Food Chemistry 158 (2014) 534–545

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Improvements in the malaxation process to enhance the aroma quality

of extra virgin olive oils
P. Reboredo-Rodríguez, C. González-Barreiro, B. Cancho-Grande, J. Simal-Gándara ⇑
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, University of Vigo, Ourense Campus, E-32004 Ourense, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The influence of olive paste preparation conditions on the standard quality parameters, as well as volatile
Received 11 January 2014 profiles of extra virgin olive oils (EVOOs) from Morisca and Manzanilla de Sevilla cultivars produced in an
Received in revised form 21 February 2014 emerging olive growing area in north-western Spain and processed in an oil mill plant were investigated.
Accepted 24 February 2014
For this purpose, two malaxation temperatures (20/30 °C), and two malaxation times (30/90 min)
Available online 6 March 2014
selected in accordance with the customs of the area producers were tested. The volatile profile of the oils
underwent a substantial change in terms of odorant series when different malaxation parameters were
Keywords:
applied.
Extra-virgin olive oil
Malaxation
Ó 2014 Elsevier Ltd. All rights reserved.
Volatile compounds
Odour activity value (OAV)
Odorant series

1. Introduction The processing and characteristics of different quality olive oils

Virgin olive oil (VOO) is the principal source of fat above all in
the Mediterranean diet (Aparicio & Harwood, 2003). Spain is a
traditionally olive-growing country of the Mediterranean which
produces and exports high quality VOO from a wide variety of cul-
tivars. The Spanish olive grove is present in 34 of the 50 Spanish
provinces and occupies an area of 2,509,677 ha. The areas of olive
production in Spain – in descending order – are Andalusia (60.4%),
Castilla-La Mancha (15.8%), Extremadura (10.2%), Catalonia (4.6%),
Valencia (3.7%), Aragon (2.3%) and others (3.1%) including Galicia
(AAO-Agencia para el aceite de oliva, 2013). Galicia (Northwestern
Spain) was centuries ago, in the Middle Ages, a great producer of
oil. Nowadays, there is a resurgence of this culture, from a family
and artisan production to half-scale production. The oils produced
in this area are thought to possess a characteristic aroma profile,
but – to our knowledge – there are scarce data on their composi-
tion (Reboredo-Rodríguez, González-Barreiro, Cancho-Grande, &
Simal-Gándara, 2012; Reboredo-Rodríguez, González-Barreiro,
Cancho-Grande, & Simal-Gándara, 2013a, 2013b).
VOO oil is highly appreciated by consumers for its healthy and
sensory properties. The olfactory attributes of VOO arise mainly
from the occurrence of C5 and C6 saturated and unsaturated alde-
hydes, alcohols and esters responsible for some typical sensory
notes (such as ‘cut grass’, ‘fruity’ and ‘floral’).
⇑ Corresponding author. Tel.: +34 988 387000; fax: +34 988 387001.
E-mail address: jsimal@uvigo.es (J. Simal-Gándara).
are controlled and defined by the European Commission Imple-
menting Regulation No 29/2012 (European Union Commission,
2012). The production of a high quality VOO is not only strongly
dependent on the nature of the cultivar and the use of healthy olive
fruit with an appropriate degree of maturity, but also it is
influenced by other several factors like edaphoclimatic conditions,
agricultural practices, extraction methods, processing techniques
and/or storage conditions (Boselli, Di Lecce, Strabbioli, Pieralisi, &
Frega, 2009; Inarejos-García, Gómez-Rico, Salvador, & Fregapane,
2009).
The extraction process of VOO, consisting only of physical
methods, includes olive crushing, malaxation of the pastes and
separation of the oil phase. Malaxation of the olive paste, obtained
previously from the olive crushing, is a crucial step of the process
where the olive paste is subjected to a slow continuous kneading,
aimed at breaking off the emulsions formed during the crushing
process and facilitating adequate coalescence (Angerosa,
Mostallino, Basti, & Vito, 2001; Stefanoudaki, Koutsaftakis, &
Harwood, 2011).
According to Clodoveo (2012) malaxation of olive paste must be
considered much more than a simple physical separation, because
a complex bioprocess takes place that is very relevant to the
quality and composition of the final product. During malaxation
considerable changes in the oil’s chemical composition occur
because of the partition phenomena between oil and water and
vice versa and the catalytic activity of fruit enzymes. Different
enzymatic reactions of oxidoreductases naturally present in olive

