A virgin olive oil (VOO) can be defined as the oil obtained from the fruit of the olive tree (
Olea europaea), using exclusively mechanical or physical procedures. To obtain a VOO, olives cannot be treated with other procedures than washing, decanting, centrifugation, and filtration [
1], thus excluding oils obtained with solvents or by re-esterification or oil mixing procedures [
2]. Among VOOs, it is possible to distinguish three main types according to their free acidity, expressed as oleic acid concentration: (i) extra virgin olive oil (EVOO), defined as a VOO whose free acidity does not exceed 0.8 g/100 g, and whose other characteristics are in accordance with those established for the category [
3]; (ii) VOO, whose free acidity exceeds 0.8 g/100 g, but whose other characteristics are still in accordance with those established for the category; and (iii) lampante olive oil, which is a VOO whose free acidity is higher than 3.3 g/100 g, but still conserves the organoleptic properties characteristic of this group [
4]. The last type of VOO is intended for refining processes or for technical uses, although it has also been used fraudulently to adulterate EVOO [
5]. On the other hand, when olives are subjected to chemical processes to obtain the oils, they cannot be denominated as VOO, which gives rise to other types of olive oils such as: (a) refined olive oil, obtained by refining VOOs and whose maximum free acidity, expressed in grams of oleic acid, cannot exceed 0.3%; and (b) olive oil, obtained by refining VOOs and whose free acidity cannot exceed 1%. Moreover, there are other by-products of the olive that cannot be considered olive oil, such as crude pomace oil, refined pomace oil, and olive pomace oil [
6].
The European Union introduced different denominations to protect EVOO according to the differential characteristics given by its origin, genetics, and phenotype: protected designation of origin (PDO), protected geographical indication, and traditional specialties guaranteed. PDO is granted to products that owe their quality or characteristics, including sensory profile, to the geographical environment [
13]. This has been corroborated by different studies [
14,
15,
16]. Numerous examples of PDO EVOOs can be found in the different producing countries, especially in Spain, Italy, and Greece. For example, in Spain, several PDO EVOOs can be described. To cite an example, EVOO “Montes de Toledo” is obtained from Cornicabra olives’ variety. This product is characterized by medium-intense bitterness and pungency, the most prominent sensory descriptors being “apple” and “almond” [
17] In Portugal, six PDO EVOOs have been registered. One of these products is “Azeite de Trás-os-Montes”, obtained from Verdeal Transmontana, Cobrançosa, Madural, Cordovil, and other olive varieties. The product characterized by a fresh and fruity taste, with notes of almond, notable sensations of sweetness, and also some bitterness and spiciness [
18]. Regarding Italy, 42 certified PDO products have been registered. An example is “Brisighella” PDO, produced with the olive variety Nostrana di Brisighella. This EVOO is characterized by medium intensity of fruity, medium-intense sensation of bitter and intense perception of pungent. Other sensory descriptors include “tomato”, “grass”, or “artichoke” [
19]. Several PDO EVOOs have been registered in Greece, one of the main examples being the “Kalamata” EVOO, obtained from Koroneiki and Matsolia varieties and characterized by moderately intense fruitiness, green fruit aroma, slight bitterness, and mild pungency [
20].
