for the detection of the adulteration of color. In this case, only the detection of pigments outside the common profile as defined for the VOO, or changes in the aforementioned quantitative ratios of certain pigments, would indicate the

In the list of colorants permitted by the EU for use as food additives are two natural colorants named "chlorophylls" or "natural green" (E-140i) and "copper complexes of chlorophyll" (E-141i), which are obtained by solvent extraction from different edible plant sources. The colorant compounds of E-140i—chlorophylls, pheophytins, and carotenoids—undergo the same transformations as the natural pigments of VOO. The additive E-141i, produced by the addition of copper (II) salts to the pigments causing the formation of copper–chlorophyll derivatives primarily, is preferentially used for the adulteration of olive oil because of its highly stable green color. It is not a pure compound but rather a heterogeneous mixture of chlorophyllic derivatives, whose variability depends on the starting material, the process used for its synthesis, and even the particular commercial batch. In any case, analysis of different commercial samples of the colorant E-141i [101] showed that more than 76% of the compounds were copper–chlorophyll derivatives, with the major component being Cu–pyropheophytin *a*. Of the chlorophyllic pigments present in the colorant E-141i, 99.6% are not found in olive oil; therefore, a simple detection of one of these compounds in olive oil would reveal the oil's

A maximum allowable level of copper-chlorophyll derivatives cannot be set for edible oils in general. This is because the detection of these copper–chlorophyll derivatives in VOO is indicative of adulteration by E-141i addition [101], yet this claim cannot be made for olive pomace oil. In this case, it is possible for copper–chlorophyll derivatives to form "natural" complexes via reactions between the chlorophyll pigments and copper from the olive pulp under acidic conditions during the storage and/or drying of the raw material (olive pomaces) (**Figure 7**). These fat-soluble compounds could be transferred to the crude oil during the extraction process and are not completely removed during the refining step (industrial sector private reports). At present, there is no regulation for these refined oils and the detection of copper–chlorophyll in refined olive pomace oil is necessary but not sufficient to certify that

color adulteration of commercialized olive oil [55,84,101].

the oil has been adulterated by the addition of colorant E-141i.

adulteration.

84 Products from Olive Tree

The qualitative pigment profile of genuine VOO is affected by the small degree of degradation of the oil that can take place during its storage, even under appropriate storage conditions—in darkness and at a controlled temperature [100, 102]. In general, all the degradation reactions that began during the oil extraction progress during its storage; the carotenoid pigment fraction is affected by geometric isomerization (*Z/E* isomerizations); 5,6-epoxide is transformed to 5,8-furanoid xanthophylls; degradation of pigments gives colorless products. Mainly in the chlorophyllic pigments fraction, pheophytinization is completed and a certain grade of allomerization continues. Beside, a new reaction commences: pyropheophytinization (**Figure 8**), which has been found to be strongly affected by temperature (Section 4.2). Pyphy *a* (PP) content is a highly variable parameter that depends not only on the operating conditions (time and temperature) but also on the initial amount of pheophytin *a* (P) in the starting oil. This compound is the precursor pigment for PP and is associated with variety and ripening degree [100]. However, when the PP content is expressed as percentage (PPP), with respect to the sum of PP+P, the parameter shows a considerably lower variation margin. In oils stored at controlled temperatures of 15°C, less than 3% of PPP are formed after a year [100], whereas at an average annual temperature of 22°C, the compound can reach values of 7–14% [102]. Therefore, the PPP parameter has been suggested as a chemical marker for monitoring the degradation of VOO [89, 100, 103].

Traditionally, the presence of pyropheophytins in foods of plant origin has been associated with heat treatments of cooking or conservation [104], and in this sense, their presence in VOOs is related with "deodorato" or deodorized olive oils [105, 106]. PP becomes the major chlorophyllic derivative in olive oil that has been thermally treated by deodorization to eliminate organoleptic defects, and its percentage content can reach values of 60% and above. Milder treatments of physical distillation to eliminate slight sensory defects—in conditions of low temperature (<100°C), and under a current of nitrogen and vacuum (2–6 mbar)—to produce what is known as "deodorato" oil, reduce this pyropheophytinization reaction while the other chemical parameters remain unaltered [107]. Therefore, the PPP has also been suggested as a chemical marker of VOO thermal treatments [108]. These results have prompted researchers to study the kinetic behavior of chlorophyll pigments [85] to establish a prediction model of PPP evolution over time as a function of temperature (**Figure 9**) [102]. The model obtained can be applied to establish a best-before date or shelf life of the VOO, a statement obligatory in the label accordance with the latest regulations [109]. Although this initiative is positive, one must not forget the complexity of putting the model into practice, given that the best-before date requires studies of the shelf life so that the legislator has elements of technical and scientific judgment to support the established timescales. Therefore, the availability of a scientific basis to determine the best-before date for the product is important for the olive oil sector.

In the aforementioned regulations, the inclusion of any special storage conditions for the VOO, such as "must be stored away from light and heat" is also obligatory on the label [84]. The chlorophyllic pigments are quite sensitive to both factors, and therefore, the pigments are good markers of the storage/conservation conditions of VOO. The best-before for VOO is the period in which the oil can be maintained—in the specified storage conditions—without losing optimal quality. Although PPP is not a parameter that is directly related to the sensory quality of VOO, it has been demonstrated to be a good chemical marker to trace the oil's storage conditions. As such, and in the absence of scientific studies that describe the kinetic behavior of the chemical components responsible for the positive and negative attributes used in the evaluation of the organoleptic quality of VOO, PPP could be a very useful tool for establishing a best-before date. Although the freshness of oil does not necessarily mean that the oil has a high quality, it is indisputable that a high-quality VOO (ExtraVOO) loses freshness during storage and, in parallel, it loses their quality because their positive or desirable sensory descriptors will also decrease in intensity over time [110, 111]. The oil can even develop a sensory defect in which case, it would lower the commercial category "extra virgin" (EVOO) to "virgin" (VOO). These sensory changes are strongly influenced by temperature and illumination degree, and PPP may be an indirect marker of such changes.

An analysis of PPP in the initial EVOO (before it is bottled) permits, in accordance with the predicted model established by Aparicio-Ruiz et al. [102] (**Figure 9**), the calculation of the storage time remaining for the oil (protected from the heat and light), to exceed a PPP limit established by the regulations, and gives a specific best-before date for each oil. What limit will be set as the standard? It could be the level that an EVOO reaches a year after its production —if it is stipulated that this is the time in which the EVOO loses its sensory quality. In accordance with this proposition, the maximum limit of PPP can be established at around 14%, in accordance with the study of storage carried out for monovarietal EVOOs obtained from different olive varieties and at distinct ripening degrees [102], and which served to validate the mathematical prediction model of PPP. In Australia, a limit for PPP ≤17 has been included in the olive oil standards [112]. A study from the Davis University aimed to correlate sensory and chemistry results [113] revealed that of 141 samples of commercial EVOO that failed the sensory standard, few samples (at most 29) failed some IOC chemical standards, while more samples (67 and 68) failed the additional chemical tests (diacylglycerols and PPP, respectively) adopted by Australia [112]. If the PPP standard ≤17 is decreased to ≤15 according to the German Society for Fat Science (DGF) [114], the number of failed samples increases to 83.

The mathematical model for PPP provides the producer and/or wholesaler with a tool to determine the speed of the pyropheophytinization reaction as a function of temperature and the storage conditions that can delay it, facilitating the distinction between aged oil and oil that has been heat-treated. If the storage location of an EVOO, and therefore the temperatures reached during the storage period, is known, it is possible to calculate the estimated PPP and compare it to the experimental value obtained by chemical analysis. An experimental value higher than the theoretical value could indicate that the VOO has not been sheltered from heat and light or has been submitted to a mild deodorization treatment.

The quantitative pigment analysis in the VOO is usually carried out by HPLC with visible detection. This subject has been reviewed by Gandul-Rojas et al [5]. PP and P show the same response signal with a UV–vis detector because both compounds have identical electronic absorption spectrum. Since PPP is a relative parameter, independent of the absolute PP amounts, it can be directly obtained from the ratio between the peak areas that correspond to P and PP in the chromatogram, and the calibration process is not essential. However, there are wide differences between the limit of quantification (LOQ) of the different methods proposed for the analysis of PPP which depend mainly on the system used for purification and concentration of the sample prior to the chromatographic analysis.

The LOQ is an important aspect when the oil is stored in adverse conditions such as unprotected from light. These conditions cause a pigment photo-oxidation to colorless products but surprisingly a rapid increase in PPP is also observed [111]. This result is not due to an increase of PP content but to a faster degradation of P than PP. It is important to note that the oil storage during long time under light conditions can decrease the PP content so much that its analytical detection is impossible, and PPP may result in an erroneous value of 0 (industrial sector private reports).

