*3.5. Phenolic Compounds*

A reduction of the total concentration of the identified phenolic compounds was detected in the oils from particular treatments, but the highest decrease compared to the control treatment was detected in the oils obtained from the fruits of both cultivars stored at −20 ◦C (Table 3). Hachicha Hbaieb et al. [9] have found that negative effects of storage time on phenolic compounds in oils were enhanced by an increase in storage temperature from 4 to 25 ◦C. Yousfi et al. [10] found that the main phenolic compounds in VOO exhibited a reduction during 15 days of fruit storage, which was in correlation with the increase in the applied temperature (from 2 to 18 ◦C). Other authors, who investigated the influence of freezing of fruits on trees, reported a decrease in the concentration of phenolic compounds in the obtained oils. They explained it as a consequence of fruit freeze injuries, which lead to cell dehydration and destruction of cell membranes, and consequently to cell death and high oxidation of cell contents as a result of the contact between enzymes and their respective substrates, which might have affected the phenolic composition of the oils [40,42]. Morelló et al. [40] investigated the influence of freezing of Arbequina fruits on trees on phenols in the obtained oils. They have found that total phenols and secoiridoids decreased after frost because ice crystals destructed olive tissues, which encouraged the oxidative degradation of phenolic compounds in reactions catalyzed by polyphenol oxidase enzyme [40]. Masella et al. [26] investigated the difference between three different methods of freezing of olives and found a significant reduction of total phenols in oils obtained from fruits after 6 months of storage at freezing temperatures (about 40% of the control oils) regardless of the freezing method used. It must be mentioned that not all the identified phenolic compounds absorb UV light equally, meaning the use of oleuropein as a standard for all secoiridoids with the response factor equal to one in the HPLC-DAD analysis in this study might have resulted with an overestimation of the reduction of the total phenol concentration in the oils of particular treatments. For example, p-HPEA-EDA (oleocanthal) has a lower response factor in comparison to 3,4-DHPEA-EDA (oleacein), and the same applies for the corresponding aglycone isomers of ligstroside and oleuropein. This difference is related to the different substitution of the aromatic ring. The underestimation of the secoiridoids bearing the tyrosol moiety might have had a notable impact on the calculated total phenol concentrations. More specifically, although the reduction noted is relative for each compound, the actual total phenolic loss might be less than reported.

Considering the secoiridoid group, a reduction was found in the case of treatments at RT and −20 ◦C in the oils from both cultivars. Since secoiridoid compounds are strongly related to the VOO shelf life [45], it can be assumed that the oils stored at +4 ◦C would have the longest shelf life among the oils obtained from the stored fruits. Reduction of secoiridoids was lower in RO oils, which initially had a lower concentration of total secoiridoids compared to IB oils. Li et al. [15] noticed that the higher the initial concentration of these phenolic compounds in oil, the faster they decrease during storage, possibly because higher concentrations are more susceptible to oxidation with respect to other antioxidants in olive oil. Guillaume et al. [42] noted a reduction of the concentration of secoiridoids in the oils obtained from the frost-damaged fruits of three olive cultivars (Frantoio, Barnea and Picual) grown in Australia. Hachicha Hbaieb et al. [9] have also observed a larger decrease in secoiridoids concentration in Arbequina and Chétoui oils obtained from fruits stored at 25 ◦C than at 4 ◦C, and related this to the lower β-glucosidase activity determined in olive fruits from the former treatment.




