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Olive Oil Composition: Volatile Compounds

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Olive Oil Composition: Volatile Compounds
Marco D.R. Gomes da Silva1, Ana M. Costa Freitas2,
Maria J. B. Cabrita2 and Raquel Garcia2
1REQUIMTE, Departamento de Química, Faculdade de Ciências e
Tecnologia/ Universidade Nova de Lisboa, Campus da Caparica,
2Escola de Ciências e Tecnologia, Departamento de Fitotecnia,
Instituto de Ciências Agrárias e Ambientais Mediterrânicas ICAAM,
Universidade de Évora, Évora,
Portugal
1. Introduction
In general olive oil is defined on the basis of its sensory characteristics. European Union
(EU) regulations establish the organoleptic quality of virgin olive oil by means of a panel
test, evaluating positive and negative descriptors (EU regulations). For the organoleptic
assessment, several volatile compounds are considered as the main responsible for negative
and positive attributes. Volatile compounds, either major or minor, are crucial to olive oil
quality; even when present below their olfactory threshold, they can still be important to
understand their formation and degradation pathways and provide useful quality marker
information.
Volatile composition of olive oils can be influenced by a number of factors, from agronomic
and climatic aspects to technological ones. Cultivar, geographic region, ripeness, harvest
and processing methods can affect the volatile composition of olive oil. Storage time is also
critical for quality. In order to evaluate the volatile profile of olive oil, sensitive analytical
techniques as well as extraction procedures were developed. The big issues on aroma
analysis are, the loss of compounds during sample preparation steps, and the knowledge
that some of the so-called “compounds of interest” (with higher aroma threshold) are,
probably, present only in trace amounts. Due to its nature, olive oil is a difficult matrix; for
these reasons several methods have been, so far, proposed. The advantages and drawbacks
of these methods will be further discussed. One dimension-Gas Chromatography (1D-GC)
analysis was, until recently, the most used method to analyze volatiles in different matrices.
The increased development of 2D-GC, allowing higher sensitivity and enhanced separation
power, is changing the 1D-GC approach. The type of 2D and/or 3D qualitative and
quantitative information, provided by 2D-GC systems, promoted the development of
powerful chemometrics tools allowing a useful, and potentially easy, way for data
interpretation. Fingerprint comparison can be used on a routine basis, providing important
and quick information concerning differences among the olive oils produced and, probably
most important, also allowing frauds detection.
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
18
This work will be divided in four main parts: 1) a brief summary of the composition and
biosynthesis of the volatile fraction of olive oil; 2) the role of volatile compounds in olive oil
quality: nutritional and sensorial quality; 3) the effect of agronomic and technological
practices on olive oil aroma; 4) analytical methodologies for quantification and identification
of volatiles compounds: new analytical methods.
2. Composition and biosynthesis of the volatile fraction of olive oil
The wide variety of volatile compounds found in high quality virgin olive oil are produced
through biogenic pathways of the olive fruit, namely the lipoxygenase (LOX) pathways
(Hatanaka, 1993), and fatty acid or aminoacid metabolism, as depicted in fig.1 (Angerosa et
al., 2004; Angerosa et al., 2002). Besides the contribution of several volatile compounds,
related with the mentioned pathways, the role of other compounds, especially aldehydes
derived from auto-oxidation processes, should also be considered to the final aroma of the
olive oils (Angerosa, 2002). Other metabolized products, originated from possible
fermentations, conversion of some aminoacids, enzymatic activities of moulds or oxidative
processes, are closely related with off-flavour of virgin olive oil. As illustrated in fig. 1,
several compounds namely carbonyl compounds, alcohols, esters and hydrocarbons
contribute to the aroma profile of olive oil (Angerosa et al., 2004).
Fig. 1. The main pathways involved in the formation of the volatile profile of high quality
virgin olive oils. Adapted from (Angerosa et al. 2004; Angerosa 2002).
The volatile compounds, responsible for virgin olive oil aroma, are usually: low molecular
weight (<300 Da); high volatility, sufficient hydrosolubility, fair liposolubility and chemical
features to bond with specific proteins (Angerosa et al., 2002).
During crushing and malaxation steps, considerable changes, in olive oil chemical
composition occurs accomplished by the activation of olive fruit enzymes due to the
Olive Oil Composition: Volatile Compounds 19
inherent disruption of cellular tissues. Consequently, the LOX pathway is initiated by the
hydrolysis of triglycerides and phospholipids, mediated by acyl hydrolase (AH), leading to
the release of fatty acids. Lypoxygenases, after their release, become immediately active and
transform the unsaturated fatty acids, produced by the action of AH, linolenic (LnA) and
linoleic (LA) acids, into their corresponding 9- and 13-hydroperoxides, as shown in figure 2.
The subsequent cleavage of fatty acids 13-hydroperoxides is catalysed by specific
hydroperoxide lyases (HPL) leading to the formation of C6 aldehydes (Z)-hex-3-enal and
hexanal from linolenic and linoleic acids, respectively) and oxoacids. The unsaturated form
of C6 aldehyde ((Z)-hex-3-enal) undergo rapid isomerisation to the more stable (E)-hex-2-
enal. The action of alcohol dehydrogenase (ADH), catalyses the reversible reduction of
aliphatic C6 aldehydes to the corresponding volatile alcohols (Benicasa et al., 2003; Angerosa
et al., 1998a). Alcohol species are further transformed into esters due to the catalytic activity of
alcohol acetyl transferase (AAT), producing acetates (Kalua et al., 2007) (figure 2). Several
factors, such as cultivar and extraction process, including operating temperature, seem to
play a relevant role on the improvement of AAT activity (Salas, 2004). When the substrate is
LnA, LOX catalyses, besides the hydroperoxide formation, also its cleavage, via an alkoxy
radical, increasing the formation of stabilized pent-1,3-diene radicals. These compounds can
suffer dimerization leading to the production of C10 hydrocarbons (pentene dimmers) or react
with a hydroxyl radical present in the medium, leading to C5 carbonyl compounds (Angerosa
et al. 1998b, Pizarro et al., 2011). The most important fraction of volatile compounds, of high
quality virgin olive oils, comprises C6 and C5 compounds, especially C6 linear unsaturated and
saturated aldehydes. The presence of other volatile compounds, namely C7-C11
monounsaturated aldehydes, C6-C10 dienals, C5 branched aldehydes and alcohols and some
C8 ketones, in relatively high concentrations, in the aroma of virgin olive oil, is associated with
unpleasant notes. The presence, or lack of defects, in the aroma of olive oils is related with the
contribution of the various pathways involved on volatiles formation.
When the most active pathway is the LOX cascade the olive oil aroma will not be defective.
LOX pathway is predominant in oils of high quality.
3. The role of volatile compounds in olive oil quality: Nutritional and
sensorial quality
The International Olive Oil Council (IOOC), European Commission (EC) and Codex
Alimentarius have defined the quality of olive oil based on several parameters, such as free
fatty acid content, peroxide value, spectrophotometric absorvances in the UV region,
halogenated solvents and sensory attributes (Boskou 2006; Kalua et al., 2007; Lopez-Feria et al.,
2007). In order to evaluate olive oil quality, the Codex Alimentarius and IOOC include also the
insoluble impurities, some metals and unsaponifiable matter determinations (Boskou 2006).
The nutritional value of olive oil arises from high levels of oleic acid and minor components,
such as phenolic compounds. It is well recognized that the consumption of some natural
antioxidant phenolic compounds produce beneficial health effects. These substances
possess strong radical scavenging capacities and can play a relevant role in protecting
against oxidative damages and cellular aging. Together with their bioactivity, olive oil
phenols have a significant role on the flavour and the bitter taste of olive oil (Boskou 2006;
Servili et al. 2002). Sensory quality plays a crucial role in the acceptability of foodstuffs and
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
20
some characteristics such as colour and flavour are the main sensations which contribute to
their acceptability among consumers. Hence, the evaluation of the sensory quality of olive
oils involves perception of both favourable and unfavourable sensory attributes.
Fig. 2. Lypoxygenase pathway for the formation of major volatile compounds. (Source:
Benincasa et al., 2003).
Olive oil possesses a highly distinctive taste and flavor due to specific volatile organic
compounds, belonging to several chemical classes, namely aliphatic and aromatic
hydrocarbons, aliphatic and triterpenic alcohols, aldehydes, ketones, ethers, esters and furan
and thiophene derivatives (Kiritsakis et al. 1998). These compounds, retained by olive oil
during the extraction process, stimulate human gustative and olfactive receptors giving rise
to olive oil balanced flavour of green and fruity attributes. Such compounds stimulate the free
endings of the terminal nerve located in all the palate and in the gustative buds promoting the
chemesthetic perceptions of pungency, astringency and metallic attributes. During olive oil
tasting, the stimulation of the olfactory epithelium, by a large number of volatile compounds
can also occur explaining all other sensations perceived by consumers (Angerosa, 2002). The
major volatile compounds of olive oil which contribute for the positive attributes of olive oil
Olive Oil Composition: Volatile Compounds 21
aroma (fruity, pungent and bitter) include hexanal, (E)- hex-2-enal, hexan-1-ol and 3-
methylbutan-1-ol. Their concentrations, except for (E)-hex-2-enal, varying widely, are
generally very low reaching minimum levels of ppb. Thus, volatile compounds, which are
responsible for most sensory properties of olive oils, play a significant role on the evaluation of
the overall oil quality having a decisive influence on acceptability. The sensory defects are also
associated with the volatile composition of the olive oil and are, usually, related with chemical
oxidation and exogenous enzymes involved in microbial activity. Chemical oxidation is
responsible for the formation of off- flavour compounds, such as pent-2-enal and hept-2-enal
The off- flavour compounds associated with unpleasant sensory notes can be assembled in five
classes- fusty, moistness- humidity, winey- vinegary, metallic and rancid (Morales et al. 1997;
Morales et al., 2005; Escuderos et al., 2007; Faria et al.; Angerosa 2002; Kalua et al. 2007).
