Effects of storage on the oxidative stability of acorn oils extracted from three different Quercus species

Abstract

BACKGROUND
Acorn fruit and its components and by‐products are receiving renewed interest due to their nutritional and phytochemical features. In particular, the oil extracted from acorns is recognized for having high nutritional quality and for being rich in bioactive compounds. Despite the growing interest, few papers are available that consider the evolution of acorn‐oil characteristics during storage. Our aim was to investigate the storage‐related changes in acorn oils extracted from three Quercus species grown in Algeria (Q. ilex, Q. suber, and Q. coccifera) 180 days after production, with a focus on polar and volatile compounds, not yet investigated. Basic quality parameters, phenolic content, antioxidant activity and induction time were also monitored.

RESULTS
The oxidation markers (peroxide value and UV absorptions) increased during storage, whereas antioxidants decreased. A distinctive volatile profile was observed at the time of production, which underwent changes during storage. Polar compounds increased, whereas induction time decreased. The oil extracted from Quercus suber L. was the most affected by storage time.

CONCLUSION
Floral and fruity volatile compounds detected in the oils’ headspace could explain the pleasant flavor of acorn oils reported by other authors. As with other vegetable oils, storage depletes both volatiles and antioxidants and produces oxidation compounds, such as oxidized triacylglycerols. However, the acorn oils that were studied were quite stable under storage in the dark at room temperature for 6 months. © 2020 Society of Chemical Industry

Full text

Research Article
Received: 26 April 2020 Revised: 11 June 2020 Accepted article published: 1 July 2020 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.10623
Effects of storage on the oxidative stability
of acorn oils extracted from three different
Quercus species
Fatima Z Makhlouf,aGiacomo Squeo,b
*
Graziana Difonzo,b
Michele Faccia,bAntonella Pasqualone,bCarmine Summo,b
Malika Barkataand Francesco Caponiob
Abstract
BACKGROUND: Acorn fruit and its components and by-products are receiving renewed interest due to their nutritional and phy-
tochemical features. In particular, the oil extracted from acorns is recognized for having high nutritional quality and for being
rich in bioactive compounds. Despite the growing interest, few papers are available that consider the evolution of acorn-oil
characteristics during storage. Our aim was to investigate the storage-related changes in acorn oils extracted from three Quer-
cus species grown in Algeria (Q. ilex,Q. suber, and Q. coccifera) 180 days after production, with a focus on polar and volatile com-
pounds, not yet investigated. Basic quality parameters, phenolic content, antioxidant activity and induction time were also
monitored.
RESULTS: The oxidation markers (peroxide value and UV absorptions) increased during storage, whereas antioxidants
decreased. A distinctive volatile profile was observed at the time of production, which underwent changes during storage. Polar
compounds increased, whereas induction time decreased. The oil extracted from Quercus suber L. was the most affected by stor-
age time.
CONCLUSION: Floral and fruity volatile compounds detected in the oils’headspace could explain the pleasant flavor of acorn
oils reported by other authors. As with other vegetable oils, storage depletes both volatiles and antioxidants and produces oxi-
dation compounds, such as oxidized triacylglycerols. However, the acorn oils that were studied were quite stable under storage
in the dark at room temperature for 6 months.
© 2020 Society of Chemical Industry
Supporting information may be found in the online version of this article.
Keywords: volatile compounds; polar compounds; oxidation; vegetable oil; antioxidants
INTRODUCTION
Acorn fruits from Quercus spp. are considered nutritionally valu-
able and have been used for thousands of years wherever oak
trees were found, being a good source of carbohydrates, proteins,
and fat.
1
Milled to flour, acorns have recently been proposed as
functional ingredients for producing bread and biscuits.
2-4
The
lipid fraction of acorns is also recognized for having high nutri-
tional quality because it is rich in unsaturated fatty acids and in
bioactive compounds, such as polyphenols, tocopherols, and ste-
rols.
5-11
Acorn oil has a long history of use in most parts of the
world as food, cooking oil and in folk medicine. It can be extracted
by boiling, crushing, or pressing. Some acorn varieties contain
more than 30% of oil, the composition and flavor of which have
been reported to be very similar to those of virgin olive oil
(VOO).
12
Like other vegetable oils, acorn oil undergoes chemical reac-
tions such as isomerization and oxidation of fatty acids.
13
Lipid
oxidation is one of the most deleterious reactions during storage
and processing, markedly affecting the quality of vegetable
oils.
