Antioxidants, Vol. 12, Pages 30: Olive Oil Produced from Olives Stored under CO2 Atmosphere: Volatile and Physicochemical Characterization

3.4. Volatile Compounds (VCs)The VCs of the oil produced from the control olives and the olives treated with CO2 were examined. In Figure 3, typical chromatograms of the VCs are presented, while in Table 2, a comparison of the analyses among the three oils (from CO2 treated and control olives, and commercial oil) is presented.

In total, 30 different VCs, were detected. A total of 18 of them (which account for 83.87 ± 0.60% of the total VCs) were identified in the CO2 atmosphere processed sample, followed by 13 in the control (64.83 ± 2.21% of the total VCs) and 11 in the sample of commercial olive oil (56.55 ± 1.76% of the total VCs).

In all cases, aldehydes such as hexanal and E-hex-2-enal and alcohols such as Z-2-hexen-1-ol and Z-3-hexen-1-ol made up the majority of the C6 VCs that were found. The primary VCs found were C6 compounds, which are produced when linoleic or α-linolenic acids are oxidized under the action of LOX [14]. Variable acyl hydrolase activity and, as a result, good or poor availability of free polyunsaturated fatty acids could be the cause of the different values of C6 aldehydes found in samples. The oil produced from control olives was characterized by the highest level of hexanal (24.09 ± 1.44%), and Z-3-hexen-1-ol (3.22 ± 0.40%). The total C6 VCs were 42.93 ± 2.89% (66.22 ± 2.10% of total VCs), and the total C5 VCs were 0.69 ± 0.12% (1.06 ± 0.21% of total VCs). Compared to the other samples, Z-2-Penten-1-ol was detected only in the control oil (0.69 ± 0.12%). In addition, an aromatic heterocyclic compound, methoxy-phenyl-oxime, was detected only in oil produced from control olives at 1.77 ± 0.08% (its appearance in virgin olive oil was reported earlier [11]). Methoxy-phenyl-oxime is formed during the growth process (fermentation) of Sorangium cellulosum, a Gram-negative bacterium of the group myxobacteria [33]. Myxobacteria, which are primarily found in soil, create compounds with known antineoplastic activity [11]. The majority of the hydrocarbons in the waxes that cover olive fruits and leaves are n-alkanes. They limit water loss and decrease the cuticle’s ability to become wet [34]. During our study, n-alkanes, such as n-decane (C10) and n-undecane (C11), were detected only in oil produced from control olives (at 1.75 ± 0.05% and 0.71 ± 0.08%, respectively) because of the slow ripening process. These compounds were detected earlier in virgin olive oils by other authors [35,36,37].As regards the commercial oil sample, it was characterized by the highest levels of E-hex-2-enal (44.55 ± 1.63%) and Z-2-hexen-1-ol (2.76 ± 0.13%). Figure 4 presents, the total C6 VCs in the commercial oil which were 50.00 ± 1.62% (88.41 ± 0.12% of total VCs detected). At the same time, no C5 VCs were detected in the commercial oil sample. In the commercial oil sample, the green attributes were positively correlated with Z-3-hexen-1-ol following the same pattern of the sum C6 alcohols and E,E-2,4-hexadienal [11]. The E,E-2,4-hexadienal has also been detected earlier in olive oil [11,36,38]. E-β-Ocimene is also present in virgin olive oil as reported in previous studies [13,35,36,37,39,40] and was detected only in commercial oil sample at a low concentration (0.81 ± 0.16%). α-Copaene is a mono-unsaturated tricyclic sesquiterpene (C15). The highest percentage appeared in the commercial oil sample (2.18 ± 0.03%), followed by the oil produced from control olives (0.87 ± 0.09%) and, finally, the oil produced from CO2-processed olives (0.55 ± 0.05%). This compound was also reported earlier in virgin olive oil [11,13,35,36,39,40,41]. E,E-α-Farnesene a tetra-unsaturated acyclic sesquiterpene (C15) was detected only in the commercial oil sample in a low amount (0.91 ± 0.02%). This compound has already been reported to be part of virgin olive oil by other authors [11,21,35,36,39,40,41]. (+)-Cyclosativene, a tetracyclic sesquiterpene (C15), was detected during our study only in commercial oil sample (0.25 ± 0.01%). It was also reported to be part of virgin olive oil [11,35,40,41]. Cyclosativene, generated through the sesquiterpene synthase enzyme under the farnesyl pyrophosphate [42], has antioxidant and anticarcinogenic properties [43]. Most terpenes have substantial pharmacological bioactivity due to their anti-inflammatory, anticancer, antidiabetic, antioxidant, or antibacterial activity [44].In the oil produced from CO2 atmosphere-processed olives, only E-hex-2-enal (1.05 ± 0.04%) out of the other C6 compounds was detected. This happened possibly because E-hex-2-enal (the main VC in most European virgin olive oils) decreases (as observed for most of the aldehydes formed from the LOX pathway) with the increase of ripeness [45]. Additionally, ethyl esters such as ethyl 3-hydroxybutyrate (0.28 ± 0.02%) were detected only in oil produced from CO2-processed olives. In the biosynthesis of cholesterol, acetoacetate is further reduced to D-3-hydroxybutyrate in the mitochondrial matrix [46]. The fresh green fruity notes are brought on by these C6 VCs, which are also present in the aroma of many other vegetable