Pyropheophytin a in Soft Deodorized Olive Oils

Mild refined olive oil obtained by neutralization and/or by soft deodorization at a low temperature and its blending with extra virgin olive oil (EVOO) is not allowed and is difficult to detect. Chlorophyll derivatives, pheophytins and pyropheophytin, and their relative proportions were proposed as parameters to detect such processes. The objective of this study is to determine changes in EVOO, in terms of pheophytins and pyropheophytin, occurring after several well-controlled mild refining processes. The changes on those chlorophyll pigments due to the processes depend on the temperature, stripping gas, acidity and oil nature. The data obtained show that, at temperatures below 100 °C, the rate at which pyropheophytin a is formed (Ra) is lower than the rate at which pheophytins a+a’ disappear (Ra+a’). As a consequence, the Ra+a’ and Ra ratios are considered to be directly linked to pheophytins a+a’ decrease instead of to pyropheophytin a formation. Stripping gas very slightly affects the transformation of the chlorophyll pigments; actually both acidity and N2 enhance the increment in the Ra+a’ and Ra ratios. In relation to the oil nature, the higher the initial pheophytin a+a’ content, the higher the increase in the Ra+a’ and Ra relations.


Introduction
Olive tree (Olea europaea) is one of the most expanded crops in the world. This has repercussions not only regarding the nutritional point of view but also with respect to the economy of, mainly, Mediterranean countries. Both the International Olive Council (IOC) and the European Union consider virgin olive oil (VOO) as just the oil obtained from the fruit of the olive tree solely by mechanical or other physical processes under conditions, particularly thermal conditions, that do not lead to alterations in the oil, and which has not undergone any treatment other than washing, decantation, centrifugation, and filtration [1,2]. If the quality of the oil does not meet a number of standards [3], it cannot be considered as 'edible' and must be refined. Refined olive oil (ROO) is a flavorless, colorless product that cannot be sold by retail and that has to be mixed with genuine VOO. Controlled olive oil blends are available in the market under the designation of olive oil (OO) composed of refined and virgin olive oils. Blends of olive oil with other vegetable oils are available too [4]. However, the high market price that VOO can reach has made it target of both mislabeling and illegal blending, and much work has been done to uncover such practices, including that on the detection of the correct proportion of olive oil in legal blends with seed oils [5,6].
Regarding adulterations, they may consist, for instance, of the addition of hazelnut oil, of oil obtained from the second extraction of the olive paste (olive pomace oil), or of soft deodorized olive oil. Hazelnut oil can be detected within an interval of 20-25% through the determination of the difference between the actual and the theoretical content of triacylglycerols (TAG) with equivalent carbon number

Chemicals
All chemical reagents were of analytical grade. The standard of chlorophyll a was purchased at Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Sodium hydroxide pellets, phenolphthalein, and diethyl ether, were from Panreac Química, S.A.U. (Castellar del Valles, Barcelona, Spain). Acetone and methanol were from Romil Chemicals Ltd. (Waterbeach, Cambridge, GB, UK). The deionized water used was obtained from a Milli-Q 50 system (Millipore Corp., Burlington, MA, USA).

Samples
Four Spanish monovarietal olive oils (Hojiblanca, Picual and two Manzanilla samples) were purchased directly from producers. A second set consisting of six olive oils of several origins and qualities (L-1, M-1, H-1, L-2, M-2 and H-2), with no varietal specifications, was purchased from local markets or directly from producers.

Qualitative Analysis of Chlorophyll Pigments
Several methods have been proposed to determine chlorophyll pigments in olive oils, including rapid and routine techniques [28,30,31].
In this work, we follow the method described by the International Standard Organization [32] and the German Society for Fat Science [33], based on the procedure previously described by Gertz and coworkers [29]. This method is currently one of the most widely used for the determination of phy and pyphy. Briefly, 300 mg of the oil samples is weighed into a 4-mL vial and introduced, with the help of 1 mL n-hexane, into a 1-g silica solid phase extraction (SPE) column, previously activated with 5 mL hexane. Subsequently the vial is rinsed twice with 1 mL n-hexane and added onto the column. A first fraction is eluted with 5 mL of a mixture consisting of n-hexane:diethyl ether (90:10, v/v) and is discarded. A second fraction is eluted with 5 mL acetone and collected. Then, it is evaporated in a rotary evaporator and re-suspended in 0.5 mL acetone for its subsequent analysis using a high-performance liquid chromatography-diode array detector (HPLC-DAD).
The HPLC analyses of the chlorophyll pigments were carried out with an HP Agilent 1100 Liquid Chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a DAD. Acquisition of data was done with the Agilent ChemStation for the HPLC System program. The conditions for the HPLC assays were: Waters Spherisorb ODS2 C18 column (250 × 4.6 mm internal diameter, 3-µm particle size) (Waters Ltd., Hertfordshire, UK), 20-µL injection volume through a Rheodyne Manual Sample Injector Valve (Idex Health & Science LLC, Rohnert Park, CA, USA), and isocratic elution conditions water:methanol:acetone (4:36:60, v/v/v), at a flow rate of 1 mL/min. Sequential detection was performed at 410 nm.