13,14
A very wide pattern of factors influences the oxidation
of edible oils, such as fatty acid composition, processing condi-
tions, exposure to heat or light, and the concentration and type
of oxygen, free fatty acids, mono- and diacylglycerols, transition
metals, peroxides, thermally oxidized compounds, pigments,
and antioxidants.
15
The main result of lipid oxidation is the modification of the sen-
sory properties of food, characterized by changes in color and
occurrence of an unpleasant flavor referred to as rancidity.
15-17
From a nutritional point of view, oil oxidation degrades essential
*Correspondence to: G Squeo, Department of Soil, Plant and Food Sciences,
Food Science and Technology Unit, University of Bari Aldo Moro, Via Amen-
dola, 165/A, 70126 Bari, Italy. E-mail: giacomo.squeo@uniba.it
aLaboratoire Bioqual, INATAA, , Université Frères Mentouri Constantine 1, Con-
stantine, Algeria
bDepartment of Soil, Plant and Food Sciences, Food Science and Technology
Unit, University of Bari Aldo Moro, Bari, Italy
J Sci Food Agric 2020 www.soci.org © 2020 Society of Chemical Industry
1

fatty acids, causes loss of essential nutrients, micronutrients, and
vitamins, and gives rise to toxic compounds and oxidized
polymers.
15
The ability of oil to resist the oxidation process is known as oxi-
dative stability and can be expressed as the time necessary to
attain the critical point of oxidation, whether it is a sensorial
change or a sudden acceleration of the oxidative process. Over
time, the oil shelf-life ends and off-flavors arise.
15
Despite the increasing interest in acorn oil, depicted as a possi-
ble substitute for the most famous extra virgin olive oil,
1
little
information is available about its stability over time or about the
evolution of its chemical and nutritional characteristics. Lopes
and Bernardo-Gil
9
reported that acorn oils from Quercus rotundifo-
lia L. and Quercus suber L. from Portugal are quite stable under
normal storage conditions. However, the authors followed only
the evolution of the peroxide value with storage time and a lack
of information about the evolution of other important parame-
ters, such as spectrophotometric absorptions, polar, and volatile
compounds, was evidenced.
The aim of this paper was to evaluate the characteristics and sta-
bility of oils obtained from three different Quercus species (Q. ilex,
Q. suber, and Q. coccifera) at extraction and after storage. It
focused particularly on polar and volatile compounds, which are
important both for nutritional and sensory quality. Two discrete
sampling points were considered: immediately after production
and after 180 days of storage.
MATERIALS AND METHODS
Extraction and storage of oils
Oil samples were obtained as reported in Makhlouf et al.
10
from
mature acorn fruits of three Quercus species: Quercus ilex L. (QI),
Quercus suber L. (QS), and Quercus coccifera L. (QC) collected in a
forest located in eastern Algeria (in the Oum El Bouaghi region).
After extraction, the oils were packaged in glass bottles and
stored in the dark at 23 ± 2 °C for 180 days. The most abundant
fatty acids in both QS, QI, and QC oils were palmitic acid (∼12%),
stearic acid (∼2.7%), oleic acid (∼67.5%), and linoleic acid (∼15%).
Oils were analyzed at the beginning of storage (T0) and after
180 days (T180).
Analysis of polar compounds
The polar compounds (PC) were separated from the oils as
described by Gomes and Caponio,
18
using column chromatogra-
phy. Briefly, oil was weighted (0.5 g), dissolved in the eluent (mix-
ture petroleum ether/diethyl ether; 87/13, v/v) and firstly the non-
polar fraction was recovered. The amount of total polar com-
pounds was calculated as the difference between the weight of
the sample added to the column and that of the non-polar frac-
tion eluted and expressed in g 100 g
−1
.
18
Subsequently, the polar
compounds were extracted by performing a second elution with
diethyl ether. The ether was then removed and the polar fraction
was recovered in tetrahydrofuran (THF) and subjected to high
performance size-exclusion chromatography (HPSEC) analysis,
using THF as eluent at 1 mL min
−1
flow rate. The HPSEC system
consisted of a series 200 pump (Perkin-Elmer, Norwalk, CT, USA)
with Rheodyne injector, a 50 μL loop, a PL-gel guard column (Per-
kin-Elmer, Beaconsfield, UK) of 5 cm length and 7.5 mm i.d., and a
series of two PL-gel columns (Perkin-Elmer, UK) of 30 cm length
and 7.5 mm i.d. each. The columns were packed with a highly
cross-linked styrene-divinylbenzene copolymer with particles of
5μm and a pore diameter of 500 Å. The detector was a series
200 refractive index (Perkin-Elmer, USA). Polar compounds were
identified by polystyrene standards (Supelco, Milan, Italy) as
reported in a previous paper.