Moistness-humidity, which possesses the highest sensory significance, is related to the
presence of C8 volatile compounds (e.g. oct-1-en-3-ol and to a lesser extent oct-1-en-3-one) and
short chain fatty acids (Morales et al., 2005). Normally they are a characteristic flavour of oils
produced from olives infested with fungi and yeasts as a result of an inappropriate storage.
Fusty sensory defect is correlated with the presence of ethyl butanoate, propanoic and
butanoic acids, a characteristic flavour of oils from olives stored in piles which have
undergone an advanced stage of anaerobic fermentation (Morales et al. 2005). Moreover, the
presence of acetic acid, ethanol, 3-methylbutan-1-ol and ethyl acetate contributes to winey-
vinegary attributes due to the olives fermentation. The rancid negative attribute is due to oils
oxidation, characterized by the absence of C6 aldehydes and alcohols produced from linolenic
acid, the absence of esters and the presence of several aldehydes with low odour threshold
(Morales et al, 1997). Metallic flavour is associated to oils that have been in prolonged contact
with metallic surfaces, during processing, and is characterized by the presence of pent-1-en-3-
one; this ketone has been proposed as a useful marker of metallic off-flavour (Venkateshwarlu
et al. 2004). The occurrence of pent-1-en-3-one is also positively correlated with bitter and
pungency taste while hexanal is negatively correlated with these characteristics, depending on
the final amounts. Z-Hex-3-en-1-ol and E-hex-2-enal are negatively correlated with bitter and
pungent characteristics, respectively. Other common defects of olive oils, such as muddy
sediment and cucumber are related with olive oil preservation.
Poor quality olive oils show remarkable modifications on their sensory basic characteristics,
namely the decrease or absence of green, bitter and pungent notes. Generally, the intensity
of stimuli elicited by volatile substances is related to their amount. Some other chemical
factors, such as volatility and hydrophobic character, size, shape and stereochemistry of
volatile molecules, type and position of functional groups as well as external factors, such as
matrix effects, seem to affect odour intensity, more than their concentration, due to the
influence of these chemical features on the interaction with olfactory and gustative receptors
(Angerosa et al., 2004). Odour activity value, evaluated by means of the ratio between its
concentration and its odour threshold, constitutes a useful tool to identify the main
contributors to the olive oil aroma. The thresholds of several of these compounds are
already presented in dedicated literature (Bouskou 2006; Angerosa 2002).
4. The effect of agronomic and technological practices on olive oil aroma
Factors affecting volatile composition of olive oils can be classified into four main groups:
environmental (soil and climate); agronomic (irrigation, fertilization); cultivation (harvesting,
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
22
ripeness) and technological procedures (post-harvest storage and extraction systems) (Aparicio
& Luna, 2002). It is generally accepted that volatile profile of virgin olive oils depends on the
level and the activity of the enzymes involved in LOX pathway. As previously referred, the
major volatile compounds responsible for odour notes of virgin olive oils are the C6 and the C5
volatile compounds which emerge from primary or secondary LOX pathway, respectively.
The enzymatic levels are determined genetically, so they differ from cultivar to cultivar, but
the enzymatic activity is influenced by all factors mentioned above. Apart endogenous plant
enzymes, responsible for the positive aroma perception in olive oils, chemical oxidation and
microbial activity (associated with sensory defects) should be considered.
4.1 Cultivars
Cultivars and harvest time must be carefully selected in order to correspond to the optimal
level of fruit maturity (Esti et al., 1998; Caponio et al., 2001). Olives ripening is quite
important for olive oil final composition. The cultivar influence depends on the activity of
enzymes and is a genetic characteristic (Tena et al, 2007). The higher LOX activity for linoleic
acid than linolenic acid supports the biogenesis of a higher amount of C6 unsaturated
volatile compounds the major constituents of olive oil aroma; usually olive fruits show the
highest LOX activity 15 weeks after anthesis; activity decreases during development and
ripening periods (Salas et al., 1999). Another enzyme involved is HPL that catalyses the
cleavage of fatty acids hydroperoxides producing volatile aldehydes. The highest level of
HPL activity is detected in green olive fruits, harvested at the initial development stages.
Although there is a slight decrease at maturity, a high activity level is maintained
throughout maturation. The decrease in C6 volatile compounds concentration, in the olive
oils of mature olives, is not attributed to HPL activity (Salas & Sanchez, 1999) rather to the
availability of substrate. The behaviour of these two enzymes supports the decrease of C6
volatile compounds content during fruit ripeness. At earlier ripening stages the amount of
C6 aldeydes and alcohols are very similar, and when olive skin colour changes from green
to purple most of the C6 aldehydes reach their maximum concentration (Angerosa & Basti,
2001). With the increase of ripeness a decrease is observed for most of the aldehydes formed
from the lipoxygenase pathway, namely E-hex-2-enal (the main volatile compound in most
European virgin olive oils), being Z-hex-3-enal an exception (Aparicio & Morales,1998).
Kalua et al (2007), however, state that the decrease in C6 aldehydes, from the lipoxygenase
pathway, might not be characteristic of all cultivars.
The olive cultivar influences also fatty acid composition and, particularly, the ratio of oleic
to linoleic acid (C18:1/C18:2), triglyceride profile, and phenolic content of olive oil (Aparicio
et al., 2002; Tovar et al., 2002; Beltran et al., 2005). Some differences can be found in the fatty
acid content of varietal virgin olive oils (Aparicio 2000); they do not vary so much, however,
as to be determinant for the volatile profile. In spite, C6 volatile compounds (aldeydes,
alcohols and acetyl esters) formed from 13-hydroperoxides of linoleic and-linolenic acids,
account for 60 to 80% of the total volatile compounds (Aparicio & Luna 2002). The
concentration of C6 volatile compounds, of 36 monovarietal virgin olive oils produced in
countries from Mediterranean basin, show that aldehydes (hexanal, Z-hex-3-enal and E-hex-
2-enal) and pent-1-en-3-one contribute, distinctly, to the sensory profile of these varietal
oils, taking into account the odour thresholds of these volatile compounds (75, 3, 1125 and
50 g Kg-1, respectively) (Aparicio & Luna 2002). These authors found high concentrations
of E-hex-2-enal in Italian cultivars, in accordance with results previously obtained by Solinas
Olive Oil Composition: Volatile Compounds 23
et al. (1988), and they all suggest that monovarietal virgin olive oils could be distinguished
by this compound. According to Solinas et al. (1988) octanal, nonanal and hex-2-enal
contents are a cultivar characteristic; the presence of propanol, amyl alcohols, hex-2-enol,
hexan-2-ol and heptanol seems also to be related to the olive cultivar. Nevertheless, olive
oils from different cultivars, produced under the same exact conditions (extraction system,
ripeness stage, pedoclimatic and agronomic conditions), exhibit different amounts of total
volatiles, ranging from 9-83 to 35 mg kg-1 (Luna et al., 2006). Baccouri et al. (2008), when
studying volatile compounds from Tunisian and Sicilian monovarietal virgin olive oils,
found that the overall amounts of C6 aldehydes were clearly higher than the sum of C6
alcohols in Chemlali and Sicilian samples, whereas, in Chetoui oils, the sum of C6 alcohols
was generally higher than the C6 aldehydes. The explanation relays again in the differential
activity of the enzymes involved. These authors also reported a decrease, in the amounts of
C5 aldehydes and alcohols, during the maturation. Morales et al. (1996) studied the influence
of ripeness on the concentration of green aroma compounds; the total content of volatile
compounds decreases with ripeness; there are markers for monovarietal virgin olive oils
obtained from unripe (E-hex-2-enal), normal ripe (hexyl acetate) and overripe olives (E-hex-
2-enol) regardless of the variety (Aparicio & Morales, 1998)
D’Imperio et al. (2010), when studying the influence of harvest, method and schedule, on
olive oil composition, found a remarkable decrease of E-hex-2-enal as was previously
reported by Aparicio et al. (1998); an increase of hexanal seems to be related to the use of
shakers for harvesting. A decrease in unsaturated fatty acids content was also observed
relating these findings to the lipoxygenase pathway.
4.2 Environmental factors
Pedoclimatic factors depends on environmental conditions, soil, type and structure, and/or
climatic conditions, temperature and rainfall (Beltran et al., 2005). Cultivars do not always
grow at the same altitude, but olive grove zones are spread over a wide range of altitudes,
where climatic conditions can be quite different. All these have impact on chemical and
sensory profiles of olive oils. Monovarietal olive oils, obtained from olives grown at higher
altitudes, are in general sweeter and have a stronger herbaceous fragrance, when compared to
the ones produced with olives grown at lower altitudes. Lower temperatures, at higher
altitudes, may influence the enzymes from lipoxygenase pathway, since hexanal (green-sweet
perception) comes from increased levels of linoleic acid, and E-hex-2-enal (green odour and
astringency taste) from lower levels of α-linolenic acids (Aparicio & Luna, 2002). Temime et al.
