Antioxidant Properties and Fatty Acid Profile of Cretan Extra
Virgin Bioolive Oils: A Pilot Study
Dariusz Nowak , MichałGośliński, and Cezary Popławski
Department of Nutrition and Dietetics, Faculty of Health Sciences, Ludwik Rydygier Collegium Medicum in Bydgoszcz,
Nicolaus Copernicus University in Toruń,Dębowa 3, 85-626 Bydgoszcz, Poland
Correspondence should be addressed to Dariusz Nowak; firstname.lastname@example.org
Received 17 February 2021; Revised 18 March 2021; Accepted 19 March 2021; Published 26 March 2021
Academic Editor: Eduard Hernández
Copyright © 2021 Dariusz Nowak et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Olive oil is considered a valuable ingredient of human diet. It is a good source of mono- and polyunsaturated fatty acids, as well as
other bioactive compounds, especially polyphenols. The composition of olive oil depends mainly on the variety of plant, cultivation
practices, and manufacturing conditions. Traditional processing methods may ensure better quality and health beneﬁts. Therefore,
the aim of the study was the evaluation of antioxidant properties and fatty acid proﬁle of Cretan extra virgin bioolive oils. These
ones were compared with commercial Spanish, Italian, and Greek extra virgin olive oils. Obtained results showed that sample
Cretan 1 had about 15% higher antioxidant capacity and about 60% higher total polyphenol content than commercial
counterparts. This one had also a favorable proﬁle of fatty acids, especially 20% more linoleic acid. We concluded that
traditional production methods, using millstones, cold pressing, and without centrifugation and ﬁltration ensure better olive oil
quality and related health beneﬁts.
Olive oil is a popular source of fats in the human diet, especially
in the Mediterranean diet. In general, olive oil is characterized
by high amounts of monounsaturated fatty acid (MUFA) and
contains minor components with biological properties (e.g.,
phenolic compounds, pigments, squalene, sitosterols, and
triterpenes) . A speciﬁc composition of olive oil fatty acids
and other bioactive compounds, such as polyphenols, has
been proven to be protective against the development of car-
diovascular diseases [2–5]. The content of these components
depends on the cultivar, climate, harvesting time, and the
manufacturing conditions [1, 6].
According to the European Union legislation, olive oil is
classiﬁed into categories reﬂecting its quality and organolep-
tic properties, namely, extra virgin olive oil (EVOO), virgin
olive oil (VOO), lampante virgin olive oil (LVOO), reﬁned
olive oil (ROO), and also olive oil (OO) [7, 8]. EVOO pro-
duced by mechanically pressing the olives is considered the
best quality and possesses the best composition of bioactive
compounds, which aﬀect its health beneﬁts [9, 10]. These olive
oils contain an array of phenolic antioxidants, from three
major chemical classes: the simple phenolics (tyrosol and
hydroxytyrosol), the secoiridoids (oleuropein aglycon and
oleocanthal), and lignans [11–13] and phenolic acids (vanillic,
chlorogenic, gallic, caﬀeic, p-coumaric, and ferulic) .
Greece is ranked third after Spain and Italy in virgin olive
oil production. In Greece and Italy, the extra virgin olive oil is
consumed in majority, whereas in Spain, this represents less
than half. The dominant olive cultivar in Greece is the cv.
Koroneiki, especially on Crete. This gives oils of medium to
high content of phenolic compounds. Greek extra virgin
bioolive oils, produced mainly with traditional, nonintensive
cultivation practices, are mostly of exceptional quality [14,
15]. Recent study shows that Greek EVOO had a high con-
tent of oleocanthal and oleacein and their derivatives .
These compounds are considered key oxidation inhibitors.
It is worthy to note that oleacein has been declared a more
potent antioxidant than hydroxytyrosol [16, 17]. A study
showed that the speciﬁc phenolic content within EVOO
can aﬀect human health [18, 19]. Literature data for Greek,
especially Cretan extra virgin olive oils, are not as popular
International Journal of Food Science
Volume 2021, Article ID 5554002, 6 pages
as those for Spanish or Italian ones. Therefore, the aim of the
study was the evaluation of antioxidant properties and the
fatty acid proﬁle of Cretan extra virgin bioolive oils. These
ones were compared with commercial Spanish, Italian, and
Greek extra virgin olive oils.
2. Materials and Methods
2.1. Materials. The analysis comprised ﬁve extra virgin olive
oils. Spanish, Italian, and Greek samples were purchased at a
local health food store and declared as extra virgin. Cretan
extra virgin bioolive oils were produced from cv. Koroneiki
(Chania region, northwest Crete) and have been certiﬁcated
by BIO HELLAS no. B-515504 (Cretan 1) and no. B-515573
(Cretan 2). Moreover, sample Cretan 1 comes from two hun-
dred years of organic cultivation (i.e., manual harvesting,
without artiﬁcial irrigation), and this olive oil was produced
by a traditional method using millstones, cold pressing, and
without centrifugation and ﬁltration. All the analyzed olive
oils were produced between April and May 2018 (bottled into
250 mL) and had at least one-year best-before date.