A marked effect of temperature on the rate of degradation reactions is also observed for the carotenoid pigments fraction, and some ratios, such as the percentage of lutein *Z* isomers, or the percentage of neochrome, could be suggested as chemical markers to monitor the degradation of VOO, as "freshness indicators" [86, 87]. The presence of certain geometric isomers of carotenoids in foods is also related with heat processing. Reaction conditions similar to those utilized in the mild deodorization of VOO are sufficient to significantly increase the percentage of lutein *Z* isomers [86] or the 5,8-furanoid isomer of neoxanthin, namely neochrome [87]. This has indicated the need for some criteria as markers of VOO heat treatment, in addition to the marker established by the chlorophyllic pigments fraction (PPP) [102]. The kinetic behavior of the isomerization of the carotenoid pigments in olive oil has been studied and mathematical models for the prediction of their evolution over time as a function of temperature has been established [86, 87].

### **Acknowledgements**

PPP evolution over time as a function of temperature (**Figure 9**) [102]. The model obtained can be applied to establish a best-before date or shelf life of the VOO, a statement obligatory in the label accordance with the latest regulations [109]. Although this initiative is positive, one must not forget the complexity of putting the model into practice, given that the best-before date requires studies of the shelf life so that the legislator has elements of technical and scientific judgment to support the established timescales. Therefore, the availability of a scientific basis

In the aforementioned regulations, the inclusion of any special storage conditions for the VOO, such as "must be stored away from light and heat" is also obligatory on the label [84]. The chlorophyllic pigments are quite sensitive to both factors, and therefore, the pigments are good markers of the storage/conservation conditions of VOO. The best-before for VOO is the period in which the oil can be maintained—in the specified storage conditions—without losing optimal quality. Although PPP is not a parameter that is directly related to the sensory quality of VOO, it has been demonstrated to be a good chemical marker to trace the oil's storage conditions. As such, and in the absence of scientific studies that describe the kinetic behavior of the chemical components responsible for the positive and negative attributes used in the evaluation of the organoleptic quality of VOO, PPP could be a very useful tool for establishing a best-before date. Although the freshness of oil does not necessarily mean that the oil has a high quality, it is indisputable that a high-quality VOO (ExtraVOO) loses freshness during storage and, in parallel, it loses their quality because their positive or desirable sensory descriptors will also decrease in intensity over time [110, 111]. The oil can even develop a sensory defect in which case, it would lower the commercial category "extra virgin" (EVOO) to "virgin" (VOO). These sensory changes are strongly influenced by temperature and

to determine the best-before date for the product is important for the olive oil sector.

86 Products from Olive Tree

illumination degree, and PPP may be an indirect marker of such changes.

Society for Fat Science (DGF) [114], the number of failed samples increases to 83.

The mathematical model for PPP provides the producer and/or wholesaler with a tool to determine the speed of the pyropheophytinization reaction as a function of temperature and

An analysis of PPP in the initial EVOO (before it is bottled) permits, in accordance with the predicted model established by Aparicio-Ruiz et al. [102] (**Figure 9**), the calculation of the storage time remaining for the oil (protected from the heat and light), to exceed a PPP limit established by the regulations, and gives a specific best-before date for each oil. What limit will be set as the standard? It could be the level that an EVOO reaches a year after its production —if it is stipulated that this is the time in which the EVOO loses its sensory quality. In accordance with this proposition, the maximum limit of PPP can be established at around 14%, in accordance with the study of storage carried out for monovarietal EVOOs obtained from different olive varieties and at distinct ripening degrees [102], and which served to validate the mathematical prediction model of PPP. In Australia, a limit for PPP ≤17 has been included in the olive oil standards [112]. A study from the Davis University aimed to correlate sensory and chemistry results [113] revealed that of 141 samples of commercial EVOO that failed the sensory standard, few samples (at most 29) failed some IOC chemical standards, while more samples (67 and 68) failed the additional chemical tests (diacylglycerols and PPP, respectively) adopted by Australia [112]. If the PPP standard ≤17 is decreased to ≤15 according to the German

> We acknowledge the financial support of the Spanish Government (Projects AGL 2012-39714/ ALI and AGL2015-63890-R) and Junta de Andalucía (Project AGR2010-6271).

### **Author details**

Beatriz Gandul-Rojas, María Roca and Lourdes Gallardo-Guerrero\*

\*Address all correspondence to<EMAIL_ADDRESS>
Food Phytochemistry Department, Instituto de la Grasa (CSIC), Pablo de Olavide University Campus, Sevilla, Spain

### **References**

**Author details**

88 Products from Olive Tree

Campus, Sevilla, Spain

**References**

Beatriz Gandul-Rojas, María Roca and Lourdes Gallardo-Guerrero\*

Mol. Sci. 2012;13:3291–3340. doi:10.3390/ijms13033291.

Food Phytochemistry Department, Instituto de la Grasa (CSIC), Pablo de Olavide University

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#### **Pigments in Extra‐Virgin Olive Oil: Authenticity and Quality Pigments in Extra**‐**Virgin Olive Oil: Authenticity and Quality**

Cristina Lazzerini, Mario Cifelli and Valentina Domenici Cristina Lazzerini, Mario Cifelli and Valentina Domenici

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64736

#### **Abstract**

Pigments, divided into carotenoids and chlorophyll derivatives, are responsible for the colour of extra‐virgin olive oil (EVOO). The concentration of pigments in EVOO depends on several factors, such as the maturity of olives before oil production, the cultivar and the geographic origin of olives. Pigments naturally degrade in olive oil (OO) during storage, and they may decompose due to light, temperature and oxygen exposure. The nature and concentration of pigments in EVOOs are different from seed oils, and this is a base of their use to reveal oil treatments and sophistication. In this chapter, the analytical methods, mainly chromatographic and spectroscopic, applied to identify and quantify pigments are overviewed. In particular, the applications of these methods to check the authenticity and the quality of extra‐virgin olive oil are discussed.

**Keywords:** carotenoids, chlorophylls, quantitative methods, adulterations, EVOOs

#### **1. Introduction**

The authenticity of edible olive oils is a subject of intense research all over the world. The aim is to guarantee the protection of consumers and to avoid frauds damaging not only the consumers but also the producers. Olive oil (OO), and in particular extra‐virgin olive oil (EVOO), is one of the most susceptible to fraud foods. There are many different frauds which can be roughly divided into [1]:

**•** *Adulteration*: at least a component is substituted with one of a lower value;

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

All the above frauds are usually intentional, and the main motivation is economical.

Most of the known frauds in the field of EVOOs concern their saponifiable fraction, which represent the 98–99% of olive oil. It is composed of saturated and unsaturated fatty acids, esterified almost entirely to glycerol to form triacylglycerols. Diacylglycerols, monoacylgly‐ cerols and free fatty acids are also components of the saponifiable fraction. The unsaponifiable fraction is composed of a very large number of minor compounds, very important for the flavour and the nutritional properties of EVOOs [2]. Minor components of olive oil, which include phenols, aliphatic and other alcohols, hydrocarbons, tocopherols, sterols and those responsible of the colour [3], namely the pigments, can be the object of alterations and frauds, too. An example is the addition of an artificial pigment, called E141, which is similar to chlorophyll, where the inner metal ion, Mg++, is substituted with the more stable Cu++. This fraud can be unmasked by a simple spectroscopic analysis of the sophisticated olive oil [4]. Since the amount of pigments is a distinct feature of EVOOs, with respect to other oils, the identification and quantification of pigments have become the subject of intense research, and several methodologies are now available [5–15].

### **2. Carotenoids and chlorophyll derivatives**

The unique colour of olive oil related to its pigment content [16] varies from a light gold to a rich green. Green olives produce a green oil because of the high chlorophyll content, while ripe olives yield a yellow oil because of the carotenoids (yellow red). The exact combination and proportions of pigments determine the final colour of the oil. Their presence in olive oil depends on olive fruits (*Olea europa, L.*), but also on genetic factors (olive cultivar), the stage of fruit ripeness, environmental conditions, the extraction processing [8, 17–19] and storage conditions. The role of chlorophylls as natural pigments accounting for greenish colours and in photosynthesis is well known. There are also some reports about the benefits of dietary chlorophylls for human health [20].

The structure of chlorophyll pigments, consisting of one tetrapyrrole macrocycle, coordinated to a Mg++ ion to form a planar complex, is responsible for the absorption in the visible region of the spectrum of olive oils. Here, both the bluish‐green chlorophyll‐a and the yellowish‐green chlorophyll‐b can be found. Chlorophylls in olive oils are mostly converted to pheophytins, due to the exchange of the central Mg++ ion with acid protons. Pheophytin‐a (see **Figure 1a**) is predominant with respect to pheophytin‐b. In the case of bad storage conditions, pheophytins are further degraded to pyropheophytins [21, 22]. The main carotenoids present in olive oils are lutein and β‐carotene (see **Figures 1b**, **c**).

