foods

Review
Bioactive Compounds and Quality of Extra Virgin
Olive Oil
Cecilia Jimenez-Lopez 1,2 , Maria Carpena 1 , Catarina Lourenço-Lopes 1 ,
Maria Gallardo-Gomez 3 , Jose M. Lorenzo 4 , Francisco J. Barba 5 , Miguel A. Prieto 1, * and
Jesus Simal-Gandara 1, *
1 Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Faculty of Food Science
and Technology, University of Vigo, Ourense Campus, E32004 Ourense, Spain;
[email protected] (C.J.-L.); [email protected] (M.C.);
[email protected] (C.L.-L.)
2 Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolonia,
5300-253 Bragança, Portugal
3 CINBIO, Universidade de Vigo, Department of Biochemistry, Genetics and Immunology,
Campus Universitario Lagoas Marcosende, 36310 Vigo, Spain; [email protected]
4 Meat Technology Centre Foundation, 32900 San Cibrao das Viñas, Spain; [email protected]
5 Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and
Forensic Medicine Department, Universitat de València, Faculty of Pharmacy, Avda, Vicent Andrés Estellés,
s/n, Burjassot, 46100 València, Spain; [email protected]
* Correspondence: [email protected] (M.A.P.); [email protected] (J.S.-G.)




Received: 30 May 2020; Accepted: 24 July 2020; Published: 28 July 2020


Abstract: (1) Background: Extra virgin olive oil (EVOO) is responsible for a large part of many health
benefits associated to Mediterranean diet as it is a fundamental ingredient of this diet. The peculiarities
of this golden, highly valued product are in part due to the requirements that must be met to achieve
this title, namely, it has to be obtained using exclusively mechanical procedures, its free acidity
cannot be greater than 0.8%, it must not show sensory defects, and it has to possess a fruity taste.
(2) Methods: All these characteristics are key factors to EVOO quality, thus the chemical composition
of these many health-promoting compounds, such as unsaturated fatty acids (which are also the
major compounds, especially oleic acid), as well as minor components such as tocopherols or phenolic
compounds (which behave as natural antioxidants) must be preserved. (3) Results: Due to the
presence of all these compounds, the daily consumption of EVOO entails health benefits such as
cardioprotective, antioxidant, anti-inflammatory, anti-tumor properties or acting as regulator of
the intestinal microbiota, among others. (4) Conclusions: Taking all together, conserving EVOO
chemical composition is essential to preserve its properties, so it is worth to control certain factors
during storage like exposure to light, temperature, oxygen presence or the chosen packaging material,
to maintain its quality and extend its shelf-life until its consumption.

Keywords: extra virgin olive oil; chemical composition; bioactive substances; EVOO
quality; applications

1. Introduction
Olea europaea L., commonly known as the olive tree, is a small tree species typically distributed
by the Mediterranean countries [1]. Olive oil, its main derived product, has experienced an increase
in its popularity due to its organoleptic characteristics and its associated beneficial health effects [2].
The olive tree is one of the species that was first cultivated. In fact, there are records for the first
cultivated trees which date from 6000 years in Asia Minor regions according to the International

Foods 2020, 9, 1014; doi:10.3390/foods9081014 www.mdpi.com/journal/foods

Foods 2020, 9, 1014 2 of 31

Olive Council (IOC) [3]. Its origin is linked to Mediterranean civilizations and regions, characterized
by soft and rainy winters and hot dry summers [4]. Although its industry uses large areas for the
cultivation of trees, nowadays, its cultivation has been spread to regions of all continents (excluding
Antarctica). Still, Mediterranean countries remain the main olive producers, led by Spain, Italy, and
Greece. In consequence, the European Union (EU) is responsible for the 70% of the global olive
production, generating a production value of 7000 million euros each year, becoming a key factor for
the agro-industrial sector development and thus, a social and economic engine for the EU southern
regions [5].
The French writer Georges Duhamel once wrote “There, where the olive tree gives up, is where the
Mediterranean ends. The tree of light is the nature and culture of the Mediterranean” [6]. This statement
corroborates the joining and simultaneous development of a culture-bound to olive products and
particularly olive oil. So much, that olive oil is a crucial part of the commonly known as Mediterranean
diet (MED). This diet consists of a balanced combination of low animal protein consumption with
a high intake of fruits, vegetables and cereals and olive oil as the principal source of fat in many
foods [3]. MED has been usually associated with a low incidence of cardiovascular diseases (CVD)
risk in EU southern regions, which likewise, show higher life expectancy when compared to EU
northern countries and the United States of America (USA) [7,8]. Several studies have been conducted
to evaluate and prove MED as the main protective agent in the primary prevention of chronic diseases.
Research has pointed out that the beneficial health effects of MED are attributed to a large extent to
olive oil.
Some of those beneficial properties are gathered up in Figure 1. Its unique composition and
biological properties are largely responsible for this association. Olive oil composition is mainly
formed by triglycerides and a variety of several compounds in small quantities. Among the glyceride
fraction, olive oil shows a high content of fatty acids and particularly, an elevated proportion of
monounsaturated fatty acids (MUFA). Unsaturated acids are up to 85% of its composition, due to its
high content in oleic acid (C18:1), which might range between 70–85% and other fatty acids as linoleic
or palmitoleic acid. In the case of saturated fats, they entail around de 14% of oil composition, basically
due to palmitic and stearic acids. Regarding minor compounds, they signify less than 2% of olive
oil composition, and the best representatives of this group are phenolic compounds, although this
minor group also includes some lipophilic compounds like α-tocopherol (vitamin E). Likewise, there
are several hydrophilic phenolic compounds, among which the simple phenol hydroxytyrosol and
the secoiridoid oleuropein must be highlighted [1,8]. Besides, olive oil is also a source of pigments
like carotenoids [3]. Phenolic compounds are known for their biological properties. In particular,
these olive oil compounds have shown potential as antioxidant, anti-inflammatory and antimicrobial
agents [9]. However, their concentration is related to different factors: olive varieties, environmental
factors, time of harvest and extraction, and storage conditions [1]. To sum up, olive oil is considered as
a superfood due to its health properties derived from its unique composition; this is, its lipid profile
and its bioactive compounds content [10]. Moreover, its singular composition has attracted so much
attention that olive oil has been studied as a source of these bioactive compounds but also their derived
residues have shown high concentrations of these molecules and thus a great potential for industrial
recovery and related applications, such as formulation of new high added value products [10].
Foods 2020, 9, x 
Foods 2020, 9, 1014 3 of 31 
3 of 31

 
Figure 1. Scheme of the beneficial and healthy effects of the olive oil inclusion in the Mediterranean
Figure 1. Scheme of the beneficial and healthy effects of the olive oil inclusion in the Mediterranean 
diet. Information adapted from [11,12].
diet. Information adapted from [11,12]. 
Figure 2 shows a detailed procedure of the extraction of olive oil from Olea europaea L. of different
Figure 2 shows a detailed procedure of the extraction of olive oil from Olea europaea L. of different 
varieties, whose extracting process follows a common line formed by different steps: collection, grading,
varieties,  whose  extracting 
washing, crushing, malaxing,process  follows 
separation anda centrifugation,
common  line  storage
formed and
by  different 
packaging. steps: 
Thiscollection, 
process is
grading, washing, crushing, malaxing, separation and centrifugation, storage and packaging. This 
not aimed at improving oil quality; however, attention must be paid to its development to not cause a
process is not aimed at improving oil quality; however, attention must be paid to its development to 
loss of quality [13,14]. Depending on the modifications of this process and the resultant organoleptic
not cause a loss of quality [13,14]. Depending on the modifications of this process and the resultant 
characteristics of the product, among others, the EU defines on the Article 118 of European Commission
organoleptic 
(EC) Regulation characteristics  of  the  product, 
(EC) No 1234/2007 among 
six main types of others, 
oils, whichthe  are
EU delineated
defines  on alsothe by
Article 
the IOC 118  of 
[15].
European  Commission  (EC)  Regulation  (EC)  No  1234/2007  six  main  types  of  oils, 
The specific attributes of each type of oil are shown in Table 1. Regarding all these groups, virgin olive oil which  are 
delineated 
(VOO) mustalso  by  the  IOC due
be highlighted [15]. 
toThe 
theirspecific  attributes 
organoleptic of  each  type 
characteristics and of  oil  are  shown 
beneficial in  Table 
properties. Among 1. 
Regarding  all  these  groups,  virgin  olive  oil  (VOO)  must  be  highlighted  due 
VOOs, the EU establishes three types of oils: extra virgin olive oil (EVOO), VOO, and lampante oliveto  their  organoleptic 
characteristics and beneficial properties. Among VOOs, the EU establishes three types of oils: extra 
oil. Furthermore, IOC adds an extra subgroup called ordinary virgin oil. All VOOs are characterized
virgin  olive 
for being oil  (EVOO), 
obtained VOO,  and 
by mechanical lampante 
processes (onlyolive  oil.  Furthermore, 
washing, decantation,IOC  adds  an  extra 
centrifugation andsubgroup 
filtration)
called ordinary virgin oil. All VOOs are characterized for being obtained by mechanical processes 
under specific thermal conditions which do not cause any alteration. Afterward, they are divided
(only washing, decantation, centrifugation and filtration) under specific thermal conditions which do 
according to their acidity, which gives an idea of the content in free fatty acids based on the percentage
not cause any alteration. Afterward, they are divided according to their acidity, which gives an idea 
of oleic acid. Lower acidity values guarantee a high-quality oil, showing it has been obtained from
of the content in free fatty acids based on the percentage of oleic acid. Lower acidity values guarantee 
healthy olives and under ideal conditions. Moreover, they are submitted to sensory analysis to asses
a  high‐quality 
some requirementsoil,  showing 
[16]. it  has  been  obtained  from  healthy  olives  and  under  ideal  conditions. 
Moreover, they are submitted to sensory analysis to asses some requirements [16]. 
Foods 2020, 9, 1014 4 of 31
Foods 2020, 9, x  4 of 31 