2.2. Methods. For the spectrophotometric assays, 1±0:01 g of
olive oil was extracted with 9.3 mL n-hexane and homoge-
nized using a vortex mixer for 30 sec. For each olive oil, ﬁve
parallel samples in three replicates were prepared.
2.3. DPPH Assay. The antioxidant capacity of the olive oils was
determined in extracts (1 g of olive oil and 9.3 mL n-hexane) by
a standard DPPH methodusing 0.1 mM methanol solution of a
1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich) .
This method is widely used to test the antioxidant capacity
of foods, including olive oils . The absorbance was mea-
sured on a Hitachi U-1900 spectrophotometer at 517 nm after
30 min of incubation in the dark at room temperature. For
each olive oil, ﬁve parallel samples in three replicates were
analyzed, from which the mean value was calculated. The
antioxidant capacity was expressed as milligrams of Trolox
per 1 liter of olive oil (mg Tx/L).
2.4. ABTS Assay. The antioxidant capacity of the olive oils
was determined by Re et al.’s  method using 2,2′-azino-
bis-(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium
salt (ABTS, Sigma-Aldrich). The absorbance was measured
on a Hitachi U-1900 spectrophotometer at 734 nm after
6 min of incubation in the dark at room temperature. For
each olive oil, ﬁve parallel samples in three replicates were
analyzed, from which the mean value was calculated. The
antioxidant capacity was expressed as milligrams of Trolox
per 1 liter of olive oil (mg Tx/L).
2.5. Folin-Ciocalteu Assay. The total polyphenols content
(TP) of the samples was determined by the Folin-Ciocalteu
assay . The absorbance was measured on a Hitachi U-
1900 spectrophotometer at 765 nm after 30 min incubation
in the dark at room temperature. The results were expressed
as milligrams of gallic acid equivalents per 1 liter of olive oil
2.6. Determination of Fatty Acid Proﬁle. The fatty acid proﬁle
was analyzed according to the European Union Commission
Regulation  using a Hewlett-Packard 6890 gas chromato-
graph, equipped with a ﬂame-ionization detector (FID) and a
SP-2560 fused silica capillary column (100 m × 0:25 mm;
0.20 μmﬁlm thickness). The injector and detector tempera-
tures were set at 220
C and 240
C, respectively. Helium was
used as a carrier gas with a ﬂow rate of 1 mL min
. The anal-
ysis was performed at the following temperature program:
C held for 5 min, then increased at rate of 4
C, and held for the subsequent 20 min. The total run
time was approximat ely 50 min. Individual fatty acids were
identiﬁed by comparing their retention times with standards
and quantiﬁed as a percentage of the total fatty acids.
2.7. Statistical Analysis. The results were statistically analyzed
by calculating the mean and standard deviation. The inter-
pretation of the results was performed with MS Excel 2010
Analysis ToolPak software, with one-way analysis of variance
(ANOVA) using Tukey’s as a posttest: diﬀerent letters in the
same row indicate statistical signiﬁcance (at p<0:05).
3. Results and Discussion
3.1. Antioxidant Properties. Obtained results showed that
among all the analyzed olive oils, the signiﬁcantly highest
antioxidant capacity, both the DPPH and ABTS assays, was
determined in sample Cretan 1, i.e., 348 ± 5:0and 623 ± 7:0
mg Tx/L, respectively (Table 1). Olive oil Cretan 1 had also
the highest total polyphenol content (658 ± 40 mg GAE/L).
Other extra virgin olive oils were characterized by TP about
400 mg GAE/L, with no signiﬁcant diﬀerences between them
(p<0:05). The sample Cretan 2 was similar in antioxidant
capacity to commercial Greek and Italian olive oils, but their
values were slightly lower than the Spanish one.
Other researchers reported a great variability in TP
ranged from 50 to 1000 mg/kg (usually 100-300 mg/kg) in
olive oils of diﬀerent origin, i.e., Greek, Italian, Spanish,
Israeli, and Turkish [14, 25, 26]. Total polyphenol content
has been repeatedly proved to be a marker for olive oil stabil-
ity, which is also related to characteristic taste. Moreover, TP
was considered a parameter which categorized olive oils as
low (50–200 mg GAE/kg), medium (200–500 mg GAE/kg),
and high (500–1000 mg GAE/kg) . In addition, literature
data stated that cv. Koroneiki gives olive oils of higher TP
content, in general . In another study, ﬁfty-ﬁve mono-
or multivarietal extra virgin olive oils from Italy, Spain,
France, Turkey, Greece, Portugal, Australia, the USA, and
South Africa were analyzed. The oil samples produced from
Italian cv. Coratina possessed the highest amount of poly-
phenols and antioxidant capacity, whilst the sample pro-
duced from French cv. Cayon contained the lowest amount.