http://dx.doi.org/10.1016/j.foodchem.2014.02.140
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545
pulp (such as polyphenoloxidases-PPO, lipoxygenases-LOX and
peroxidases-POD) involving in the transformation of volatile and
phenolic compounds take place (Boselli et al., 2009; Taticchi
et al., 2013). The rate and the extent of these reactions are greatly
affected by malaxation time and temperature, two technological
parameters that can markedly modify not only the oil yield but
also the composition and quality of the final VOO produced
(Inarejos-García et al., 2009).
In the present work, we have examined the effect of different
malaxation operating conditions commonly used by Galician pro-
ducers on the standard quality parameters, as well as the volatile
profile of EVOOs obtained from two olive fruits, Manzanilla de
Sevilla and Morisca. The aim of this work was to find the right com-
bination of malaxation time and temperature in order to ensure
the best quality of the resulting oils with a characteristic aroma.
2. Material and methods
2.1. Oil samples
For this study, olives from two olive orchards with a different
variety each were collected in November 2011 in the Southeast
of Galicia (NW Spain). Each variety presented one maturation in-
dex (MI) according to the method proposed by the International
Olive Oil Council (IOOC-International Olive Oil Council, 1984),
based on the evaluation of the olive skin and pulp colours of the
fruit. Two different oils were done: a mixture of Morisca and Ver-
dial de Badajoz cultivars (90:10%) (MI = 3.4) and a mixture of Man-
zanilla de Sevilla and ‘unknown’ cultivars (95:5%) (MI = 2.1), with
one in a higher proportion than the other. It should be noted the
huge difficulty to obtain monovarietal oils in the olive oil mills of
this area; a little percentage of a different olive variety is usually
found in the olive batches processed.
The oils were elaborated under identical conditions at a semi-
industrial scale. Thus, all oil samples were processed in an oil mill
plant (Almazara Profy, Industrias Céspedes e Hijos, S.L.) with a
production capacity of 200 kg/h equipped with an olive washing
machine, a hammer crusher, a horizontal kneader with a non-
hermetic closure and a two-phase horizontal decanter. Leaves
and dirt were removed by washing under cold running water
before extraction. The olive paste corresponding to a mixture of
Morisca and Verdial de Badajoz (90:10%) cultivars was kneaded
at 20 ± 3 °C during 30 and 90 min, as well as 30 ± 2 °C during 30
and 90 min. On the other hand, the olive paste corresponding to
a mixture of Manzanilla de Sevilla and ‘unknown’ (95:5%) cultivars
were kneaded only at 30 ± 2 °C during 30 and 90 min. The temper-
ature at three different points (left, centre and right of the malaxer)
was checked by using an infrared thermometer at 10 min intervals.
The conditions used in terms of temperature and time of malaxa-
tion were selected according to producers customs.
Four oil samples were obtained for each set of conditions and
were stored in dark-brown glass bottles without headspace at
10 °C in the dark. The samples were allowed to settle and racked
for about 4 months. This is the procedure typically used by local
producers before marketing their oil (Reboredo-Rodríguez et al.,
2013b).
Genetic and morphological determinations of representative
olive samples were performed by the Pomology Group of the
Department of Agronomy at the University of Cordoba (Spain),
using fingerprinting based on Simple Sequence Repeat (SSR) mark-
ers. The results were used to characterise the studied cultivars.
Accurately identified varieties included in the database of the
World Olive Germplasm Bank of Cordoba (WOGB), which is the
main repository of olive genotypes in Spain, were used as reference
samples.
535
2.2. Analytical methods
2.2.1. Quality indices, fatty acids, sterols and erythrodiol+uvaol assays
Standard quality indices (viz., free acidity; peroxide value; UV
absorption characteristic, K270, K232; waxes, trilinolein and DECN),
fatty acids, sterols and triterpenic dialcohols composition were car-
ried out, following the analytical methods described by European
Commission’s Regulation EEC/2568/91 and subsequent amend-
ments (European Union Commission, 1991, 2003, 2007). The val-
ues of these parameters in different olive oils can be limited by
regulations established by the European Union.
Fatty acid assays were determined according to EEC/2568/91
and subsequent amendments (European Union Commission,
1991, 2003).
Sterols and erythrodiol+uvaol were determined by following the
procedures set out in Annexes V and VI of Regulation EEC/2568/91
and subsequent amendments (European Union Commission, 1991,
2003).
2.2.2. Extraction and identification of volatiles
Volatile compounds were extracted from the EVOO samples by
Dynamic Headspace (DHS) with an automatic sampler device, the
Master DHS (DANI Instruments S.p.A., Cologno Monzese, Milan,
Italy), following our previous work (Reboredo-Rodríguez et al.,
2012). In short, the samples (volume: 9.0 mL of EVOO, fast shaking)
were directly placed into the DHS sampler in standard 20 mL vials
that can be heated at a chosen temperature (40 °C). An inert gas
flow (He, flow: 150 mL/min) was used to purge the sample (time:
90 min) in order to carry out the volatile compounds; then the
purged gas passed through a cooled trap (0 °C) where the com-
pounds were concentrated. The trap was quickly heated in back-
flush to a high preset temperature (250 °C) transferring the
compounds to the chromatographic column in a narrow band
and a reduced volume of gas. The Master DHS has a device espe-
cially designed to remove humidity from the desorbed gas before
entering into the GC–MS system. It was kept at low temperature
during the injection phase and was heated during the baking step
to eliminate retained water.
Afterwards, volatile compounds were separated and identified
on a Trace GC gas chromatograph with a PolarisQ ion trap mass
selective detector (ITMS) interfaced to a PC computer running the
software Xcalibur 1.4, from Thermo Finnigan (Rodano, Italy).
Chromatographic separations were done with a ZB-WAX fused-
silica capillary column (60 m  0.32 mm ID, 0.50 lm film thick-
ness, Phenomenex, Torrance, CA, USA). The carrier gas, helium,
was circulated at 1 ml/min in the constant flow mode. A split/split-
less injector in the split mode was used (split ratio, 1:10). The injec-
tor temperature was 200 °C. The oven temperature programme was
as follows: 40 °C for 5 min; 2 °C/min ramp to 125 °C; 10 °C/min
ramp to 250 °C and holding for 5 min. The transfer line temperature
was 250 °C, and the ion trap manifold temperature 200 °C. The ion
energy for electron impact (EI) was set constantly at 70 eV. Identi-
fication of the volatile compounds was achieved by comparing the
GC retention times and mass spectra over the mass range 35–300
amu for the samples with those for pure standards analysed under
the same conditions. Mass detection was performed in the selected
ion recording (SIR) mode for quantification. The concentration of
volatile compounds in EVOO samples was calculated taking into
account the method recoveries (Reboredo-Rodríguez et al., 2012).
2.3. Calculation of the odorant series values
An odorant series is defined as a group of volatile compounds
with similar aroma descriptors (Peinado, Mauricio, & Moreno,
2006). The total intensities for every odorant series were calculated
as sum of the odour activity value (OAV) (defined as concentration
536 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545

Table 1
Standard quality indices (free acidity; peroxide value; UV absorption characteristic, K270, K232, DK; waxes, trilinolein and DECN), fatty acids, sterols and triterpenic dialcohols
composition of the studied EVOOs.