Quantitative Analysis of Chlorophyll Pigments
Pigments were quantified with a calibration curve obtained by least-squares linear regression analysis. The concentration range of the curve fitted the expected level of chlorophyll in VOO. We proceed as follows: From a 0.01% chlorophyll standard solution, we prepared five different diluted solutions in acetone (concentrations between 0.1 and 0.5 mg/kg) and we injected them, in duplicate, in the HPLC system.

Sensitivity and Method Repeatability
Tests to assess the repeatability of the method and trials to establish the limit of detection (LOD) were performed according to published procedures [34].
The LOD can be defined as the minimum concentration of an analyte that can be detected, although not necessarily quantified, with an acceptable confidence through a given analytical procedure. These concentration values should produce sharp, symmetrical analyte peaks with no tailing or shoulders and with a signal-to-noise ratio of at least 3. That is a concentration whose signal equals the blank signal (Y) plus three times (k = 3) its standard deviation (S): LOD = Y + k × S.
In order to calculate the LOD, five olive oil solutions prepared at different dilutions from a sample with low chlorophyll pigments content were taken to the HPLC, their areas measured and the respective standard deviations calculated.
The repeatability of the method was assessed with three VOO samples of different chlorophyll pigment concentrations (L-1, M-1 and H-1 with low, medium and high chlorophyll concentration, respectively). We determined the phy a+a' and of pyphy a concentrations (in mg/kg) together with the R a+a' percentage.
Measurements were done in triplicate. The statistical analysis of the repeatability was carried out following the ISO 5725 Norm [35] and AOAC Regulation [36].
The statistical parameters used were: The statistical study of the results was carried out by one-way analysis of variance (one-way ANOVA) of a number of repeated samples. The minimum significant level was set at 5%. The analysis was performed using the SPSS 12.0 program (SPSS Inc., Chicago, WI, USA).

Olive Oil Soft Neutralization Process
We carried out the soft neutralization procedure using an aqueous sodium hydroxide solution at 12 % (w/v). In order to know the volume needed for the free fatty acid neutralization, we first determined the free acidity of the starting oil according to the method published by the IOC. This method drives to the calculation of the free acidity expressed as the percentage of oleic acid and its performance had already been tested according to the corresponding collaborative tests [37].
Next, we placed 10 ± 0.001 g of each olive oil sample in test tubes and added a volume of the 12 % (w/v) aqueous sodium hydroxide solution corresponding to the free acidity, plus a 5% excess (2 mL approximately). We shook the tubes for 20 min and then centrifuged them (10 min, 3000 rpm, 16 cm centrifugation diameter). On each case, we discarded the aqueous phase and washed the remaining oily phase with 5-6 portions distilled water for 5 min. We repeated this last step until we had made sure there were no free-soaps (the pink color of the phenolphthalein disappeared completely). Finally, we centrifuge them for 10 min in the described conditions.

Olive Oil Mild Deodorization Procedure
Soft deodorization is a technique utilized to eliminate unpleasant odors in olive oil, getting a matrix that keeps its chemical composition unaltered. We carried out the process under soft thermal conditions, vacuum, and a certain stripping agent (N 2 or Air), in a way that such gas passed for a given period of time through a volume of relatively hot oil at a low pressure. In order to do this, we prepared our own laboratory equipment, mimicking industrial conditions as much as possible. Such equipment consisted of the following parts:

2.
Kitasato flask to prevent the sucking back of the sample.

3.
Beaker with glycerine as thermal liquid and stirring magnet. 4.
Vacuum pump with vacuum control.
We took the olive oil samples through different mild deodorization processes (vacuum at 22.5 mmHg; 600 mL/min stripping gas), and studied the influence of the following factors (Table 1): 1.

4.
Stripping gas (using hojiblanca variety: Treatment #3). Moreover, the effect of combining neutralization and deodorization was considered. In order to do that, three olive oils with a low, medium and high content of chlorophyll pigments (L-2, M-2, and H-2, respectively) were used. After neutralization with sodium hydroxide (Section 2.6) and filtering, oils were soft deodorized under N 2 , at 98 • C, for three hours.