**Figure 1.** Molecular structure of (a) pheophytin‐a, (b) lutein and (c) β‐carotene.

**•** *Tampering*: legitimate products are used in a fraudulent way;

**•** *Counterfeit*: all aspects of the fraudulent product are fully replicated.

several methodologies are now available [5–15].

chlorophylls for human health [20].

**2. Carotenoids and chlorophyll derivatives**

product agreement;

product;

100 Products from Olive Tree

**•** *Overrun*: a legitimate product is produced with a certain amount in excess with respect to

**•** *Simulation*: illegitimate products are designed to look like but not exactly copy the original

Most of the known frauds in the field of EVOOs concern their saponifiable fraction, which represent the 98–99% of olive oil. It is composed of saturated and unsaturated fatty acids, esterified almost entirely to glycerol to form triacylglycerols. Diacylglycerols, monoacylgly‐ cerols and free fatty acids are also components of the saponifiable fraction. The unsaponifiable fraction is composed of a very large number of minor compounds, very important for the flavour and the nutritional properties of EVOOs [2]. Minor components of olive oil, which include phenols, aliphatic and other alcohols, hydrocarbons, tocopherols, sterols and those responsible of the colour [3], namely the pigments, can be the object of alterations and frauds, too. An example is the addition of an artificial pigment, called E141, which is similar to chlorophyll, where the inner metal ion, Mg++, is substituted with the more stable Cu++. This fraud can be unmasked by a simple spectroscopic analysis of the sophisticated olive oil [4]. Since the amount of pigments is a distinct feature of EVOOs, with respect to other oils, the identification and quantification of pigments have become the subject of intense research, and

The unique colour of olive oil related to its pigment content [16] varies from a light gold to a rich green. Green olives produce a green oil because of the high chlorophyll content, while ripe olives yield a yellow oil because of the carotenoids (yellow red). The exact combination and proportions of pigments determine the final colour of the oil. Their presence in olive oil depends on olive fruits (*Olea europa, L.*), but also on genetic factors (olive cultivar), the stage of fruit ripeness, environmental conditions, the extraction processing [8, 17–19] and storage conditions. The role of chlorophylls as natural pigments accounting for greenish colours and in photosynthesis is well known. There are also some reports about the benefits of dietary

The structure of chlorophyll pigments, consisting of one tetrapyrrole macrocycle, coordinated to a Mg++ ion to form a planar complex, is responsible for the absorption in the visible region of the spectrum of olive oils. Here, both the bluish‐green chlorophyll‐a and the yellowish‐green chlorophyll‐b can be found. Chlorophylls in olive oils are mostly converted to pheophytins, due to the exchange of the central Mg++ ion with acid protons. Pheophytin‐a (see **Figure 1a**) is

**•** *Diversion*: the sale or distribution of legitimate products out of the intended market;

All the above frauds are usually intentional, and the main motivation is economical.

The level of pigments ranges from a few ppm to almost 100 ppm. Fresh olive oils usually have a higher pigments content. The major components are pheophytin‐a (from few ppm up to 25 ppm), followed by β‐carotene (from few ppm up to 15 ppm) and lutein (from few ppm up to 10 ppm).

Carotenoids are isoprenoid compounds having a hydrocarbon structure with carbon double bonds that are responsible for many of their properties [23, 24]. Carotenoids can be divided into carotenes (carotenoids containing only carbon and hydrogen) and xanthophylls (carote‐ noids that also contain oxygenated functions, such as epoxide, hydroxyl, acetate, carbonyl and carboxylic groups, among others). In EVOOs, the main carotenoids are β‐carotene and lutein [25]. The carotenoid fraction of olive oil also includes other xantophylls [15, 16].

See also Chapter entitled "Chlorophylls and Carotenoids in Food Products from Olive Tree" by Beatriz Gandul‐Rojas, María Roca and Lourdes Gallardo‐Guerrero.

### **3. Methods to identify and quantify pigments in olive oils**

In the literature, several works have been published about analytical methods able to identify and quantify pigments in oil matrices. These methods can be divided into two main categories based on the physical principle: (1) chromatographic techniques (characterized by pretreat‐ ment of the samples, such as extraction and/or saponification) and (2) spectroscopic techniques (without pretreatment of the samples). In the following, these two main classes of methods are overviewed.

#### **3.1. Chromatographic techniques**

The most successful chromatographic technique for pigments quantification is the *high‐ performance liquid chromatography,* HPLC, coupled with mass spectroscopy or UV‐vis absorp‐

**Figure 2.** Basic treatment schemes to analyse the pigments' content in EVOOs according to (a) the (LPD) [26] and (b) the SPE [27] chromatographic methods.

tion through the diode array detection (DAD). These methods consist of three steps: (a) extraction/purification of the pigments from olive oil sample; (b) chromatographic separation of pigments; and (c) fraction analysis. The extraction/purification step can be done by *liquid phase distribution* (LPD) [26] or by *solid phase extraction* (SPE) [27]. The LPD method includes the separation of pigments between two phases, one in hexane and the other one in N,N‐ dimethyl formaldehyde (DMF), as shown in **Figure 2a**. On the contrary, the SPE method is based on the use of C18 columns, which are previously conditioned with a mixture of methanol and hexane. Details of the procedure are reported in **Figure 2b**. The comparison between the two procedures shows that the recoveries of lutein, β‐carotene and pheophytin‐a are much better in the SPE than LPD method [28]. The chromatographic separation of pigments is highly dependent on the column: direct columns are normally very sensitive to the eventual presence of water and temperature gradients, and they require longer retention times [29]. Inverse column gives better results. The use of C18 inverse column allows optimal separation of chlorophylls and their derivatives, as well as separation of short chain and low molecular weight carotenoids [30].

based on the physical principle: (1) chromatographic techniques (characterized by pretreat‐ ment of the samples, such as extraction and/or saponification) and (2) spectroscopic techniques (without pretreatment of the samples). In the following, these two main classes of methods are

The most successful chromatographic technique for pigments quantification is the *high‐ performance liquid chromatography,* HPLC, coupled with mass spectroscopy or UV‐vis absorp‐

**Figure 2.** Basic treatment schemes to analyse the pigments' content in EVOOs according to (a) the (LPD) [26] and (b)

overviewed.

102 Products from Olive Tree

**3.1. Chromatographic techniques**

the SPE [27] chromatographic methods.

The recent introduction of C30 inverse columns allowed a good separation for long chain carotenoids and their isomers with lower polarity, such as lycopene and β‐carotene [15, 31]. A disadvantage of C30, with respect to C18, is the longer retention times. Normally, the better choice of the column depends on the starting matrix [32‐34].

Solvent mixtures to analyse an oil matrix have been optimized [33], and they consist of eluent A, based on water/reagent ion pair/methanol (1:1:8, v/v), and eluent B, based on acetone/ methanol (1:1, v/v). The reagent *ion pair* is represented by a solution of tetrabutylammonium (0.05 M) and ammonium acetate (1 M).

**Table 1.** Elution gradient for pigments separation commonly used in HPLC methods.

The gradient scheme used for pigments separation is reported in **Table 1**.

Mass spectroscopy (MS) has been used less frequently to analyse pigments in olive oils. This technique is very useful to understand the structural features and degradation of chlorophylls due to oxidative effects. Recently, a new methodology based on HPLC coupled with high‐ resolution time‐of‐flight (hrTOF) mass spectrometry has been developed, and thanks to the computer‐assisted analysis, the completion of MS fragmentation of chlorophylls and their derivatives has been reached [35]. No applications of this technique in terms of authentication and quality aspects of olive oils have been reported so far.

This table is modified from **Table 2** in Ref. [33] (reproduced with permission from Wiley Company).

**Table 2.** Parameters commonly used for the HPLC‐DAD detection of pigments in EVOOs. The retention factor, k, the peak positions of the UV‐vis spectra for DAD detection and the ratios between relevant peaks are reported (see reference 33).

#### **3.2. Spectroscopic techniques**

Spectroscopic techniques, such as UV‐vis absorption, nuclear magnetic resonance (NMR), Fourier transform infrared (FT‐IR) spectroscopy and fluorescence, are used to investigate olive oil chemical composition and to differentiate oils according to cultivar, variety and storage conditions. The main feature of these techniques is the possibility to use them directly on olive oil samples, without any pretreatment. In most of the cases, these techniques are suitable for investigating the saponifiable fraction of olive oil, as is the case of FT‐IR [36], and NMR spectroscopy [37, 38].

due to oxidative effects. Recently, a new methodology based on HPLC coupled with high‐ resolution time‐of‐flight (hrTOF) mass spectrometry has been developed, and thanks to the computer‐assisted analysis, the completion of MS fragmentation of chlorophylls and their derivatives has been reached [35]. No applications of this technique in terms of authentication

**I II III 100 III/II I/II**

**Pigments k Peak positions (nm) Ratio between peaks**

This table is modified from **Table 2** in Ref. [33] (reproduced with permission from Wiley Company).

reference 33).