Parameter 20 °C 30 °C
A. Morisca+Verdial de Badajoz (90:10%) 30 min 90 min 30 min 90 min
Acidity (%) 0.45 ± 0.01a 0.37 ± 0.01b 0.42 ± 0.00ab 0.52 ± 0.02c
Peroxides (meq. O2/kg oil)* 6.11–9.11 5.90–9.38 6.66–8.79 7.48–14.70
K232 1.68 ± 0.19a 1.69 ± 0.31a 1.71 ± 0.22a 1.86 ± 0.39a
K270 0.16 ± 0.03a 0.19 ± 0.01a 0.15 ± 0.01a 0.20 ± 0.01a
DK 0.0021 ± 0.0000a 0.0054 ± 0.0047ab 0.0022 ± 0.0001a 0.0130 ± 0.0000b
Waxes (mg/kg) 44.1 ± 1.6a 49.6 ± 4.9a 50.7 ± 6.2a 54.0 ± 0.0a
Trilinolein (%) 0.33 ± 0.12a 0.40 ± 0.04a 0.40 ± 0.06a 0.33 ± 0.07a
DECN 0.17 ± 0.02a 0.14 ± 0.01a 0.16 ± 0.05a 0.10 ± 0.13a
Fatty acid composition by GC (% m/m methyl esters)
Myristic C14:0 0.013 ± 0.002a 0.009 ± 0.001a 0.010 ± 0.001a 0.011 ± 0.001a
Palmitic C16:0 12.88 ± 0.00a 12.76 ± 0.19a 12.88 ± 0.23a 12.98 ± 0.08a
Palmitoleic C16:1 0.92 ± 0.02ab 0.88 ± 0.01a 0.94 ± 0.01ab 0.96 ± 0.01b
Margaric C17:0 0.061 ± 0.001a 0.056 ± 0.006a 0.067 ± 0.004a 0.064 ± 0.005a
Margaroleic C17:1 0.098 ± 0.003a 0.088 ± 0.004a 0.097 ± 0.005a 0.101 ± 0.001a
Stearic C18:0 3.04 ± 0.13a 2.97 ± 0.04a 2.99 ± 0.04a 2.98 ± 0.00a
Oleic C18:1 67.99 ± 0.31a 68.14 ± 0.25a 67.74 ± 0.28a 67.92 ± 0.04a
Linoleic C18:2 13.10 ± 0.07a 13.24 ± 0.02ab 13.41 ± 0.01b 13.16 ± 0.06a
Linolenic C18:3 0.91 ± 0.04a 0.92 ± 0.01a 0.91 ± 0.01a 0.89 ± 0.01a
Arachidic C20:0 0.48 ± 0.03a 0.48 ± 0.00a 0.49 ± 0.01a 0.46 ± 0.00a
Eicosenoic C20:1 0.29 ± 0.01a 0.28 ± 0.01a 0.28 ± 0.00a 0.28 ± 0.00a
Behenic C22:0 0.17 ± 0.02a 0.15 ± 0.00a 0.15 ± 0.00a 0.15 ± 0.01a
Lignoceric C24:0 0.063 ± 0.010a 0.053 ± 0.010a 0.068 ± 0.004a 0.059 ± 0.001a
trans oleics 0.015 ± 0.007a Traces 0.020 ± 0.000a 0.011 ± 0.000a
trans L+Ln 0.025 ± 0.007a 0.020 ± 0.000a 0.027 ± 0.004a 0.020 ± 0.000a
Sterols by GC relative amount (%)
Cholesterol 0.053 ± 0.010a 0.061 ± 0.027a 0.069 ± 0.001a 0.062 ± 0.002a
Brassicasterol Traces Traces Traces Traces
Campesterol 2.33 ± 0.01a 2.39 ± 0.02b 2.35 ± 0.01ab 2.32 ± 0.01a
Stigmasterol 1.14 ± 0.01a 1.02 ± 0.01b 1.12 ± 0.01a 1.33 ± 0.03c
Apparent b-Sitosterol 95.60 ± 0.31a 95.64 ± 0.20a 95.46 ± 0.24a 95.46 ± 0.23a
D7-Stigmastenol 0.12 ± 0.05a 0.15 ± 0.01b 0.18 ± 0.08a 0.10 ± 0.00c
Total sterols (lg/g) 1600.0 ± 34.0a 1342.6 ± 146.6a 1616.1 ± 55.1a 1596.5 ± 40.3a
Erythrodiol+Uvaol 3.29 ± 0.08a 3.13 ± 0.01a 3.48 ± 0.16a 3.63 ± 0.19a
Parameter 30 °C
B. Manzanilla de Sevilla+Unknown (95:5%) 30 min 90 min
Acidity (%) 0.25 ± 0.01a 0.28 ± 0.05a
Peroxides (meq. O2/kg oil)* 2.24–5.23 1.94–4.35
K232 1.45 ± 0.11a 1.38 ± 0.13a
K270 0.12 ± 0.03a 0.13 ± 0.01a
DK 0.0036 ± 0.0002a 0.0034 ± 0.0001a
Waxes (mg/kg) 34.5 ± 3.5a 42.9 ± 4.5a
Trilinolein (%) 0.07 ± 0.01a 0.11 ± 0.01a
DECN 0.06 ± 0.03a 0.13 ± 0.06a
Fatty acid composition by GC (% m/m methyl esters)
Myristic C14:0 0.014 ± 0.002a 0.011 ± 0.001a
Palmitic C16:0 10.80 ± 0.15a 11.15 ± 0.05a
Palmitoleic C16:1 0.86 ± 0.01a 0.87 ± 0.01a
Margaric C17:0 0.180 ± 0.057a 0.210 ± 0.014a
Margaroleic C17:1 0.280 ± 0.141a 0.285 ± 0.134a
Stearic C18:0 3.23 ± 0.08a 3.05 ± 0.08a
Oleic C18:1 78.80 ± 0.00a 77.66 ± 0.03b
Linoleic C18:2 4.11 ± 0.08a 4.96 ± 0.06b
Linolenic C18:3 0.80 ± 0.06a 0.81 ± 0.01a
Arachidic C20:0 0.43 ± 0.15a 0.43 ± 0.12a
Eicosenoic C20:1 0.30 ± 0.01a 0.33 ± 0.01a
Behenic C22:0 0.14 ± 0.04a 0.14 ± 0.01a
Lignoceric C24:0 0.072 ± 0.005a 0.116 ± 0.062a
trans oleics 0.023 ± 0.003a 0.022 ± 0.013a
trans L + Ln Traces Traces
Sterols by GC relative amount (%)
Cholesterol 0.050 ± 0.013a 0.068 ± 0.025a
Brassicasterol Traces Traces
Campesterol 2.30 ± 0.01a 2.61 ± 0.01b
Stigmasterol 0.54 ± 0.02a 0.62 ± 0.00b
Apparent b-Sitosterol 96.52 ± 0.08a 96.06 ± 0.09b
D7-Stigmastenol 0.12 ± 0.01a 0.15 ± 0.01a
Total sterols (lg/g) 1443.3 ± 54.2a 1503.4 ± 133.5a
Erythrodiol+Uvaol 2.02 ± 0.02a 2.00 ± 0.00a