2. EVOO’s Flavor
As mentioned before, 98 to 99% of olive oil is composed of triacylglycerols and monounsaturated fatty acids (MUFAs), while the remaining 1–2% are the minor compounds, which are responsible for the organoleptic qualities of EVOO [
21,
22]. These compounds are present in the pulp and pits of olives and are transferred to the EVOO during the fabrication process. Some compounds are hydrocarbons, like squalene and β-carotene, fatty alcohols, triterpenic alcohols and dialcohols, tocopherols, sterols, 4-methylseterols, pigments like chlorophylls and pheophitins, and many phenols, like phenolic alcohols (hydroxytyrosol (HT) and tyrosol), secoiridoids (oleuropein (OLE) derivatives, oleacein, and oleocanthal), and lignans (e.g., pinoresinol) [
22]. Flavor of oils is a field in which researchers continue to investigate, since it is a complex matrix. Although aroma and flavor of numerous food products are caused by one or a few compounds, in the case of oils, there are thousands of chemical compounds that influence these organoleptic characteristics. Furthermore, those compounds can also interact with each other, hindering tastings or even the establishment of good organoleptic quality parameters [
23]. However, not all the contained compounds have the same importance and influence in the flavor. This characteristic is determined by the compound’s odor threshold value, which is the minimum concentration of a compound able to develop an olfactory response [
24]. Identification of the flavored molecules of EVOO is possible through the application of chromatographic techniques such as gas or liquid chromatography (GC or LC, respectively) coupled to a mass spectrometer (MS). In this sense, the compounds responsible for the flavor can be classified into large chemical groups, among which it is worth highlighting aldehydes, alcohols, esters, ketones carboxylic acids, and phenolic compounds. Several studies have reported the analysis and identification of EVOO aroma compounds, but some suggest that the quantitative ratios among volatiles are more correlated with the organoleptic characteristics, rather than their absolute quantities [
25]. Although the presence of most of these flavored compounds has a desirable effect, there are also a series of molecules that provide negative attributes such as rancid, fusty, winey, vinegary, and frozen, a fact recognized by the International Olive Council [
26]. This type of metabolite constitutes a category called “off-flavor” and is usually formed by oxidation, which may be initiated in the olive fruit.
Some of the most important flavored molecules of EVOO are guaiacol (olive paste, soapy), 1-penten-3-ol (grassy, green plants), hexanal (cut grass), octanal (citrus, lemon), (
Z)-3-hexenyl acetate (fruity) [
27], (
E)-2-hexenal (green), 6-methyl-5-hepten-2-one (nutty), and (
E)-2-decenal (soapy, fatty) [
28]. However, the composition of olive oil and the compounds that provide its organoleptic properties varies according to various factors. Some of these factors include olive variety, ripeness (green, green/ripe, or fully ripe), geographical and climatic conditions, pest and diseases, maturation process, cultivation, processing, time and conditions of their storage, technological aspects of oil extraction, or enzyme levels [
23,
29]. All this contributes to the complexity and balance of olive oils, which is defined as harmony [
30]. The evaluation of sensory quality allows to classify the oils into various quality and sensorial grades [
31].
2.1. Flavor Compounds
Mainly, the sensorial and organoleptic properties of EVOO are provided by the aromas and volatile compounds present in EVOO. Volatile compounds formed during the growth and processing of olive fruit contribute to a combined sensation of smell and taste of the resulting oil, commonly called flavor [
31]. Volatile compounds are molecules with less than 300 Da, which vaporize easily at room temperature. These compounds that are released into the headspace, stimulate the olfactory receptors in the nasal cavity by dissolving into the mucus and bonding to olfactory receptors, after passing through the external nostrils, giving an odor sensation [
32]. Furthermore, while tasting the EVOO, the aromas are also perceived when the compounds interact with the receptors in the nasal cavity after migrating from the mouth through the nasopharynx. Aldehydes, alcohols, esters, ketones, hydrocarbons, furans, and carboxylic acids, among others, are some of the identified volatile compounds present in EVOO responsible for its aroma attributes [
21] (
Figure 1 and
Figure 2), (see also
Supplementary Material Tables S1–S4).
Figure 1. Odor thresholds and sensory descriptors of aldehydes and alcohols in olive oil.
Figure 2. Odor thresholds and sensory descriptors of esters, ketones, carboxylic acids, and other compounds in olive oil.