104 Products from Olive Tree

**3.2. Spectroscopic techniques**

**Table 2.** Parameters commonly used for the HPLC‐DAD detection of pigments in EVOOs. The retention factor, k, the peak positions of the UV‐vis spectra for DAD detection and the ratios between relevant peaks are reported (see

Spectroscopic techniques, such as UV‐vis absorption, nuclear magnetic resonance (NMR), Fourier transform infrared (FT‐IR) spectroscopy and fluorescence, are used to investigate olive oil chemical composition and to differentiate oils according to cultivar, variety and storage conditions. The main feature of these techniques is the possibility to use them directly on olive oil samples, without any pretreatment. In most of the cases, these techniques are suitable for

Neoxanthin 2.4 415 438 467 88 – Neoxanthin 2.8 415 438 467 88 – Violaxanthin 3.1 413 434 464 93 – Luteoxanthin 3.4 398 420 447 100 – Auroxanthin 3.6 379 400 425 94 – Antheraxanthin 4.1 421 445 470 45 – Mutatoxanthin 4.4 414 438 464 60 – Auroxanthin isomer 4.7 379 400 425 92 – Chlorophyll‐b 4.8 465 602 650 – 3.3 Lutein 5.1 423 444 472 63 – b‐Cryptoxanthin 5.8 431 450 477 28 – cis‐Lutein 6.3 416 438 466 38 – Chlorophyll‐a 6.7 430 620 666 – 1.1 Neoxanthin 7.3 415 438 467 88 – Pyropheophytin‐a 9.7 406 506 666 – 2.2 Pheophytin‐a′ 12.1 406 506 665 – 2.1 Pheophytin‐a 12.4 406 506 665 – 2.1 Pheophytin‐a isomer 12.7 406 506 665 – 2.1

and quality aspects of olive oils have been reported so far.

The broadness of the electromagnetic spectrum allows to get information from different spectral regions; those ones mostly used to study food and relative frauds are three: (1) the ultraviolet (UV) region (200–400 nm), (2) the visible (Vis) region (400–779 nm) and (3) the near‐ infrared (NIR) region (780–2500 nm).

In particular, the UV region is characterized by high signal to noise ratio and high sensitivity, due to the presence of many signals from tocopherols, anthocyanins, phenols and diene/triene compounds [39].

As it is shown in **Figure 3**, the UV region is characterized by very intense signals in extra‐virgin olive oils, due to the variety of minor components.

**Figure 3.** (A) Full experimental spectrum in the UV‐vis region of an extra‐virgin olive oil, showing the two separated regions: UV and Vis one. (B) Enlargement of the spectrum in the sole Vis region showing the typical shape of the Vis absorption spectrum of extra‐virgin olive oils, due to the pigments content and responsible of the colour.

Concerning the NIR absorption region, most of the applications on olive oils concern the acidic part, allowing a differentiation among different botanic origin of seeds oils [40], but with this technique, pigments cannot be investigated.

A useful technique to study chlorophylls and carotenoids is fluorescence spectroscopy [41], which is a photoluminescence process. Fluorescence spectroscopy can be performed either by right‐angle and front‐face techniques, with several possibilities of investigation. Different types of signals can be indeed acquired: emission spectra, excitation spectra and synchronous spectra [42]. Several works have been published based on fluorescence spectroscopy applied to extra‐virgin olive oils [43–49], since several chemical constituents, including pigments, give fluorescence in specific conditions and they can be identified. The quantification of fluoro‐ phores is less straightforward than in absorption spectroscopies, as deeply addressed in Ref. [49]. In particular, this work shows that the right‐angle method gives several artefacts, while front face technique is more appropriate for quantification of fluorophores. In emission spectra of olive oils, the band centred at 670–680 nm is clearly due to chlorophyll‐like chromophores. In particular, the pheophytins' signals are expected around 670 nm, but the maximum of the emission band can be slightly shifted depending on the used technique (right‐angle or front‐ face). Interestingly, a weak emission band, centred at about 666 nm in the spectrum of sunflower oil, reveals the presence of fluorescent chlorophyll derivatives. Such a band is undetectable in peanut oil [49]. Carotenoids, present in olive oil with a relatively high con‐ centration, are characterized by a less intense emission. In fact, carotenoids strongly compete for incident light with other chromophores present in olive oil due to their concentration and high extinction coefficient. Carotenoids signals can be observed in the 430–480 nm region, but their quantification remains very difficult by fluorescence techniques.

The synchronous fluorescence technique has been developed to analyse large sets of olive oil samples with the aim of differentiate them on the basis of botanic origin. This technique consists in acquiring emission and excitation spectra simultaneously, by fixing a constant distance between the emission and excitation wavelengths [45–48]. This spectroscopic method is coupled with a multivariate statistical analysis, such as PLS, *partial least squares*, PCA, *principal components analysis*, or PARAFAC, *PARAllel FACtor analysis*. These methods have been proved to be satisfactory for discriminating oils of different botanic origin based on chloro‐ phylls and their derivatives quantification, but not for carotenoids. Nevertheless, despite of good potentialities, no works are known about applications in the field of frauds and authen‐ tication of EVOOs.

#### *3.2.1. Methods based on Vis absorption for pigments identification and quantification*

As previously stated, Vis absorption spectra of extra‐virgin olive oils have characteristic features [50, 51]: a three‐peak band in the range 390–520 nm and a sharper band around 660– 675 nm. This last absorption band is due to the electronic transition of chlorophylls and their derivatives, while the first band is more complex, since it is due to the overlap among carote‐ noids and chlorophylls absorption signals. As it can been seen in **Figure 4**, the visible absorp‐ tion spectrum of extra‐virgin olive oils is very different from the absorption spectra of other seeds and fruits oils. This evidence is at the basis of several works [5, 11, 52–54] devoted to the authentication of EVOOs and the identification of specific frauds, such as the mixing between olive oils and oils obtained from other seeds.

Visible (vis) light absorption of EVOOs is associated with the pigments' content, and this specificity is at the origin of several research works aiming to substitute the chromatographic methods with much faster and direct spectrophotometric methods [5, 11, 12, 39].

A recent methodology proposed by Cayuela et al. [12] associates the absorbance measured at specific wavelengths in the visible region, namely the K470 and K670 indexes, to the amount of carotenoids and chlorophyll derivatives, respectively. This method has the ad‐ vantages to be fast, non‐destructive and inexpensive. However, the single absorbance val‐ ues at specific wavelengths in the Vis spectrum of EVOOs do not allow a reliable and

unambiguous quantification of the pigments' content, in particular for the superposition of carotenoids and pheophytins signals in the 390–520 nm region. Moreover, this method is able to quantify only the total amount of carotenoids and chlorophyll derivatives, and not the single compounds.

phores is less straightforward than in absorption spectroscopies, as deeply addressed in Ref. [49]. In particular, this work shows that the right‐angle method gives several artefacts, while front face technique is more appropriate for quantification of fluorophores. In emission spectra of olive oils, the band centred at 670–680 nm is clearly due to chlorophyll‐like chromophores. In particular, the pheophytins' signals are expected around 670 nm, but the maximum of the emission band can be slightly shifted depending on the used technique (right‐angle or front‐ face). Interestingly, a weak emission band, centred at about 666 nm in the spectrum of sunflower oil, reveals the presence of fluorescent chlorophyll derivatives. Such a band is undetectable in peanut oil [49]. Carotenoids, present in olive oil with a relatively high con‐ centration, are characterized by a less intense emission. In fact, carotenoids strongly compete for incident light with other chromophores present in olive oil due to their concentration and high extinction coefficient. Carotenoids signals can be observed in the 430–480 nm region, but

The synchronous fluorescence technique has been developed to analyse large sets of olive oil samples with the aim of differentiate them on the basis of botanic origin. This technique consists in acquiring emission and excitation spectra simultaneously, by fixing a constant distance between the emission and excitation wavelengths [45–48]. This spectroscopic method is coupled with a multivariate statistical analysis, such as PLS, *partial least squares*, PCA, *principal components analysis*, or PARAFAC, *PARAllel FACtor analysis*. These methods have been proved to be satisfactory for discriminating oils of different botanic origin based on chloro‐ phylls and their derivatives quantification, but not for carotenoids. Nevertheless, despite of good potentialities, no works are known about applications in the field of frauds and authen‐

As previously stated, Vis absorption spectra of extra‐virgin olive oils have characteristic features [50, 51]: a three‐peak band in the range 390–520 nm and a sharper band around 660– 675 nm. This last absorption band is due to the electronic transition of chlorophylls and their derivatives, while the first band is more complex, since it is due to the overlap among carote‐ noids and chlorophylls absorption signals. As it can been seen in **Figure 4**, the visible absorp‐ tion spectrum of extra‐virgin olive oils is very different from the absorption spectra of other seeds and fruits oils. This evidence is at the basis of several works [5, 11, 52–54] devoted to the authentication of EVOOs and the identification of specific frauds, such as the mixing between

Visible (vis) light absorption of EVOOs is associated with the pigments' content, and this specificity is at the origin of several research works aiming to substitute the chromatographic

A recent methodology proposed by Cayuela et al. [12] associates the absorbance measured at specific wavelengths in the visible region, namely the K470 and K670 indexes, to the amount of carotenoids and chlorophyll derivatives, respectively. This method has the ad‐ vantages to be fast, non‐destructive and inexpensive. However, the single absorbance val‐ ues at specific wavelengths in the Vis spectrum of EVOOs do not allow a reliable and

methods with much faster and direct spectrophotometric methods [5, 11, 12, 39].

their quantification remains very difficult by fluorescence techniques.