Sensitivity and Method Repeatability
The lowest detectable concentration of pyphy a was 0.07 mg/kg. The data obtained in two consecutive determinations of the same sample, using the same analytical method, did not differ in more than the value of 'r' (Table 2). From those data (RSD r = 0.34-6.59%, RSD r = 2.5-10%, and RSD r = 1.82-4.62%, for phy (a+a') and pyphy a, and R a+a' , respectively) one may consider the method to have a good repeatability.

Qualitative Analysis of Chlorophyll Pigments
The selected conditions lead to the separation of individual pigments. The HPLC chromatograms consist of a series of peaks, three of them well resolved, whose retention times appear within the range from 15 to 25 min ( Figure 1). They correspond to phy b and b', phy a, phy a', and pyphy a. After, pyphy a' might also appear. Those peaks were identified according to the published bibliography [28,33].

Quantitative Analysis of Chlorophyll Pigments
We calculated the analyte concentration corresponding to each peak (phy a, phy a', and pyphy a) using the chlorophyll calibration curve: concentration (mg/kg) = −0.00319 + 0.0111 × peak area. We used chlorophyll as a standard instead of pyphy a because pyphy a is not commercialized as such and its synthesis is laborious.

Quantitative Analysis of Chlorophyll Pigments
We calculated the analyte concentration corresponding to each peak (phy a, phy a', and pyphy a) using the chlorophyll calibration curve: concentration (mg/kg) = −0.00319 + 0.0111 × peak area. We used chlorophyll as a standard instead of pyphy a because pyphy a is not commercialized as such and its synthesis is laborious.

Olive Oil Mild Deodorization Procedure
As shown in Table 3, the olive oil samples under study presented a wide variation in their chlorophyll content and, as observed in previous studies on different olive varieties [38], phy a was always the dominant pigment, being particularly high in the case of Manzanilla 1. According to our experience in the last ten years, where we have been analyzing 150 samples a year, on average, such a value may be considered to be very high. However, we have to keep in mind that it is not possible to give an expected average value (and therefore a reference value) for this parameter since the total content of chlorophyll compounds depends, among other things, on the characteristics of the starting samples and on the storage conditions [27]. Interestingly, the analyte concentrations seem to be dependent on the cultivar, which is the opposite to those observed by previous researchers over studies in which a higher number of cultivars were considered [39]. Therefore, the small number of samples advise us to be cautious regarding such a statement. We are aware that our assertion may seem contradictory to that observed in the cases of Manzanilla 1 and Manzanilla 2 (same cultivar but totally different results). In such a circumstance we have to take into consideration that the pigment composition and content of a certain oil is highly conditioned by the oil's initial quality, light exposure, temperature, etc., and not only by the cultivar. This is indeed a line of research to be focused on during our next endeavors, where a wider number of varieties are being systematically analyzed.

Effect of Deodorization Time
In order to consider the effect of the deodorization time, samples of monovarietal VOO hojiblanca, manzanilla, (manzanilla 1 and manzanilla 2) and picual were subjected to different deodorization timespans, at 98 • C, using N 2 as a carrier gas (Table 1, Treatment #1), for which results are shown in Figure 2. Under such accelerated conditions, the rate of evolution per hour is around 50% for hojiblanca and 25% for the others cultivars, which is very high in comparison with the normal 5-6% evolution per year observed during non-accelerated conditions [39].
In all cases, there was a quick rise in phy a+a' during the first hours of treatment, which slowed down later (Figure 2A). In the case of hojiblanca cultivar, the R a+a' relation reaches 17% after around 2.5 h, whereas in the cases of picual, manzanilla 1 and manzanilla 2, at least 4.5 h are needed to exceed the 17% threshold. Such a 17% limit is the one proposed by Australian and Californian regulatory bodies and corresponds to the minimum pyphy a content accepted for fresh EVOO [40,41]. Differences among cultivars (Table 3) may be due to the low initial pyphy a content, 0.70 mg/kg, in comparison with the phy a+a' presence, 10.90 mg/kg, observed in hojiblanca, which in turn gives an already higher initial R a+a' in comparison to the others.