(2006) studied the volatile compounds from Chétoui olive oils, the second variety cultivated in
Tunisia, and reported significant differences on volatile compounds when, just, environmental
conditions were different. Dabbou et al. (2010) studied the quality and the chemical
composition of monovarietal virgin olive oil, from the Sigoise variety, grown in two different
locations in Tunisia, a sub-humid zone (Béjaoua, Tunis) and an arid zone (Boughrara, Sfax).
Olive oils produced from olives grown at the higher altitude were characterized by higher
contents of E-hex-2-enal (11.92 mg kg-1) and hexanal (1.24 mg kg-1), whereas the oils, from the
lower altitude, were distinguishable by the higher content of Z-hex-2-en-1-ol (8.78 mg kg-1)
and hexan-1-ol (2.17 mg kg-1). The sum of the products of the lipoxygenase oxidation
pathways was higher in oils from Béjaoua (15.92 mg kg-1) than in those from Boughrara (15.20
mg kg-1). Among the LOX oxidation products, the amount of hexanal was higher in Béjaoua
oils (1.24 mg kg-1), whereas the content of Z-hex-2-en-1-ol was considerably lower.
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
24
In a recent study, concerning the behaviour of super-intensive Spanish and Greek olive
cultivars grown in northern Tunisia, Allalout et al. (2011) found significant differences
between oils; they consider, the majority of the studied analytical parameters, to be deeply
influenced by the cultivar-environment interaction.
It seems there is an effect of genotype-environment interaction, responsible for olive oils
characteristics.
4.3 Agronomic factors
Irrigation, a practice that has been adequately studied, seems to produce a decrease in the
oxidative stability of olive oil volatiles due to a simultaneous reduction in oleic acid and
phenolic compounds contents (Tovar et al., 2002).
According to Servili et al. (2007) the olive tree water status has a remarkable effect on the
concentration of volatile compounds, such as the C6-saturated and unsaturated aldehydes,
alcohols, and esters. Put simply, deficit irrigation of olive trees appears to be beneficial not
only due to its well-known positive effects on water use efficiency, but also by optimizing
olive oil volatile quality. Baccouri et al. (2008) reported an enhancement of the whole aroma
concentration of Chetouil oils obtained from trees under irrigation conditions when
compared to similar ones from non-irrigated trees.
The effect of agronomic practices in oil quality is still controversial: data from Gutierrez et
al. (1999) supports the hypothesis that organic olive oils have better intrinsic qualities than
conventional ones. These olive oils usually present lower acidity and peroxide index, higher
rancimat induction time, higher concentrations of tocopherols, polyphenols, o-diphenols
and oleic acid. However, this work was carried out during 1 year, with one olive cultivar
only, and results can not be generalized. Ninfali et al. (2008) in a 3-year study, comparing
organic versus conventional practice did not observe any consistent effect on virgin olive oil
quality. Genotype and year-to-year climate changes seem to have a proved influence.
4.4 Technogical factors
Volatile compounds are predominantly generated during virgin olive oil extraction, and are
important contributors to olive oil sensory quality. Virgin olive oil quality is intimately
related to the characteristics and composition of the olive fruit at crushing. Changes in olive
fruit quality during post-harvest is considered determinant to the final sensory quality.
Kalua et al. (2008) reported that low-temperature storage of fruits can produce poor sensory
quality of the final oil. This decrease in quality might be due to lower levels of E-hex-2-enal
and hexanal, associated with a decrease in enzyme activity, and a concurrent increase in E-
hex-2-enol, which might indicate a possible enzymatic reduction by alcohol dehydrogenase
(Olias et al., 1993,Salas et al. 2000) and reduced chemical oxidation (Morales et al. 1997).
Inarejos-Garcia et al. (2010) studied the olive oils from Cornicabra olives stored at different
conditions (from monolayer up to 60 cm thicknesses at 10 ºC (20 days) and 20 ºC (15 days)).
E-hex-2-enal showed a Gaussian-type curve trend during storage that can be related to the
decrease of hydroperoxide lyase activity. C6 alcohols showed different trends, during
storage, with a strongly decrease of the initial content of Z-hex-3-en-1-ol after 15 and 8
storage days at 20ºC and 10ºC under the different storage layers, whilst an increase of E-hex-
2-en-1-ol was observed (except for mono-layer). Differences might be related to the
Olive Oil Composition: Volatile Compounds 25
enhancement of alcohol dehydrogenase activity during storage. Besides the evolution and
changes observed in the desirable LOX pathway, C6 fraction, storage may give rise to
undesirable volatile compounds, from metabolic action of yeasts, which was more evident
when olive were stored at 20 ºC. The effect of the extraction process on olive oil quality is
also well documented (Ranalli et al., 1996; Montedoro et al., 1992; Di Giovacchino, 1996;
Koutsaftakis et al., 1999; Servili et al., 2004).
Technological operations include several preliminary steps, leaf and soil removal, washing,
followed by crushing malaxation and separation of the oil (and water) from the olive paste.
This last step can be achieved by pressing (the oldest system), centrifugation (the most
widespread continuous system), or percolation (based on the different surface tensions of
the liquid phases in the paste).
Ranalli et al. (2008) studied the effect of adding a natural enzyme extract (Bioliva) during
processing of four Italian olive cultivars (Leccino, Caroleo, Dritta and Coratina) carried out
with a percolation-centrifugation extraction system. The improved rheological
characteristics of the treated olive paste resulted in a reduced extraction cycle with good
effects concerning olive oil aroma characteristics. Results have shown that enzyme-treated
olive pastes always release higher amounts of total pleasant volatiles (E-hex-2-enal, E-hex-2-
en-1-ol, Z-hex-3-enyl acetate, Z-hex-3-en-1-ol, pent-1-en-3-one, Z-pent-2-enal, E-pent-2-enal
and others). For the individual C6 metabolites, from the LOX pathway, a similar trend was
generally observed, while for the total unpleasant volatiles, n-octane, ethyl acetate, isobutyl
alcohol, n-amyl alcohol, isoamyl alcohol and ethanol, an opposite behaviour was found.
The fundamental step is, however, olive crushing. The release of oil from olives can be
achieved by mechanical methods (granite millstones or metal crushers) or centrifugation
systems. These different systems affect the characteristics of the pastes and the final oil (Di
Giovacchino et al., 2002). Almirante et al. (2006) reported that the oils obtained from de-stoned
pastes had a higher amount of C5 and C6 volatile compounds, when compared to oils obtained
by stone-mills. This increment is due to stones removal, which possess enzymatic activities,
metabolizing 13-hydroperoxides other than hydroperoxide lyase, giving rise to a net decrease
in the content of C6 unsaturated aldehydes during the olive oil extraction process. Servili et al.
(2007) demonstrate that the enzymes involved in the LPO pathway have different activity in
the pulp or in the stone. Stones seem to have a lower hydroperoxide lyase activity and a higher
alcohol dehydrogenase activity when compared to the pulp. These authors also found higher
amounts of C6 unsaturated aldehydes olive oils volatiles (VOOs) obtained with the stoning
process; the stone presence in traditional extraction procedure increases the concentration of
C6 alcohols (for Coratina and Frantoio cultivars).
The next step is the malaxation. Malaxation is performed to maximize the amount of oil that
is extracted from the paste, by breaking up the oil/water emulsion and forming larger oil
droplets. The efficiency of this operation depends upon time and temperature. Pressing,
percolation, or centrifugation, are finally used to separate the liquid and solid phases.
Temperature and time of exposure of olive pastes to air contact (TEOPAC), during
malaxation, affect volatile and phenolic composition of virgin olive oil, and consequently its
sensory and healthy qualities. Cultivar still plays a fundamental role for the final
composition (Servili et al, 2003). These authors showed that TEOPAC can be used to
perform a selective control of deleterious enzymes, such as polyphenol oxidase (PPO) and
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
26
peroxidase (POD), preserving the activity of LPO. High malaxation temperature (> 25 ºC)
reduces the activity of enzymes, involved in LOP pathway, reducing the formation of C6
saturated and unsaturated aldehydes. A similar result is described by Tura et al. (2004).
These authors found that changes in malaxation time and temperature produces differences
in the volatile profile of olive oils. Increasing temperature and decreasing time led to a
reduction in the amount of volatiles produced, but they also describe cultivar as the single
most important factor in determining volatile profile of olive oils. The decrease of olive oil
flavour, produced by high malaxation temperature, is due to the inactivation of
hidroperoxide lyase (HPL) rather than lipoxygenase (LOX), as both enzymes have different
behaviour regarding temperature (Salas & Sánchez, 1999b). LOX, when assayed with
linoleic acid as the substrate, displayed a rather broad optimum temperature around 25 ºC
and maintained a high activity at temperatures as high as 35 ºC, but HPL activity peaked at
15 ºC and showed a clear decrease at 35 ºC, in assays using 13-hydroperoxylinoleic acid as
substrate. Similar results were obtained by Gomez-Rico et al. (2009) who observed a
significant increase in C6 aldehydes, in the final oil, as malaxation time increased; almost no
changes in the content of C6 alcohols were observed. Opposite results were found for the
influence of the kneading temperature, where a drop in the C6 aldehydes content as
malaxation temperature increases is observed, especially for E-hex-2-enal and a slight
increase in C6 alcohols, mainly hexan-1-ol and Z-hex-3-en-1-ol.