*3.2.1. Methods based on Vis absorption for pigments identification and quantification*

tication of EVOOs.

106 Products from Olive Tree

olive oils and oils obtained from other seeds.

In order to get a more robust and accurate method for pigments quantification, fast and rapid at the same time, a mathematical approach based on the spectral deconvolution of the Vis absorption of EVOOs in terms of its main pigment components can be performed. A first mathematical approach was proposed by Ayuso et al. [50]: the experimental Vis spectrum is reproduced by a combination of two signals, from pure β‐carotene and chlorophyll‐a spectra. More recently, a new mathematical approach has been developed by using four orthogonal functions derived from the experimental spectra of two carotenoids (β‐carotene and lutein) and two chlorophyll derivatives (pheophytin‐a and pheophytin‐b). This procedure, shown in **Figure 5**, consists of the following steps: (1) the acquisition of the experimental UV‐vis spectrum of the sample; (2) the fitting of the experimental spectrum as a linear combination of the four orthogonal functions; and (3) the calculation of pigments' concentrations and relevant statistical parameters. Steps 2 and 3 are done automatically by a home‐made program compatible with Excel software [5].

**Figure 4.** Visible absorption spectra of several vegetable oils obtained from olives, peanuts, sunflower beans, soya beans and corn beans.

This method has been applied to more than 150 EVOOs having different cultivars and with different geographic origin [5, 51, 52, 54], showing a very high quality of the fitting in all cases (R2 ranges from 0.996 to 0.998) and good possibilities of discriminating EVOOs from not EVOOs.

Additional methods to analyse olive oils from UV‐vis absorption spectra base on statistical multivariate approaches. In such cases, the discriminating factors are derived from the absorption spectra, but they are not directly related to the pigments concentration. An example is the method based on the calculation of chaotic parameters (Lyapunov exponent, autocorre‐ lation coefficients and fractal dimensions), further treated to find correlations index [10, 11, 55] and the application of PCA on large sets of data, including UV‐vis spectral ones, further treated with class‐modelling methods [56].

**Figure 5.** Scheme of the mathematical method develop [5] to obtain the concentration of four main pigments by analy‐ sing the Near UV‐vis absorption spectrum of an EVOO.

### **4. Authenticity and quality studies based on pigments quantification**

As seen in the previous sections, there are several analytical methods, both chromatographic and spectroscopic ones, able to identify and quantify pigments in EVOOs. Since the concen‐ tration of pigments strongly differs depending on several variables, their use was discouraged. It is well known that chlorophylls completely degrade into pheophytins after few months after the bottling and that the pheophytins themselves evolve into pyropheophytins depending on storage conditions. However, as reported in several works [5, 51–60], typical range of concen‐ trations of various pigments can be associated with EVOOs and these quantities can be used to discriminate them from non‐EVOOs. Most of the applications of the chromatographic methods refer to Spanish EVOOs [56–58], while the recently developed UV‐vis based mathe‐ matical approach [5] has been mainly applied to Italian [5, 51, 52, 54] and Spanish EVOOs [5, 53] as well as Greek and Tunisian EVOOs [53, 54].

The analytical methodologies described in the previous section can be roughly divided into two types: methods able to characterize the olive varieties and cultivars and methods devel‐ oped to identify the geographic origin of the olives. Some parameters used for EVOOs authentication are the ratio between the total amount of chlorophylls' derivatives and the total amount of carotenoids, the ratio between the carotenoids and lutein contents, the percentage of violaxhantin and the percentage of lutein [57–59].

For instance, according to Ref. [56], Spanish EVOOs typically have a ratio between chloro‐ phylls' derivatives and carotenoids close to 1, in case of Italian EVOOs, a higher variability of this ratio has been found [15, 49–52]. According to Ref. [57], the ratio between carotenoids and lutein varies in a range between 0.4 and 1.5, depending on the variety and cultivar and this factor could be used as authentication parameter. Another parameter to be taken into account for the authenticity of EVOOs is the ratio between the concentration of lutein and β‐carotene, which typically varies between 0.15 and 5 [15, 53, 54, 60]. However, as also reported in Refs. [19, 54], the amount of pigments in EVOOs depend strongly by olives variety, by the moment when the olives are picked and by the storage conditions [13, 57, 59].

### **5. Conclusions**

Additional methods to analyse olive oils from UV‐vis absorption spectra base on statistical multivariate approaches. In such cases, the discriminating factors are derived from the absorption spectra, but they are not directly related to the pigments concentration. An example is the method based on the calculation of chaotic parameters (Lyapunov exponent, autocorre‐ lation coefficients and fractal dimensions), further treated to find correlations index [10, 11, 55] and the application of PCA on large sets of data, including UV‐vis spectral ones, further

**Figure 5.** Scheme of the mathematical method develop [5] to obtain the concentration of four main pigments by analy‐

As seen in the previous sections, there are several analytical methods, both chromatographic and spectroscopic ones, able to identify and quantify pigments in EVOOs. Since the concen‐ tration of pigments strongly differs depending on several variables, their use was discouraged. It is well known that chlorophylls completely degrade into pheophytins after few months after the bottling and that the pheophytins themselves evolve into pyropheophytins depending on storage conditions. However, as reported in several works [5, 51–60], typical range of concen‐ trations of various pigments can be associated with EVOOs and these quantities can be used to discriminate them from non‐EVOOs. Most of the applications of the chromatographic methods refer to Spanish EVOOs [56–58], while the recently developed UV‐vis based mathe‐ matical approach [5] has been mainly applied to Italian [5, 51, 52, 54] and Spanish EVOOs [5,

**4. Authenticity and quality studies based on pigments quantification**

treated with class‐modelling methods [56].

108 Products from Olive Tree

sing the Near UV‐vis absorption spectrum of an EVOO.

53] as well as Greek and Tunisian EVOOs [53, 54].

In this chapter, the main analytical methods developed and applied to the identification and quantification of pigments in edible oils, and in particular, extra‐virgin olive oils are described. New methodologies, divided between chromatographic and spectroscopic ones, are discussed putting in evidence advantages and disadvantages.

Pigments' concentration and their relative ratios may indicate the age and storage conditions (i.e. temperature, light and oxygen exposure), but they are also a good parameter to check authenticity and quality and to reveal mixtures with other seed oils, as deeply addressed in this chapter.

### **Acknowledgements**

We would like to thank Dr. Andrea Serani and Prof. Maurizio Zandomeneghi for helpful discussions during our researches on olive oils.

### **Author details**

Cristina Lazzerini, Mario Cifelli and Valentina Domenici\*

\*Address all correspondence to<EMAIL_ADDRESS>
University of Pisa, Pisa, Italy

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#### **DNA-Based Approaches for Traceability and Authentication of Olive Oil DNA-Based Approaches for Traceability and Authentication of Olive Oil**

Christos Bazakos, Stelios Spaniolas and Panagiotis Kalaitzis Christos Bazakos, Stelios Spaniolas and Panagiotis Kalaitzis

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64494

#### **Abstract**

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[60] Roca M., Minguez‐Mosquera M.I. Change in the natural ratio between chlorophylls and carotenoids in olive fruit during processing for virgine olive oil. Journal of the Authentication and traceability of extra virgin olive oil is a challenging research task due to the complexity of fraudulent practices. Various chemical and biochemical techniques have been developed for determining the authenticity of olive oil and in recent years non-conventional methods based on DNA analysis have gained attention, due to high specificity, sensitivity and reliability. DNA analyses have very high discriminating power because ultimately the unique identity of a variety or species is to a great extent genetically dependent. Polymorphisms are genetic variations which refer to the variation in populations or species. Molecular markers provide information on genetic variations and are valuable tools to determine olive oil authenticity. Recently several DNA-based methods have been developed to authenticate olive oil, since analysis of the residual oil DNA with the use of molecular markers can lead to the identification of the variety or the plant species from which it was extracted. The aim of this chapter is to provide an overview of the current trends and critical issues on DNAtargeted approaches used for traceability and authenticity of olive oil. This is considered a rapidly expanding field with significant challenges and prospects which shall be discussed thoroughly.