Effect of Deodorization Time
In order to consider the effect of the deodorization time, samples of monovarietal VOO hojiblanca, manzanilla, (manzanilla 1 and manzanilla 2) and picual were subjected to different deodorization timespans, at 98 °C, using N2 as a carrier gas (Table 1, Treatment #1), for which results are shown in Figure 2. Under such accelerated conditions, the rate of evolution per hour is around 50% for hojiblanca and 25% for the others cultivars, which is very high in comparison with the normal 5-6% evolution per year observed during non-accelerated conditions [39]. The evolution observed in our study agrees with that previously published according to which the parameters of 100 • C and 60 min were considered as the optima, since they allowed negative volatiles removal and low pyphy formation (11.83%) [21].
If the phy a' content is not taken into account, that is, only R a is calculated ( Figure 2B), hojiblanca exceeds the 17% limit after around 1.5-2 h, whereas picual, manzanilla 1 and manzanilla 2 hold 4-4.5 h. It is then clear that the R a relation may evidence the presence of soft deodorized oils in a better way than the R a+a' relation does, meaning that phy a and pyphy a would reveal as key compounds to detect this kind of practice.
In any case, the increase in the R a+a' and R a relations is due to the phy a+a' reduction and not so much to pyphy a formation. This may be due to the phy a+a' destruction because of the effect of the deodorization conditions (98 • C), whereas pyphy a increases little after a certain time. We have to point out that we cannot expect an intensive phy a+a' destruction to be translated in an intensive pyphy a formation, since the concentrations of such derivatives do not keep a lineal relationship. Actually, previous studies show how, after the thermal treatment of olive oils, the disappearance of phy a+a' did not only correspond to the formation of pyphy a (and therefore to a lineal relationship) but also to that of other three products: 13 2 OH-phy a, 15 1 OH-lactone-phy a, and a colorless derivative [27], giving a more exact glimpse on the fate of phy a+a'.

Effect of Deodorization Temperature
The effect of the deodorization temperature was studied with the monovarietal EVOO hojiblanca, manzanilla 1 and manzanilla 2. In this case, oils were subjected to two-hour length deodorizations at 50, 75, 100, 130, and 150 • C using N 2 as a stripping gas (Table 1, Treatment #2).
Samples of hojiblanca and manzanilla 2 oils had relatively low initial concentrations of chlorophyll pigments (11.60 and 15.74 mg/kg, respectively), whereas, in the case of manzanilla 1, the pigment concentration was much higher (118.97 mg/kg).
The results are shown in Figure 3. When one compares the R a+a' and R a relations between the three different samples, it is clear that the higher the initial pigment concentration, the higher the R a+a' and R a increments. Besides, increases in temperature lead to increases in both R a+a' and R a proportions, the latter being steeper than the former, meaning that not all phy a+a' turn into pyphy a. Besides, it is clear that there is not a linear correlation with temperature and that at temperatures below 100 • C the formation of pyphy a takes place slowly, as has already been observed before [21], although we demonstrate that a two-hour deodorization versus a one-hour timespan, as stated earlier [21], has no effect on pyphy a formation, temperature being the key factor. From 100 • C on, pyphy a formation goes up notably.

Effect of Free Acidity
The study of the influence of the free acidity on the chlorophyll pigments during deodorization was carried out on samples of VOO from the picual variety. This parameter was chosen because of its relationship with the oil's initial quality. Picual samples had 0.19% free acidity and were spiked with oleic acid in order to get aliquots with 2.0 and 5.0% free acidity. Samples were subjected to 2 to 5 h length deodorizations at 98 °C, using N2 as a stripping gas (Table 1, lines 11-13).
According to the data obtained, the higher the acidity, the higher the increase in the Ra and Ra+a' relations, the effect being more pronounced when phy a' is left aside (Figure 4), since in this case the

Effect of Free Acidity
The study of the influence of the free acidity on the chlorophyll pigments during deodorization was carried out on samples of VOO from the picual variety. This parameter was chosen because of its relationship with the oil's initial quality. Picual samples had 0.19% free acidity and were spiked with oleic acid in order to get aliquots with 2.0 and 5.0% free acidity. Samples were subjected to 2 to 5 h length deodorizations at 98 • C, using N 2 as a stripping gas (Table 1, lines [11][12][13]. According to the data obtained, the higher the acidity, the higher the increase in the R a and R a+a' relations, the effect being more pronounced when phy a' is left aside (Figure 4), since in this case the 17% limit is reached after 1.25-2 h from the highest to the lowest acidity, instead of 1.5-2.25 h, respectively. Therefore, it is clear that high acidity enhances phy a+a' losses and pyphy a formation. Furthermore, pyphy a formation is clearly bound to oil quality expressed over its free fatty acid content, which contrasts with that indicated in the literature, in which a prediction model focused on olive oil shelf life stated that even if pyphy a is strongly related with light exposure and storage temperature, it does not show any association with oil quality nor with its chemical composition [42].
Foods 2020, 9, x FOR PEER REVIEW 11 of 15 17% limit is reached after 1.25-2 h from the highest to the lowest acidity, instead of 1.5-2.25 h, respectively. Therefore, it is clear that high acidity enhances phy a+a' losses and pyphy a formation. Furthermore, pyphy a formation is clearly bound to oil quality expressed over its free fatty acid content, which contrasts with that indicated in the literature, in which a prediction model focused on olive oil shelf life stated that even if pyphy a is strongly related with light exposure and storage temperature, it does not show any association with oil quality nor with its chemical composition [42].