The final step of olive oil production also affects olive oil quality. Separation of oil from
water can be achieved using a two-phase or a three phase centrifugation system. Comparing
monovarietal virgin oils obtained by both processes, the oils from two-phase decanters have
higher content of E-hex-2-enal and total aroma substances but lower values of aliphatic and
triterpenic alcohols (Ranalli & Angerosa, 1996).
Masella et al. (2009), when studying the influence of vertical centrifugation on olive oil
quality, observed significant differences both in the total volatile concentration and in the
two volatile classes from the LOX pathway involving LnA conversion. The observed
decreased of C6/LnA and C5/LnA compounds can be explained by the volatiles partition
between oil and water phases during vertical centrifugation.
Storage conditions also affect final quality. Light exposure, temperature and oxygen
concentration, storage time and container materials are also determinant. A study by
Stefanoudaki et al. (2010) evaluating storage under extreme conditions, showed subtle
differences, in the pattern of volatile compounds, in bottled olive oils stored indoors or
outdoors. When stored with air exposure the levels of some negative sensory components,
such as penten-3-ol and hexanal, increased while other positives, like E-hex-2-enal were
reduced. Filling the headspace with an inert gas can reduce spoilage.
5. Analytical methodologies for quantitation and identification of volatiles
compounds: New analytical methods
5.1 Olive oil volatile compounds
In the volatile fraction of olive oils, approximately three hundred compounds have already
been detected and identified by means of gas chromatography/mass spectrometry
(GC/MS) methods (Boskou, 2006). Among these compounds, only a small fraction
Olive Oil Composition: Volatile Compounds 27
contributes to the aroma of olive oil (Angerosa et al., 2004). The most common olive oil
volatiles have 5 to 20 carbon atoms and include short-chain alcohols, aldehydes, esters,
ketones, phenols, lactones, terpenoids and some furan derivatives (Reiners & Grosh, 1998;
Delarue & Giampaoli, 2000; Kiritsakis, 1992; Boskou, 2006; Vichi et al., 2003a, 2003b, 2003c;
Aparicio et al., 1996; Morales et al., 1994; Flath et al, 1973; Morales et al, 1995;
Bortolomeazzi et al., 2001; Bentivenga et al., 2002; Bocci et al., 1992; Servili et al., 1995;
Fedeli et al., 1973; Fedeli, 1977; Jiménez et al., 1978; Kao et al., 1998; Guth & Grosch, 1991).
As all vegetable oils, olive oil comprises a saponifiable and a non-saponifiable fraction
and both contribute for the aroma impact. As a result of oxidative degradation of surface
lipids (Reddy & Guerrero, 2004) a blend of saturated and mono-unsaturated six-carbon
aldehydes, alcohols, and their esters (Reddy & Guerrero, 2004; Matsui, 2006) are
produced. As already mentioned they are formed from linolenic and linoleic acids
through the LOX pathway, and are commonly emitted due to defence mechanism
developed by the plant in order to survive to mechanical damage, extreme temperature
conditions, presence of pathogenic agents, among others (Delarue & Giampaoli, 2000;
Noordermeer et al., 2001; Pérez et al., 2003; Angerosa et al., 2000; Angerosa et al., 1998b).
Volatile phenols are also reported as aroma contributors for olive oil and can play a
significant organoleptical role (Vichi et al., 2008; Kalua et al., 2005).
5.2 Analytical methodologies
5.2.1 Sample preparation procedures
When the analysis of a volatile fraction, of complex matrices, is considered sample
preparation cannot be underestimated. In biological samples, a wide chemical diversity,
in a wide range of concentrations, must be expected (Salas et al., 2005; Wilkes et al., 2000).
The chemical nature, and the amount of the detected compounds, strongly depends on the
extraction technique used, to remove and isolate them from their matrices. The choice of
a suitable extraction methodology depends on sample original composition and target
compounds. However, an ideal sampling method does not exist and no single isolation
technique produces an extract that replicates the original sample. In order to have enough
quantity of each compound to be detected by chromatography, a concentration step must,
usually, be considered. Sample preparation can be responsible for the appearance of
artefacts, due to the chemical nature of the compounds extracted, and thus detected and
quantified, and to a total or partial loss of compounds; this issues can, very strongly,
determine the precision, reproducibility, time and cost of a result and/or analysis (Wilkes
et al., 2000; Belitz et al., 2004; Buttery 1988; van Willige et al., 2000). These methods are
revised in a recent manuscript (Costa Freitas et al.) where sample preparation procedures
for volatile compounds are discussed as well as the advantages and drawbacks of each
method.
In olive oil analysis, its oily nature strongly influences the choice of the extraction
procedure. There are various techniques that can be used for the preparation of the sample
analytes in biological material. From those so far applied, liquid extraction with or without
the use of ultrasounds (Kok et al., 1987; Fernandes et al., 2003; Cocito et al., 1995) is probably
the most used. Besides liquid extraction, simultaneous distillation extraction (SDE) (Flath et
al., 1973) has also been widely used. The drawback of these methods is the use of solvents
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
28
and consequently the need of compounds isolation from the solvent which represents an
extra preparation step, as well as the dilutions steps during the extraction procedure. To
avoid these steps, supercritical fluid extraction (SFE) (Morales et al., 1998) was also used for
the isolation of volatile constituents of olive oil.
The methods based on extraction from the headspace are an elegant choice (Swinnerton et
al., 1962). The more often used procedures are the so called “purge and trap” techniques
(Morales et al., 1998; Servili et al., 1995; Aparicio & Morales, 1994) in which the compounds
of interest are trapped in a suitable adsorbent, from which they can be taken either directly
(using a special “thermal desorber” injector) or after retro-extraction into a suitable solvent
which, once again, includes an extra extraction step. Another choice is direct injection of the
headspace into the injection port of a GC chromatograph. This possibility does not include a
concentration step, and consequently, the minor compounds are usually missing or not
detected (Del Barrio et al., 1983; Gasparoli et al., 1986). A direct thermal desorption
technique can also be applied, avoiding the use of any types of adsorbents, by just heating
the target olive oil sample to a suitable temperature in order to promote the simultaneous,
extraction, isolation and injection of the volatile fraction into the analytical column (Zunin et
al. 2004, de Koning et al., 2008). The main advantage of this technique is its simplicity,
although a special injection system is mandatory, which can be expensive. When SPME was
introduced (Belardi & Pawliszyn, 1989; Arthur & Pawliszyn, 1990) several authors have
focused their attention on adapting the technique for aroma compounds analysis (D’Auria
et al., 2004; Vichi et al., 2003; Vichi et al., 2005; Ribeiro et al., 2008). The main advantages of
this technique are: a) it does not involve sample manipulations; b) it is an easy and clean
extraction method able to include, in just one step, all the steps usually needed for aroma
extraction. The extraction step, in SPME, can be made either by headspace sampling or
liquid sampling. Headspace sampling (HS) is usually the method of choice for olive oil
aroma analysis. The fibre chemical composition is of main interest and determines the
chemical nature of the compounds extracted and further analyzed. There are several
coatings commercially available. Polydimethylsiloxane (PDMS) and polyacrylate (PA)
coatings extract the compounds by means of an absorption mechanism (Ribeiro et al., 2008)
whereas PDMS is a more apolar coating then PA. Polydimethylsiloxane/divinylbenzene
(PDMS/DVB), polydimethylsiloxane/carboxene (PDMS/CAR), carbowax/divinylbenzene
(CW/DVB), and divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS)
extract by an adsorptive mechanism. These second group of fibres have usually a lower
mechanic stability but present higher efficiency to extract compounds with low molecular
weight (Augusto et al., 2001). In both extraction mechanisms, once the compounds are
expelled form the matrix, they will remain in the headspace and a thermodynamic
equilibrium is established between these two phases (Zhang & Pawliszyn, 1993). When
the fibre is introduced a third phase is present and mass transfer will take place in both
interphases (sample matrix/headspace and headspace/fibre). When quantification is a
requirement, equilibrium has usually to be achieved. Time and temperature are also very
important issues to take in consideration, since they will affect equilibrium (Vas & Vékey,
2004) and thus extraction efficiency. Methods that consider quantification in non-
equilibrium have also been developed (Ai, 1997; Ribeiro et al., 2008). In order to optimize
the extraction procedures by HS-SPME, the efficiency, accuracy and precision of the
extraction is also directly dependent on operational parameters like extraction time,
sample agitation, pH adjustment, salting out, sample and/or headspace volume,
Olive Oil Composition: Volatile Compounds 29
temperature of operation, adsorption on container walls and desorption conditions
(Pawliszyn, 1997).