 
Figure 2. Summary of the oil extraction process focused on extra virgin olive oil (EVOO) production.
Figure 2. Summary of the oil extraction process focused on extra virgin olive oil (EVOO) production. 
The boxes with discontinuous outline refer to the obtainment of other type of oils. Modified from
The  boxes  with  discontinuous  outline  refer to  the  obtainment  of  other  type  of  oils.  Modified  from 
different schemes of [10,17].
different schemes of [10,17]. 
Table 1. Types of olive oils. Modified from EU Council Regulation (EC) No 1234/2007 [15].
 
Free
Type of Oil Characteristics
Acidity

They are characterized for being obtained by mechanical or other physical <0.8 g per
EVOO
processes under specific thermal conditions that do not cause alterations in 100 g
Virgin olive the oil and have not suffered any treatment other than washing, decantation, ≤2 g per
Virgin olive oils
oil centrifugation or filtration. Excluded are oils obtained using solvents or 100 g
adjuvants with chemical actions, by re-esterification process or any mixture
Lampante >2 g per
with oils of other types.
olive oil 100 g
≤0.3 g per
Refined olive oil In this case, virgin olive oil is submitted to a refining process.
100 g
Olive oil (composed of refined It is the result of the blending of the two previous oils: virgin olive oils (not ≤1 g per
olive oils and virgin olive oils) lampante oil) with refined olive oil. 100 g
This type refers to oil obtained from olive pomace by using solvents, physical
treatments or oil corresponding to lampante olive oil type, except for certain
Crude olive pomace oil specified characteristics.
As well as in the case of virgin olive oils, excluded are oils obtained by means
of re-esterification and mixtures with oils of other types.
≤0.3 g per
Refined olive pomace oil This type is obtained from refining crude olive pomace oil.
10 g
It if the resultant oil from mixing refined olive pomace oil and virgin olive oil ≤1 g per
Olive pomace oil
different than lampante oil. 100 g
NOTES: (1) The International Olive Council (IOC) establishes olive oil standards in terms of sensory analysis and
chemical composition for each category. (2) In the case of Lampante oil, it is also referred as “virgin olive oil not fit
for consumption” by the IOC and with a free acidity higher than 3.3 g per 100 g.
Foods 2020, 9, 1014 5 of 31

Particularly, EVOO is obtained directly from olives, that is, pure olive juice. It is considered
the highest quality oil, and in general, it is characterized for having a low acidity, up to 0.8% and
a sensory grade higher than 6.5 points, thus, having perfect aroma and flavor [3,10]. Regarding
sensory aspects, EVOO must show a fruity note higher than 0 and, more importantly, a median of
zero defects [18]. In addition, and to offer the most possible information to the consumer, these oils
are usually labeled as intense, medium or light depending on their positive attributes [19]. However,
sensory analysis is always more difficult to unify. On the other hand, EVOO is susceptible to different
chemical alterations during its production. Hydrolytic and oxidative degradation are the main causes
of undesirable product formation so that water plays an important role since lipases (naturally present
at olive pulp or seeds) are active in an aqueous phase. Lipolytic reactions lead to the formation of
free fatty acids, thus increasing the acidity of the oil. To avoid these reactions, the filtration step is
of high importance, as well as paying attention to other factors such as avoiding microorganisms
or mechanical crushing, among others. Moreover, water may affect the transformations of different
phenolic compounds [18,20]. Regarding oxygen, EVOO must have a peroxide index (mEqO2 /kg) ≤20.
Oxygen is crucial for the lipoxygenase cascade reactions and its consequential positive sensory notes,
but its excess can cause defects [20]. Moreover, EVOO must also show specific spectrophotometric
values: absorption λ = 233 nm, ≤ 2.50 and λ = 270 nm, ≤ 0.22, which are quality and authenticity
indicators, respectively [18]. Besides, contaminants (i.e., mycotoxins or pesticides, among others)
are another crucial factor in avoiding associated risks [21]. Considering all the characteristics that
EVOO is required to fulfill, quality control is a must. Both IOC and EC have approved different
standards in matters of purity (oil composition) and quality (organoleptic profile, acidity, peroxide
value, among others) as well as sensory analysis to ensure that alterations have not occurred during
the process [10]. Additionally, another remarkable aspect is traceability. Traceability must be assured
during all the obtaining process of EVOO, including the four basic steps (harvesting, milling, storage,
and packaging batches) of the procedure to guarantee the identity of the product and its production
chain. This approach also leads to the next step, this is certification, carried out by normalization
and certification quality organisms in order to prove the rigorous process system employed at EVOO
production [22,23].
Regarding its composition, EVOO is mainly composed of triglycerides (97–99%) and minor
compounds (1–3%), which are the principal responsible for its biological properties and sensory
attributes. It has a high content of MUFA (65–83%), especially oleic acid, and some polyunsaturated
fatty acids (PUFA) such as linoleic acid, which is considered a potent fatty acid on the reduction of
low-density lipoprotein (LDL) cholesterol. This lipid profile and also high ω6/ω3 ratio have been linked
to protective effects on coronary, autoimmune and inflammatory disorders but also as anti-thrombotic and
regulators of blood pressure [24–26]. Concerning bioactive compounds, their main representatives are the
same of oil in general, namely phenolic compounds such as hydroxytyrosol and derivatives (oleuropein
and tyrosol), tocopherols but also other compounds as hydrocarbons (i.e., squalene) or pigments like
provitamin A compounds [3,26]. However, it must be mentioned that some of these compounds such as
squalene might be lost during refinery, so they can only be found on this type of oil [25].
As mentioned, all these bioactive compounds are known for their biological properties and
positive effects on human health. EVOO inclusion in the diet and its bioactive molecules have been
studied to identify its effects. EVOO is known for having a high content of antioxidant compounds with
protective properties against free radicals. Therefore, it has been pointed out that its high consumption
is related to a generally low risk of suffering colon, breast or skin cancer as well as beneficial effects on
aging and coronary diseases [1]. It has also been proposed as a preventing tool of Alzheimer’s and
other neurodegenerative diseases [3], as anti-inflammatory [7] and also as immune-stimulating [24].
Another study proved that rats fed with EVOO in substitution of lipids and complemented with
physical exercise, could avert cartilage diseases as osteoarthritis [9]. Additionally, EVOO consumption
has shown positive effects on gut microbiota [27]. Some studies have researched the bioavailability
of phenolic compounds of EVOO and found that 55–60% of them can be absorbed, most of them at
Foods 2020, 9, 1014 6 of 31

small intestine [11]. Moreover, their different compounds have shown other beneficial properties like
antimicrobial, antitumor or protective agents against cellular damage [26,28]. More specific studies
have also related EVOO treatment with positive gene regulation and with micro ribonucleic acid
(miRNA) modulation of target genes associated with synaptic plasticity as well as to motor and
cognitive behavior [29]. There are multiple bibliographic references directed towards proving all these
promoting effects. Nevertheless, a specific study must be highlighted, the PREDIMED trial (prevention
through MED, in Spanish). This is one of the largest nutritional studies ever conducted in Spain,
which evaluated the effects on primary prevention of CVD when following a MED supplemented
by EVOO or nuts mix [30]. This project groups together different studies, which have brought to
light several positive consequences: reduction of CVD risk, reduction of C-reactive protein, reduction
of atrial fibrillation, prevention of diabetes and metabolic syndrome, reduction of diastolic blood
pressure, higher protection against breast cancer or lower prevalence of non-alcoholic fatty acid
liver disease [25,31–33]. However, more epidemiologic studies and controlled trials are necessary
to better validate and understand the beneficial effects of EVOO consumption. At last, it is worth
mentioning that new disciplines (encompassed as nutrigenomics) are also working on new approaches
for evaluating the health-promoting effects, characterizing new markers, and understanding their
action mechanisms [29,34].
Regarding all the presented information, this article aims to review the current knowledge
about the unique EVOO composition and its related bioactivities. Therefore, a revision focused
on its beneficial and health-promoting properties, its chemical composition as well as some quality
parameters is presented. To achieve this purpose, a systematic search in several databases was carried
out based on the topic described, all in all, ~5000 documents have been published. Most of the
publications correspond to research articles and book chapters, 56.3% and 15.6%, respectively, followed
by reviews (12.5%). Among them, bibliographic references were chosen according to their concordance
with the covered topics. The extensive information compiled was further classified into the different
sections presented in this review. After analyzing the considerable amount of data available on this
field, it can be said that EVOO is a current topic; although it has been known and used for centuries, it is
still under study, which means EVOO has potential properties and/or applications yet to be discovered.