Values are mean ± standard deviation (n = 2). Different letters within rows indicate statistical differences as per ANOVA (p < 0.05) and Tukey’s HSD test.
*
Peroxide values were determined three months apart.
 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545 537

Table 2
Concentrations of volatile compounds of the studied EVOOs.

A. Morisca:Verdial de Badajoz (90:10%)

Volatile compound Odour descriptor* Odorant series (Odour threshold; ng/g) Concentration (ng/g)
20 °C/ 20 °C/ 30 °C/ 30 °C/
30 min 90 min 30 min 90 min
C6 compounds
trans-2-Hexenal Lawn; green-apple like; bitter Grass (1125); apple (424); bitter-like (420) 2037 ± 207a 1867 ± 217a 2204 ± 193a 1096 ± 87b
almonds
C6/LnA-Aldehydes 2037 1867 2204 1096
cis-3-Hexen-1-ol Apple; leaf-like Apple (6000); leaf (1100) 1003 ± 74ab 1104 ± 160a 1011 ± 43ab 897 ± 58b
trans-3-Hexen-1-ol Green, bitter Bitter-like (1500) 12.2 ± 1.1a 13.3 ± 1.6a 11.8 ± 0.8a 13.7 ± 1.0a
trans-2-Hexen-1-ol Green grass, leaves; green, grassy, Grass (8000); leaf (5000) 2162 ± 147a 1787 ± 188b 531 ± 29c 2586 ± 105d
sweet
cis-2-Hexen-1-ol n.f. 7.7 ± 0.8a 11.0 ± 1.5b 4.7 ± 0.3c 14.1 ± 1.2d
C6/LnA-Alcohols 3185 2915 1559 3511
cis-3-Hexenyl Olive fruity; green notes; banana Grass (750); olive fruit (750); banana (200) 667 ± 43a 764 ± 94a 764 ± 80a 210 ± 29b
acetate
trans-2-Hexenyl n.f. 4.9 ± 0.5ab 5.9 ± 0.9bc 4.7 ± 0.4a 6.0 ± 0.4c
acetate
C6/LnA-Esters 672 770 769 216
Hexanal Grass; green apple; green-sweet Grass (300); apple (80); sweet-like (75) 415 ± 38a 412 ± 48a 455 ± 56a 284 ± 28b
C6/LA-Aldehydes 415 412 455 284
1-Hexanol Fruit; banana, soft Olive fruit (400); banana (400) 1574 ± 120a 2360 ± 229b 986 ± 34c 1840 ± 137a
C6/LA-Alcohols 1574 2360 986 1840
Hexyl acetate Green; fruity; sweet Grass (1040); olive fruit (1040); sweet-like 113 ± 9a 134 ± 9b 118 ± 7a 57.4 ± 3.4c
(1040)
C6/LA-Esters 113 134 118 57.4
C5 compounds
trans-2-Pentenal Green, apple; green, bitter almond Apple (300); bitter-like (300) 17.7 ± 1.5a 15.5 ± 1.7a 24.4 ± 1.3b 12.1 ± 0.7c
C5/LnA-Aldehydes 17.7 15.5 24.4 12.1
1-Penten-3-ol Lawn; olives; leaf; pungent Grass (400); olive fruit (400); leaf (400); 650 ± 84a 624 ± 64a 765 ± 88a 634 ± 40a
pungent-like (400)
cis-2-Penten-1-ol Banana; sweet; green fruity; fresh Banana (250); sweet-like (250); olive fruit (250) 160 ± 7a 168 ± 16a 156 ± 27a 167 ± 19a
olive fruits
trans-2-Penten-1- n.f. 44.3 ± 1.1ac 40.9 ± 2.8a 52.1 ± 2.3b 49.7 ± 5.3bc
ol
C5/LnA-Alcohols 854 833 973 851
1-Penten-3-one Leaf; bitter; pungent Leaf (50); bitter-like (50); Pungent-like (50) 205 ± 6a 173 ± 14a 589 ± 49b 24.2 ± 3.7c
C5/LnA-Ketones 205 173 589 24.2