5.2.2 Chromatographic methods for the analysis of olive oil volatiles
Capillary gas chromatography (GC) is the most used technique for the separation and
analysis of volatile and semivolatile organic compounds (Beesley et al., 2001) in biological
samples. GC allows to separate and detect compounds present in a wide range of
concentrations in very complex samples, and can be used as a routine basis for qualitative
and quantitative analysis (Beesley et al., 2001; Majors, 2003). Enantioselective separations
can also be performed when chiral columns are used (Bicchi et al., 1999). The most common
detector used is the flame ionization detector (FID), known by its sensitivity and wide
linear dynamic range (Scott, 1996; Braithwaite & Smith, 1999). When coupled with Fourier
transform infrared spectroscopy (GC/FTIR) or mass spectrometry (GC/MS) (Gomes da
Silva & Chaves das Neves, 1997; Gomes da Silva & Chaves das Neves, 1999 ), compounds
tentative identification can be achieved.
The most widely used ionization techniques employed in GC/MS is electron ionization (EI
normally at 70 eV) and the more frequently used mass analysers, in olive oil volatile
research, are quadrupole filters (qMS), ion traps (ITD) and time of flight instruments
(TOFMS). The GC/TOFMS instruments allow the simultaneous acquisition of complete
spectra with a constant mass spectral m/z profile for the whole chromatographic peak,
while in qMS instruments the skewing effect is unavoidable. This fact enables the
application of spectral deconvolution (Smith, 2004), and, potentially, a more accurate use of
reference libraries for identification and confirmation of analytes may be possible.
Nevertheless, for routine laboratory the development of TOFMS dedicated mass spectral
libraries, to complement the libraries now generated by using qMS, should be considered.
Spectral matching is usually better when qMS data are compared in some instances
(Cardeal et al., 2006; Gomes da Silva et al., 2008).
In an ongoing research in our lab, HS-SPME was performed in order to identify volatile
compounds in Galega Vulgar variety. Four fibres were used and the HS-SPME-GC/TOFMS
system operated with a DB-wax column. In table 1 the complete list of compounds
identified (using the four different fibres) is provided as well as fragmentation patterns
obtained for those not yet reported in olive oils (table 2). Analysis were performed in two
columns: a polar column (DB-WAX), usually recommended for volatiles analysis, and an
apolar based column DB-5. The use of these two columns, of different polarity, was also
very useful to detect co-elutions, occuring when the polar column was used, and helped the
identification task, when associated to mass spectrometric and linear retention indices (LRI)
data confrontation. Most identification were performed by comparing retention time and
fragmentations patterns, obtained for standards, analysed under the same conditions, or by
fragmentation studies, when standards were not available. The differences observed, in the
LRI experimentally obtained for the DB-WAX column, compared to the literature were
expectable since polar columns are known as being much more unstable, then apolar
columns, and cross-over phenomena occur (Mateus et al. 2010). Their retention
characteristics varies significantly among different suppliers, which suggest the need of LRI
probability regions. This fact explains why few LRI data is available for polar columns.
These results aims to fullfill some part of this gap.
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
30
Compound name
LRI
Experimental
[Literature]
SPME
Fibres
Compound
name
LRI
Experimental
[Literature]
SPME
Fibres
Hexane n.d.
[600] D-C-P E-Pent-2-enal 1060
[1127-1131] D-C-P
Heptane n.d.
[700]
PA
D-C-P p-Xilene 1067
[1133-1147]
PA
D-C-P
Octane 800
[800]
PA
D-C-P Butan-1-ol 1074
[1147]
PA
D-C-P
Propanone 808
[820]
PA
CDVB
D-C-P
m-Xilene 1077
[1133-1147] D-C-P
E-Oct-2-ene 818
[n.f.] PA Pent-1-en-3-ol 1093
[1130-1157]
PA
D-C-P
Ethyl acetate 832
[892] D-C-P
2,6-Dimethyl-
hepta-1,5-diene
(isomer)
1101
[n.f.] D-C-P
2-Methyl-butanal 850
[915] D-C-P Cis-hex-3-enal 1113
[1072-1137] D-C-P
Dichloromethane 859
[n.f.]
PA
CDVB Heptan-2-one 1123
[1170-1181]
PA
CDVB
D-C-P
Ethanol 883
[900-929]
PA
D-C-P Heptanal 1126
[1174-1186]
PA
CDVB
D-C-P
1-Methoxy-hexane 889
[941] D-C-P o-Xilene 1128
[1174-1191] D-C-P
4-Hydroxy-butan-2-
one
892
[n.f.] PA Limonene 1139
[1178-1206]
PA
D-C-P
Pentanal 896
[935-1002] PA 3-Methyl-butan-
1-ol
1141
[1205-1211] D-C-P
3-Ethyl-octa-1,5-diene
(isomer)
907
[n.f.] D-C-P 2-Methyl-butan-
1-ol
1142
[1208-1211]
PA
PDMS
CDVB
D-C-P
3-Methyl-butanal 912
[910-937] D-C-P 2,2-Dimethyl-
oct-3-ene
1144
[n.f.] D-C-P
Propan-2-ol 918
[n.f.]
PA
CDVB
D-C-P
E-Hex-2-enal 1160
[1207-1220]
PA
CDVB
D-C-P
3-Ethyl-octa-1,5-diene
(isomer)
930
[1018]
PA
D-C-P Dodecene 1164
[n.f.]
PA
D-C-P
Pent-1-en-3-one
(isomer)
932
[973-1016] D-C-P Ethyl hexanoate 1170
[1223-1224]
PA
CDVB
D-C-P
Olive Oil Composition: Volatile Compounds 31
Compound name
LRI
Experimental
[Literature]
SPME
Fibres
Compound
name
LRI
Experimental
[Literature]
SPME
Fibres
Ethyl butanoate 946
[1023]
PA
D-C-P Pentan-1-ol 1184
[1250-1255]
PA
CDVB
D-C-P
Toluene 952
[1030-1042] D-C-P
-Ocimene 1186
[1242-1250]
CDVB
D-C-P
Ethyl 2-methyl-
butanoate
963
[n.f.] D-C-P Tridec-6-ene
(isomer)
1187
[n.f.] D-C-P
Deca-3,7-diene
(isomer)
985
[1077] D-C-P Styrene 1199
[1265]
PA
CDVB
D-C-P
Deca-3,7-diene
(isomer)
994
[1079] D-C-P Hexyl acetate 1209
[1274-1307]
PA
CDVB
D-C-P
Hexanal 1000
[1024-1084]
PA
CDVB
D-C-P
1,2,4-
Trimethylbenzene
1223
[1274]
PA
PDMS
CDVB
D-C-P
3-Methylbutyl-acetate 1037
[1110-1120] D-C-P Octanal 1231
[1278-1288]
PA
PDMS
CDVB
D-C-P
2-Methyl-propan-1-ol 1054
[1089] PA E-4,8-Dimethyl-
nona-1,3,7-triene
1247
[1306]
PA
PDMS
CDVB
D-C-P
Ethylbenzene 1056
[1119]
PA
CDVB
D-C-P
E-Pent-2-en-1-ol 1250
[n.f.] D-C-P
Z-Hex-3-enyl acetate 1258
[1300-1338]
PA
CDVB
D-C-P
Hepta-2,4-dienal
(isomer)
1453
[1463-1487]
PA
CDVB
D-C-P
E-Hept-2-enal 1272
[1320]
CDVB
D-C-P Decanal 1456
[1484-1485]
PA
CDVB
Z-Pent-2-en-1-ol 1281
[1320]
PA
D-C-P
-Humulene 1472
[n.f.] PA
6-Methyl-hept-5-en-2-
one (isomer)
1285
[1335-1337]
PA
CDVB
D-C-P
Benzaldehyde
1488
[1513]
PA
CDVB
D-C-P
Hexan-1-ol 1290
[1316-1360]
PA
CDVB
D-C-P
-Terpineol 1493
[1694] D-C-P
4-Hidroxy-4-methyl-
pentan-2-one
1313
[n.f.] D-C-P E-Non-2-enal 1494
[1502-1540]
PA
D-C-P
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
32
Compound name
LRI
Experimental
[Literature]
SPME
Fibres
Compound
name
LRI
Experimental
[Literature]
SPME
Fibres
E-Hex-3-en-1-ol 1320
[1356-1366]
PA
CDVB
D-C-P
Propanoic acid 1495
[1527] D-C-P
Z-Hex-3-en-1-ol 1322
[1351-1385]
PA
D-C-P Octan-1-ol 1504
[1519-1559]
PA
CDVB
D-C-P
4-Methyl-pent-1-en-3-
ol
1330
[n.f.]
PA
D-C-P
2-Diethoxy-
ethanol
1565
[n.f.]
PA
D-C-P
Methyl Octanoate 1331
[1386] D-C-P E,E-Nona-2,4-
dienal
1574
[n.f.]
PA
Nonan-2-one 1340
[1382]
PA
D-C-P Methyl benzoate 1587
[n.f.] D-C-P
Nonanal 1344
[1382-1396]
PA
CDVB
D-C-P
Butanoic acid 1588
[1634]
PA
D-C-P
E-Hex-2-en-1-ol 1348
[1368-1408]
CDVB
D-C-P
4-
Hydroxybutanoi
c acid
1593
[n.f.] D-C-P
Z-2-Hex-2-en-1-ol 1348
[1410-1417]
PA
D-C-P E-Dec-2-enal 1606
[1590]
PA
CDVB
D-C-P
Oct-3-en-2-one
(isomer)
1349
[1455] D-C-P Acetophenone 1617
[1624] D-C-P
Hexa-2,4-dienal
(E,E), (E,Z) or (Z,Z)
1349
[1397-1402] D-C-P 2-Methyl-
butanoic acid
1621
[1675] D-C-P
Ethyl octanoate 1353
[1428] D-C-P Nonan-1-ol 1628
[1658]
PA
CDVB
D-C-P
Hexa-2,4-dienal
(isomer)
1360
[1397-1402] D-C-P
-Muurolene 1680
[n.f.] D-C-P
E-Oct-2-enal 1367
[1425]
PA
D-C-P Aromadendrene 1681
[n.f.]