The major volatile compounds found in EVOO belong to C6 and C5 compounds [
21]. C6 aldehydes and alcohols and their corresponding esters are considered, both qualitatively and quantitatively, the most crucial and influential aroma compounds of EVOO [
33]. They are related to sweetness and green notes and contribute favorably to the aroma [
21]. C6 aldehydes (hexanal, 3(
Z)-hexenal and 2(
E)-hexenal), alcohols (hexanol, 3(
Z)-hexenol and 2(
E)-hexenol), and their acetyl esters (hexylacetate and 3(
Z)-hexenyl acetate) constitute 60–80% of total volatile fraction, 2(
E)-hexenal being the most remarkable component. All of them contribute with green notes to the oil and are formed from polyunsaturated fatty acids (PUFAs) through a cascade of biochemical reactions (lipoxygenase pathway) in which enzymes transform PUFAs to aldehydes, which are subsequently reduced to alcohols and esterified to produce esters, another large group of compounds with relevance to the sensory quality of EVOO [
34]. Although alcohols are usually contained in higher percentages than aldehydes, the latter are more relevant in EVOO’s flavor because they present lower detection thresholds [
35]. To give an example, in a study comparing the volatile compounds of EVOOs from different origins, it was observed that 6700 µg/g of trans-2-hexenal only have an odor activity of 16 whereas 26 µg/g of 1-penten-3-one had a higher odor activity value of 36 [
36]. Regarding C5 compounds, they are also contained in EVOO in reasonable amounts, contributing to its flavor [
37]. C5 aldehydes and alcohols provide pungent sensations in correlation with bitterness [
21]. Taking into account the information reported by numerous studies, it can be concluded that the C6 and C5 volatile compounds are powerful odorants, but they can be found in EVOO in a wide range of concentrations, according to differences in olive varieties and extraction methods applied [
37].
Nevertheless, minor and major volatile compounds are crucial to olive oil quality. Even the volatiles that are below the olfactory level of detection are important, because they can explain the formation of future degradation products, which will later have a significant importance in the organoleptic characteristics of the oil and they also provide useful quality markers [
21].
Another group of molecules of great influence in EVOO’s flavor are certain phenolic compounds, as there is a positive correlation between EVOO’s aroma and flavor and its polyphenol content. The phenolic composition of EVOO was found to be one of the most diverse because it depends on all of the factors mentioned previously, such as olive variety, climatic conditions, maturity, production processes, etc. [
38]. The variations in the phenolic composition are responsible for some of the different organoleptic characteristics found in EVOO [
22]. According to a few studies, HT is the phenolic compound present in most varieties of EVOO, even though it is found at considerably lower levels that secoiridoids. It varies from less than 1 mg/kg [
39] to 232 mg/kg [
40].
A study reported that the most representative complex phenols in EVOO were three oleuropein aglycones (OleA), four ligstroside aglycones, and their derivatives, belonging to the secoiridoid group. Other secoiridoids described were decarboxymethylated, hydroxylated, and methylated forms of ligstroside aglycone. Regarding lignans and flavones, pinoresinol, syringaresinol, acetoxypinorexinol, luteolin, and apigenin were identified in EVOO samples. Quinic acid was also found in all the analyzed samples [
41]. Sensory descriptors such as fruitiness, bitterness, pungency, and astringency are flavors linked to the concentration of phenolic compounds [
22,
42]. For a comprehensive specification, single flavor descriptors such as freshly cut grass, tomato, artichoke, leaves, nuts, apple, banana, tropical fruits, and herbs that are part of the main attribute fruitiness, are considered [
30].