Pentanal Woody; bitter; oily Wood (240); Bitter-like (240) 102 ± 8ac 121 ± 9a 176 ± 16b 92.9 ± 6.0c
C5/LA-Aldehydes 102 121 176 92.9
1-Pentanol Fruity; strong, sticky, balsamic Olive fruit (470); pungent-like (3000) 55.7 ± 5.9a 36.9 ± 4.7b 30.1 ± 2.7b 89.1 ± 6.7c
C5/LA-Alcohols 55.7 36.9 30.1 89.1
3-Pentanone Fruity; green; sweet Grass (70,000); olive fruit (70,000); sweet-like 517 ± 37ac 448 ± 43a 340 ± 17b 565 ± 64c
(70,000)
C5/LA-Ketones 517 448 340 565
R Total compounds 9747 10,085 8223 8639
B. Manzanilla de Sevilla:Unknown (95:5%)
Volatile compound Odorant series (Odour threshold; ng/g) Concentration (ng/g)
30 °C/ 30 min 30 °C/ 90 min
C6 compounds
trans-2-Hexenal Grass (1125); apple (424); bitter-like (420) 820 ± 73a 1733 ± 28b
C6/LnA-Aldehydes 820 1733
cis-3-Hexen-1-ol Apple (6000); leaf (1100) 2564 ± 103a 2743 ± 132a
trans-3-Hexen-1-ol Bitter-like (1500) 112 ± 4a 142 ± 9b
trans-2-Hexen-1-ol Grass (8000); leaf (5000) 2713 ± 132a 3985 ± 189b
cis-2-Hexen-1-ol 21.0 ± 2.3a 33.3 ± 3.3b
C6/LnA-Alcohols 5410 6903
cis-3-Hexenyl acetate Grass (750); olive fruit (750); banana (200) 2237 ± 63a 121 ± 11b
trans-2-Hexenyl acetate 12.3 ± 1.3a 5.4 ± 1.0b
C6/LnA-Esters 2250 127
Hexanal Grass (300); apple (80); sweet-like (75) 537 ± 11a 751 ± 32b
C6/LA-Aldehydes 537 751
1-Hexanol Olive fruit (400); banana (400) 5781 ± 172a 5857 ± 112a

(continued on next page)

538 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545

Table 2 (continued)

B. Manzanilla de Sevilla:Unknown (95:5%)

Volatile compound Odorant series (Odour threshold; ng/g) Concentration (ng/g)
30 °C/ 30 min 30 °C/ 90 min
C6/LA-Alcohols 5781 5857
Hexyl acetate Grass (1040); olive fruit (1040); sweet-like (1040) 216 ± 8a 31.3 ± 0.8b
C6/LA-Esters 216 31
C5 compounds
trans-2-Pentenal Apple (300); bitter (300) 8.1 ± 0.4a 15.7 ± 0.7b
C5/LnA-Aldehydes 8 16
1-Penten-3-ol Grass (400); olive fruit (400); leaf (400); pungent-like (400) 885 ± 125a 907 ± 143a
cis-2-Penten-1-ol Banana (250); sweet-like (250); olive fruit (250) 262 ± 36a 358 ± 59b
trans-2-Penten-1-ol 54.5 ± 7.4a 83.9 ± 12.3b
C5/LnA-Alcohols 1201 1348
1-Penten-3-one Leaf (50); bitter-like (50); pungent-like (50) 41.0 ± 4.2a 53.6 ± 2.5b
C5/LnA-Ketones 41 54
Pentanal Wood (240); bitter-like (240) 156 ± 3a 74.3 ± 4.7b
C5/LA-Aldehydes 156 74
1-Pentanol Olive fruit (470); pungent-like (3000) 115 ± 12a 130 ± 6a
C5/LA-Alcohols 115 130
3-Pentanone Grass (70,000); olive fruit (70,000); sweet-like (70,000) 996 ± 83a 942 ± 74a
C5/LA-Ketones 996 942
R Total compounds 17,531 17,996

Values are mean ± standard deviation (n = 4). Different letters within rows indicate statistical differences as per ANOVA (p < 0.05) and Tukey’s HSD test. n.f.: not found.