PA
PDMS
CDVB
D-C-P
1-Ethenyl-3-ethyl-
benzene
1378
[n.f.] D-C-P 1,2-Dimethoxy-
benzene
1686
[n.f.]
PA
PDMS
D-C-P
Oct-1-en-3-ol
(isomer)
1392
[1394-1450]
PA
CDVB
D-C-P
4-Methyl-
benzaldehyde
1690
[n.f.] D-C-P
Heptan-1-ol 1400
[n.f.]
PA
CDVB
D-C-P
Pentanoic acid 1700
[1746]
PA
CDVB
C-C-P
Olive Oil Composition: Volatile Compounds 33
Compound name
LRI
Experimental
[Literature]
SPME
Fibres
Compound
name
LRI
Experimental
[Literature]
SPME
Fibres
Linalool 1403
[1550] CDVB Butyl heptanoate 1717
[n.f.] D-C-P
Acetic acid 1408
[1434-1450]
CDVB
D-C-P E-Undec-2-enal 1726
[n.f.]
PA
CDVB
D-C-P
Hepta-2,4-dienal
(isomer)
1421
[1488-1519] D-C-P Methyl salycilate 1758
[1762] D-C-P
2-Ethyl-hexan-1-ol 1436
[1491]
PA
CDVB
D-C-P
E, E-Deca-2,4-
dienal
1780
[1710]
PA
CDVB
D-C-P
-Copaene 1440
[1481-1519]
PA
CDVB
D-C-P
2-Methoxy-
phenol (guaicol)
1836
[1855]
PA
CDVB
D-C-P
-Cubebene 1442
[n.f.] D-C-P 2-Methyl-
naphthalene
1839
[n.f.] D-C-P
Benzyl alcohol 1846
[1822-1883]
PA
CDVB
D-C-P
Octanoic acid 2047
[2069]
PA
D-C-P
Phenylethyl alcohol 1890
[1859-1919]
PA
CDVB
D-C-P
Nonanoic acid 2198
[n.f.]
PA
CDVB
D-C-P
Heptanoic acid 1900
[1962]
PA
D-C-P 4-Ethyl-phenol 2212
[n.f.] D-C-P
n.d. denote not determined; n.f. denote not found;
LRI denote linear retention indices for DB-Wax column. LRI between brackets represents the data range
found in literature: Angerosa, 2002; Contini & Esti 2006; Flath et al., 1973; Kanavouras et al., 2005;
Ledauphin et al,. 2004; Morales et al., 1994; Morales et al., 1995; Morales et al., 2005; Reiners & Grosch,
1998; Tabanca et al., 2006; Vichi et al., 2003a., 2003b; Vichi et al., 2005; Zunin et al., 2004.
Table 1. Compounds identified in olive oil samples of Galega Vulgar by means of HS-SPME-
GC/TOFMS. The fibres used are polydimethylsiloxane (PDMS), polyacrylate (PA),
carbowax/divinylbenzene (CDVB), and divinylbenzene/carboxene/polidimethylsiloxane
(D-C-P). The extraction and analysis procedure for all fibres was: 15 g of olive oil sample in
22 mL vial immersed into a water bath at 38 ºC. Extraction time was 30 min. Fibre
desorption time was 300 seconds into an injection port heated at 260 ºC. Splitless time of 1
min. A GC System 6890N Series from Agilent coupled to a Time of Flight (TOF) mass
detector GCT from Micromass using the acquisition software MassLynx 3.5, MassLynx 4.0
and ChromaLynx The system was equipped with a 60 m × 0.32 mm i.d. with 0,5 m df DB-
Wax column or a 30 m × 0.32 mm i.d. with 1 m df DB-5 column, both purchased from J&W
Scientific (Folsom USA). Acquisition was carried out using a mass range of 40-400 u.;
transfer line temperature was set at 230 ºC; ion source 250 ºC. Helium was used as carrier at
100 kPa; Oven temperature was programmed from 50 ºC for three minutes and a
temperature increase of 2 ºC/min up to 210 ºC hold for 15 minutes and a rate of 10 ºC/min
up to 215 ºC and hold.
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
34
Compound name
LRI
Experimental
[Literature]
m/z –fragmentation pattern SPME
Fibres
Ethyl pentanoate 1050
[1127]
57(66%); 60(36%); 71(5%); 73(31%);
85(100%); 88(87%); 101(30%); 115 (2%) 130
(1%) M+
D-C-P
2-Methyl-heptan-4-one 1063
[n.f.]
41(41%); 43(45%); 55(10%); 57(100%);
69(18%); 71(63%); 85(79%); 95(2%);
100
(
3%
)
; 113
(
10%
)
; 128(23%) M+
PA
D-C-P
2,6-Dimethyl-oct-2-ene
(isomer)
1181
[n.f.]
41(87%); 55(100%); 67(11%); 69(73%);
83(25%); 93(12%); 97(25,74%); 111(16%);
126
(
9,86%
)
; 140(1%) M+
D-C-P
3-Methyl-pent-3-en-1-ol
(isomer)
1306
[n.f.]
4
1(100%); 42(16%); 55(52%); 56(12%);
67(93%); 69(49%); 70(19%); 82(72%);
83
(
4%
)
; 100(3%) M+
CDVB
D-C-P
2,6-Dimethyl-octa-
2,4,6-triene (isomer)
1318
[n.f.]
77(15%); 79(38%); 91(3%); 93(22%);
95(10%); 105(55%); 121(100%); 122(10%);
136(43%) M+
D-C-P
1-Methox
y
-2-
(methoxymethyl)-
benzene
1346
[n.f.]
51(15%); 65(18%); 77(33%); 79(20%);
91(100%); 21(96%); 137 (17%); 152(6%) M+ D-C-P
Hex-4-en
y
l propanoate
(
isomer
)
1350
[n.f.]
41(42%); 55(29%); 57(25%); 67(100%);
82
51%
PDMS
D-C-P
Decan-2-one 1428
[n.f.]
41(11%); 42(10%); 43(82%); 55(4%);
57(6%); 58(100%); 59(24%); 60 (6%);
71(24%); 85(2%); 98(4%); 113 (2%);
127
(
2%
)
; 156(2%) M+
PA
D-C-P
Nonyl acetate 1526
[n.f.]
4
3(100%); 56(39%); 61(33%); 70(24%);
83
(
16%
)
; 98
(
19%
)
; 126
(
10%
)
PA
D-C-P
Z-Dec-2-enal 1608
[n.f.]
41(64%); 43(55%); 55(100%); 56(98%);
69(71%); 70(94%); 83(57%); 98(34%);
110
(
5%
)
; 136
(
2%
)
PA
D-C-P
Phenyl acetate 1964
[n.f.]
43(39%); 65(22%); 66(28%); 77(8%);
89(16%); 94(100%); 95(6%);103(8%);
117
(
9%
)
; 136(15%) M+
D-C-P
2-Methyl-phenol 2065
[n.f.]
45(7%); 50(5%); 51(9%); 52(4%); 53(8%);
54(4%); 63(3%); 77(24%); 79(19%); 80(8%);
89(4%); 90(8%); 91(3%); 107(100%);
108(98%) M+; 109
(
5%
)(
M+H
)
+
D-C-P
4-Methyl-byphenyl 2091
[n.f.]
51(6%); 63(5%); 82(10%); 83(12%);
84(11%); 115(10%); 152(21%); 153(17%);
65(32%); 167(71%); 168(100%) M+ ;
169
(
17%
)(
M+H
)
+
D-C-P
Table 2. New tentatively identified compound in olive oil samples of Galela vulgar by means
of HS-SPME-GC/TOFMS. Extraction and analytical conditions according to described in
table 1. m/z fragmentation patterns are presented; n.f. denote not found; LRI denotes linear
retention indices as in table 1. LRI between brackets represents the data range found in
literature, according to table 1.