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Considering the particular secoiridoid compounds in IB oils, most of the concentrations decreased in the oils obtained from stored fruits. Quantitatively the highest reduction with respect to IB-control was determined for the concentration of 3,4-DHPEA-EDA in IB-RT and IB-20 oils. In RO oils from the stored fruits the highest decrease with respect to RO-control oil was detected in the case of 3,4-DHPEA-EDA and oleuropein aglycone (isomer I) after fruit storage at −20 ◦C and at RT In the oils from both cultivars obtained from fruits stored at +4 ◦C the profile of secoiridoids was more similar to the control oils than that of the other two treatments. It is probable that the cold storage conditions slowed down the rate of enzymatic and biochemical reactions, which lead to the degradation of these particular phenols, as noticed in RT oils, and at the same time avoided the negative effects caused by freezing, observed in the oils obtained from the fruits stored at −20 ◦C. These findings are in agreement with the results of Hachicha Hbaieb et al. [9], who found more similar phenolic profiles of the oils obtained from Arbequina fruits stored at 4 ◦C and the freshly harvested ones, in comparison to the oil obtained from fruits stored at 20 ◦C, which was explained by the similar endogenous enzyme activity patterns detected in the fruits of the former treatments. Romero et al. [27] characterized the phenolic profile of Spanish olive oils (Cornicabra, Hojiblanca, and Picual cultivars) with "frostbitten olives" sensory defect and found that the concentrations of all the investigated groups of phenols decreased in defective oils, except secoiridoids. The authors [27] explained these differences by considering the action of enzymes that are affected by frost; physical damage of olive fruits by ice crystals formed during freezing leads to cellular destruction, allowing phenolic substrates to mix with polyphenol oxidase (PPO), which degraded them. In this study, lower concentrations of the majority of phenols, even secoiridoids, were found in oils obtained from the fruits frozen at −20 ◦C in comparison to control oils. This was probably due to the controlled freezing process applied, which did not include freezing and thawing cycles that would correspond to those occurring naturally in the olive orchard.

In both cultivars, a significant increase in the concentrations of simple phenolic compounds, hydroxytyrosol and tyrosol, was found in the oils obtained from the fruits stored at RT and +4 ◦C compared to the control oils (Table 3). The increase observed was proportional to the storage temperature applied, which was as expected, since it can be explained by increased hydrolysis of complex phenols into simple phenols at higher temperatures [15]. On the other hand, after storage at −20 ◦C, no significant change in hydroxytyrosol and tyrosol concentrations was found when compared to the control oils. Such an outcome could have possibly been connected to partial inactivation or the lower ability of PPO and peroxidases (PODs) to oxidize biophenolic glucosides at lower storage temperature, as reported earlier [46].

The concentration of total lignans decreased in the case of both monovarietal oils obtained from fruits at −20 ◦C, which was in agreement with the findings of Masella et al. [26], while in IB+4 oil the increase of total lignans was mainly a consequence of an increase in pinoresinol concentration. Guillaume et al. [42] reported that the concentration of lignans was strongly positively correlated with the intensity of the "frostbitten olives" defect, and that the concentration of acetoxypinoresinol increased after the freezing of olive fruits on the trees, which was not confirmed by the findings of this study in the case of controlled frozen storage.

The concentration of total flavonoids decreased in all the IB treatments, while for RO a decrease was detected only in the case of RO-20 oil. In the oils of both cultivars obtained after storage of fruits at +4 ◦C the profile of individual flavonoids was more similar to the one observed in the control oils than in the other two treatments. Other authors reported different trends in flavonoids behavior under various storage conditions. Hachicha Hbaieb et al. [9] reported higher flavonoids content in the oils extracted from olives stored at 4 ◦C than at 20 ◦C, probably due to the accelerated process of ripening of fruits at the higher temperature. The content of flavonoids in Cornicabra oils obtained from fruits stored at 10 ◦C and 20 ◦C did not show a clear trend at the beginning of storage, probably because of their stable structure and high oxidation resistance, while an increase of particular flavonoids was determined after a prolonged storage, probably because of the destruction of the cell structure and the release of bound phenols [37].

The concentration of total phenolic acids only increased after the RT treatment in oils of both cultivars, as a consequence of a sharp increase in p-coumaric acid concentration. Storage at lower temperatures had no influence on the concentration of phenolic acids, which was not in agreement with the results of Masella et al. [26], who reported a decrease in the concentration of p-coumaric acid after 6 months of frozen storage of olive fruits. The discrepancy observed was possibly related to the difference in storage time between the two studies.