of volatile compound in oil sample/odour threshold) of each com-
pound assigned to this series. According to its aroma descriptors, a
volatile compound can be included in one or several odorant series.
The odour descriptors of C6 and C5 compounds that contribute
to the sensory properties of EVOOs were taken from literature
(Angerosa, Mostallino, Basti, & Vito, 2000; Kalua et al., 2007; Tura,
Failla, Bassi, Pedò, & Serraiocco, 2008); and grouped in 9 different
odorant series: grass, leaf, wood, bitter-like, sweet-like, pungent-
like (or rasping), olive fruit, apple and banana. Some compounds
were included in two or more odorant series.
2.4. Statistical analysis
Analysis of variance (ANOVA) was carried out using the
statistical package Statgraphics Centurion XV for windows Version
15.2.06 (Statistical Graphics Corp., Herndon, Va, USA). Tukey’s HSD
test was used as a single-step multiple comparison method in con-
junction with ANOVA to identify significantly different means.
Partial least squares (PLS) regression was implemented by using
the statistical package Unscrambler v. 9.1 for Windows (CAMO
Software, Oslo, Norway).
3. Results and discussion
3.1. Quality indices, fatty acid, sterol and triterpene dialcohols
composition of EVOOs extracted at different malaxation conditions
It is of great importance to deepen in the effect of the malaxa-
tion conditions on the quality of the VOO produced in order to
strike an appropriate balance between the economic turnover
and the quality of the commercial product (Inarejos-García et al.,
2009).
The analysed quality indices of the olive oil samples in the differ-
ent malaxation conditions studied met the standards of the Euro-
pean Community for the classification as ‘extra virgin’ category
(European Union Commission, 1991, 2011). All the oils from Morisca
and Manzanilla de Sevilla analysed showed values below the upper
limits established by EU Regulations for the extra virgin olive oil
category (acidity 6 0.8°; peroxide index 6 20 meq O2/kg;
K270 6 0.22; K232 6 2.5; DK 6 0.01; Waxes 6 250 mg/kg; Trilino-
lein < 0.5%; DECN 6 0.2).
There was an increase, in some cases significant, when either
the malaxation temperature and/or time was risen (Table 1a, b)
such it was observed in the case of Cornicabra variety (Inarejos-
García et al., 2009). Ranalli, Contento, Schiavone, and Simone
(2001) attributed this behaviour to the increase in activity of the
lipase enzymes (responsible for the increase of free acidity) and
to an intensification of the primary oxidation processes (responsi-
ble for the increase of the K232 and peroxide index values) and the
secondary oxidation processes (responsible for the increase of the
K270 and carbonyl index values) when malaxation temperature
rises.
The fatty acid levels, as well as being in accordance with the limits
mentioned in the EU Regulation for ‘extra virgin’ category (European
Union Commission, 1991, 2003), were not significantly influenced
by both malaxation parameters (time and temperature) for both
oil varieties (Morisca and Manzanilla de Sevilla) (Table 1a, b).
Concerning sterols and the sum of erythrodiol and uvaol, ex-
pressed as sum of percentage of total sterols, it should be noted
that for both cultivars, Morisca and Manzanilla de Sevilla, no signif-
icant differences in total sterol composition and erythrodiol+uvaol
of oil samples were detected (Table 1a, b). Nevertheless, a trend is
observed by evaluating the effect of malaxation temperature in
Morisca oils, higher contents of total sterols and triterpene dialco-
hols were observed when olive paste was malaxed at the highest
temperature (30 vs. 20 °C) (Table 1a). According to Allouche et al.
(2010), this rise could be explained by the fact that higher temper-
atures decrease the oil viscosity, thus the extraction of these com-
pounds from the olive paste is favoured.
3.2. Effect of malaxation process on the volatile profile of EVOOs
Three branches of volatile C6 metabolites are generated from
linolenic acid (LnA) and linoleic acid (LA) through the lipoxygenase
(LOX) pathway. LOX transforms LnA and LA into their correspond-
ing 9- and 13-hydroperoxides, in a ratio ranging between 65:35
 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545 539

(a) C6 compounds 20 ºC
9000

8000 30 ºC

7000 20 ºC

6000
30 ºC

5000
ng/g oil

4000 20 ºC 30 ºC

3000

2000

1000

0
30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min
LnA compounds LA compounds Total compounds

Aldehydes Alcohols Esters

2500 (b) C5 compounds 30 ºC

2000 20 ºC
30 ºC

1500
20 ºC
ng/g oil

20 ºC 30 ºC
1000

500

0
30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min 30 min 90 min
LnA compounds LA compounds Total compounds

Aldehydes Alcohols Ketones

Fig. 1. (a) Sum of C6 volatile compounds in Morisca:Verdial de Badajoz (90:10%) oils at different malaxation process conditions: Temperature: 20/30 °C; Time: 30/90 min. (b)
Sum of C5 volatile compounds in Morisca:Verdial de Badajoz (90:10%) oils at different malaxation process conditions: Temperature: 20/30 °C; Time: 30/90 min. (c) Sum of C6
volatile compounds in Manzanilla de Sevilla:Unknown (95:5%) oils at different malaxation process conditions: Temperature: 30 °C; Time: 30/90 min. (d) Sum of C5 volatile
compounds in Manzanilla de Sevilla:Unknown (95:5%) oils at different malaxation process conditions: Temperature: 30 °C; Time: 30/90 min. Volatile content of C6/LnA
aldehydes is trans-2-hexen-1-al. Volatile contents of C6/LnA alcohols are the sum of: cis-2-hexen-1-ol, trans-2-hexen-1-ol, cis-3-hexen-1-ol and trans-3-hexen-1-ol. Volatile
contents of C6/LnA esters are the sum of: cis-3-hexenyl acetate and trans-2-hexenyl acetate. Volatile content of C6/LA aldehydes is hexanal. Volatile content of C6/LA alcohols
is hexanol. Volatile content of C6/LA esters is hexyl acetate. Volatile content of C5/LnA aldehydes is trans-2-pentenal. Volatile contents of C5/LnA alcohols are the sum of: 1-
penten-3-ol, cis-2-penten-1-ol and trans-2-penten-1-ol. A volatile content of C5/LnA ketones is 1-penten-3-one. Volatile content of C5/LA aldehydes is pentanal. Volatile
content of C5/LA alcohols is 1-pentanol. Volatile content of C5/LA ketones is 3-pentanone.