Olive Oil Composition: Volatile Compounds 35
Co-elutions are often impossible to detect and identify with some GC/MS instruments, in
spite of the use of selective single ion monitoring mode (SIM), or complex deconvolution
processes. The development of new analytical techniques, that maximize analyte separation,
has always been a target. Multidimensional chromatography and comprehensive two-
dimensional chromatography (David & Sandra, 1987; Bertsch, 1999) are an example of such
achievements. The high complexity of the chromatograms points out new ways of
chromatography, such as multidimensional-gas chromatography systems (MD-GC), where
the analytes are submitted to two or more independent separation steps, in order to achieve
separation. In spite of its efficiency, MD-GC is a time consuming technique, with long
analysis times, which does not fit with the demands of routine analysis. Additionally, it is
technically difficult to carry out sequential transfers in a narrow window of retention times,
since co-elutions are foreseen (Poole, 2003). Nevertheless, MD-GC is a precious tool in peak
identification for olive oil analysis when co-elutions occur (Reiners & Grosch, 1998). In 1991,
comprehensive two-dimensional gas chromatography (GC × GC) was introduced by Liu &
Phillips. The GC × GC system consists of two columns with different selectivities; the first
and second dimension columns are serially connected through a suitable interface, usually
is a thermal modulator (Phillips & Beens, 1999; Marriott & Shellie, 2002). When performing
GC × GC technique the entire sample, separated on the first column, is transferred to the
second one, resulting in an enhanced chromatographic resolution into two independent
dimensions, where the analytes are separated by two independent mechanisms (orthogonal
separation) (Venkatramani et al., 1996; Phillips & Beens, 1999; Marriott & Shellie, 2002;
Dallüge et al., 2003). The modulated zones of a peak are thermally focused before the
separation on the second column, in a mass conservative process; the resulting segments
(peaks), of the modulation, are much narrower with higher S/N ratios, than in
conventional GC (Lee et al., 2001; Dallüge et al., 2002), improving the detection of trace
analytes and the chromatographic resolution. Fast acquisition TOF spectrometers are the
suitable detectors for this technique and have considerably enlarged the application of GC
× GC. Few applications are still reported for olive oil analysis, nevertheless, they already
showed its potential. GC × GC techniques allowed identification of olive oil key flavour
compounds, present in very low concentrations (Adahchour et al. 2005); it has also been
used as a flexible technique for the screening of flavours and other classes of (semi-)polar
compounds, using the conventional orthogonal approach and the reverse, non-orthogonal
approach in order to obtain ordered structures that can simplify the identification task
(Adahchour et al. 2004); finally this separation technique can allow easy fingerprint
analysis of several olive oil matrices directly, or using image processing statistics (Vaz-Freire
et al., 2009).
5.3 Future perspectives for olive oil volatile analysis: Identification tools and
fingerprinting
A limitation of electron ionization (EI) in MS analysis is due to the fact that, too often, the
molecular ions do not survive fragmentation and, consequently, are not "seen". One way to
overcome this problem is to use a complementary technique, that provides "soft" ionization
of the molecules, allowing molecular ions detection. Chemical ionization (CI) performs this
task (McMaster and McMaster, 1998; Herbert and Johnstone, 2003). The mass spectra
obtained by CI are simpler than EI, though most of the interpretable structural information
is missing. However the compound´s molecular ions appears as a high intensity fragment
Olive Oil – Constituents, Quality, Health Properties and Bioconversions
36
and sometimes is the major fragment of the spectra. Thus, molecular weight determination
of an analyte becomes possible. Other soft ionization techniques are field ionization (FI) and
field desorption (FD). Both produce abundant molecular ions with minimal fragmentation
(Herbert and Johnstone, 2003). FI and FD are appliable to volatile and thermally stable
samples (Niessen, 2001; Dass, 2007). If high resolution mass analysers are coupled with
these ionization techniques, high capability of identification can be achieved. Together with
GC × GC a potentially new tool in olive oil compound identification is reachable and
desirable.
The application of a multimolecular marker approach to fingerprint allows, in an easy way,
the identification of certain sample characteristics. Chromatographic profiles can be
processed as continuous and non-specific signals through multivariate analysis techniques.
This allow to select and identify the most discriminant volatile marker compounds (Pizarro
et al., 2011). The quantity and variety of information, provided by two-dimensional-GC (2D-
GC) systems, promoted the increasingly application of chemometrics in order to achieve
data interpretation in a usefull and, potentially, easy way. Linear discriminant analysis
(LDA) and artificial neural networks (ANN), among other statistical classification methods,
can be applied in order to control economic fraud. These applications have been carefully
reviewed recently (Cajka et al., 2010). Together with 2D-GC systems the advantage is clear,
since, instead of a time consuming trial to determine which variables should be considered
for the statistical classification method, the selection may now become as simple as
inspecting the 2D contour plots obtained (Cardeal et al 2008, de Koning et al., 2008). Also the
use of statistical image treatment, of 2D-GC generated contour plots, can be applied for
fingerprint recognitions, precluding the alignment of the contour plots obtained, which
already allowed the identification of varieties as well as extraction technologies used to
produce high quality Portuguese olive oils (Vaz Freire et al., 2009).
6. Conclusion
A final word should also be addressed to spectral libraries. Commercial spectral libraries are
becoming increasingly more complete and specific, making GC/MS one of the most used
techniques for routine identifications. However, several compounds are not yet described in
library databases and, in spite of better algorithmic calculations, databases are only reliable
for target analysis, or when the compounds under study are known, and already
characterized with a known mass spectra. Additionally, the full separation of peaks to
ensure clean mass spectra, in order to achieve a reliable peak analyte confirmation, is still a
necessary goal.
Until now most of the analytical systems used to analyse olive oil volatile compounds are
performed in 1D-GC systems with polar or apolar column phases. Since olive oil volatile
fraction is very complex, frequent co-elutions occur. Mass spectra obtained are,
consequently, not pure, which should preclude the possibility to compare the spectra
obtained with the, claimed pure, spectra in the databases. However, tentative identifications
are reported in the literature, and it is not rare that some inconsistencies occur, even when
linear retention indices LRIs are presented. Because of their nature, the LRIs obtained in
apolar columns are more reliable. Nevertheless, a better separation is obtained in 1D-GC
systems when polar stationary phases are used, because of the wide chemical variety
Olive Oil Composition: Volatile Compounds 37
comprised in the volatile fraction of olive oils. Unfortunately, these columns present a high
variability, at least, among different purchasers, which do not facilitate LRIs comparison
with literature data. Multidimensional techniques, hyphenated with mass-spectrometry, are
now fullfiling this gap also in the separation of optical active compounds, when chiral
column phases are used. Clean mass spectra together with compound LRIs in polar, apolar
and chiral column phases represents an improved tool in compound identification and thus
in olive oil matrices characterization. LRIs considering probability regions in the 2D
resulting plot of a GC × GC experiment (with different column set combinations, e.g. polar ×
apolar, polar × chiral, etc.), can enable comparing standard compounds with the sample
compounds retention indices and thus a more reliable peak identification can be achieved, if
mass spectrometric data are simultaneously recorded. In the future, for 2D systems, more
comprehensive mass spectral libraries should include retention index probability regions for
different column sets in order to allow correlation of the results obtained in the used
systems with spectral matches and literature LRIs.
7. Acknowledgment
Authors wish to thank Fundação para a Ciência e Tecnologia, Ministério da Ciência,
Tecnologia e Ensino Superior and Programa Operacional Ciência e Inovação for financial
support (Projects PTDC/AGR-AAM/103377/2008 and PTDC/QUI-QUI/100672/2008).
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The Mediterranean diet is appraised as the premier dietary regimen and its espousal is correlated with the prevention of degenerative diseases and extended longevity. The consumption of olive oil stands out as the most peculiar feature of the Mediterranean diet. Olive oil rich in various bioactive compounds like oleanolic acid, oleuropein, oleocanthal, and hydroxytyrosol is known for its anti-inflammatory as well as cardioprotective property. Recently in silico studies have indicated that phytochemicals present in olive oil are a potential candidate to act against SARS-CoV-2. Although extensive studies on olive oil and its phytochemical composition; still, some lacunas persist in understanding how the phytochemical composition of olive oil is dependent on upstream processing. The signaling pathways regulated by olive oil in the restriction of various diseases is also not clear. To answer these queries, a detailed search of research and review articles published between 1990 to 2019 were reviewed in this effect. Olive oil consumption was found to be advantageous for various chronic non-communicable diseases. Olive oil’s constituents are having potent anti-inflammatory activities and thus restrict the progression of various inflammation-linked diseases ranging from arthritis to cancer. But it is also notable that the amount and nature of phytochemical composition of household olive oil are regulated by its upstream processing and the physicochemical properties of this oil can give a hint regarding the manufacturing method as well as its therapeutic. Moreover, daily uptake of olive oil should be monitored as excessive intake can cause body weight gain and change in the basal metabolic index. So, it can be concluded that olive oil consumption is beneficial for human health, and particularly for the prevention of cardiovascular diseases, breast cancer, and inflammation. The simple way of processing olive oil maintains the polyphenol constituents and provides more protection against non-communicable diseases and SARS-CoV-2.