2. Main Components of EVOO

Virgin olive oils are oils obtained from the fruit of the olive tree (Olea europaea L.) solely by
mechanical or other physical means under conditions, particularly thermal conditions, that do not lead
to alterations in the oil, and which have not undergone any treatment other than washing, decantation,
centrifugation and filtration [35]. The use of said physical techniques allows the preservation of many
compounds that make EVOO one of a kind among plant oils. Its uniqueness is due to the abundance
of fatty acids, PUFA and MUFA but also the occurrence of many bioactive molecules, like hydrophilic
phenols, phytosterols, tocopherols and carotenes that provide several functional properties as well
as a long storage time due to their high oxidative stability [36–38]. Other vegetable oils, like palm
oil, are rich in saturated fats, which are more stable during the cooking or frying processes than the
unsaturated ones, avoiding degradation to toxic compounds, but they do not have beneficial properties
for the human health as the unsaturated one. On the other hand, sunflower oil is rich in unsaturated
fats, especially in linoleic and oleic acids that enhance its healthy properties but decrease its thermal
stability [39]. EVOO has a good PUFA:MUFA balance, which confers it stability properties against
oxidative thermal degradation, particularly regarding the formation of volatile aldehydes, so EVOO is
a proper and recommendable oil to use in food frying [40]. This relation between PUFA and MUFA and
the low content of saturated fats also makes EVOO one of the healthiest vegetable oils to be consumed
raw because it helps reduce LDL cholesterol levels in the human body [41].
The composition of EVOO is a result of several factors like genotypic potential, environmental
factors, fruit ripening, harvest time, agricultural factors (irrigation, sunlight, orchard management)
and also technological factors like the method applied for oil extraction or the storage conditions [42].
Foods 2020, 9, 1014 7 of 31

The concentration of the minor and major fruit components changes and depends on all those variables.
Apart from that, the olives ripening process lasts a few months in which the atmospheric, environmental
and agricultural conditions play a very important role despite the strict genetic control that can be
applied [43,44]. During the maturation and ripening process, the photosynthetic activity decreases
as the oil content in the fruits increases [45]. In the first stage of ripening, denominated green stage,
the ripe fruits have already acquired their final size, so the maturation proceeds, and the chlorophylls
in the skin are slowly swapped by anthocyanins, turning the olives from green to dark violet or purple
until the end of the ripping process. These changes in color define the spotted, purple and black
stages [43,46]. Olives have the highest phenolic compound content at the phase between green and
darker skin, and therefore, the degree of maturation is an important factor to define the right harvest
time that will originate the best quality olive oil [47]. Figure 3 shows a summary of representative
chemical structures of some relevant compounds present in EVOO.
Foods 2020, 9, x  8 of 31 

Lipids 

   
Carbohydrate 

 
 
Pigments 

 
Vitamins 

   
   
Figure 3. Representative chemical structures of some relevant compounds present in EVOO.
Figure 3. Representative chemical structures of some relevant compounds present in EVOO. 
2.1. Primary Metabolites
2.1. Primary Metabolites 
2.1.1. Lipids
2.1.1. Lipids 
Lipids are one of the principal sources of energy for all living beings and additionally, they are also
involvedLipids are one of the principal sources of energy for all living beings and additionally, they are 
in many physiological functions, as their role as a structural component of cell membranes,
also  involved  in  many  physiological  functions,  as  their  role  as  a  structural  component  of  cell 
membranes,  nervous  system,  the  production  of  hormones,  brain  development  and  also  on  the 
promotion of liposoluble vitamins absorption.   
EVOO is widely used in the human diet, especially in the MED and has been long renowned for 
its  many  health‐promoting  properties.  Its  consumption  is  associated  with  reduced  risk  of  several 
chronic illnesses, like diabetes, hypertension, obesity and CVD [48,49]. These health properties are 
Foods 2020, 9, 1014 8 of 31

nervous system, the production of hormones, brain development and also on the promotion of
liposoluble vitamins absorption.
EVOO is widely used in the human diet, especially in the MED and has been long renowned
for its many health-promoting properties. Its consumption is associated with reduced risk of several
chronic illnesses, like diabetes, hypertension, obesity and CVD [48,49]. These health properties are
related to the presence of bioactive compounds like phenolic compounds but also with the high content
in MUFA. Olive oil has a high content in oleic acid transforming it into a healthy fat, especially when
compared with other vegetable oils [40]. This lipid can decrease the risk of CVD due to its effects on
the lipids present in the blood vessels [50]. According to available data, there is 65.2–80.8% of MUFA
in the lipidic fraction of olive oil [51]. Other fatty acids found in the total fatty acids composition of
olive oils are palmitic acid, palmitoleic acid, stearic acid, linoleic acid, α-linolenic acid, and other minor
ones that are listed in Table 2.
Triacylglycerols constitute a big part of the edible oil and a high percentage of the saponifiable
fraction is constituted by MUFA [27]. The principal triacylglycerol detected in olive oil is
oleic–oleic–oleic (OOO), representing about half of the total triacylglycerol portion found in
EVOO. Other triacyclglycerols also present are palmitic–oleic–oleic (POO), oleic–oleic–linoleic
(OOL), palmitic–oleic–linoleic (POL) and stearic–oleic–oleic (SOO) [43,52]. Diacylglycerols and
monoacylglycerols have been identified in VOO at concentrations of 1–2.8% and 0.25, respectively [53].

Table 2. Major EVOO components.

Component Concentration References

Lipids
Fatty acids (%)
Myristic acid C14:0 0.05 [53]
Palmitic acid C16:0 9.4–19.5 [51,54]
Palmitoleic acid C16:1 0.6–3.2 [51,54]
Heptadecanoic acid C17:0 0.07–0.13 [51]
Heptadecenoic acid C17:1 0.17–0.24 [51]
Stearic acid C18:0 1.4–3.0 [51,54]
Oleic acid C18:1 63.1–79.7 [51,54]
Linoleic acid C18:2 6.6–14.8 [51,54]
α-Linolenic acid C18:3 0.46–0.69 [51,54]
Arachidic acid C20:0 0.3–0.4 [51,54]
Eicosenoic acid C20:1 0.2–0.3 [51,54]
Docosanoic acid C22:0 0.09–0.12 [51,54]
Lignoceric acid C24:0 0.04–0.05 [51]
MUFA 65.2–80.8 [51]
PUFA 7.0-15.5 [51]
Other lipids
Diacylglycerols (%) 1–2.8 [53]
Monoacylglycerols (%) 0.25 [53]
Total sterol content (mg/kg) 1000–3040 [43,55]
Tocopherols (mg/kg)
α- Tocopherol 10.2–208 [51,54,56]
β- Tocopherol 0.75–1.05 [51]
γ- Tocopherol 0.7–2.1 [51]
Carbohydrates (mg/kg)
Squalene 200–8260 [43,54,56,57]
Pigments (mg/kg)
Total chlorophylls (mg/kg) 0.15–61.96 [51,58]
Pheophytin-a (mg/kg) 0.08–0.49 [56]
Total carotenoids (mg/kg) 0.53–31.51 [51,54,58]
β-carotene (mg/kg) 0.15–0.67 [56]
Lutein (mg/kg) 0.65–3.60 [56]
Other Compounds
Total phenolic compounds (mg/kg) 213–450 [54]
Triterpene dialcohols (% of total sterols) 0.9–2.8 [55]
β-sitosterol (mg/kg) 530.2–2638.6 [56]
Foods 2020, 9, 1014 9 of 31