19
For quantitative determination
of the single polar compounds, known amounts of triacylglycerol
oligopolymers (TAGP), oxidized triacylglycerols (ox-TAG), and dia-
cylglycerols (DAG) were obtained by preparative gel permeation
chromatography of PC derived from a refined peanut oil and then
used as standards in the HPSEC. The results were reported as g of
each fraction per 100 g
−1
of oil.
Oxidative stability measurement
Induction time (IT) was determined using the Rapidoxy (Anton
Paar, Graz, Austria) oxidative stability instrument, which is a micro-
processor-controlled automatic testing device for the quick mea-
surement of the oxidative stability of lipid matrices in response to
forced oxidation by increasing temperature and O
2
pressure.
20
The oxidative stability was evaluated by measuring the IT, which
is expressed as the time needed for a 10% drop of the oxygen
pressure under following conditions: T =140 °C, P =700 kPa.
Analysis of volatiles compounds
Volatile compounds were analyzed by headspace solid phase
micro-extraction coupled with gas chromatography / mass spec-
trometry (HS-SPME-GC/MS) as described by Caponio et al.
21
The
sample (1 g of oil) was weighed in a 20 mL screw cap vial fitted with
a silicon/Polytetrafluoroethylene (PTFE) septum (VWR International,
Radnor, PA, USA). After 2 min for temperature equilibration, volatiles
were extracted by exposing a solid phase micro-extraction (SPME)
fiber 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane
(DVB/CAR/PDMS) (Supelco, Bellefonte, PA, USA) in the headspace
ofthesampleat40°C for 20 min and then desorbed for 2 min
into the injector port of an Agilent 6850 series gas chromato-
graph equipped with an Agilent 5975 mass spectrometer (Agilent
Technologies Inc., Santa Clara, CA, USA).
The volatile compounds were separated using a HP-Innowax
polar capillary column (60 m length ×0.25 mm i.d. ×0.25 μmfilm
thickness), under previously reported conditions.
21
The compounds were identified by comparison of their mass
spectra with the mass spectra present in the National Institute
of Standards and Technology (NIST) and Wiley libraries. Only
those with a match quality above 70% were considered. The
results were expressed as total ion count areas.
Conventional analyses of oils
The determinations of the free fatty acids (FFA), peroxide value
(PV), and spectrophotometric constants (K
232
,K
270
,ΔK) were as
described in Conte et al.
22
Extraction and analysis of phenolic fractions from oils
Polyphenols were extracted from Quercus oils by liquid–liquid
extraction using a methanol / water mixture (70:30, v/v) and fol-
lowing the procedure reported by Caponio et al.,
23
with the addi-
tion of 250 μL of a gallic acid solution as an internal standard for
quantification at a concentration of 100 mg L
−1
, prepared in
methanol / water (70:30, v/v).
Phenolic compounds were separated, tentatively identified
using mass spectra (MS
2
) and ⊗
max
and quantified as described
by Makhlouf et al.
10
The total phenolic content (TPC) was consid-
ered as the sum of the single phenolic compounds quantified.
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2

Evaluation of the antioxidant activity
The antioxidant activity of the oil phenolic fraction was evaluated
on the basis of the scavenging activity of the ABTS (2,20-azinobis
(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt),
and also for their capacity to scavenge the DPPH radical (1,1-
diphenyl 2-picrylhydrazyl) compared with a reference antioxidant
standard Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid) as described in Difonzo et al.
24
The results were
expressed in μmol Trolox equivalents (TE) per g of oil.
Statistical analysis
Analyses of variance (one-way and two-way ANOVAs), followed
by Fisher’s least significance difference (LSD) post hoc test for mul-
tiple comparisons, and correlation analysis, were carried out on
the experimental data by means of Minitab 17 (Minitab Inc., State
College, PA, USA). Principal component analysis (PCA) was carried
out by means of the same software on the correlation matrix.
Results were considered significant when P≤0.05.