Thesis
Olive oil is a vital component of the Mediterranean diet, hence Lebanese, owed to its well-known economic and nutritional value. Several environmental, agricultural, and technological factors play an essential role in defining olive oil's quality. In Lebanon, preliminary studies on the quality of extra virgin olive oil have shown that certain quality criteria exceed the International Olive Council's (IOC) standards. However, the causes of such non-conformities have not been clearly identified. Accordingly, ninety-six olive oil samples have been harvested from two seasons, processed using different extraction methods, and collected from eight locations (Akkar, Chouf, Hasbaya, Koura, Tyr, Nabatiyeh, Zgharta, and Hermel). These locations are identified by the European Union to have potentials for Protected Geographical Indications (PGI). In this perspective, and to meet the European framework's requirements, the analyzed oil will be subjected to conventional chemical analysis as suggested by the IOC and to ultra-fast analysis using 3D-front face spectroscopy (3D-FFFS) and ultra-flash gas chromatography (Ultra-FGC).A correlation between the fatty acid profile and the pedoclimatic conditions of the main olive growing regions in Lebanon was noticed. Three main pedoclimatic conditions, altitude, temperature, and relative humidity, were the major influencers and the reason for the distinctive fatty acid profile of the Lebanese olive oil. Lebanese areas with high altitudes, low average temperature, and low relative humidity have high oleic acid content. As for areas with lower altitudes, higher average temperature, and higher relative humidity, the fatty acid profile was characterized by linoleic, linolenic, palmitoleic, and palmitic acids. In addition to the environmental factors, agricultural ones, particularly the harvest date, had affected the chemical constituents of olive oil. The results obtained showed that the harvest date strongly influenced acidity and total polyphenols. A change in the fatty acid profile characterized by a higher linoleic and lower oleic content, an increase in ∆^7-stigmastenol exceeding the limit set by the IOC standards, and a dominating off-flavor compound (ethanol) was noticed as a result of delaying the harvesting time. Besides, two technological factors, particularly improper fruit storage, and bad hygienic practices, significantly affected olive oil’s quality parameters and fatty acid content.3D-FFFS and Ultra-FGC were used in-line with conventional analysis, and they both showed an undeniable performance. 3D-FFFS coupled with chemometric tools, namely multiple linear regression (MLR) applied on parallel factor (PARAFAC) scores and partial least squares (PLS), was tested on inconsistent qualities of olive oil samples to predict quality parameters. Twenty-two MLR models were generated, the majority of which showed a good correlation coefficient (R>0.7). A second model using PLS on the unfolded emission-excitation matrices was also conducted to improve the regression and assess whether the variability can be handled successfully. However, similar results, with a slight improvement over the MLR model, were obtained. As for Ultra Flash GC, it made it possible to identify, in only a few minutes (< 2 min), ethanol, (E,E)-2,4-decadienal (organoleptic defect), and 1-hexanol (fruity, grassy) as the main volatiles characterizing the Soury variety.This study offers the potential to disseminate an analytical control plan that links environmental aspects in Lebanon and cultivation/harvesting techniques to olive oil's resulting physicochemical characteristics. Such a matrix incorporating rapid analysis techniques will facilitate governance over the end product's final quality and, subsequently, conformity to IOC standards. Furthermore, this work will set the ground through a detailed identification fiche for PGI.
Article
Attention to the quantitative and qualitative characteristics of the fruit is very important due to its effect on the yield and quality of olive oil. The aim of this study was to compare the three cultivars Picual, Coratina, Zard and T18 genotype in terms of physiological, quantitative and qualitative indices of olive fruit and oil in 2016 at the Tarom Olive Research Station and in the completely randomized block design based on the four level of treatment in three replications planned and implemented. After evaluating the results of traits such as length and diameter of fruit and kernel, ratio of length to diameter of fruit, rate of fruit flesh to kernel, Average fruits weight and Average fruit wet and dried flesh weight, product, oil percentage, total oil phenol, acidity and peroxide, carotenoid, chlorophyll and oil fatty acid profile, it was found that Picual had the highest fruit and kernel among these cultivars and genotype. Rate of fruit flesh to kernel was higher in Picual cultivar without significant difference with T18 genotype. In the wake of the larger fruits in Picual, in terms of weight traits, the fruits of these cultivars showed more value. This cultivar was the highest product with 116.75 kg production. After examining the quantitative and qualitative characteristics of the oil, it found that the Picual cultivar had the highest amount of total phenol, carotenoid and chlorophyll in the oil and, in contrast, the oil acidity was lower than the others were. Coratina showed the highest amount of oleic acid (72.53%), the highest ratio of oleic acid to palmitic acid and stearic acid, the highest amount of unsaturated fatty acids (84.03%) and the lowest palmitic acid. Therefore, according to the results of this study, it can be concluded that among three cultivars Picual, Coratina, Zard and T18 genotype, Coratina cultivar due to its higher content of phenolic compounds as well as higher amount of unsaturated fatty acids and proper balance between saturated and unsaturated fatty acids, it has better quality oil, but the percentage of oil and yield in Picual and Zard cultivars is higher than Coratina, so the combined cultivation of these three cultivars should be used to maintain quality and higher yield.
Article
Full-text available
The impact of harvest period on the quality parameters, polyphenols, fatty acids, sterols, and volatile compounds of Lebanese olive oil from the Soury variety was investigated in this study. Two groups of olive oil were compared, each with a specific harvest date. HD1 was harvested in October, whereas HD2 was picked in November. The analysis of both olive oil categories showed that HD2 witnessed a significant increase in all quality parameters except K270 and a decrease in total polyphenol content from 138 mg/mL to 44 mg/mL. Oleic and linoleic acids had an inverse relation, where the former decreased and the latter increased with the harvest date’s advancement. Palmitic acid in both groups was higher than the standards set for extra virgin olive oil. The relative amount of β -Sitosterol was mainly found to decrease, while those of stigmasterol, ∆5,24 -stigmastadienol, ∆7 -stigmastenol, and ∆7 -avenasterol increased with delaying harvest time. As for the volatile compounds, principle component analysis was used on the flash GC data to differentiate HD1 from HD2. Ethanol was found mostly characterizing HD2, whereas HD1 was influenced by 1-hexanol and (E,E)-2,4-decadienal. It can be concluded that the Soury variety should be harvested early, and a delay would result in the declassification of Lebanese olive oil quality from extra virgin to virgin olive oil.
Book
The Handbook of Olive Oil presents an up-to-date view of all aspects of olive oil. It is written from an inter-disciplinary point of view and will be of use in research and development as well as in routine laboratory and process operations. This second edition includes new chapters devoted to genetic studies and agronomic aspects of new orchards and cultivars, which, in combination with the most recent biochemical studies and technological developments, explain the unique chemical composition of olive oil. The analytical aspects of the first edition are now described in six new chapters focused on the chemical compounds responsible for olive oil traceability and sensory perceptions (odor, color, and taste) utilizing chromatographic, spectroscopic, and in-tandem techniques. Nutritional and sensory aspects are the basis for the current success of virgin olive oil among consumers, and this new edition re-analyzes in two new chapters the role of lipids, in general, and olive oil, in particular, in nutrition and health. In addition, the methodologies developed for determining sensory quality, olive oil oxidation, and deep-frying are extensively described and discussed. The role of consumers in olive oil studies of marketing and acceptability is covered in a new chapter. This second edition has not ignored the fact that the popularity of olive oil has made it a preferred target for fraudsters. Deliberate mislabeling or mixtures containing less expensive edible oils are topics described in depth in two chapters devoted to traceability and adulteration. There is also a new chapter focused on the olive refining process, which is a relevant activity in the olive oil world, and another chapter displaying tables of chemical and sensory information from olive oils produced all over the world. The book is written at two levels: the main level is structured as a tutorial on the practical aspects of olive oil. A second, more methodological level, is intended for specialists in the different sciences that contribute to olive oil studies (biochemistry, chemistry, physics, statistics etc). This edition also details changes that are needed in different disciplines in order to overcome current problems and challenges. © Springer Science+Business Media New York 2013. All rights reserved.
Article
In this month's column, Ron Majors examines trends in gas chromatography column usage based on user surveys. He notes increased use of capillary columns over packed columns, greater popularity of columns with smaller diameters, and longer lives for cross-linked and bonded phases.
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A technique for water sample analysis is presented which uses chemically modified fused silica optical fibers as micro solid phase extractors. The small size of the fibers (100 μm o.d.) allows for direct introduction into the on column injector port of a high resolution gas chromatograph (GC), where the analytes are thermally desorbed. This eliminates the need for solvents and syringes which are used in liquid-liquid or solid phase extraction techniques. Thus, this method lowers cost and analysis time per sample, as well as eliminates possible sources of error. Results show that the efficiency and selectivity of this technique are dependent upon the thickness and polarity of the stationary phase. Initial results indicate that the limit of detection for 2-naphthol and FID detection is approximately 5 ng/g with a linear response range of 5 orders of magnitude.
Chapter
Modern mass spectrometry - the instrumentation and applications in diverse fields Mass spectrometry has played a pivotal role in a variety of scientific disciplines. Today it is an integral part of proteomics and drug discovery process. Fundamentals of Contemporary Mass Spectrometry gives readers a concise and authoritative overview of modern mass spectrometry instrumentation, techniques, and applications, including the latest developments. After an introduction to the history of mass spectrometry and the basic underlying concepts, it covers: Instrumentation, including modes of ionization, condensed phase ionization techniques, mass analysis and ion detection, tandem mass spectrometry, and hyphenated separation techniques Organic and inorganic mass spectrometry Biological mass spectrometry, including the analysis of proteins and peptides, oligosaccharides, lipids, oligonucleotides, and other biological materials Applications to quantitative analysis Based on proven teaching principles, each chapter is complete with a concise overview, highlighted key points, practice exercises, and references to additional resources. Hints and solutions to the exercises are provided in an appendix. To facilitate learning and improve problem-solving skills, several worked-out examples are included. This is a great textbook for graduate students in chemistry, and a robust, practical resource for researchers and scientists, professors, laboratory managers, technicians, and others. It gives scientists in diverse disciplines a practical foundation in modern mass spectrometry.