products [15,18]. According to Figure 4, in the oil from CO2-processed olives, the total C6 VCs were 1.82 ± 0.02% (2.17 ± 0.04% of total VCs). The oil from CO2-processed olives greatly differs (p6 VCs content. The aldehyde and ester levels in the olives, which bestow a pleasant aroma, reduce when olives are stored. VCs that cause bad odors are produced when olives or oil are stored for an extended time [12,20].C5 VCs have a sensory behavior quite similar to that of C6 VCs. The level of C5 compounds (such as Z-2-penten-1-ol) was found to be lower in comparison to the C6 compounds. One of the amyl alcohol isomers, 3-methylbutan-1-ol was detected only in the oil produced from CO2-processed olives (3.63 ± 0.11%). The n-6(S)-hydroperoxylinolenic acid is cleaved anaerobically by the LOX to produce a C13-oxoacid (13-oxo-12,9-tridecadienoic acid) and a C5 alcohol (Z-2-penten-1-ol), according to the study of Salas et al. [18]. An integral component of the olive oil aroma is the C5 VCs that bestow fruity and sweet aromas [18]. Hexanal, E-hex-2-enal, hexan-1-ol, and 3-methylbutan-1-ol are the main VCs of olive oil that contribute to the positive aroma characteristics (fruity, spicy, and bitter) [14]. In addition, Figure 4 presents the total C5 VCs in the oil from CO2-processed olives which were 3.63 ± 0.11% (4.33 ± 0.17% of total VCs detected). The quality of the oil is also directly correlated with the presence of short-chain alcohols in virgin olive oil. Since small amounts of these alcohols may occur during the ripening of olives, low levels of methanol and ethanol are acceptable. Especially, ethanol has also been detected in low concentrations [12,36,37,47]. On the other hand, significant amounts of ethanol are produced during the fermentation processes, which primarily occur during the storage of olive fruit [48,49]. Additionally, hydroxytyrosol, tyrosol, and ethanol are produced as a result of the hydrolysis of oleuropein during the preservation of olive oil [50]. During our experiments, ethanol was detected only in oil produced from CO2-processed olives. It had higher concentrations than reported in previous studies and the highest concentration among the VCs (50.75 ± 2.07%). Substantially, ethanol is a cellular fermentation product from continuous emissions of CO2 in olive fruit storage [51].Aromatic alcohols have been also detected earlier in olive oil [47,52]. Benzyl alcohol and 2-phenylethan-1-ol are constituents of the olive oil volatile fraction [2]. In our case, benzyl alcohol (2.12 ± 0.17%) was detected only in the oil produced from CO2-processed olives. 2-Phenylethan-1-ol was detected at a significantly higher level (p2-processed olives (14.69 ± 0.21%) in comparison to the oil produced from control olives (0.54 ± 0.02%). Another odorant, guaiacol (2-methoxyphenol), is already found in green olives but appears in higher concentration as the fruit ripens [52]. Guaiacol and 2-phenylethan-1-ol are the aroma compounds that are important for the fusty flavor [47]. These specific volatile phenols were found in high concentrations in olive oils that had strong fusty, musty, and muddy defects. Their concentration is significantly correlated with the duration of storage and with sensory evaluation, suggesting that they could be used as analytical indices of the oxidation of olive fruits during storage, most likely reflecting the activity of microorganisms [53]. Perhaps this is why it was detected only in oil produced from CO2-processed olives but in a very low concentration (0.84 ± 0.13%). Olive-pomace oil contains many more aliphatic alcohols than other olive oils, including dodecan-1-ol [54] and, therefore, it was also detected in oil produced from CO2-processed olives in a very low concentration (0.16 ± 0.02%).Hexanoic acid and ethanol condense to form the ester known as ethanol hexanoate. The formed ethyl hexanoate was detected only in oil produced from CO2-processed olives in a low amount (1.47 ± 0.07%). The fatty acid ester ethyl octanoate, sometimes referred to as ethyl caprylate, is created when ethanol reacts with the caprylic acid (a saturated fatty acid labeled 8:0). Ethyl octanoate was detected only in oil produced from CO2-processed olives and also in a low percentage (1.46 ± 0.04%). A fatty acid ester called ethyl decanoate, commonly referred as ethyl caprate, is created when ethanol reacts with capric acid (a saturated fatty acid labeled C10:0). Ethyl decanoate was detected only in oil produced from CO2-processed olives in a low concentration (0.26 ± 0.07%), as well. These compounds were also detected earlier in virgin olive oil [16,47,52].Ethenylbenzene, also known as styrene, is a volatile aromatic hydrocarbon that can be produced artificially (industrial paints, adhesives, packages, combustion products) or naturally (plant wax, amino acid fermentation). Ethenylbenzene was detected only in oil produced from CO2-processed olives at a low concentration (4.10 ± 0.42%). It has also been reported by other authors as a component of virgin olive oil [38]. Another aromatic heterocyclic compound, 1,3-benzothiazole, was detected in all oils examined in this study at a low percentage (0.42–0.31%). However, no reports on its appearance in olive oils exists in the literature. 