RESULTS AND DISCUSSION
Polar compounds and oxidative stability
In our study, a comparison of the acorn oils characteristics before
and after storage was carried out. Six months of storage were con-
sidered a suitable and realistic timeframe to monitor the storage-
related changes given the initial characteristics of the oils –i.e. a
high amount of unsaturated fatty acid and low oleic / linoleic acid
ratio –which make these oils especially prone to oxidation.
25,26
Polar compounds are important quality parameters for asses-
sing the level of lipid degradation of heated or stored oils.
27,28
They are generated during autoxidation and thermo-oxidation
processes and provide information on primary and secondary oxi-
dation.
15,29
Polar compounds are substances with a polarity
greater than that of unaltered triglycerides, and comprise TAGP,
ox-TAG, DAG, sterols, triterpene diols, and FFA.
18
In particular,
ox-TAG and TAGP are indicative of oil oxidation and thermal alter-
ation, respectively, while DAG and FFA are related to hydrolytic
alteration.
29
As shown in Table 1, at T0 the oils had low levels of
total PC content, with significant differences between QS and
the other Quercus species considered. In more detail, TAGP were
found only in traces, as expected, these oxidative products usually
being formed after intense thermal stress, such as after frying or
refining processes.
30
Ox-TAG ranged between 0.36% and 0.40%,
with a significantly higher content in QS than QI. However, the
most consistent part of PC was represented by FFA (Table 3) and
DAG (Table 1), the latter accounting for approximatively 45% of
the total, on average. The DAG content was found to be similar
to that reported for other vegetable oils such as rapeseed, sun-
flower, soybean oils or safflower oil.
31,32
At the time of production,
QS oil had the highest level of DAG and PC, compared with the
other Quercus species. After 180 days of storage, a significant
increase in PC was observed in all samples, rising by around
40% for QC and QI oils, while QS oil presented the greatest varia-
tion, increasing from 3.24 to 5.36% (about 65%). The increase in
the PC level was mainly due to the accumulation of DAG, FFA
(Table 3), and ox-TAG resulting from hydrolytic and oxidative
alteration. Regarding ox-TAG, a general increase was observed,
although it was statistically significant only for QI oil. The DAG
increase was significant for both QI and QS samples. Overall, QS
oil showed the highest amount of total PC, even after storage. In
contrast with what is reported for sunflower oil,
29
quite intense
hydrolytic activity was observed in acorn oils during storage. An
increase in DAG was already reported for other vegetable oils,
such as extra virgin olive oil stored in darkness,
25
and it depends
on storage temperature and initial oil acidity.
33
The induction time (IT) of fresh oils, determined by the Rapidoxy
test, varied significantly among the species from about 215 min
(QI) to about 381 min (QC). After storage, the IT decreased signif-
icantly, at an extent between −22.5% (QI) and −31% (QS). The
significant reduction was likely due to a depletion of the natural
antioxidants of acorn oil during storage (Table 3). As expected,
considering the whole dataset (T0 and T180 data), an accordance
between IT and TPC was observed, as evidenced by the Pearson's
correlation coefficient (r =0.973, P=0.001).
Volatile profile
Volatile compounds of oils are of great interest because they are
related to quality and are responsible for the sensory attributes
of foods and oils.
34
Table 2 reports the volatile profile of the acorn
oils obtained both immediately after extraction and after
180 days of storage, as well as the results of a two-way ANOVA.
Although the acorn oils were mildly evaporated after solvent
extraction, likely reducing the total amount of volatiles, significant
differences among the oils were kept. Overall, 29 volatile com-
pounds were identified, belonging to different chemical classes:
terpenes, esters, acids, ketones, aldehydes, and alcohols. The vol-
atile profile varied widely, and significant differences were
highlighted. Some compounds were detected exclusively in the
oil of certain acorn species, indicating peculiar sensorial character-
istics as a function of the genotype. Quercus coccifera L. was the
Table 1 Polar compounds and oxidative stability of acorn oils at extraction (T0) and after 180 days storage (T180)
QI QS QC
T0 T180 T0 T180 T0 T180
TAGP (g 100 g
−1
) Traces Traces Traces Traces Traces Traces
ox-TAG (g 100 g
−1
) 0.36 ±0.01 D 0.50 ±0.02 A 0.40 ±0.01 BC 0.41 ±0.01 B 0.36 ±0.02 CD 0.40 ±0.03 BCD
DAG (g 100 g
−1
) 1.08 ±0.04 D 1.49 ±0.06 C 1.73 ±0.05 B 2.00 ±0.01 A 0.86 ±0.03 E 0.96 ±0.08 DE
PC (g 100 g
−1
) 2.39 ±0.09 C 3.34 ±0.22 B 3.24 ±0.21 B 5.36 ±0.21 A 2.13 ±0.07 C 3.01 ±0.02 B
IT (min) 215.63 ±4.69 D 166.95 ±2.93 F 284.04 ±5.78 B 195.88 ±3.18 E 381.24 ±7.71 A 265.58 ±7.82 C
QI, Quercus ilex L.; QS, Quercus suber L.; QC, Quercus coccifera L.
