3.1. Total Polyphenols
The concentrations of different polyphenols and of the groups found in raw and cooked EVOO samples are presented in Table 2
When EVOO was heated in a pan, the sumatory of polyphenolic content decreased by around 40% at the low temperature (120 °C) and 75% at the high temperature (170 °C). Casal et al. (2010) reported a decrease of 50% in the total phenolic content, measured by the Folin-Ciocalteu method, after heating olive oil in a domestic deep-fat fryer at 170 °C for 3 h. [23
]. However, in this study, the oil was deep fried so the samples were less exposed to oxygen and light, which may explain why the results are different to those presented here. Moreover, Folin-Ciocalteau methods are not selective, so this variation is not measuring only the phenol content, but also other reducing compounds. For this reason, it is also difficult to compare the results with those showed by one recent study, in which the degradation of the total phenolic content during a sautéing process was evaluated. The authors showed a decrease of approximately 50% of the antioxidant capacity measured by the Folin-Ciocalteau method after sautéing typical Mediterranean vegetables (potato, eggplant, tomato, and pumkin) for 10 min at 100 °C [33
For the ANOVA and multiple regression models, the normality of residuals was verified. To check this assumption, normal probability plots of the residuals were plotted for each compound. The graph for the sum of polyphenols is shown in Figure 1
. The results of the ANOVA test and the linear regression models are shown in Table 3
. The temperature was mainly responsible for the polyphenols depletion and there were no significant effects from time or the interaction. These results are in accordance with those reported by Goulas et al. (2015), who showed that heating the oil at 180 °C for 1 h or for 5 h made no difference in polyphenol content decrease [8
The model for the sum of polyphenols was great fitted, with a R2 of 0.97, in which 97% of the variance is explained by the model. The slope for temperature was significantly different from 0, suggesting that a longer cooking period does not change the polyphenolic fraction when the EVOO is processed only once. As the low level is −1 and the high level is +1, and the β of the temperature is −131, then cooking using a high temperature decreased the polyphenol content 232 mg/kg more than applying a moderate temperature, which represents 27% of the raw EVOO concentration.
Secoiridoids are the largest group of EVOO polyphenols. Secoiridoids include oleuropein, ligstrosides, and their derivatives. Some of them have been reported to have important benefits to health, such as oleocanthal or oleacein [34
]. Oleocanthal has demonstrated anti-inflammatory effects [35
] and a protective role against some diseases, such as Alzheimer disease [34
], and oleacein has proven to protect against cardiovascular diseases, reducing hypertension [36
] and inhibiting neutrophils adhesion [37
During the cooking process, secoiridoids decreased 45% at the low temperature and 70% at the high temperature. Among this group, a different behavior was observed in hydroxyelenolic acid, which is not a polyphenol but a related compound produced by the ester breakdown of ligstroside, oleuropein, and their aglycones [38
]. Thus, the formation of hydroxyelenolic acid was enhanced by processing at a moderate temperature. However, a longer cooking period and a higher temperature promoted its degradation, giving a lower concentration.
According to ANOVA analysis, the factor responsible for the depletion of secoiridoids was temperature. However, different results were found for each secoiridoid, in which oleacein and oleuropein aglycone were also affected by the interaction of time and temperature, and hydroxydecarboxymethyl oleuropein aglycone and hydroxyoleuropein aglycon were affected by all of the evaluated factors. These results are in accordance with those reported by Attya et al. (2010), in which heating at 90 °C was shown to cause almost no degradation of oleocanthal and oleacein in EVOO, but at 170 °C the concentration of both compounds was reduced by half, reflecting the major role played by temperature in polyphenol degradation [16
The models were properly fitted for most of the secoiridoids analyzed, however, oleocanthal showed a R2
of 0.7 because of its high reactivity, which also prompted the development of a new method for its specific analysis [29
]. Oleocanthal presents keto-enolic tautomerism, which impeded its proper analysis, as it reacts with the solvent during the chromatographic separation. Oleocanthal may have reacted more in some samples than others, but even with only a 70% model fit, the result indicates that the cooking time did not change the oleocanthal concentration. In contrast, it decreased by 100 mg/kg of oil after cooking at a high temperature compared to the moderate temperature.