Four classes of sterols also take place in olive oil and are commonly used to check its
genuineness because their presence is linked to the quality of the oil. These four classes are
common sterols (4-Desmethylsterols), 4α-Methylsterols, triterpene alcohols (4, 4-Dimethylsterols)
and triterpene dialcohols [43]. Common sterols in EVOO are present in both free and esterified
forms [59]. The leading components of this sterol fraction are campesterol, β-Sitosterol and
∆5-Avenasterol [60,61], and in smaller amounts, it is also possible to find stigmasterol, cholesterol,
cholesterol, brassicasterol, sitostanol, ergosterol, campestanol, ∆7-Cholestenol, ∆7-Avenasterol,
∆7-Stigmasterol, ∆7-Campesterol, ∆5,24-Stigmastadienol, ∆5,23-Stigmastadienol, ∆7,24-Ergostadienol,
∆7,22-Ergostadienol, 22,23-Dihydrobrassicasterol and 24-Methylene-cholesterol [62,63]. The total sterol
content of EVOO varies between 1000 and 2000 mg/kg, being the first value the inferior limit set by the
EU Commission [43]. β-Sitosterol is the main compound in the sterol fraction with values between 75%
and 90% of the total sterol fraction, while ∆5-Avenasterol has values between 5% and 20% [62]. Crop year,
cultivar, ripeness of the fruit, storage time of the olives before oil extraction and geographic influences all
contribute to sterol composition of the final EVOO obtained [64–66]. At the same time, storage time and
conditions of the final product are also factors that can originate several important changes particularly
in the concentrations of each individual sterol [43]. 4-Monomethylsterols are present in smaller amounts
and signify part of sterol biosynthesis as intermediates. They can be found in their free and esterified
forms [67]. The most common are gramisterol, obtusifoliol, cycloeucalenol and citrostadienol [60,62],
and their concentrations vary between 50 and 360 mg/kg of oil [60,68]. Triterpene alcohols, also identified
as 4,4-Dimethylsterol, are a very complex fraction that can be in free and esterified form, and whose
main compounds are butyrospermol, β-Amyrin, cycloartenol and 24-Methylenecycloartanol. In smaller
amounts or trace quantities, cyclosadol, cyclobranol, dammaradienol, germanicol, 24-Tirucalladienol,
24-Methylene-24-Dihydroparkeol, α-Amyrin, taraxerol, 7, parkeol and tirucallol can also be found [62].
Total triterpene alcohol levels range from values of 350 to 1500 mg/kg [68,69]. Lastly, among the
triterpene dialcohols class, erythrodiol (5α-olean-12-ene-3β, 28-diol, homo-olestranol) in free and
esterified form and uvaol (∆12-Ursen-3β,28-diol) are the major triterpene dialcohols found in EVOO [70],
and their presence is mainly affected by cultivation characteristics [68]. EVOO contains levels of total
erythrodiol from 19 to 69 mg/kg of oil, and the free form is inferior to 50 mg/kg [59,68].

2.1.2. Tocopherols
Three isoforms of tocopherols are present in EVOO: α-, β- and γ-tocopherol. α-Tocopherol can be
found in its free form and represents more than 90% of the identified section with ranges from 206.5
to 270.9 mg/kg of oil to 191.5 to 292.7 mg/kg of oil, values that fluctuate with variables as the year of
harvest and spacing between olive trees [71]. Both, the distance between plants and the crop year
influenced statistically tocopherols amount [43,71]. Besides, the high levels of this type of tocopherol
may be linked to the high levels of chlorophyll pigments and the simultaneous necessity for singlet
oxygen deactivation [72].
Lower quantities of β-Tocopherol (~10 mg/kg), γ-Tocopherol (~20 mg/kg) and δ-Tocopherol
(~10 mg/kg) can also be found on EVOO. The total tocopherol concentration seems to decrease in the
ripping of the fruits, and the refining or the hydrogenation process causes their degradation, so they
are only found in the EVOO and VOO [73].

2.1.3. Carbohydrates
There are two hydrocarbons mainly present in olive oil, 2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-
Tetracosahexaene also known as squalene and β-Carotene, which will be addressed in the pigments
section of this review. Squalene is the last metabolite synthesized before the sterol ring formation.
Some of the beneficial health effects of olive oil are partially linked with the presence of squalene, and
it has also demonstrated antitumoral effects against certain types of cancer [74,75]. This compound
constitutes more than 90% of the hydrocarbon fraction and is the most abundant compound in the
unsaponifiable matter, with concentrations ranging from 200 to 7500 mg/kg oil [57]. In a different
Foods 2020, 9, 1014 10 of 31

study, squalene was reported in even higher concentrations, up to 12,000 mg/kg oil. Squalene content
depends on several variables like the type of olive cultivation and the oil extraction technique applied,
and it decreases largely during the refining process so it is present in larger quantities in EVOO and
VOO [76].
The remaining fraction of carbohydrates in EVOO is composed of triterpene and diterpene,
isoprenoid polyolefins, hydrocarbons and n-paraffins [43,76].

2.2. Secondary Metabolites

2.2.1. Phenolic Compounds

The principal group of antioxidants in EVOO are hydrophilic phenols, and these compounds
are extremely relevant when it comes to determining the quality of the oil regarding their sensory
characteristics, like bitterness, pungency and stability [38,77], as well as determining the organoleptic
characteristics of aroma and flavor of each EVOO [53]. The oxidative stability of EVOO depends not
only on the olive variety and quality but also on the harvesting time; cultivation area; the degree of
unsaturation and the levels of antioxidants present from tocopherols, hydrophilic phenols and carotenes.
Besides, factors like oil extraction system and storage conditions also influence its conservation [78].
The correlation of the phenolic content of olive oil and oxidative stability was studied showing
that these two are interconnected [79]. Furthermore, EVOO phenolic compounds provide benefits for
human health in the prevention of several chronic diseases [80,81]. Various studies indicate that EVOO
phenolic compounds have antioxidant, anti-inflammatory, antimicrobial and antitumoral activities, and
they can also modulate gene expression to protect proteins that take part in the cellular mechanisms
involved in the inflammation process, the oxidative stress resistance and in lipid metabolism [82,83].
Therefore, the major antioxidant substances found in EVOO are polar phenolic compounds that can be
present in free, bound or esterified forms [43], and usually, its total phenolic content ranges between 50
and 1000 mg/kg [84], being more common in concentrations between 100 and 300 mg/kg [43]. Likewise,
each EVOO has a different phenolic profile, content and composition due to the differences discussed
above [78].
Phenolic compounds have been largely reported in EVOO composition, with more than 30 different
compounds identified [47,85], being the major phenolic acids present in EVOO hydroxybenzoic,
p-Coumaric, ferulic, gallic, syringic, vanillic, caffeic, o-coumaric and sinapic acids [53]. Other types
of polyphenols that can also be found in EVOO are flavonoids, lignans, hydroxy-isocromans,
secoiridoids and phenolic alcohols. The major flavonoids found in EVOO are luteolin, apigenin
and many of their derivates [86,87], whereas the main lignans present are (+)-pinoresinol and
(+)-1-Acetoxypinoresinol [88], being the usual lignan content in EVOO between 1 and 100 mg/kg [89].
Secoiridoids are rare phenolic compounds present in plant species, nevertheless, they are found in
abundance in Oleaceae species, particularly in O. europaea leaves and fruits. However, they are insoluble
in oil and therefore only a small percentage of these compounds ends up in the final EVOO after the
mechanic extraction process. Nevertheless, they are one of the most important micronutrients on EVOO
for their sensorial and heath properties [38,80]. The most common secoiridoids are demethyloleuropein,
oleuropein, ligstroside and their aglycones, the last ones accounting for approximately 90% of the
phenolic compounds in EVOO [90]. Secoiridoids are hydrolyzed through crushing and malaxation
by enzymatic reactions catalyzed by endogenous b-glucosidases yielding secoiridoid aglycons [91].
The bitterness of olive oil is due to the secoiridoids present, especially the dialdehydic form of
oleuropein aglycone [92].
Isochromans are only found at low concentration in EVOO, and the two mainly found are 1-Phenyl-
6,7-dihydroxy-isochroman and 1-(30 Methoxy-40 -hydroxy)phenyl-6,7-dihydroxy-isochroman [93].
The concentration of these compounds increases during the extraction process because of the hydrolytic
process that originates carbonyl compounds and hydroxytyrosol, which are isocromans derivatives [88].
Finally, the principal phenolic alcohols found in EVOO are tyrosol (p-Hydroxyphenyl ethanol) and
Foods 2020, 9, 1014 11 of 31

hydroxytyrosol (2-[3,4-Dihydroxyphenyl] ethanol). These are present in small concentrations in

fresh olive oil but tend to increase along the storage process because of the hydrolysis of olive oil
secoiridoids [94].