**Keywords:** olive oil and plant oils authentication, traceability, DNA, molecular markers, identification of varietal origin, SNPs, SSRs, biotechnological approaches

### **1. Introduction**

Olive oil is the main component of the Mediterranean diet and one of the most valuable food products of the agro-food industry, not only due to taste, but also due to high nutritional value

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[1]. Authentication and traceability of extra-virgin olive oil is a challenging endeavour that requires persistent efforts and continuous progress due to the complexity and advancement of novel fraudulent practices. Authentication uses methodologies, tools and technological platforms aiming at detection, prevention and exposure of adulteration and mislabelling of food commodities. Traceability is the ability to trace a food commodity from the production until the distribution stage (European Council regulation-EC 178/2002).

The various ways of olive oil adulteration mainly comprise (i) economic adulteration that refers to either the mixing of lower-grade vegetable oils with extra-virgin olive oil or with minimally processed olive oils such as non-refined or cold-pressed olive oils and (ii) mislabelling and misleading origin of protected designation of origin (PDO) and protected geographical indication (PGI) olive oils that are legally protected. One of the aims of PDO and PGI is to provide added market value high-quality olive oils that are derived from well-defined geographical regions. In most of the cases, the higher quality might be attributed to either the cultivar from which the olive oil was extracted or the edaphoclimatic conditions of the area, which is known as the 'terroir' in wine industry.

Therefore, adulteration of extra-virgin olive oil occurs not only by accidental contamination during the stages of oil processing, but is an act of deliberate addition of less expensive olive oils by fraudsters for financial profit.

Plethora of chemical and biochemical techniques have been developed for detecting the adulteration while ensuring authenticity and traceability of extra-virgin olive oil [2–6]. However, neither conventional analytical chemistry methods nor the analysis of biomorphological traits is always able to accurately detect the region of origin and/or the olive oil cultivar due to the diverse climatic conditions in which olive trees are exposed every year and their impact on the chemical composition of olive oil [7, 8].

The authenticity of olive oil, especially the extra-virgin, has been extensively studied by using several analytical approaches such as chromatography, stable isotope analysis, spectroscopy and nuclear magnetic resonance [9, 10]. In an interesting review [11], an extensive analysis of the most relevant compounds used as target analytes for olive oil characterisation and authentication was presented. Among others, the division of all analytical methods for olive oil authentication into 'targeted analysis' and 'profiling or non-targeted analysis' was suggested. The targeted analysis is based on the analysis of chemical compounds that appear only in the adulterant oil species (i.e. seed oils) and not in olive oil samples. Whereas the nontargeted analysis refers to the simultaneous detection of many known or unknown analytes belonging to a pre-defined metabolic pathway; usually, there is no differentiation among them. Although the aim is to rapidly determine the genuineness of olive oils, complicated multivariate statistical procedures are needed.

Recently, non-conventional methods based on DNA analysis have gained attention due to their high specificity, sensitivity and accuracy to detect the varietal origin of olive oil as well as the botanical origin of plant oils [12–15]. Moreover, DNA-based methods overcome deficiencies of conventional methods such as denaturation of proteins due to the heating and processing of food commodities [16]. Therefore, non-conventional methods offer an alternative, complementary approach since they rely only on the analysis of the DNA. Plenty of biomolecular methodologies have been developed for the authentication of olive oil using DNA markers. Molecular markers provide information on polymorphisms within DNA regions and are considered valuable tools to determine olive oil authenticity. Polymorphisms are genetic variations that can be detected either in nuclear, ribosomal or mitochondrial genomes as well as in the genomes of other organelles, such as chloroplasts.

[1]. Authentication and traceability of extra-virgin olive oil is a challenging endeavour that requires persistent efforts and continuous progress due to the complexity and advancement of novel fraudulent practices. Authentication uses methodologies, tools and technological platforms aiming at detection, prevention and exposure of adulteration and mislabelling of food commodities. Traceability is the ability to trace a food commodity from the production until the

The various ways of olive oil adulteration mainly comprise (i) economic adulteration that refers to either the mixing of lower-grade vegetable oils with extra-virgin olive oil or with minimally processed olive oils such as non-refined or cold-pressed olive oils and (ii) mislabelling and misleading origin of protected designation of origin (PDO) and protected geographical indication (PGI) olive oils that are legally protected. One of the aims of PDO and PGI is to provide added market value high-quality olive oils that are derived from well-defined geographical regions. In most of the cases, the higher quality might be attributed to either the cultivar from which the olive oil was extracted or the edaphoclimatic conditions of the area,

Therefore, adulteration of extra-virgin olive oil occurs not only by accidental contamination during the stages of oil processing, but is an act of deliberate addition of less expensive olive

Plethora of chemical and biochemical techniques have been developed for detecting the adulteration while ensuring authenticity and traceability of extra-virgin olive oil [2–6]. However, neither conventional analytical chemistry methods nor the analysis of biomorphological traits is always able to accurately detect the region of origin and/or the olive oil cultivar due to the diverse climatic conditions in which olive trees are exposed every year and their

The authenticity of olive oil, especially the extra-virgin, has been extensively studied by using several analytical approaches such as chromatography, stable isotope analysis, spectroscopy and nuclear magnetic resonance [9, 10]. In an interesting review [11], an extensive analysis of the most relevant compounds used as target analytes for olive oil characterisation and authentication was presented. Among others, the division of all analytical methods for olive oil authentication into 'targeted analysis' and 'profiling or non-targeted analysis' was suggested. The targeted analysis is based on the analysis of chemical compounds that appear only in the adulterant oil species (i.e. seed oils) and not in olive oil samples. Whereas the nontargeted analysis refers to the simultaneous detection of many known or unknown analytes belonging to a pre-defined metabolic pathway; usually, there is no differentiation among them. Although the aim is to rapidly determine the genuineness of olive oils, complicated multi-

Recently, non-conventional methods based on DNA analysis have gained attention due to their high specificity, sensitivity and accuracy to detect the varietal origin of olive oil as well as the botanical origin of plant oils [12–15]. Moreover, DNA-based methods overcome deficiencies of conventional methods such as denaturation of proteins due to the heating and processing of food commodities [16]. Therefore, non-conventional methods offer an alternative, comple-

distribution stage (European Council regulation-EC 178/2002).

which is known as the 'terroir' in wine industry.

impact on the chemical composition of olive oil [7, 8].

oils by fraudsters for financial profit.

116 Products from Olive Tree

variate statistical procedures are needed.

Several DNA-based methods have been developed to authenticate olive oil, since analysis of the residual oil DNA with the use of molecular markers can lead to the identification of the variety or the species from which it was extracted regardless of environmental conditions during olive fruit's growth [13]. Plethora of studies took advantage of molecular markers for the identification of the varietal origin of olive oil such as SNPs [14, 17], microsatellites [18– 20], SCARs [21] and AFLPs [22, 23]. Recent advances in olive genome and transcriptome sequencing increased the analytical targets and enriched the olive molecular markers database.

However, the reliability and reproducibility of these techniques are strictly dependent on the quality of the DNA extracted from oil samples [12, 19, 24, 25]. Trace amount of DNA is found in olive oil, which is highly degradable. Therefore, a high number of DNA isolation protocols were published while several commercial kits dedicated to olive oil DNA extraction are available [26].

DNA-based reaction chemistry was combined with existing detection methods such as capillary electrophoresis, high-resolution melting (HRM), TaqMan probes, qRT-PCR and single-base extension (SNaPshot) resulting in numerous analytical approaches with particular advantages and disadvantages.

The chapter provides an overview of the current trends and critical issues on DNA-targeted approaches used for traceability and authenticity of olive oil. This is a rapidly expanding field with significant challenges and prospects that shall be discussed thoroughly. Moreover, issues of adulteration with oils of plant origin will be thoroughly discussed while recent advances in authentication and traceability of herbs and medicinal plants will be taken into consideration and compared with oil matrices.

### **2. Recent advances in DNA extraction from olive and other plant oil matrices**

A pre-requisite for the development and improvement of polymerase chain reaction (PCR) based DNA fingerprinting methods is the isolation of adequate quality and quantity DNA. Although the quantity of recoverable DNA from plant oils is hardly detectable by any means, either nano-spectrophotometer or agarose gel electrophoresis, most of the times it is sufficient for molecular markers analysis. However, nuclease activity and the presence of PCR inhibitors such as fats, residual polysaccharides and polyphenols might inhibit PCR amplification [19, 21, 24, 27, 28].

The purity of extracted DNA is the most crucial and significant step in DNA fingerprinting methods ensuring the validity and the reproducibility of plant oil forensic methodologies. Therefore, the need for a reliable and reproducible protocol for DNA isolation from plant oils is mandatory for either research or industrial applications. The ideal plant oil DNA extraction method should be reproducible, simple, relatively cheap and capable to recover stable, free of PCR inhibitors DNA either from filtered and/or heavily processed plant oils or from unfiltered olive oils.