TAGP, triacylglycerol oligopolymers; ox-TAG, oxidized triacylglycerols; DAG, diacylglycerols; PC, total polar compounds; IT, induction time.
Values are expressed as means ±standard deviation, n=3.
Different letters in a row indicate significant differences at P≤0.05 with a two-way ANOVA followed by a Fisher's LSD test.
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3

richest in terpenic compounds. In fact, in fresh QC oil, around 87%
of the total identified volatiles was represented by terpenic com-
pounds. Eucalyptol, d-limonene and p-menthone were some of
the most abundant compounds. d-Limonene was the most
abundant terpene in QI and QS oils, too. It is the principal compo-
nent of citrus fruit essential oils and its biological and chemical
properties have been studied in depth.
35,36
The QC oil was charac-
terized by the highest concentrations of terpenes and ketones,
Table 2 Volatile compounds of acorn oils at extraction (T0) and after 180 days storage (T180)
QI QS QC
T0 T180 T0 T180 T0 T180
Aldehydes
Hexanal 0.73 ±0.03 D 0.99 ±0.06 C 2.21 ±0.03 A 2.05 ±0.19 A 0.00 ±0.00 E 1.35 ±0.03 B
(E)-2-Hexenal 0.50 ±0.03 BC 0.00 ±0.00 D 1.00 ±0.19 A 0.69 ±0.12 B 0.40 ±0.13 C 0.00 ±0.00 D
Benzaldehyde 8.35 ±0.16 A 3.25 ±0.49 C 5.06 ±0.86 B 4.84 ±0.16 B 5.02 ±0.24 B 5.27 ±0.13 B
Phenylacetaldehyde 2.89 ±0.66 B 0.00 ±0.00 C 3.55 ±0.37
AB
0.00 ±0.00 C 4.06 ±0.04 A 0.00 ±0.00 C
∑Aldehydes 12.46 ±0.77
A
4.25 ±0.55
D
11.82 ±1.46
A
7.58 ±0.23
C
9.49 ±0.15 B 6.62 ±0.10 C
Alcohols
Ethanol 13.30 ±3.76 A 0.00 ±0.00 C 0.00 ±0.00 C 0.00 ±0.00 C 7.16 ±2.64 B 0.00 ±0.00 C
Benzene ethanol 58.34 ±8.23 A 3.33 ±0.08 D 16.89 ±3.86
C
8.00 ±1.44
CD
29.39 ±1.48 B 10.05 ±0.41
CD
∑Alcohols 71.64 ±11.98
A
3.33 ±0.08
D
16.89 ±3.86
C
8.00 ±1.44
CD
36.54 ±1.17 B 10.05 ±0.41
CD
Esters
Butyl acetate 33.08 ±1.27 A 0.00 ±0.00 D 4.60 ±0.08 C 0.00 ±0.00 D 19.09 ±0.46 B 0.00 ±0.00 D
Ethyl hexanoate 0.40 ±0.06 A 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B
Hexyl butyrate 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.71 ±0.05 A 0.00 ±0.00 B
∑Esters 33.49 ±1.21
A
0.00 ±0.00
D
4.60 ±0.08
C
0.00 ±0.00
D
19.80 ±0.51 B 0.00 ±0.00 D
Terpenes
Camphene 0.00 ±0.00 -B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 1.19 ±0.05 A 0.00 ±0.00 B
⊎-Pinene 0.72 ±0.04 B 0.83 ±0.25 B 0.00 ±0.00 C 0.00 ±0.00 C 4.85 ±0.32 A 0.00 ±0.00 C
d-Limonene 19.84 ±1.55 C 22.64 ±0.30
C
24.27 ±1.11
C
36.39 ±6.12
B
32.13 ±0.79 B 64.80 ±0.45 A
Eucalyptol 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 388.08 ±18.54
A
0.00 ±0.00 B
3,7-Dimethyl-1,3,6-octatriene 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 1.51 ±0.06 A 0.00 ±0.00 B
p-Menthone 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 37.58 ±1.49 A 0.00 ±0.00 B
Linalool 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 27.31 ±0.05 A 0.00 ±0.00 B
⊎-Ocimene 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 12.17 ±1.45 A 0.00 ±0.00 B
(+)-trans-Carane 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 1.79 ±0.06 A 0.00 ±0.00 B
γ-Terpinene 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 5.88 ±0.24 A 0.00 ±0.00 B
Cyclohexanol 5-methyl-
2-(1-methylethyl)
0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 36.01 ±3.24 A 0.00 ±0.00 B
∑Terpenes 20.56 ±1.59
C
23.47 ±0.05
C
24.27 ±1.11
C
36.39 ±6.12
C
548.49 ±16.30
A
64.80 ±0.45
B
Ketones
Pentan-2-one 1.07 ±0.09 A 0.00 ±0.00 C 0.62 ±0.00 B 0.00 ±0.00 C 0.99 ±0.01 A 0.00 ±0.00 C