2.2.2. Pigments
The lipophilic carotenoid and chlorophyll pigments occurring in olive oil are responsible for its
characteristic color [95]. The coloration of EVOO is greener in the presence of green olives that have
higher chlorophyll content whereas using mature olives with higher carotenoid content we obtain a
more yellowish oil, so the final color is a result of the proportions of these pigments [96]. EVOO has a
large variety of carotenoids and chlorophylls, from β-Carotene, violaxanthin, neoxanthin, lutein and
other xanthophylls to chlorophyll a and b, pheophytin a and b and other minor derivatives [97,98].
These pigments can be found in amounts up to 100 ppm of total carotenoids and major pigments like
pheophytin up to 25 ppm, β-carotene up to 15 ppm and lutein up to 10 ppm [96], although these values
depend on various factors. The final concentration of each pigment in the final EVOO relies on the
physicochemical characteristics of the fruit, the geographic origin, climate and irrigation conditions
and the mechanic extraction process used. Storage conditions and final packaging also play a role in
pigment concentration and type [96,99,100].
Quality and adulteration of EVOO are sometimes analyzed through the measuring of pigment
compounds because they are correlated with EVOO nutritional value, freshness and antioxidant
properties [99,101]. In addition, pigments can also be used for the authentication of EVOO, by
measuring the chlorophyll and carotenoid pigments of EVOO and comparing them through a quality
index, in which the total chlorophylls to total carotenoids ratio must be around 1 and the ratio of minor
carotenoids to lutein must be around 0.5, to declare it an authentic olive oil [102]. These parameters are
valid for any olive oil regardless of the studied variety. Furthermore, other pigments like violaxanthin,
lutein and total pigment content can be useful as a tool to identify a monovarietal EVOO [102].
Chlorophylls, carotenoids and other minor pigments like lutein and violaxanthin can be stable for
more than one year in storage regardless of the degree of ripeness and variety of the olives used to
produce that oil [103].
The degradation of chlorophylls occurs as a consequence of a pheophytinization reaction that starts
from the malaxation step during the extraction of the EVOO and increases throughout storage time [104].
During that process, the chlorophylls naturally present (a and b) are slowly but irreversibly converted
into pheophytins a and b, where the central Mg+2 ion of the porphyrin ring is exchanged with two
hydrogen atoms making the molecules more stable. These eventually turn to pyropheophytins by the
removal of the carboxymethyl group, which are the ultimate products of chlorophyll degradation [105].

3. Biological Properties of EVOO

The Seven Country Study conducted in the middle of the 20th century first demonstrated the
cardioprotective capacities and health benefits of MED [106], olive oil being the hallmark of this
dietary pattern. Since then, plenty of observational and epidemiological studies have demonstrated
the health-promoting effects of consuming olive oil.
Many health benefits of following a MED enriched in EVOO have been reported by the PREDIMED
trial [107], such as protection against CVD [30] or oxidative damage [108,109] and prevention of breast
cancer [110] and type 2 diabetes mellitus [111]. Many other randomized controlled trials, prospective
study cohorts and meta-analysis, supported by in vitro experiments, indicate that EVOO possesses
interesting biological activities and pharmaceutical-nutritional properties (Table 3) that exert a beneficial
health impact that stands out over other fats and oils. Nevertheless, many attributes of EVOO are also
related to the MED, the context in which its beneficial effects have been mainly evaluated.
Foods 2020, 9, 1014 12 of 31

Table 3. Main bioactivities associated with EVOO consumption.

Bioactivity Studies Description Main Results Ref

RCT, PREDIMED (n = 7447 participants at Following a MED enriched with EVOO
[30,107]
high CVD risk) decreases CVD risk by 30%
PREDIMED observational study For each 10g EVOO/day intake, CVD risk
[112]
(n = 7216 participants) decreases by 10%
10–50 mL/day EVOO reduced diastolic
Systematic review of 15 RCTs [113]
blood pressure by 0.7 mm Hg
Cardioprotection
For every increase of 25 g of olive oil
Meta-analysis of 9 studies (38,673 stroke
consumption the risk of CVD, stroke and
and 101,460 CHD cases from RCT, [114]
CHD was reduced by 18%, 26% and
case-control and prospective studies)
4% respectively
High polyphenol olive oil intake
Meta-analysis of 26 RCTs significantly reduced CVD and [115]
inflammatory markers
European Food Safety Authority 5 mg/day of olive oils polyphenols protects
[116]
health claim. blood lipids from oxidation
RCTs evaluating the effect of olive oils EVOO and high-phenolic olive oils
consumption on blood lipids consumption reduces LDL oxidation in a [117–120]
oxidative state. dose-dependent manner
Antioxidant properties
Adherence to a MED enriched with EVOO
Controlled trials with sub-samples of
improves HDL function and protects [108,109]
PREDIMED cohort (n = 296) and (n = 210)
against LDL oxidation
Lignans present in EVOO show antioxidant
In vitro studies review. [121]
activity in vitro
Regular consumption of EVOO reduces
Meta-analysis of 13 studies based on 9 RCTs [122]
IL-6, CRP and TNF-α levels
Diet supplemented or enriched in olive oil
Anti-inflammatory Meta-analysis of RCTs (3106 participants) [123]
reduces IL-6 and CRP plasmatic levels
capacity
High-phenolic virgin olive oil in breakfast
Randomized crossover study (49 patients) reduces postprandial [124]
inflammatory response.
Meta-analysis of 19 case-control studies Olive oil consumption is associated with
(comprising 13,800 cancer cases and lower odds of developing digestive and [125]
23,340 controls) breast cancers
Cohort-study follow up (2321 breast cancer Inverse association between adherence to
[126]
cases and 1665 controls) and meta-analysis MED and ERN breast cancer
Women following MED enriched in EVOO
RCT with a sub-sample of the PREDIMED
Antitumoral activity showed 62% relatively lower risk of breast [110]
cohort (n = 4152 women)
cancer compared to control low-fat diet
The adherence to MED is associated with
Systematic review and meta-analysis of 83
lower risk of cancer mortality and lower
studies, comprising a total of [127]
risk of breast, colorectal, gastric and liver
2,130,753 subjects
cancers, among others
The phenolic fraction of EVOO, as well as
In vitro experiments of antitumoral activity isolated phenolic compounds, shows
[128–130]
of phenolic compounds on cancer cell lines antitumoral and cytotoxic effect on different
cancer cell lines
Virgin olive oil enriched in phenolic
RCT with 12 hypercholesterolemic compounds consumption favors gut
Gut microbiota [131]
participants bifidobacteria growth and decreases serum
modulation
levels of oxidized LDL
Systematic review and meta-analysis of Polyphenols exert a prebiotic action on gut
[132]
17 RCTs microbiota, improving also CVD and CRC
EVOO: extra virgin olive oil; CVD: cardiovascular disease; CHD: coronary heart disease; RCT: randomized controlled
trial; MED: Mediterranean diet; HDL: high-density lipoprotein; LDL: low-density lipoprotein; IL-6: interleukin-6;
CRP: C-reactive protein; TNF-α: tumor necrosis factor alpha; IBD: inflammatory bowel disease; ERN: estrogen
receptor negative; CRC: colorectal cancer.

3.1. Cardioprotective Properties

The Seven Country Study started in the 1950s first demonstrated the cardioprotective abilities
of MED [106] and has been supported by numerous further studies based both on MED and olive
oil consumption.
Foods 2020, 9, 1014 13 of 31

More recently, the cardioprotective benefits of a MED enriched with EVOO have been proven by
the PREDIMED study. This multicenter, randomized, controlled trial involved ~ 7500 subjects with
potential cardiovascular risk, showing no CVD at enrolment. The PREDIMED trial resulted in a 30%
decrease of a major CVD development, such as stroke or myocardial infarction, in comparison to a
control group that followed a low-fat diet [30,107]. An observational study based on the PREDIMED
cohort indicated that consumptions of 10g EVOO/day are related to CVD risk diminutions up to
10% [112]. A recent systematic review evaluating clinical trials reported that diets enriched with
10–50 mL/day of EVOO (but not diets supplemented with EVOO capsules) significantly decreased
diastolic blood pressure by 0.73mm Hg [113].
In another meta-analysis of randomized controlled trials, case-control and prospective cohort
studies including ~40,000 cases of stroke and ~100,000 cases of coronary heart disease (CHD), it was
reported that for each increase of 25 g of olive oil intake, stroke and CHD risk was reduced by 26% and
4%, respectively. When combined stroke and CHD, olive oil consumption also showed preventing
effects, decreasing the risk of a CVD event by approximately 18% [114].
The preventive role of EVOO polyphenols against CVD was also documented in a meta-analysis
of controlled trials that evaluated the effect of low versus high polyphenol olive oil on markers of CVD
risk. Olive oil consumption ranged from 25–75 mL/day. High polyphenol olive oil significantly reduced
the CVD-risk markers malondialdehyde, oxidized LDL, total cholesterol, high-density lipoprotein
(HDL) cholesterol and also some inflammatory indicators like C-reactive protein (CRP) or interleukin-6
(IL-6) [115].