Effect of the Stripping Gas
The study of the influence of the carrier gas on the chlorophyll pigments was carried out on hojiblanca VOO. Those samples were subjected to 2 to 5 h length deodorizations at 98 • C, using either N 2 or air as a stripping gas (Table 1, Treatment #3).
The results are shown in Figure 5. When N 2 is utilized as a stripping gas, the R a+a' relation is around 4.6-5% higher than when air is chosen ( Figure 5A), meaning that the 17% limit is exceeded after 2.0 h in the case of N 2 , and after 2.7 h if air is applied. After three hours, there is not a statistically significant difference on the R a+a' relation between both stripping gases. The same tendency is observed for the R a relation, although the time to surpass the limit is 1.5 and 2.4 h, respectively ( Figure 5B).

Effect of the Stripping Gas
The study of the influence of the carrier gas on the chlorophyll pigments was carried out on hojiblanca VOO. Those samples were subjected to 2 to 5 h length deodorizations at 98 °C, using either N2 or air as a stripping gas (Table 1, Treatment #3).
The results are shown in Figure 5. When N2 is utilized as a stripping gas, the Ra+a' relation is around 4.6-5% higher than when air is chosen ( Figure 5A), meaning that the 17% limit is exceeded after 2.0 h in the case of N2, and after 2.7 h if air is applied. After three hours, there is not a statistically significant difference on the Ra+a' relation between both stripping gases. The same tendency is observed for the Ra relation, although the time to surpass the limit is 1.5 and 2.4 h, respectively ( Figure  5B).   Table 4 shows the results of applying neutralization, and neutralization followed by soft deodorization (3 h, 98 • C, N 2 stripping gas), together with the initial pigment contents in the samples under study. VOO sample L-2 possesses the lowest amount; therefore, pyphy a is not formed in a substantial way. Consequently, R a+a' and R a equal zero. After neutralizing VOO sample M-2, phy a+a' and pyphy a content decrease minimally, which turns into an increase in R a+a' and R a , although without substantial meaning. This is in the way round for sample H-2 but, as it may be expected, the subsequent deodorization resulted in phy a+a' decrease and pyphy a increase, with the corresponding change in the R a+a' and R a relations. Table 4. Chlorophyll derivative concentrations (mg/kg) present in olive oil samples with low (L-2), medium (M-2) and high (H-2) pigment contents after different treatments: neutralization, filtration, and soft deodorization under N 2 , at 98 • C, for 3 h. R a+a' and R a (both in %) are also given. In no case the R a+a' and R a relations exceed the 17% value established as a limit from which an oil may be suspected to be soft deodorized.

Conclusions
In this pilot study, we observed that changes in chlorophyll pigments, due to soft the deodorization process, depend on the temperature, the limit of which was 100 • C. Below such ceiling, the rate at which pyphy a is formed is lower than the rate at which phy a+a' disappear. This indicates that, besides pyphy a formation, there exist parallel processes through which other non-detected compounds are formed. As a consequence, the R a+a' and R a relations are considered to be more directly linked to phy a+a' decrease than to pyphy a formation.
Stripping gas slightly affects the transformation of chlorophyll pigments; in fact, N 2 enhances the increment in the R a+a' and R a relations.
Acidity also boosts the increment in the R a+a' and R a relations. Regarding the oil nature, the higher the initial phy a+a' content, the higher the increase in the R a+a' and R a relations. If the initial phy a+a' presence is too low, the value of the R a+a' and R a relations will be zero.
Finally, we are sensitive to the fact that the number of samples under study was too limited to draw definite conclusions, yet it is our intention through this approach to offer a new insight in the detection of the soft deodorization oils in virgin olive oils. Indeed we will continue developing this line of research to answer open questions such as the fate of phy (a+a') or the actual influence of the cultivar on the chlorophyll profiles.