2.2. Influencing Factors in EVOO’s Flavor Compounds
As previously stated, numerous variables are responsible for the presence of volatile compounds, phenolic compounds, and their concentration in EVOO, such as, cultivar, fruit maturity, geographic region, climate conditions, processing methods, and storage of the oil. Starting with cultivars, several studies have reported that different cultivars conduce to EVOOs with characteristic volatile profiles. For example, an analytical study tested several EVOO samples from diverse cultivars of Tunisia, which showed different volatile profiles. Some of the compounds analyzed appeared exclusively in certain oils, so they may be used to establish compositional differences [
43]. A more recent study evaluated Greek olive cultivars, obtaining similar conclusions [
44]. In addition, olive tree species present great variability in terms of their volatile composition and phenolic compounds, consequently affecting oils sensory quality [
45]. The degree of the fruit maturity before producing the oil is also a known variable and the aroma compounds tend to increase with it, as proven by several studies [
46,
47]. The storage of the fruits before the EVOO production also changes the volatile composition of the produced oil, leading to the apparition of off-flavors [
21,
48]. Different studies reported that the quality of EVOO is similar whether produced with freshly harvested fruits or with fruits stored for a week. However, if the fruits are stored for a long time, there is an OleA and ligstroside aglycone degradation, a decrease in the lignan and flavonoid groups, while the phenolic acids fraction showed a notable increase [
1].
The volatile profile was characterized by a decrease in lipoxygenase products (formed by the lipoxygenase pathway, shown in Figure 3 and the biosynthesis of volatile molecular markers of oxidation of C7–C10 aldehydes (nonanal, decanal, (Z) and (E)-2-heptenal, (E)-2-octenal and (E, E)-2,4-heptadienal) and hydrocarbons (n-dodecane and n-tetradecane).
Figure 3. Pathways for the formation of the principal volatile compounds present in extra virgin olive oils (EVOOs).
The mono and sesquiterpenes also showed an increase during the storage time [
49]. Considering this, it is necessary to process the olives as soon as possible after harvesting, with a maximum storage time of one week to avoid loss of quality. A study showed that linalool and ethyl esters were not found in the headspace being only present in the oil in trace amounts. Furthermore, ethyl esters, at low concentrations, contribute to the EVOO flavor, while they are associated with the turbidity of olive oils at higher concentrations. It was also determined that the presence of linalool and terpenic compounds depended on the EVOO variety. Furthermore, aldehydes, ketones, acetate compounds, and alcohols were also observed [
42].
3. Degradation of EVOO’s Organoleptic Properties
The study of the carbonyl compounds responsible for the flavor of EVOO (linear saturated and unsaturated aldehydes and alcohols, esters, and hydrocarbons) is carried out with the aim of avoiding modifications, either due to the aging or degradation of the product, but also due to attempted fraud. One of the most accepted techniques for this purpose is the head space solid phase micro-extraction coupled to a gas or mass spectrometry system (HS-SPME-GC/MS) which, due to its speed and precision, is used to analyze the compounds responsible for the aroma of various food matrices [
54]. This methodology is based on the absorption of volatile compounds from EVOO in silica, and their identification by MS detection, commonly coupled to a chromatography instrument, either liquid or gas. This technique is a good indication of the organoleptic quality of olive oils, since changes in the composition of the volatile fraction of an EVOO also lead to changes in its aroma. [
55]. On the other hand, certain phenolic compounds also play an aromatic function in EVOO. The high performance liquid chromatography (HPLC) technique is one of the most used to detect phenolic compounds. Through it, tests can be carried out on the effect of certain factors, whether intrinsic (composition of the matrix itself, presence of antioxidant compounds) or extrinsic (adulterations, time, temperature, exposure to light, and oxygen), on the quality of a certain EVOO [
56].