3.2. Antioxidant Activity

The antioxidant effects of EVOO have been deeply analyzed given the correlation between
oxidative stress and CVD or atherosclerosis. Evidence from several meta-analyses and randomized
controlled trials, such as the EUROLIVE study [117,119], demonstrated in their analyses the reduction
of lipid oxidative damage, the LDL capacity to suffer oxidation and a decrease in oxidized LDL
concentration after high-phenolic VOO and EVOO intake, in a dose-dependent way [118,120]. It is
also worth noting the health claim allowed by the European Food Safety Authority (EFSA) concerning
the protective effects of 5mg/day of olive oil phenolic compounds against LDL oxidation [116].
The PREDIMED cohort was also used to evaluate the antioxidant effects of EVOO. The intervention
group with MED enriched with EVOO reported an improvement of HDL atheroprotective functions,
oxidative status and composition and also increased resistance to LDL oxidation and low grade of LDL
oxidative alterations in comparison to the control low-fat diet [108,109].
Pinoresinol and acetoxypinoresinol, phenolic compounds present in EVOO but not in olive
fruits or refined oils, isolated from EVOO or other sources such as sesame seed, have reported
in vitro antioxidant capacity [121]. The enzymatic hypoxanthine/xanthine oxidase assay reported
a higher antioxidant potential of acetoxypinoresinol, compared to the classic antioxidants, vitamin
E and dimethylsulfoxide (IC50 of 0.91, 12.4 and 2.30 nM, respectively). Pinoresinol possesses the
ability to inhibit LDL oxidation but has shown inconsistent results (IC50 ranging from 24.6–558 µM,
2,2-Diphenyl-1-picryl-hydrazyl-hydrate free radical assay (DPPH) colorimetric assay).

3.3. Anti-Inflammatory Activity

Recurrent or chronic inflammation is a main etiologic factor of several non-communicable
pathologies, whose prevalence is promptly increasing. Thus, the anti-inflammatory effects of EVOO
have gained attention and so have been widely evaluated.
A recent meta-analysis of randomized controlled trials evaluated regular olive oil intake effects
on inflammation [122]. The authors reported a decrease in the levels of IL-6, tumor necrosis factor-α
(TNF-α) and CRP, the three plasmatic inflammatory indicators considered. Such beneficial effects were
shown in studies when EVOO was regularly consumed for more than 3 months. The overall health
status of participants should also be taken into account, as the strongest positive effects were reported
Foods 2020, 9, 1014 14 of 31

among unhealthy groups (with type 2 diabetes mellitus or at risk of CVD). Another meta-analysis
comprising 3106 participants also showed a significant reduction of IL-6 and CRP levels, when olive
oil was consumed as a supplementary or natural intake [123]. The adherence to a high-phenol
VOO breakfast decreased the postprandial inflammatory response, reducing the levels of plasma
lipopolysaccharides in patients with metabolic syndrome [124]. The higher polyphenol content of
EVOO may mediate the mentioned favorable effect as it has demonstrated anti-inflammatory effects
in vitro [133]. The anti-inflammatory effect of phenolic compounds-enriched EVOO has also been
reported in the adipose tissue in mice, with anti-atherosclerotic effects [134].
Due to these mentioned capacities, EVOO has also been proposed as a potential therapeutic product,
reducing inflammation in inflammatory bowel diseases, including ulcerative colitis and Crohn’s
disease, being both related to chronic inflammation of the intestinal mucosa [135,136]. The benefits
of EVOO consumption were evaluated in other autoimmune and chronic inflammatory diseases
such as rheumatoid arthritis [137], systemic lupus erythematosus [138] or multiple sclerosis [139,140]
with promising results in murine models. Besides, both in vitro and in vivo studies outline that the
anti-inflammatory activity of EVOO provides a neuroprotective effects that could prevent cognitive
decline and, therefore, the development of Alzheimer’s disease or elderly dementia [3,141].

3.4. Antitumoral Activity

Traditionally, a lower incidence of cancers such as breast, colorectal, endometrium and prostate
cancer has been observed in Mediterranean countries linked to dietary factors, when compared to the
USA or other European countries [142]. The antitumoral and anticancer activities of EVOO, as well as
of specific fractions or isolated compounds, have been widely studied and evidenced both in vitro
with cell cultures and in vivo with animal models, observational cohort studies and clinical trials [143].
Evidence from 19 case-control observational studies, including in total 13,800 cancer cases and
23,340 controls, suggests that olive oil intake is inversely associated with the risk of having any type of
cancer (34% lower likelihood of cancer for high olive oil intake) [125]. More precisely, this meta-analysis
associated lower odds for developing breast and digestive cancer with olive oil consumption (log odds
ratio of −0.45 and −0.36, respectively).
The strongest beneficial effects of EVOO concerning cancer have been described in breast cancer
prevention. A meta-analysis reported a statistically significant inverse association between estrogen
receptor-negative postmenopausal breast cancer and the adherence to MED [126]. Breast cancer
incidence was also included in the PREDIMED trial, which included ~ 4200 women. Those allocated
to the MED enriched with EVOO showed a 62% relatively lower risk of breast cancer, compared to
women who followed a low-fat diet [107,110]. To our knowledge, no recent large case-control or
prospective cohort studies have been conducted about the relationship between colorectal cancer risk
and EVOO consumption. Nevertheless, a meta-analysis has determined that MED consumption is
related to a 14% lower risk or developing colorectal cancer [127]. Recently, it has been suggested that
the antitumoral activity of EVOO, lowering colorectal tumor incidence in rats, could be mediated by
epigenetic mechanisms, such as miRNA and deoxyribonucleic acid (DNA) methylation [144].
In vitro experiments have shown that both the phenolic fraction of EVOO and specific compounds
such as hydroxytyrosol, caffeic acid, p-Coumaric acid, 1-acetoxypinoresinol and pinoresinol, among
others, have antitumoral activity against breast cancer cell lines [128,129]. Other in vitro studies about
the cytotoxic effect of the EVOO lignan pinoresinol have reported variable results, depending on
the cancer cell line tested. Pinoresinol shows a cytotoxic effect against breast, lung and prostate cell
lines, and it inhibits cell viability of colon cancer cells. A synergic effect with other EVOO phenolic
compounds have been reported [121,130].

3.5. Positive Modulation of Gut Microbiota

Much of the health benefits of olive oil consumption are attributed to the metabolism of the
phenolic compounds carried out by the gut microbiota [145]. It is estimated that 90–95% of total
Foods 2020, 9, 1014 15 of 31

phenolic compounds intake is not absorbed in the small intestine; therefore, they remain in the large
intestinal lumen where they are subjected to gut microbiota metabolic activities. As a consequence,
polyphenols are converted to low-molecular-weight compounds that are absorbed and responsible for
the health benefits derived from polyphenol-rich food, such as EVOO [132,146,147].
A recent review and meta-analysis of randomized controlled trials supports the prebiotic action
of polyphenols, capable of modulating and improving intestinal microbe populations, which affects to
CVD and colorectal cancer markers [132]. Furthermore, another randomized controlled trial showed
that the ingestion of VOO enriched with phenolic compounds decreases the serum levels of oxidized
LDL in hypercholesterolemic participants as well as increases the presence of Bifidobacterium spp in
feces. Slight changes in the profile of fecal microbial metabolites were also reported. These data
suggest that the cardioprotective effect of phenolic compounds could be mediated by the populations
of bifidobacteria present in the gut microbiota [131]. The possible modulation of gut microbiota by
olive oil and its role in cancer prevention, especially colorectal cancer, has also been suggested [148].
However, the complexity of the human diet, the lack of accuracy in the measurement of dietary
intake and the extensive variation on microbiota between individuals challenges the evaluation of how
diet changes modulate gut microbiota and its metabolic activity [149].

3.6. Other Bioactivities

The detailed description above was limited to the main bioactivities attributed to EVOO and
its specific components. However, some other activities of biological relevance are under study.
A recent extensive review highlighted the anti-aging properties of the major phenolic compound in
EVOO, hydroxytyrosol, suggesting that it can contribute to the correct regulation of mechanisms that
maintain cell homeostasis, such as mitogen-activated protein kinase (MAPK) and mammalian target of
rapamycin (mTOR) pathways, whose imbalance is a hallmark of aging [150]. Moreover, this phenolic
compound also modulates the metabolism of adipose tissue, stimulating mitochondrial biosynthesis
and increasing the function of the mitochondrial respiratory chain in vitro [151].
EVOO consumption has also been associated with the enhancement of blood circulation and
coagulation, by reducing platelet aggregation (mechanism related to CVD) and decreasing the levels of
coagulation factor VII, effects attributed to minor components of EVOO [12]. Interestingly, a study
carried out in murine models reported that polyphenols present in EVOO may improve learning and
memory, by reversing the oxidative damage in the brain associated with aging and diseases related to
the production of amyloid-β protein [152].
As a final remark, hydroxytyrosol, pinoresinol and oleuropein from EVOO have been reported to
possess antimicrobial capacity. Pinoresinol has shown antifungal activity against several pathogenic
fungi such as Fusarium verticillioides, Fusarium graminearum and Candida albicans [121]. Additionally,
oleuropein and hydroxytyrosol were found to be effective against fungi and several strains of bacteria,
viruses, including human immunodeficiency viruses (HIV) and parasites [151,153].