Among the analyzed olive oils was also some Cretan of cv.
Koroneiki, of which its antioxidant properties were average
in relation to the others . Moreover, Sicari  deter-
mined the antioxidant capacity, TP, and structure of poly-
phenolic compounds in three diﬀerent Italian extra virgin
olive oils from the province of Reggio Calabria. The analyzed
samples had a high content of polyphenols ranged 370-
2 International Journal of Food Science
530 mg GAE/kg. Moreover, in this study, a positive correla-
tion was observed between the antioxidant activity (deter-
mined by DPPH and ABTS assay) and the concentration of
total polyphenols . Our results also conﬁrmed positive
correlation between the DPPH and ABTS methods
(r=0:902). Furthermore, the ABTS assay resulted in a much
higher value of antioxidant capacity, which was also con-
ﬁrmed in other studies [30, 31]. De Bruno et al.  reported
that it could be due to the diﬀerent composition of analyzed
samples containing hydrophilic and lipophilic antioxidant
compounds. The ABTS assay is more applicable to both
hydrophilic and lipophilic antioxidant systems, whereas
DPPH assay is more related to hydrophobic system response
. In addition, our results showed a higher correlation
between ABTS and TP (r=0:912) than between DPPH and
On the other hand, Condelli et al.  reported that the
diﬀerences in antioxidant capacity may depend on the com-
position and proﬁle of phenolic compounds, rather than total
polyphenol content. This study was conducted on 75 Italian
commercial extra virgin olive oils. Finally, Jimenez-Lopez
et al.  pointed out various factors that aﬀect the quality
of EVOO and its bioactive compounds. Bruno et al.  con-
cluded that harvesting time and climate conditions inﬂuence
the phenol composition as ratios of phenol compounds and
their total amount.
3.2. Fatty Acid Proﬁle. The appropriate proﬁle of fatty acids
determines the quality and health beneﬁts of olive oil. The
analyses showed that the tested olives had a typical content
of oleic acid C18 : 1 (above 72%) . Particularly important for
health are polyunsaturated fatty acids (PUFA). Sample Cre-
tan 1 had about 20% more linoleic acid C18 : 2, whereas the
content of α-linolenic acid C18 : 3 did not diﬀer signiﬁcant
between all the samples (Table 2).
Table 1: Antioxidant capacity and total polyphenols of extra virgin olive oils.
Spanish Italian Greek Cretan 1 Cretan 2
DPPH (mg Tx/L) 324 ± 5:0
313 ± 3:0
290 ± 4:0
348 ± 5:0
282 ± 1:0
ABTS (mg Tx/L) 552 ± 7:0
513 ± 5:0
501 ± 5:0
623 ± 7:0
517 ± 6:0
TP (mg GAE/L) 409 ± 36
393 ± 62
403 ± 86
658 ± 40
423 ± 14
Data are mean ± SD (n=5). Diﬀerent letters in the same row indicate statistical signiﬁcance at p<0:05; Tx: Trolox equivalents; GAE: gallic acid equivalents.
Table 2: Fatty acid proﬁle of extra virgin olive oils (%).