Lipid oxidation is the main process that leads to EVOO degradation, which has been associated with the reduction of the nutritional and the beneficial effects of the original product. It comprises a complex chain of reactions catalyzed due to the presence of oxygen in the final product. Oxygen leads to the oxidation of EVOO compounds (including fatty acids or liposoluble vitamins such as A, D, E, and K) and creates unstable products which ultimately trigger further degradation reactions that cause off-flavors and rancid odor [
57,
58]. During the primary oxidation, unstable lipid hydroperoxides are produced as result of the oxidation of unsaturated fatty acids by action of the oxygen or several catalyst agents (iron, copper, enzymes, heat or light). Lipid hydroperoxides do not affect the organoleptic properties of EVOO since they do not provide color, odor, or taste. However, during the second step, named auto-oxidation, the peroxides react with other low molecular weight molecules present in the food matrix to oxidize them and create secondary products such as alkanes, alkenes, aldehydes, and ketones. These products of the second step are volatiles and responsible for the rancid odor and taste of EVOO. Major compounds formed in oxidized olive oil are pentanal (woody, bitter, oily), hexanal (green, sweet, grassy), octanal (fatty), and nonanal (fatty, waxy), trans 2-pentenal (green, bitter almond), and 2-heptenal (green, bitter almond), and thus, they are mainly responsible for the off-flavor [
32]. Some investigations carried out in this regard have concluded that certain C7-C12 unsaturated aldehydes, 2, 4-heptadienal (fatty, rancid), (
E)-2-nonenal (paper-like, fatty), and (
E)-2-octenal (grassy, spicy) are responsible for rancidity; (
Z)-3-hexenyl acetate (green, banana) and nonanoic acid (fat, must, sweat, sour) for fusty defects, acetic acid (sour, vinegary) and butyric acid (rancid, cheese) are responsible for winey-vinegary defects, and limonene was found as a relevant compound in frozen oil [
26]. Hexanal, for example, is formed during the lipid oxidation of linoleic, gamma-linolenic, and arachidonic acids and gets increased along the storage. Concentrations around 5–10 μg/mL have been considered to induce rancid odor which makes them sensorially unacceptable [
59,
60]. These products continue reacting with other molecules triggering structural and organoleptic alterations.
A study evaluating the conditions that led to the oxidative alterations of EVOO conclude that the presence of fluorescent light, followed by elevated temperatures were the principal factors responsible for the EVOO oxidation [
61]. The effect of temperature is one of the most studied factors affecting EVOO’s sensory quality, since high temperatures normally lead to degradation of compounds, favoring isomerization reactions, etc. During the distribution and storage of EVOOs until they are consumed, the product goes through various thermal conditions that can alter its chemical composition, putting its quality into question. One of the most used indicators to check the quality of EVOO with respect to its aroma is (
E)-2-hexenal, which is the most representative compound (28.3%) of the fraction of volatile organic compounds [
62]. A study carried out an analysis of the changes suffered by the volatile fraction of an EVOO in the Calabria region (Italy) due to exposure to high temperatures, finding that the concentration of (
E)-2-hexenal decreased significantly and proportionally with the application of heat, reaching minimum values of 1.7% after applying 220 °C for 120 min. The control sample contained 21 compounds in said fraction, among which α-farnesene, α-cubebene, and nonanal should also be highlighted, which account for approximately 15.3%, 14.4%, and 8.9%, respectively, of the volatile fraction of the EVOO, although these values may vary depending on the olive variety and its growing conditions (climate, irrigation, pesticides, and/or applied treatments, etc.) [
63,
64]. With the application of heat, the α-farnesene and α-cubebene contents decreased to 0.1% and 0.2%, respectively; while the nonanal content increased to 12.6%, when 220 °C was applied for 120 min. Likewise, the time of application of heat is another determining factor of the changes observed in the composition of the EVOO, since the results of exposure to 220 °C during lower times (30 and 60 min) reported small losses of (
E)-2-hexenal (3.2% and 2.7%, respectively). On the other hand, compounds that were not part of the original composition of fresh EVOO appear after heat treatment, specifically, aldehydes derived from the oxidation of the oleic acid such as (
Z)-2-heptanal, (
E)-2-octenal, 2-nonenal, and (
Z)-2-decenal [
65], so they could be used as indicators of EVOO exposure at high temperatures, as some of them can be detected in high concentrations (up to 30% of the volatile fraction) when heat is applied [
55]. The appearance of aldehydes is detrimental, not only because they negatively alter the taste of EVOO but also because they show toxicity related to the development of cancer, especially in the case of (
E)-2,4 decadienal. Thus, it is advisable and healthy to use lower temperatures and shorter times during culinary processes that involve the use of EVOO [
66].