In the last 15 years, many extraction methods and commercial kits were used and further improved valuable tools were provided. Commercial kits were used in several reports with positive results in most cases. Testolin and Lain compared the performance of a variety of commercial kits on DNA isolation from filtered and non-filtered olive oil and concluded that most consistent results were obtained using the QIAamp DNA Stool Mini Kit [24]. The potential of this commercial kit to isolate DNA of acceptable quality from oils of plant origin such as sesame oil was validated by Spaniolas et al. [29]. However, the high cost of this commercial kit motivated researchers to develop or modify CTAB (Cetyltrimethylammonium bromide)-based protocols. Muzzalupo and Perri [28] introduced the use of Proteinase K during malaxation to prevent DNA degradation. Intact DNA was then isolated from non-filtered olive oil sediments using the CTAB method of Doyle and Doyle [30]. The main limitation of this method was the use of Proteinase K during malaxation that is not applicable commercially.

The addition of this enzyme prior to the storage of olive oil improved the amount of recovered DNA [24], although this is not recommended for commercial application. Therefore, Consolandi et al. tested the efficiency of Proteinase K by adding it to the pellet and the oily phase after hexane treatment and centrifugation of oil sample, with positive results [31]. However, the innovation of this method relies on the disruption of neutral micelles by adding the surfactant Tween-20 after hexane extraction. Busconi et al. isolated intact DNA from unfiltered laboratory and commercial olive oils, using a CTAB method on 0.5 g of pellet [32]. They were able to amplify up to 1942 bp fragments. Martins-Lopes et al. used the same method, but their starting material was 6 ml unfiltered olive oil and not 0.5 g oil sediment [33]. The various CTABbased methodologies indicated that the strategy of using common plant DNA extraction protocols can lead to oil matrix DNA isolates of adequate quality.

Alternatively, Breton et al. extracted DNA from variable amounts of olive oil using several techniques such as magnetic beads, silica extraction, spun column and hydroxyapatite [25]. The most efficient approach which that provided free of PCR inhibitors DNA was the magnetic beads. This technique was further applied in many commercial virgin and crude olive oils leading to the claim by the authors that although it needs further improvement, it could be routinely applied in any olive oil sample. Doveri et al. [34] used the official Swiss method for lecithin extraction [35] from 15 ml unfiltered olive oils, resulting to less than 50% successful amplifications due to the low quantity and quality of extracted DNA.

Giménez et al. compared four DNA isolation methods for commercial mono-varietal olive oils [26]. The study included a CTAB-based [36] and a hexane method [31] as well as two modified CTAB-methods, CTAB-hexane and CTAB-hexane-chloroform. The CTAB-hexane-chloroform methodology comprising a washing step with chloroform/isoamyl alcohol in combination with the addition of linear acrylamide prior to the precipitation proved to have a beneficial effect on recovering DNA of adequate quality and quantity. It is worth mentioning that the starting olive oil volume was reduced to less than 1 ml. This protocol was successfully used to extract DNA from several plant oils indicating the potential of this method for oil DNA forensic applications [37, 38]. Ramos-Gomes et al. improved this protocol by increasing the concentration of Tween-20 in the lysis buffer and reducing the lysis time to 15 minutes while the precipitation time was limited to 1 h without the addition of linear acrylamide [38]. This improved protocol increased the yield and the PCR amplification efficiency of olive and seed oils DNA [38]. Raieta et al. [39] further modified the CTAB-hexane-chloroform protocol [26] by adding more purification steps with more critical excision of major DNA band from 1% agarose gel aiming to remove most of the PCR inhibitors such as residues of organic components or other contaminants. This protocol was successfully applied on various mono- and multi-varietal commercial filtered olive oils.

Recently, Muzzalupo et al. developed an innovative and simple method that bypasses the DNA extraction/purification steps by using KAPA3G Plant DNA polymerase that enables the direct DNA amplification from virgin filtered and unfiltered olive oils [40]. Definitely, this method seems more efficient and faster than the traditionally DNA isolation protocols. However, the success of this approach remains to be tested in lab and industrial applications.

Considering that in the 1990s only a couple of olive oil DNA extraction protocols were reported, the current availability of numerous protocols ensures the positive prospects of DNA-based olive oil authentication and traceability.

### **3. Olive oil and varietal origin**

The purity of extracted DNA is the most crucial and significant step in DNA fingerprinting methods ensuring the validity and the reproducibility of plant oil forensic methodologies. Therefore, the need for a reliable and reproducible protocol for DNA isolation from plant oils is mandatory for either research or industrial applications. The ideal plant oil DNA extraction method should be reproducible, simple, relatively cheap and capable to recover stable, free of PCR inhibitors DNA either from filtered and/or heavily processed plant oils or from unfiltered

In the last 15 years, many extraction methods and commercial kits were used and further improved valuable tools were provided. Commercial kits were used in several reports with positive results in most cases. Testolin and Lain compared the performance of a variety of commercial kits on DNA isolation from filtered and non-filtered olive oil and concluded that most consistent results were obtained using the QIAamp DNA Stool Mini Kit [24]. The potential of this commercial kit to isolate DNA of acceptable quality from oils of plant origin such as sesame oil was validated by Spaniolas et al. [29]. However, the high cost of this commercial kit motivated researchers to develop or modify CTAB (Cetyltrimethylammonium bromide)-based protocols. Muzzalupo and Perri [28] introduced the use of Proteinase K during malaxation to prevent DNA degradation. Intact DNA was then isolated from non-filtered olive oil sediments using the CTAB method of Doyle and Doyle [30]. The main limitation of this method was the use of Proteinase K during malaxation that is not applicable commercially. The addition of this enzyme prior to the storage of olive oil improved the amount of recovered DNA [24], although this is not recommended for commercial application. Therefore, Consolandi et al. tested the efficiency of Proteinase K by adding it to the pellet and the oily phase after hexane treatment and centrifugation of oil sample, with positive results [31]. However, the innovation of this method relies on the disruption of neutral micelles by adding the surfactant Tween-20 after hexane extraction. Busconi et al. isolated intact DNA from unfiltered laboratory and commercial olive oils, using a CTAB method on 0.5 g of pellet [32]. They were able to amplify up to 1942 bp fragments. Martins-Lopes et al. used the same method, but their starting material was 6 ml unfiltered olive oil and not 0.5 g oil sediment [33]. The various CTABbased methodologies indicated that the strategy of using common plant DNA extraction

protocols can lead to oil matrix DNA isolates of adequate quality.

amplifications due to the low quantity and quality of extracted DNA.

Alternatively, Breton et al. extracted DNA from variable amounts of olive oil using several techniques such as magnetic beads, silica extraction, spun column and hydroxyapatite [25]. The most efficient approach which that provided free of PCR inhibitors DNA was the magnetic beads. This technique was further applied in many commercial virgin and crude olive oils leading to the claim by the authors that although it needs further improvement, it could be routinely applied in any olive oil sample. Doveri et al. [34] used the official Swiss method for lecithin extraction [35] from 15 ml unfiltered olive oils, resulting to less than 50% successful

Giménez et al. compared four DNA isolation methods for commercial mono-varietal olive oils [26]. The study included a CTAB-based [36] and a hexane method [31] as well as two modified CTAB-methods, CTAB-hexane and CTAB-hexane-chloroform. The CTAB-hexane-chloroform methodology comprising a washing step with chloroform/isoamyl alcohol in combination

olive oils.

118 Products from Olive Tree

The olive cultivar together with the region of production directly affects the quality traits of olive oil. Therefore, mono-varietal extra-virgin olive oils or blends of specific cultivars grown in certain regions are considered as premium olive oils of higher value due to specific quality characteristics. These premium olive oils are protected by the European Commission through certification labels of PDO and PGI in order to ensure authenticity and protection of consumers. Therefore, there is a pressing need for reliable identification of the genetic identity of extravirgin olive oils.

Several attempts were carried out to relate chemical composition of olive oils with the cultivar of origin such as the mono-saturated fatty acids that are major constituents of olive oils and confer high nutritional value. In this effort, many reports used the fatty acids content to discriminate olive cultivars [41–43] while Mannina et al. performed a study in a well-defined and limited geographical region achieving a relationship between the fatty acid composition and some Sicilian cultivars [42]. These studies revealed that although the varietal effect was important for olive oil discrimination purposes based on fatty acid composition, the geographical and environmental effects were strongly affecting this approach [13]. Recently, Laroussi-Mezghani et al. were able to predict the varietal origin of six Tunisian cultivars by their fatty acid composition and near-infrared spectra associated with chemometric treatment. Other olive oil constituents such as the triacylglycerols have also been widely studied for their discriminatory efficiency on cultivar origin [41, 44–46]. In addition, several attempts were also made to correlate a long list of minor components such as sterols, pigments, phenolic and volatile compounds, hydrocarbons and tocopherols of olive oils with the cultivar origin of olive oil. Although Matos et al. [47] were able to discriminate three olive cultivars based on sterol composition; sterol was mostly studied in combination with other chemical compounds such as fatty acids and triacylglycerols [48, 49].