3-Penten-2-one 5.24 ±0.19 A 0.00 ±0.00 C 2.49 ±0.84 B 0.00 ±0.00 C 2.26 ±0.53 B 0.00 ±0.00 C
Camphor 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 0.00 ±0.00 B 10.61 ±0.18 A 0.00 ±0.00 B
∑Ketones 6.31 ±0.27 B 0.00 ±0.00
D
3.11 ±0.84
C
0.00 ±0.00
D
13.87 ±0.34 A 0.00 ±0.00 D
Acids
Acetic acid 40.97 ±4.39
AB
48.85 ±8.01
A
1.53 ±0.21 D 15.79 ±0.10
C
37.74 ±4.22 B 15.39 ±0.95 C
QI, Quercus ilex L.; QS, Quercus suber L.; QC, Quercus coccifera L.
Values are expressed as means (total ion count area ×10
6
)±standard deviation, n=2.
Different letters in rows indicate significant differences at P≤0.05 with a two-way ANOVA followed by a Fisher's LSD test.
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4

and by the lowest amounts of aldehydes. Quercus ilex L. oil
showed a significantly higher level of esters, alcohols, and acids
of the acetic series, which was likely indicative of over-mature or
degraded raw material. Similar compounds from fermentation
processes were also found in biscuits enriched with acorn flour.
4
Finally, QS had the lowest amount of alcohols, esters, and
acetic acid.
Several authors reported that acorn oil has a pleasant odor.
1
Even though we did not perform a flavoromic study, this state-
ment is consistent with the pattern of aromatic compounds
observed, in particular considering the oil from Quercus coccifera.
For example, linalol was reported to have a ‘floral’aroma and is
found in grapes and essential oils;
37
phenyl acetaldehyde is an
important pleasant compound in many fruits and flowers;
38
cam-
phene is one of the major constituents of Valeriana officinalis
essential oil;
39
pinene and eucalyptol are found in several plant
and oils such as Eucalyptus globulus;
40
p-menthone is found in
several essential oils from herbal medicines.
41
The variations in the profiles of volatile compounds observed
between Quercus species might be related to the genetic charac-
teristics, environmental conditions, and degree of maturity of the
acorns. However, it is difficult to point out the source of these dif-
ferences as this experimental work represents, as far as we know,
the first report about the volatile composition of acorn oils.
Storage caused noteworthy changes in the volatile profile of
acorn oils due to the loss of the majority of the volatiles. In fact,
a significantly lower amount of aldehydes, alcohols, esters, and
ketones was observed. The QC oil, which was the richest in ter-
penes at the time of production, experienced a very important
loss of such compounds. Interestingly, among terpenes, d-limo-
nene was the only one found in all the oils after 180 days of stor-
age. No volatile markers of oxidation were found, probably due to
the mild storage conditions (room temperature in darkness). The
exception was represented by hexanal, which was absent in QC
at T0 but significantly increased after storage, likely deriving from
oxidation.
15
On the whole, QC oils behaved very differently from the other
Quercus species. Indeed, both the huge amount of eucalyptol at
T0 and its total disappearance after storage seemed a quite
unusual observation. Moreover, the lack of information about
acorn volatiles did not help in finding a clear explanation, which
might be furnished by further studies. In other matrices it has
been reported that eucalyptol accumulates during the early
stages of fruit maturity and then progressively disappears.