and 55:45, respectively. Only the 13-hydroperoxides, from both
LnA (13-HPOT) and LA (13-HPOD), are cleaved by hydroperoxide
lyase (FAHL) into C12 oxo-acids, cis-3-hexenal and hexanal, as
the enzyme has a high substrate specificity. Enzymatic transforma-
tion of the two aldehydes mediated by isomerases (IR), alcohol
dehydrogenases (ADH) and alcohol acetyl transferases (AAT) yields
the corresponding C6 esters and C6 alcohols. An additional branch
of short-chain green volatiles, including oxygenated C5 com-
pounds, is biosynthesised through another LOX pathway. In this
case, 13-HPOT undergoes a b-scission yielding pentene dimers
and pentenols through the alkoxyl radical. The subsequent oxida-
tion of pentenols catalysed by an alcohol dehydrogenase yields
C5 carbonyl compounds (Ranalli, Pollastri, Contento, Iannucci, &
Lucera, 2003).
3.2.1. Effect of malaxation time
In our oils, an opposite behaviour for the most abundant C6 alde-
hydes, trans-2-hexenal and hexanal, is observed taking account the
effect of time: in Morisca oils C6 aldehydes decreased in concentra-
tion with increasing times of olive paste kneading, such decreases
were statistically significant (p < 0.05) at 30 °C (Table 2a, Fig. 1a).
In the case of Manzanilla de Sevilla oils, the trend differed com-
pletely (Table 2b, Fig. 1c) and was in good agreement with other
varieties such as the Italian Coratina and Frantoio (Angerosa et al.,
2001), Leccino, Dritta and Caroleo (Ranalli et al., 2003), the Spanish
Cornicabra (Gómez-Rico, Inarejos-García, Salvador, & Fregapane,
2009) and the Tunisian Chemlali and Chetoui (Youssef et al.,
2013). With these results, it is clear that the behaviour of enzymes
pattern during malaxation is linked to the variety.
540 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545

C6 compounds
18000 (c)
Total compounds
16000

14000

12000 LnA compounds LA compounds

ng/ g oil

10000

8000

6000

4000

2000

0
30 min 90 min 30 min 90 min 30 min 90 min

Aldehydes Alcohols Esters

C5 compounds
3000 (d)
Total compounds

2500

2000
LnA compounds LA compounds
ng/ g oil

1500

1000

500

0
30 min 90 min 30 min 90 min 30 min 90 min

Aldehydes Alcohols Ketones

Fig. 1 (continued)

The content of C6 alcohols also experienced an increase with
lengthy times for both varieties except trans-2-hexen-1-ol at low
temperatures (20 °C) for Morisca oils (Table 2a, b, Fig. 1a, c).
A considerable decrease was detected for C6 esters at 30 °C in
both cultivars, especially for cis-3-hexenyl acetate (Table 2a, b,
Fig. 1a, c). Ranalli et al. (2003) attributed this behaviour to a pro-
gressive inactivation of AAT.
The concentrations of individually C5 compounds were influ-
enced differently by time adopted during the malaxation for both
varieties: for Morisca oils, ketones were the compounds that expe-
rienced the most significant changes, only at 30 °C. Thus, the con-
centrations of 1-penten-3-one diminished drastically while the
amount of 3-pentanone increased (Table 2a). Nevertheless, the
concentration of ketones in Manzanilla de Sevilla oils rarely varied;
on the other hand, the amount of LnA-alcohols in these oils,
especially cis-2-penten-1-ol, was higher at prolonged malaxation
times (Table 2b).
In general terms, the total concentration of C6 volatiles in-
creased with extended times of olive paste kneading for both stud-
ied oils regardless of temperature (Fig. 1a, c). For C5 volatiles, this
behaviour was maintained in the case of Manzanilla de Sevilla oils
(Fig. 1d) but not in the case of Morisca oils (Fig. 1b): C5 compounds
tended to decrease at high times.
3.2.2. Effect of malaxation temperature
Changes in malaxation temperature (at 20 and 30 °C) were eval-
uated only with one cultivar (Morisca). According to Salas and
Sánchez (1999) the malaxation temperature generally causes a
decrease of levels of volatile compounds from LOX pathways, as
a consequence of proved inactivation of hydroperoxide lyases. This
 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545 541

Fig. 2. (a) One-dimension PLS-2: scores plot for Morisca:Verdial de Badajoz (90:10%) oils extracted at 20/30 °C for 30/90 min (x), together with loadings plot of X-variables for
the 18 volatile descriptors (y) and loadings plot of Y-variables for the 9 aroma descriptors (z). One principal component accounted for 86% and 78% of the variance in X and Y
data, respectively. (b) One-dimension PLS-2: scores plot for Manzanilla de Sevilla:Unknown (95:5%) oils extracted at 30 °C for 30/90 min (x), together with loadings plot of X-
variables for the 18 volatile descriptors (y) and loadings plot of Y-variables for the 9 aroma descriptors (z). One principal component accounted for 97% of the variance in both
X and Y data, respectively.