In the case of the sum of secoiridoids, the ANOVA results showed that the interaction factor was not significant, although in the multiple regression model the β for the interaction was different to 0, indicating that there was an effect. This difference occurred because the test used for ANOVA and the test used for the regression model were different: ANOVA applies a F-test, and, for the regression models, a t-test is used. Despite the ANOVA result giving a p-value of over 0.05, it showed a trend that this factor had an effect on secoiridoid degradation (p-value = 0.052).
According to the slopes of the models, the temperature was mainly responsible for the depletion of seicoiridoids during a domestic sautéing process. Slopes were analyzed as a percentage of the initial concentration (in raw EVOO), as the initial concentration for each polyphenol differed substantially, making it difficult to compare the models between the different polyphenols. The results are shown in Table 3
. The slopes represent the values ranging from 12% (ligstroside aglycone) to 20% (oleuropein aglycone) of their original concentrations. The most different one was oleocanthal, which showed just a 6.5% depletion and withstands better the temperature than oleuropein aglycone. The compounds with a o
-diphenol group were the most reactive, with a slope representing between 18% (hydroxydecarboxymethyl oleuropein aglycone) an 20% (oleacein) of their initial concentration. On the other hand, oleocanthal and ligstroside aglycone (compounds with just one hydroxyl group) showed less reactivity. ortho
-Diphenols are the most reactive, as they can be converted easily to ortho
-quinones through a radical reaction [39
]. Also, the intermediates of the reaction are radicals too, so they stabilized by the hydroxyl in the ortho position [40
]. This rapid conversion may be responsible for the higher degradation compared to single phenols. This difference in the reactivity is also reflected in the activation energy, in which oleocanthal presents lower than oleacein because a higher temperature change is needed to degrade oleocanthal at the same rate as oleacein [16
As mentioned above, hydroxyelenolic acid was an exception, its concentration was affected by the cooking time and the interaction between time and temperature, but not the temperature alone. For this compound and for elenolic acid, the time factor had a positive effect as the slopes were positive, indicating that frying with EVOO for longer periods may increase their concentration. Although the hydroxyelenolic acid model was not well fitted, the elenolic acid model showed an 82% fitness result. Like hydroxyelenolic acid, elenolic acid is not a phenol, but a derivative of oleuropein and ligstroside aglycones. Thus, despite a long cooking process degrading some of the polyphenols, it can enhance some related and new compounds.
3.3. Phenolic Alcohols and Others
Phenolic alcohols, mainly hydroxytyrosol and hydroxytyrosol acetate, are derivates from oleuropein, like the secoiridoids, but as their chemical behaviors are different, they are classified in a different group [27
When frying at a low temperature for a short time, only 9% of phenolic alcohols decreased, although a depletion of 85% (90% in the case of hydroxytyrosol), when applying a high temperature and a long cooking time, was observed. At a low or moderate temperature, the hydroxytyrosol degradation, formed by the ester breakdown of oleuropein and its aglycones (Figure 2
), may be counteracted by the rate of its generation. However, at a high temperature, its degradation is more likely to occur, resulting in a substantial reduction. Similar results are observed by Ramírez-Anaya et al. (2019), who showed that after sautéing typical Mediterranean vegetables at 100 °C, the hydroxytyrosol content only decreased between 25% and 50% [33
]. Furthermore, a similar behavior was found by Krichene et al. (2015), who showed an increase in hytroxytyrosol concentration during the first months of storage due to the transformation of oleuropein and derivates in the compound, but after some months there was a high decrease in its concentration [42
The concentration of phenolic alcohols was affected by temperature and time and hydroxytyrosol was also affected by their interaction, however, the β value for the temperature factor was higher, indicating that it is mainly responsible for their degradation.
Hydroxytyrosol was the most degraded compound by temperature, its slope represents 30% of its initial concentration. Thus, the amount of hydroxytyrosol diminished greatly—about 60% of the hydroxytyrosol concentration in the raw EVOO cooked at a high temperature compared to the EVOO cooked at a low temperature. So, cooking at low temperature should be recommended due to the fact that, according to the European Food and Safe Authority, it protects low-density lipoproteins (LDL) from oxidative damage [43
] that have proven health effects.
The minor groups of polyphenols in EVOO are phenolic acids, lignans, and flavones. There was no possibility to build a model for those groups because of their low concentration. The only model properly fitted (>80%) for flavones was luteolin that was mainly affected by temperature. In the case of lignans, the only compound present in quantifiable amounts was pinoresinol, which increased during cooking probably because of the transformation of 1-acetoxypinoresinol and because of its high temperature stability [44