4. EVOO Quality Regarding the Chemical Composition

As aforementioned, olive oil qualification as “extra virgin” demands various requisites. On one
hand, it must be obtained using exclusively mechanical procedures, which grants the “virgin” label.
On the other hand, it must show a low free acidity (<0.8%), low peroxide value, high antioxidant
activity, a perceptible fruity taste and no sensory defects, acquiring in this way the label “extra”.
However, once those badges are achieved, it is important to attend to some storage factors to maintain
and assure these quality characteristics until the oil is used [154,155]. Figure 4 shows a summary
concerning the quality-involved parameters.
Foods
  2020, 9, 1014 16 of 31

 
Figure 4. Parameters and factors concerning EVOO quality and its preservation.

4.1. Analytical Techniques

Parameters usually taken as EVOO quality indicators, as well as authenticity and fraud control
markers are acidity, peroxide value, absorption coefficients K232 and K270 , color, total phenolic content
(TPC), fatty acids composition and volatile compounds’ profile. Spectrophotometric techniques are the
most used to perform stability and quality analysis of EVOO since they are based on quick, simple,
cheap and non-destructive methodologies offering the possibility of executing real-time controls.
Several compounds are studied through these methodologies, such as certain pigments (chlorophylls,
pheophytins, carotenoids, etc.) which are an easy and quick way to assess the degradation of EVOOs
caused by temperature and/or light. The absorbance range analyzed for this purpose is between 350
and 750 nm [154,156].
Phenolic compounds, in addition of being responsible for a large part of the beneficial effects of
EVOO, actively intervene in its quality, extending its useful life thanks to its antioxidant power [157].
This is the reason why its concentration is analyzed as a quality measure since the loss of these
compounds is parallel to the degradation of EVOO. They are usually analyzed spectrophotometrically
following the Folin–Ciocalteu method, as TPC (at absorbances of ~750 nm) and using gallic acid as
standard, a methodology that offers speed, ease and reproducibility [155].
For the subsequent analysis of visible spectroscopy data and the prediction of optimal storage
conditions, it is possible to use certain mathematical tools, such as multilayer perceptrons, capable of
finding non-linear relationships between the variables involved, whether dependent or independent,
based on artificial neural networks. Despite being a tool with many chemical applications including
the food industry, it is important to add that it does not provide very reliable extrapolations since the
model is based reliably on the breadth of the experimentally studied data [154,158].
Other analytical techniques that can be used are high-performance liquid chromatography or gas
chromatography (HPLC, GC), nuclear magnetic resonance (NMR), differential scanning calorimetry
(DSC) or vibrational spectrometry, which includes Raman and infrared spectrometry. These processes
are more accurate when quantification is desired, although they are also more time-consuming, they
require more specific types of equipment and are usually aimed at determining the origins of the
EVOO or performing anti-fraud controls [159,160].1 
  As an innovative and emerging methodology, it is worth highlighting the use of chemosensors
and  biosensors. These devices were initially based on electrochemical detection but have evolved in
the direction of being coupled to smartphones, allowing to combine spectrophotometric techniques
Foods 2020, 9, 1014 17 of 31

with optical image detection from the smartphone’s camera, resulting in on-site analysis of samples
without requiring any pretreatment and without the need for a laboratory environment [155,161].

4.2. Effect of Olive Variety

As expected, the variety of olives used to produce EVOO determines its final composition,
constituting an internal factor that influences stability and quality [162,163]. Not only the variety but
also the geographical location and the growing conditions, as well as the applied mechanical production
process, affect its quality [51,164,165]. Taking all these variables into account, it is possible to obtain
EVOOs with different concentrations of compounds in such wide ranges as 50.5–80.5% for oleic acid,
91–665 mg/kg for α-tocopherol and 50–900 mg/kg for secoiridoid derivatives and lignans [166]. As an
example, two varieties are discussed in this section, Empeltre varietal and Manzanilla Cacereña varietal.
Based on the analyzes performed by visible spectrophotometry Manzanilla varietal contains a major
amount of pigments (chlorophylls and their derivatives such as pheophytins, as well as carotenoids),
and greater absorbance is observed on EVOO produced using Manzanilla Cacereña varietal. This fact
also affects EVOO’s coloration, since the greater the number of pigments, the more yellowish-greenish
the tonality. Likewise, a higher density is also observed in the liquid produced with the variety of
olives that present a higher concentration of pigments [154].
Another study based on the comparison of EVOOs from two different varieties, Manzanilla and
Picual, determined that the Manzanilla variety has a higher PUFA content and a lower MUFA content
than the Picual variety, although a decrease in MUFA was found proportional to the increase in PUFA
during degradation of both types of EVOO. Similarly, the Picual variety shows a higher content of
polyphenols than Manzanilla, and therefore, a higher antioxidant activity. According to these authors,
the compounds most influenced by the variety of olive used are fatty acids [167].
Therefore, the amount of oleic acid and natural antioxidant compounds, namely tocopherols
and phenolic compounds and particularly, oleuropein, that are present in the final composition of the
EVOO, is directly proportional to its better resistance to degradation. This fact is due to their capacity
to avoid the formation of volatile compounds, responsible for the rancidness defect, and consequently
enhance its shelf-life [166,168].

4.3. Effect of Light

Exposure to sunlight is one of the main factors that promote and accelerate the degradation of
chemical compounds, such as pigments or phenolic compounds. Besides, it is one of the key factors as
the EVOO is highly exposed to light during its stay in supermarkets and points of sale [169]. A study
that analyzed the effect of light in 14 commercial varieties of EVOOs, recreating the conditions to which
they are subjected in stores for 6 months, showed that light has a greater degrading effect on those
compounds that contribute to the health-promoting properties, such as phenolic compounds, than on
the compounds responsible for its quality, like acidity or oleic acid content. The quantification of the
different analyzed compounds: oleic acid, total phenolic compounds, MUFA, PUFA, α-tocopherol,
lignans and oleuropein, was carried out using HPLC and concluded that the loss of the “extra”
condition can be predicted in time according to the initial concentration of oleuropein that a certain
EVOO presents, thus this compound shows a strong antioxidative power [166]. Another fast and
efficient way to perform light degradation analysis is by measuring pigments, such as chlorophyll.
Nevertheless, light exposure is not recommended during the storage period if extending EVOO’s
shelf-life and preserving its properties is desired [170,171].

4.4. Effect of Temperature

Another factor to consider during EVOO conservation is temperature. Several studies carried
out with different EVOOs from different sources (Almazara del Ebro and As Pontis) highlight this
fact. Comparing the absorption spectra in the visible ultraviolet of the oils, temperatures close to
Foods 2020, 9, 1014 18 of 31

40 ◦ C cause greater adulterations in the composition of pigments than low temperatures, such as 3 ◦ C,
although the ideal temperature for its conservation is around 23 ◦ C [154,172].
As a curious fact, when EVOO is stored at low temperatures, a crystallization process caused by
freezing can be observed. In this process, triacylglycerols start to freeze through the methyl fraction,
which is just the part that melts first in the opposite process. Through the analysis of the freezing and
melting kinetics, information related to the geographical origin of the EVOO can be obtained [160,173].
Moreover, low temperatures application during EVOO processing has shown to be an effective strategy
to improve its yield and quality [174].

4.5. Effect of Time

Time, as in most aspects of life, also plays a key role in the quality of the EVOO. Generally
speaking, the shelf life of EVOOs is between 9 and 18 months, depending on other concomitant factors
such as temperature or chemical composition, since when it is stored at 3 ◦ C or room temperature, less
degradation is observed than at 40 ◦ C during the same period analyzed (88 days). In the same way,
depending on the nature of the compounds that are present, they will suffer more or less degradation
over time. Going back to previous examples, EVOOs obtained from the Empeltre and Manzanilla
Cacereña varieties suffered different degradations under the same conditions: the Manzanilla Cacereña
variety generated an EVOO whose visible absorption spectrum suffered a drastic decline in the first
10–12 days of storage after opening the bottle, regardless of temperature, while the Empeltre variety
maintained a more linear spectrum over time, suggesting that the Manzanilla variety contained more
easily degradable compounds [154]. Likewise, a longer study in time, in which the degradation
suffered by 14 varieties of commercial EVOOs during 22 months of storage in the absence of light
and at room temperature was analyzed, showed that peroxide values increase proportionally with
storage time, so it is recommended that consumption occurs as close as possible to the production
process [175].

4.6. Effect of Oxygen Presence

Oxygen presence is an essential factor in the oxidative degrading process. Moreover, EVOO
oxygen content once produced, oxygen permeability while packaging and headspace oxygen are
important factors influencing deterioration of lipids and overall EVOO quality and shelf-life during
storage [169]. A study conducted on the effect of headspace oxygen concentration and the presence of
light on the quality of EVOO over time determined that the shelf life of EVOO could be maximized,
exceeding 12 months of stability, by applying headspace oxygen between 2% and 5%. However,
intending to preserve the majority of beneficial compounds possible, such as pigments and polyphenols,
it is preferable to carry out preservation in the absence of light and at low temperatures, around 10 ◦ C,
as well as using headspace oxygen of 2%. For this purpose, modified atmosphere packaging can be
used, in which oxygen is replaced by other inert gases such as argon or helium [176].