Spanish Italian Greek Cretan 1 Cretan 2
Caprylic acid C8 : 0 nd nd nd nd nd
Capric acid C10 : 0 nd nd nd nd nd
Lauric acid C12 : 0 0:03 ± 0:04 nd nd nd nd
Tridecanoic acid C13 : 0 nd nd nd nd nd
Myristic C14 : 0 nd nd nd nd nd
Myristoleic acid C14 : 1 nd nd nd nd nd
Pentadecanoic acid C15 : 0 nd nd nd nd nd
Palmitic acid C16:0 13:08 ± 0:55 12:98 ± 0:08 12:93 ± 0:16 13:20 ± 0:25 12:94 ± 0:14
Palmitoleic acid C16 : 1 1:05 ± 0:23
0:67 ± 0:72
0:53 ± 0:57
0:12 ± 0:02
0:12 ± 0:01
Heptadecanoic acid C17 : 0 0:15 ± 0:04
0:63 ± 0:75
0:47 ± 0:58
0:84 ± 0:02
0:77 ± 0:01
Stearic acid C18 : 0 3:25 ± 0:02
2:86 ± 0:07
2:76 ± 0:06
2:87 ± 0:01
2:91 ± 0:03
Elaidic acid C18 : 1 n9t nd nd nd nd nd
Oleic acid C18 : 1 n9c 72:77 ± 0:14
73:74 ± 0:16
75:29 ± 0:01
72:49 ± 1:29
74:18 ± 0:10
Linoleic acid C18:2 8:13 ± 0:04
7:60 ± 0:10
6:44 ± 0:06
8:94 ± 0:62
7:56 ± 0:03
α-Linolenic acid C18:3 0:76 ± 0:04 0:72 ± 0:06 0:74 ± 0:01 0:73 ± 0:01 0:72 ± 0:01
Arachidic acid C20 : 0 0:39 ± 0:01
0:48 ± 0:11
0:41 ± 0:01
0:41 ± 0:03
0:44 ± 0:01
11-Eicosenoic acid C20 : 1 0:27 ± 0:02 0:27 ± 0:01 0:30 ± 0:04 0:26 ± 0:01 0:28 ± 0:01
Behenic acid C22 : 0 0:11 ± 0:01
0:06 ± 0:08
0:13 ± 0:01
0:14 ± 0:01
0:07 ± 0:11
Erucic acid C22 : 1 nd nd nd nd nd
MUFA (%) 74.09
PUFA (%) 8.89
SFA (%) 17.01 17.00 16.7 17.46 17.14
Data are mean ± SD (n=4). Diﬀerent letters in the same row indicate statistical signiﬁcance at p<0:05; nd: not detected; MUFA: monounsaturated fatty acids;
PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids.
3International Journal of Food Science
The mean values of fatty acid proﬁle found in the pres-
ent study were the within limits established by the Interna-
tional Olive Oil Council for purity criteria of olive oils .
Stefanoudaki et al.  reported that Greek olive oils of cv.
Koroneiki were characterized by a higher concentration of
oleic acid C18 : 1 (74.7–79.9%), but the concentration of lino-
leic acid C18 : 2 and α-linolenic acid C18 : 3 was slightly lower
(ca. 5.0–7.0% and 0.55–0.76%, respectively). Mikrou et al.
 studied 68 monovarietal EVOOs, originating from three
regions of Greece and two local cultivars (Koroneiki and
Kolovi), and reported similar concentration oleic, linoleic,
and α-linolenic acid. The exception was the cultivar Kolovi,
which had a higher linoleic acid content of 11.63% .
Other studies showed that Italian and Spanish olive oils had
even lower values of C18 : 3, i.e., 0.49–0.54%  and 0.48%
, respectively. These values were much lower than
obtained for commercial olive oils in our studies, whereas
Morello et al.  reported similar to our values of C18 : 3.
Taking into account the content of PUFA, the tested samples
present the upper range of these compounds, which could be
found in literature data. Furthermore, Stefanoudaki et al. 
stated that lower concentration of oleic acid resulted in a
higher concentration of heptadecanoic C17 : 0 and linoleic
C18 : 2 acids, which was also conﬁrmed in our research.
Therefore, Kosma et al.  concluded that variations in
the fatty acid composition may be owed to factors such as
cultivar and other factors for example climatic conditions
and geographical origin. The reported statement for Greek
oils has been also conﬁrmed in diﬀerent studies on Italian
olive oils. Moreover, Piscopo et al.  demonstrated the
eﬀect of olive cultivar and the environmental inﬂuences on
the fatty acid composition of monovarietal olive oils, consid-
ering also the quality of a same cultivar in diﬀerent areas.
We are aware that our study has some limitations. In the
next studies, the proﬁle of polyphenolic compounds and
other valuable parameters such as tocopherol, squalene, oleo-
canthal, and oleacein should be examined.
The results of our research showed that the sample Cretan 1
had about 15% higher antioxidant capacity and about 60%
higher total polyphenol content than Spanish, Italian, and
Greek extra virgin olive oils. These olive oil had also a favor-
able composition of fatty acids, especially linoleic and α-lino-
lenic acid. The sample Cretan 2 did not diﬀer signiﬁcant from
the commercial counterparts.
In conclusion, the antioxidant properties depend on the
manufacturing conditions. Oils from olives grown on organic
farms (manual harvesting, without artiﬁcial irrigation) and
produced with traditional methods, i.e., using millstones, cold
pressing, and without centrifugation and ﬁltration, had higher
antioxidant properties and favorable proﬁle of fatty acids.
The results are in the article and in the corresponding author.
Conflicts of Interest
The authors declare no conﬂict of interest.
D.N. is responsible for the design of experiments, experi-
ments, data analysis, and writing of manuscript; M.G. is for
the experiments and statistical analysis—support; and C.P
critically revised the paper. All the authors have approved
the ﬁnal version of the manuscript.
We are grateful to the management of the company GUEST
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