1,3-Benzothiazole belongs to benzothiazoles which are a class of high-production volume chemicals with various applications in the industry [55,56]. Benzothiazoles are used in agriculture to prevent and control soil-borne phytopathogenic fungi which affect crops [57].The measurement of nonanal may be a suitable technique to identify the onset of oxidation. Even if hexanal is present in the original flavor, it was discovered that the hexanal/nonanal ratio is a suitable indicator to identify the start of oxidation and follow its progress [38]. This happened in oil produced from control olives (hexanal/nonanal, 24.09%/1.06%). However, in oil produced from CO2-processed olives nonanal was detected at 0.58 ± 0.10%, while hexanal was not detected. The major compounds formed in oxidized olive oil are saturated carbonyl compounds, including pentanal, hexanal, octanal, and nonanal [12], and were not detected in commercial oil. The most frequently appearing volatile markers of oxidation of virgin olive oil during storage are nonanal and E-2-decenal [49]. Τhe autoxidation of oleic acid can be exclusively responsible for the appearance of nonanal [58].Vegetable oil hydrocarbon fraction composition exhibits significant variations that may be used to characterize the oil. Despite their low concentration in olive oil, terpenic hydrocarbons (mono- and sesquiterpenes) exhibit significant variation depending on the variety and the region of production [40]. Limonene is a hydrocarbon classified as a monocyclic monoterpene (C10) and generated by alcohol α-terpineol with a loss of a proton. (+)-Limonene smells of oranges, whilst (−)-limonene resembles the smell of lemons [59]. (+)-Limonene was detected in all samples. The highest quantity appeared in oil produced from control olives (13.11 ± 0.70%), followed by commercial oil (1.60 ± 0.06%), while the lowest concentration was detected in the oil produced from CO2-processed olives (0.90 ± 0.04%). The presence of (+)-limonene was previously reported in virgin olive oils [21,35,36,37,39,41,52]. α-Myrcene is a structural isomer of β-myrcene and during our study was detected both in oil produced from control olives and commercial oil at the concentration of 0.98 ± 0.12% and 0.50 ± 0.11%, respectively. Virgin olive oil has previously been reported to contain this compound [39,40]. The acyclic monoterpenes β-myrcene (C10) and E-β-ocimene (C10) are generated through the monoterpene synthase enzyme under the geranyl pyrophosphate [59]. β-Guaiene is a bicyclic sesquiterpene (C15)—a compound from the azulene group. It was detected only in oil produced from CO2-processed olives, in low concentrations (0.12 ± 0.01%). Vichi et al. [40] have identified a different natural chemical compound, δ-guaiene. According to the above results, it appears that the oil produced from CO2-processed olives presents a much different volatile profile which is attributed to the different procedure of debittering olives. These VCs resulted in the development of a much different flavor, which was highly valued during sensory evaluation by many panelists. Therefore, the storage of olives under the CO2 atmosphere can be used not only for their debittering but also for the production of a series of new products with highly valued sensory attributes. It is worth noting that β-guaiene was first detected in an extra virgin olive oil. It is usually found as a component of many plants and mushrooms [44]. Azulene derivatives have been known for centuries for their biological activities and are widely applied in medicine and pharmacy [60]. Guaiene has a delicate, woody flavor and an earthy, spicy aroma [61]. 3.5. Physicochemical CharacteristicsOne of the main criteria used for the differentiation among the various varieties of olive oil is acidity, which assesses the free fatty acid (FFA) content. Extra virgin olive oil (EVOO), the most expensive grade of olive oil, is required by Commission Implementing Regulation (EU) No 299/2013 [26] and Commission Delegated Regulation (EU) No 2022/2104 [62] to contain no more than 0.8% FFA. However, all the olive oil characteristics laid down by the Regulation for each category must be met. Three requirements must also be met for olive oil to be certified as EVOO, in addition to the FFA content; it must be manufactured using mechanical extraction techniques without the use of chemicals or hot water, come from first-cold pressing, and have the best flavor possible [63]. In addition, another quality control analysis is the measurement of conjugated dienes and trienes. According to Regulations (EU) No 299/2013 [26] and No 2022/2104 [62], EVOO must not contain more than 2.50 conjugated dienes (K232), no more than 0.22 conjugated trienes (K270) and the variation of the specific extinction (ΔΚ) must not be more than 0.01. Taking into account the regulations, it was observed (Table 3) that the oil produced from the control sample is out of specification, shows advanced oxidation and is, therefore, of poor quality. The oil produced from CO2-processed olives and the commercial oil sample appears to be of good quality and within the legislation requirements for the EVOO category.