The volatile compounds are known to be strongly related to the genotype and the geographic origin of olive fruit, giving the unique flavour and quality of olive oil [13]. The volatile fraction in virgin olive oil consists of more than 100 compounds, while the most important substances for olive variety discrimination are the products of the lipoxygenase pathway (LOX) [50].

Despite the plethora of biochemical methods and analytical tools that were developed for the identification of olive oil cultivar, the main issue is the significant effect of climatic conditions and olive oil processing on chemical composition of virgin olive oil.

DNA-based methods can be used as complementary approaches for olive oil authentication and traceability considering a number of advantages over conventional methods such as reliability, specificity and sensitivity without any influence by the environmental conditions.

Currently, there are plenty of reports on the discrimination of olive cultivars using various molecular markers such as amplified-fragment length polymorphism [51, 52], random amplified polymorphic DNA (RAPD) [53], sequence-characterised amplified regions [54], simple sequence repeats (SSRs) [55, 56], inter-simple sequence repeats (ISSRs) [57] and singlenucleotide polymorphisms [58, 59]. However, only few of them were tested on the identification of the varietal origin of olive oil. This can be attributed to the low quality of olive oil DNA due to severe fragmentation as a result of the degradation process.

A study showed that the DNA quality is affected during storage of filtered olive oil in retail store conditions [27]. The amplification of 107 bp DNA fragments was successful for olive oil samples stored up to 1 year, whereas DNA fragments longer than 415 bp were successfully amplified only after 20 days of storage and no longer [27]. Moreover, Montemurro et al. and Pafundo et al. employed AFLP molecular markers to study the effect of olive oil storage length on the use of DNA as an analyte for molecular traceability [28, 60]. They showed a significant deterioration of DNA quality within a month while the AFLP profiles of leaf and oil DNA were not similar after 9 and 12 months of storage. These reports indicate the absolute requirement for short DNA template molecular markers in order to be used for olive oil DNA forensic applications.

One of the first efforts to fingerprint four Italian olive oils was made by Pafundo et al. using AFLP markers [23]. AFLP markers are based on the detection of restriction fragments by PCR amplification using a genome as template [61]. The origin of polymorphism in AFLP is based on base substitutions within restriction sites or deletions/insertions between two adjacent restriction sites. Therefore, the high-quality DNA is a pre-requisite for such a molecular marker. The identity between AFLP profiles in leaves and oils reached a maximum of 70% due to differences in the level of DNA degradation between leave and oil samples [23]. Three years later, Montemurro et al. reported enhanced AFLP profiles of 10 Italian cultivars by optimising the DNA extraction protocol and restriction/ligation conditions [22]. They also suggested the setting up of an olive oil reference data bank with AFLP profiles, which could be used as an identity card of mono-varietal olive oils. However, the requirements of high-quality DNA and short-storage time for reliable traceability lead to the advancement of other molecular markers such as SSRs and SNPs in combination with high-performance analytical platforms.

their fatty acid composition and near-infrared spectra associated with chemometric treatment. Other olive oil constituents such as the triacylglycerols have also been widely studied for their discriminatory efficiency on cultivar origin [41, 44–46]. In addition, several attempts were also made to correlate a long list of minor components such as sterols, pigments, phenolic and volatile compounds, hydrocarbons and tocopherols of olive oils with the cultivar origin of olive oil. Although Matos et al. [47] were able to discriminate three olive cultivars based on sterol composition; sterol was mostly studied in combination with other chemical compounds such

The volatile compounds are known to be strongly related to the genotype and the geographic origin of olive fruit, giving the unique flavour and quality of olive oil [13]. The volatile fraction in virgin olive oil consists of more than 100 compounds, while the most important substances for olive variety discrimination are the products of the lipoxygenase pathway (LOX) [50].

Despite the plethora of biochemical methods and analytical tools that were developed for the identification of olive oil cultivar, the main issue is the significant effect of climatic conditions

DNA-based methods can be used as complementary approaches for olive oil authentication and traceability considering a number of advantages over conventional methods such as reliability, specificity and sensitivity without any influence by the environmental conditions. Currently, there are plenty of reports on the discrimination of olive cultivars using various molecular markers such as amplified-fragment length polymorphism [51, 52], random amplified polymorphic DNA (RAPD) [53], sequence-characterised amplified regions [54], simple sequence repeats (SSRs) [55, 56], inter-simple sequence repeats (ISSRs) [57] and singlenucleotide polymorphisms [58, 59]. However, only few of them were tested on the identification of the varietal origin of olive oil. This can be attributed to the low quality of olive oil DNA

A study showed that the DNA quality is affected during storage of filtered olive oil in retail store conditions [27]. The amplification of 107 bp DNA fragments was successful for olive oil samples stored up to 1 year, whereas DNA fragments longer than 415 bp were successfully amplified only after 20 days of storage and no longer [27]. Moreover, Montemurro et al. and Pafundo et al. employed AFLP molecular markers to study the effect of olive oil storage length on the use of DNA as an analyte for molecular traceability [28, 60]. They showed a significant deterioration of DNA quality within a month while the AFLP profiles of leaf and oil DNA were not similar after 9 and 12 months of storage. These reports indicate the absolute requirement for short DNA template molecular markers in order to be used for olive oil DNA forensic

One of the first efforts to fingerprint four Italian olive oils was made by Pafundo et al. using AFLP markers [23]. AFLP markers are based on the detection of restriction fragments by PCR amplification using a genome as template [61]. The origin of polymorphism in AFLP is based on base substitutions within restriction sites or deletions/insertions between two adjacent restriction sites. Therefore, the high-quality DNA is a pre-requisite for such a molecular marker. The identity between AFLP profiles in leaves and oils reached a maximum of 70% due

and olive oil processing on chemical composition of virgin olive oil.

due to severe fragmentation as a result of the degradation process.

as fatty acids and triacylglycerols [48, 49].

120 Products from Olive Tree

applications.

The efficiency of RAPD, ISSR and SSR molecular markers for varietal identification of olive oil was evaluated by Martins-Lopes et al. in 23 Portuguese olive oil samples [33]. This study demonstrated that the ISSR marker system was more informative as compared to the inadequate efficiency of RAPD primers in olive oil samples. SSR analysis was performed to compare the profile of DNA samples isolated from olive oil with that of leaves with satisfactory results.

SSRs are also called microsatellites and consist of 1–10 bp tandem repeats, with a variable number of repetitions [62]. They are highly polymorphic due to the variation in the number of repeats. Microsatellites can be detected by PCR amplification using specific primers annealed to the unique flanking sequences providing high discrimination power. So far they are the most popular molecular markers for olive oil fingerprinting purposes.

Although many studies reported identical olive oil and leaf profiles [18, 24, 34, 63–65], the validity of this approach due to occurrence of repeatability problems is mostly dependent on the SSR sequence and the olive oil DNA quality. Another significant concern on the interpretation of the results is related to differences in amplicon size and allele drop-out in olive oil DNA analysis. These issues might be probably attributed to the low quality and quantity of olive oil DNA, which affect the allele amplification.

In several studies, the appearance of additional than the expected alleles was reported. Muzzalupo et al. and Ben Ayed et al. showed the allele contribution of the pollinator cultivar that is present in seed embryo [12, 19]. This finding contradicts many studies that appeared thereafter. However, additional alleles might appear either by accidental mixing with other cultivars during harvesting or by mixing with traces of olive oils originated from other cultivars during oil-milling processes.

The initial attempts to identify olive cultivars in commercial olive oil samples using SSR markers were reported by Pasqualone et al. and Breton et al. [25, 65]. The SSR profiles of olive oil DNA were identical to those obtained from leaves and drupes, showing the potential of SSR marker to be used for forensic applications. Pasqualone et al. demonstrated the effectiveness of SSR analysis in verifying the identity of a PDO olive oil by the genotyping of a limited number of DNA microsatellites [20]. More recently, Pasqualone et al. showed that a single microsatellite marker was sufficient to discriminate Leucocarpa olive oil from six other monovarietal olive oils providing an identification key based on the PCR amplification profiles [63]. Additionally, Alba et al. demonstrated the potential of a single, highly polymorphic SSR marker to discriminate seven Italian PDO mono-varietal olive oils [18]. Vietina et al. studied the traceability of 21 mono-varietal olive oils using microsatellites, concluding that in parallel with the improvement of olive oil DNA extraction methods, the implementation of authentication procedures might require selection of highly polymorphic SSR markers that display the same alleles in different laboratories and the robustness of the method should be assessed in an inter-laboratory ring trial [66].