4.7. Effect of Packaging Material

Finally, another external factor that influences the quality of the EVOO over time is the packaging
material chosen. Some of the materials of choice for this purpose are tinted glass, polyethylene
terephthalate (PET), tinplate, aluminum, tetra bricks or bag-in-box packages [177]. Various studies
position glass as one of the materials that best preserves EVOO, since it is an inert material and,
being tinted, reduces the passage of light. On the other hand, polypropylene and polyethylene are
materials that show high oxygen permeability, so they are not recommended when preserving EVOO
properties for a long time [169,178]. However, as happened before, this factor also depends on other
conditions. The use of bag-in-box packages at home environment between 22 and 37 ◦ C seems to
benefit the preservation and lengthening of the EVOO shelf-life, as demonstrated in a study in which
this packaging method was compared to tinplate steel containers [179].
Foods 2020, 9, 1014 19 of 31

5. Possible Applications of EVOO beyond Nutritional Purposes

5.1. Extractive Solvent

EVOO used in preparing culinary dishes offers numerous advantages, whether the implicit
biological activities listed above or certain physicochemical characteristics that become indirectly
beneficial. An example is the extractive capacity of some lipophilic compounds contained in the rest of
the ingredients present in the recipes, managing to isolate them from the protection of the original
matrix and, therefore, increase their bioavailability in the body, as occurs with lycopene present in
tomato, the largest known source of this compound. This transfer of the lycopene from the tomato to
the EVOO is achieved with a simple application of microwave heat to the food which additionally
favors its isomerization, obtaining Z-Lycopene, the isomer that is mostly absorbed and is found in
plasma. Hence, this EVOO capacity could be used to maximize extractive processes of certain lipophilic
molecules, allowing their safe application in the food industry and avoiding the use of contaminating
organic solvents [180].

5.2. Therapeutic Application

As previously mentioned, the bioactivities associated with EVOO are numerous, many related to
its antioxidant capacity. The inclusion of EVOO in the daily diet supposes health benefits and helps to
prevent the appearance of certain diseases such as heart and vascular, neurodegenerative, cytotoxic,
metabolic and inflammatory diseases, among others, [3,181] due to its content in functional compounds
such as polyphenols, tocopherols, carotenoids, sterols, fatty acids, squalene, etc. Therefore, EVOO
becomes an attractive candidate for possible applications in therapeutics, constituting a potential
strategy to use it in certain treatments that require its properties [182]. An example of this is a study
conducted on the ability of EVOO to counteract the effect of chromium poisoning, a compound
classified among the 20 most dangerous for humans and for the environment by the Agency for Toxic
Substances and Disease Registry (ATSDR), which is capable of causing both acute and chronic toxicity,
depending on the exposure. Some of the damages it causes in humans are neurotoxicity, genotoxicity,
carcinogenicity, dermatotoxicity, or hepatotoxicity. So the oral administration of 300 µL of EVOO
in animal models of rats previously intoxicated with chromium manages to reduce the genotoxic,
immunotoxic and cytotoxic effects of the most harmful form of this compound, its hexavalent derivative,
reducing the consequences of punctual or prolonged exposure [183].
Another way of approaching this point is the addition of more beneficial compounds to EVOO
since it is a product daily consumed in many countries, which means transforming it into a vehicle
enriched with functional compounds so that the health-promoting effects that EVOO already owns
per se are increased [184]. An effective approach to incorporate such compounds into EVOO is using
encapsulation, which preserves them from becoming rancid while improving the stability of those
functional compounds such as vitamins or calcium [185–187].

6. Conclusions
EVOO, also colloquially known as “liquid gold” is a natural product of unquestionable value
and not only in the monetary sense of the term but also for its recognized properties and advantages
on health. It is considered food for its nutritional value and is practically a requirement of the MED,
contributing to the benefits associated with it. Its chemical composition allows EVOO to be classified
as a lipophilic product since lipids are the main compounds, especially MUFA, followed by PUFA.
This lipid fraction is responsible for protective properties on coronary, autoimmune and inflammatory
disorders, granting anti-thrombotic and regulation effects of blood pressure Although in a smaller
quantity, other compounds such as tocopherols or polyphenols are also present, which are associated
with the powerful antioxidant and inflammatory activity of EVOO, among other qualities. For all
these reasons, the inclusion of this golden ingredient in the diet, in addition to offering characteristic
organoleptic properties, provides substances capable of preventing the appearance or development
Foods 2020, 9, 1014 20 of 31

of diseases of various nature, from heart and circulatory diseases to metabolic disorders, including
carcinogenic processes. Compared to other types of olive oil, EVOO must meet more stringent
requirements that give it the right to possess that nomenclature. However, it is a shame that, during
distribution and storage, the previously achieved quality is corrupted, leaving some of its most
distinguished and desirable properties on the way. Therefore, the factors that promote the degradation
of its components must be known, intending to reduce the negative impact that may originate in
EVOO and thus increasing its shelf-life. These factors can be divided into intrinsic factors, such as
the variety of olives and their cultivation conditions, about which little can be done once EVOO has
been produced; and external factors, which include exposure to light, temperature, time or the type
of material used for packaging. Due to the functional properties it presents, other applications for
EVOO could also be considered, such as its use in therapies in which other treatments are not very
effective, like certain neurodegenerative diseases or as a vehicle for the administration of certain
pharmacological compounds in a comfortable way, by simply adding them to the diet. Future studies
aimed at optimizing and maximizing the capabilities and applications of this product will, without a
doubt, be welcomed and well received by the industry, not only food and agriculture but also by the
pharmaceutical and cosmetics industries.

Author Contributions: Formal analysis, M.A.P., J.S.-G., C.J.-L., M.C., C.L.-L., M.G.-G., J.M.L. and F.J.B.;
investigation, C.J.-L., M.C., C.L.-L., M.G.G. and F.J.B.; methodology, C.J.-L., M.C., C.L.-L., M.G.-G., J.M.L.
and F.J.B.; supervision, M.A.P. and J.S.G.; validation, M.A.P. and J.S.G.; writing—original draft, M.A.P., J.S.-G.,
C.J.-L., M.C., C.L.-L., M.G.-G. and F.J.B.; writing—review and editing, M.A.P. and J.S.-G. All authors have read
and agreed to the published version of the manuscript.
Funding: The research leading to these results received financial support from Programa de Cooperación
Interreg V-A España—Portugal (POCTEP) 2014–2020 (projects Ref.: 0181_NANOEATERS_01_E and Ref:
0377_IBERPHENOL_6_E) supporting the pre-doctoral work of C.J.L. To MICINN supporting the Ramón&Cajal
grant for M.A.P. (RYC-2017-22891); to the company AlgaMar (www.algamar.com) and Axudas Conecta Peme
(Xunta de Galicia) supporting the IN852A 2018/58 NeuroFood Project that financed the pre-doctoral grant for
C.L.L.; to EcoChestnut Project (Erasmus+ KA202) for supporting the work of M.C.R.; to Ibero-American Program
on Science and Technology (CYTED-AQUA-CIBUS, P317RT0003) for financial support. This project has received
funding from the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH
Project (H2020-BBI-JTI-2019), the JU receives support from the European Union’s Horizon 2020 research and
innovation program and the Bio Based Industries Consortium.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations List
Generic
CHD Coronary Heart Disease
CVD Cardiovascular Diseases
DSC Differential Scanning Calorimetry
DPPH 2,2-Diphenyl-1-picryl-hydrazyl-hydrate free radical assay
EFSA European Food Safety Authority
EU European Union
EVOO Extra Virgin Olive Oil
GC Gas Chromatography
HPLC High Performance Liquid Chromatography
HIV Human Immunodeficiency Viruses
IC50 Half Maximal Inhibitory Concentration
IOC International Olive Council
MED Mediterranean Diet
NMR Nuclear Magnetic Resonance
PREDIMED Prevention Through Mediterranean Diet
USA United States of America
VOO Virgin Olive Oil
Foods 2020, 9, 1014 21 of 31

Biological and Chemical Compounds

CRP C-Reactive Protein
DNA Deoxyribonucleic Acid
HDL High-Density Lipoprotein
Hg Mercury
IL-6 Interleukin-6
LDL Low-Density Lipoprotein
MAPK Mitogen-Activated Protein Kinase
miRNA Micro Ribonucleic Acid
mTOR Mammalian Target of Rapamycin
MUFA Monounsaturated Fatty Acids
OOL Oleic–Oleic–Linoleic Triacylglycerol
OOO Oleic–Oleic–Oleic Triacylglycerol
PET Polyethylene Terephthalate
POO Palmitic-Oleic-Oleic Triacylglycerol
POL Palmitic–Oleic–Linoleic Triacylglycerol
PUFA Polyunsaturated Fatty Acids
SOO Stearic–Oleic–Oleic Triacylglycerol
TPC Total Phenolic Content
TNF-α Tumor Necrosis Factor-A

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