Regarding the color measurement, the lightness (L*) of the oil produced from the CO2-processed olives was darker than the other samples. Moreover, the oil produced from the control sample had a very light color. In addition, determining the Chroma (Cab*), i.e., color density, showed that the oil produced from CO2-processed olives was less colorful than the other samples. Determination of hue angle (habo) showed that all samples had a hue of yellow color. In conclusion, the oil produced from CO2-processed olives and commercial oil samples appeared to be slightly reddish.

Moreover, the concentration of total carotenoids and total chlorophylls was evaluated for the determination of pigments. In olive oils, these bioactive substances are responsible for their color [27,64,65,66]. According to the results (Table 3 and Figure 5), it is observed that the oil produced from CO2-processed olives had the highest concentration in both these bioactive substances. In Figure 5, the absorption spectra obtained for chlorophylls and carotenoids in different olive oils are presented. The oil produced from control olives appeared to be very poor in these compounds. These results were also verified by the colorimetry technique. Total pigments were measured for the oil produced from the CO2-processed sample and were found to be 6.021 ± 0.056 mg/Kg as opposed to the commercial oil sample containing 3.493 ± 0.097 mg/Kg and the oil produced from the control sample (0.782 ± 0.015 mg/Kg).Subsequently, the oils’ total polyphenol content was determined. The total polyphenols for the oil produced from the CO2-processed olives sample were 209.09 ± 18.86 mg of gallic acid equivalents (GAE)/Kg, while for the commercial oil sample was 132.20 ± 8.68 mg GAE/Kg. The oil produced from the control sample was found to have a low content in total polyphenols and specifically 44.52 ± 18.36 mg GAE/Kg. In addition, the determination of α-tocopherol content of the samples was carried out. The concentration of α-tocopherol for the oil produced from the CO2-processed sample was 180.89 ± 22.91 mg/Kg, while for the commercial oil sample was 102.04 ± 12.26 mg/Kg. The oil produced from the control sample was found to have a low α-tocopherol concentration of 32.12 ± 5.17 mg/Kg. The Rancimat method was performed to evaluate the oils’ susceptibility to oxidation [67,68]. The oil produced from the CO2-processed sample showed an induction period of 13.70 ± 0.14 h, while the commercial oil and the oil produced from control olives samples were 6.35 ± 0.21 and 3.50 ± 0.14 h, respectively. The oxidative stability of the oil deriving from the olives stored under the CO2 atmosphere was significantly higher (pThe fatty acid (FA) profile is shown in Table 4. It is observed that the main FA in all samples was oleic acid (C18:1ω-9c). There were statistically significant differences (p2-processed olives, respectively. Based on the results, regarding olive oils, the overall percentage of FAs and their profile is in accordance with the literature [36,69]. The FAs content of the oils is the primary factor used to establish their quality indices during production, storage, and trading [70]. As a result, the quantity of FAs in oil closely corresponded to the oil’s authenticity and quality.All the above results indicate that the oil produced from the CO2-processed olive sample had all characteristics rendering it suitable to be labeled as EVOO. In addition, the unique organoleptic characteristics such as the aroma, flavor, and color of the product, the high antioxidant activity, and the high content of bioactive compounds (polyphenols, α-tocopherol, carotenoids, and chlorophylls) resulted in an innovative olive oil with excellent chemical characteristics and probable beneficial effects on health [14,65].

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