42
Thus,
the high amount of eucalyptol in QC could be explained by differ-
ences in the level of maturity of some of the acorns used for pro-
ducing the examined oils, even though, in our study, fruits were
collected when globally considered mature. On the other hand,
a similar progressive depletion of eucalyptol during 6 months
storage was evidenced in packaged herbs.
43
To help in exploring the volatile pattern of the acorn oils, a prin-
cipal component analysis was performed and the results are
reported in Fig. 1. The first two principal components accounted
for more than 85% of the total data variability. The score plot
(Fig. 1(a)) shows that the fresh oils had very different characteris-
tics in terms of volatiles. However, their features became very sim-
ilar after storage, then all the stored oils were in the same area of
the score plot. The QC and QI oils were the most affected by stor-
age –i.e. they showed the greatest distance between the corre-
sponding fresh oils in the score plot –although in the former
this result was due to the severe loss of terpenes, while in the lat-
ter it was due to the decrease in alcohols and esters.
Quality parameters, phenolic content, and antioxidant
activity
Table 3 shows the quality characteristics of the oils, the total phe-
nolic content, and the antioxidant activity of acorn oils after stor-
age, together with the percentage differences compared with
T0.
10
The QS oil had the highest level of FFA, which was about
twice as high as was observed in QI and QC, which were, in turn,
very similar to each other. After storage the acidity values
increased in all samples, although less sharply in QI and QC
(+34% and + 82%, respectively) than QS, whose increase in acid-
ity was very consistent (+154%). The increase in FFA was ascrib-
able to the hydrolytic degradation of acylglycerols.
The PV was not significantly different among the Quercus spe-
cies, and remained rather low even after 180 days of storage. In
comparison, Lopes and Bernando-Gil
9
reported a PV value of
Table 3 Quality parameters, phenolic content and antioxidant activity of acorn oils after 180 days storage and percentage variation respect to the
moment of extraction (T0)
a
QI
Δ%QI
T0-T180 QS
Δ%QS
T0-T180 QC
Δ%QC
T0-T180
FFA (g 100 g
−1
) 1.30 ±0.19 C +34 2.87 ±0.28 A +154 1.67 ±0.18 B +82
PV (meq O
2
kg
−1
) 1.94 ±0.71 A +29 1.46 ±0.30 A +76 1.68 ±0.38 A +68
K
232
1.56 ±0.00 C +1 2.00 ±0.01 B +23 2.59 ±0.00 A +8
K
270
0.50 ±0.06 C +2 0.66 ±0.00 B +29 1.34 ±0.02 A +18
ΔK 0.17 ±0.03 C —0.24 ±0.02 B +50 0.38 ±0.02 A +12
TPC (mg gallic acid equivalent kg
−1
oil)
95.38 ±7.41
C
−21 145.50 ±7.60
B
−22 201.20 ±9.42
A
−33
DPPH assay (μmol TE g
−1
oil) 0.94 ±0.05 C −26 1.95 ±0.16 B −28 2.33 ±0.15 A −30
ABTS assay (μmol TE g
−1
oil) 0.80 ±0.02 C −49 2.01 ±0.08 B −38 2.78 ±0.01 A −27
a
Original data at T0 are reported in Makhlouf et al. (2018)
10
.
QI, Quercus ilex L.; QS, Quercus suber L.; QC, Quercus coccifera L.
FFA, free fatty acids; PV, peroxide value; K
232
, specific extinction at 232 nm; K
270
, specific extinction at 270 nm; TPC, total phenolic content.
Values are expressed as means ±standard deviation, n=3.
Different letters among the varieties indicate significant differences at T180 at p≤0.05 with a one-way ANOVA followed by a Fisher's LSD test.
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5

about 6 meq O
2
kg
−1
in acorn oils from Portugal at the moment of
production. The PV increase after storage in comparison with T0
was relevant, ranging from about +30% to +76% but, on the
whole, was not crucial for the oil quality.
Regarding the spectrophotometric constants (K
232
,K
270
and
ΔK), QC oil had the highest value followed by QS and, finally, by
QI. Considering the evolution after storage, the relative increase
in absorptions at 232 and 270 nm, and of ΔK, was definitely more
marked in QS respect to the other oils. Compared with T0, ΔK
values remained unvaried for QI while a slight increment (+12%)
was observed for QC.
The amount of total phenolic compounds is an important factor
in the evaluation of the quality of edible oil because they improve
oil resistance to oxidation and are positively correlated with shelf
life.
44
After storage, the QC oil showed the highest TPC and antiox-
idant activity (assessed by the DPPH and ABTS tests) followed, in
decreasing order, by the QS and QI oils. The QC oil experienced
the greatest decrease of TPC (about 33%), while QS and QI
showed a decrease of about 22%. The reduction in TPC after
180 days of storage was likely due to the activity of phenolic com-
pounds against oxidation.
45
Considering the antioxidant activity,
it is noteworthy that, while a similar decrement in the values of
DPPH test was observed in all the oils, more marked differences
were observed considering the ABTS test. The DPPH assay there-
fore reflected more realistically the behavior of TPC while the
ABTS test seemed to be not in accordance.
Concerning the phenolic profiles (supporting information,
Table S1), the most representative compounds in the three oils
that were studied were those already evidenced in fresh oils,
10
namely trigalloyl-hexahydrodiphenoyl-glucose followed by tetra-
galloyl-pentoside (787/861) and pentagalloyl-glucose. Only in QS
and QC was a remarkable amount of tetragalloyl-pentoside (393/
468/787/973) reported. The other compounds were detected in
lower amounts. The evolution of single compounds after storage
was different in the oils. The most affected by storage were ped-
unculagin, tetragalloyl-pentoside, trigalloyl-hexahydrodiphe-
noyl-glucose (787/861), trigalloyl-hexahydrodiphenoyl-glucose
and pentagalloyl-glucose in QI and QS oils, and digalloyl-
10.07.55.02.50.0-2.5-5.0
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
QC
QI
QS
sampl e
T180
T180
T180
T180
T180
T180
T0
T0
T0
T0
T0
T0
0.30. 20.10.0-0.1-0.2-0. 3-0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
Acetic ac id ∑ Keto n es
Camph or
3-Penten-2-one
Pentan-2-one
∑ Terpenes
Cyclohexanol 5-methyl-2-(1-meth
γ-Terpinene
(+)-trans-Carane
β-Ocimene
Linalool
p-Menthone
3,7-Dimethyl-1,3,6-o ctatriene
Eucalyptol
d-Limonene
β-Pinene
Camph ene
∑ Esters
Hex yl bu tyrate
Ethyl hexan oate
Butyl acetate
∑ Alcoho ls
Benz ene eth anol
Ethanol
∑ Aldehydes
Pheny lacetaldehyde
Benzaldeh yde
(E)-2-Hexenal
Hexanal
(A)
(B)
Figure 1 Score plot (a) and loading plot (b) of the principal component analysis carried out on volatile compounds.
www.soci.org FZ Makhlouf et al.
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6

hexahydorxydiphenoyl-glucose, digalloyl-hexahydorxydiphe-
noyl-glucose (387/785), trigalloyl-glucose, tetragalloyl-pentoside,
and trigalloyl-hexahydrodiphenoyl-glucose (393/468/787/973) in
QS and QC.
On the whole, after 180 days of storage in the dark, the basic
quality of acorn oils was not jeopardized, but a significant differ-
ence in acorn oil degradation as a function of the Quercus species
was observed.
CONCLUSION
New insights about the characteristics and the oxidative evolution
of acorn oil after storage have been reported. Acorn oils obtained
from three different Quercus species were subjected to oxidation
during 180 days of storage in the dark, with an increase in polar
compounds over time, although their quality was not extensively
compromised. Thus, a quite good stability against oxidation in the
tested conditions was evidenced.
An interesting volatile composition, not yet investigated previ-
ously, was observed. Terpenic compounds, responsible for floral
and fruit flavors were the most abundant volatiles in the head-
space of Quercus coccifera oil. Acorn oil from Quercus suber
showed the highest amount of polar compounds after storage
and experienced the greatest increase in spectrophotometric
absorption constants. Our results suggested that acorn oils from
different species can have peculiar characteristics and thus could
be used for different purposes.
ACKNOWLEDGEMENTS
This work was carried out during the stay of Dr Fatima Z. Makhlouf
at the Department of Soil, Plant and Food Sciences of the Univer-
sity of Bari Aldo Moro, Italy. The authors acknowledge the Univer-
sity Frères Mentouri Constantine 1 –INATAA, for financial support
of the scholarship of Dr Fatima Z. Makhlouf.
AUTHORSHIP
All the authors have contributed equally to the conceptualization,
development and writing of the work.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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