Fig. 2 (continued)

trend was evidenced by other authors as well (Angerosa et al.,
2001; Gómez-Rico et al., 2009; Kalua, Bedgood, Bishop, & Prenzler,
2006; Ranalli et al., 2001).
With prolonged times (90 min) most C6-compounds under-
went statistically significant variations when temperature is in-
creased. In this sense, C6-aldehydes, alcohols and esters coming
from LnA (trans-2-hexenal, cis-3-hexen-1-ol, cis-3-hexenyl ace-
tate, respectively) and LA (hexanal, 1-hexanol and hexyl acetate,
respectively) decreased. Only an increase in C6/LnA-alcohols was
observed, the amount of trans-2-hexen-1-ol was higher at tested
extreme temperatures and times: 30 °C and 90 min (Table 2a,
Fig. 1a).
542 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545

Fig. 2 (continued)

Fig. 2 (continued)

Nevertheless, interactions between malaxation time and
temperature were observed for C5-compounds: the behaviour
of the majority compounds at short times (30 min) was opposite
to high times (90 min) when temperature was changed
(Table 2a).
In conclusion, the total concentration of C6 volatiles decreased
with high temperatures regardless of paste kneading time
(Fig. 1a). For C5 volatiles, when malaxation time was set to
30 min an increase in the total amount of C5 compounds was
observed at 30 °C, when time was set to 90 min C5/LnA compounds
diminished and C5/LA compounds increased slightly (Fig. 1b).
Angerosa et al. (2001) found the same trend for Coratina oils, with
the exception of 1-penten-3-one, although these authors assessed
only C5/LnA compounds. In a previous paper, the same authors
(Morales, Angerosa, & Aparicio, 1999) highlighted that the
production of 1-penten-3-one increased with temperature regard-
less malaxation time, suggesting this volatile is produced through
an oxidation process.
 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545 543

Fig. 2 (continued)

Fig. 2 (continued)

3.3. One-dimension partial least squares regression (PLS2) between
volatile compounds and odorant series
To estimate quantitatively the overall oil aroma, a method
based in the addition of OAVs was employed. This procedure
makes it possible to relate quantitative information obtained by
chemical analysis to sensory perception, providing a tentative aro-
ma profile (Sánchez-Palomo E & Alonso-Villegas R, 2010).
Two PLS2 modelling (one for Morisca and another for Manzanil-
la de Sevilla oils) between two data matrices (both volatile com-
pounds and odorant series) were performed providing in both
cases one-factor or 1D model. The models were evaluated by cross
validation via the root mean square error for predictions (RMSEP),
which was calculated to be lower than 10 in both cases. In general,
there were found significant positive and negative correlations
(r = |0.7–1.0|) amongst many of the data of both matrices.
544 P. Reboredo-Rodríguez et al. / Food Chemistry 158 (2014) 534–545
Connecting 18 volatile compounds with 9 aromatic series in
Morisca oils extracted at different malaxation conditions (20 or
30 °C for 30 or 90 min) resulted in two different groups of oils.
The other two groups of oils were remaining together in the middle
of both extreme groups (Fig. 2a(x)). The findings obtained can be
summarised as follows:
1. According to the scores figure (Fig. 2a(x)), the upper half only
included the Morisca oils extracted at 30 °C for 30 min, while
the lower half mainly included the Morisca oils extracted at
30 °C for longer times (90 min). However, along the middle
baseline of 0 were placed the non-differentiated groups of
Morisca oils extracted at 20 °C for both 30 and 90 min.
2. According to the X- and Y-loadings figures (Fig. 2a(y), Fig. 2a(z)),
for the highest values of PC1, Morisca/30 °C/30 min oils
were mainly associated to the following volatile compounds:
1-penten-3-one, trans-2-hexenal and cis-3-hexenyl acetate,
and to the odorant series: bitter-like, pungent-like, leaf, apple
and grass. For the lowest values of PC1, Morisca/30 °C/90 min
oils were mainly associated to the volatiles trans-2-hexen-1-
ol, 1-hexanol and 3-pentanone, and to the odorant series olive
and banana fruits. Morisca/20 °C/30 or 90 min oils were more
similar to Morisca/30 °C/90 min oils.
A clear separation of two groups of Manzanilla de Sevilla oils
was obtained (Fig. 2b) by linking 18 volatile compounds with 9
aromatic series:
1. According to scores figure (Fig. 2b(x)), the upper half only
included Manzanilla de Sevilla oils extracted at 30 °C for
30 min, while the lower half mainly included Manzanilla de
Sevilla oils extracted at the same temperature for longer times
(90 min).
2. According to the X- and Y-loadings figures (Fig. 2b(y), Fig. 2b(z))
for the highest values of PC1, Manzanilla de Sevilla/30 °C/30 min
oils were mainly associated to the volatiles cis-3-hexenyl acetate,
hexyl acetate and pentanal, and to the odorant series banana,
olive, grass and wood. For the lowest values of PC1, Manzanilla
de Sevilla/30 °C/90 min oils were mainly associated to the vola-
tiles trans-2-hexen-1-ol, trans-2-hexenal and hexanal, and to
the aromatic series apple, sweet-like, bitter-like and leaf.
4. Conclusions
The results obtained in this work shows that it is possible to
modulate the volatile fraction of an oil in accordance with malax-
ation temperature ranging from 20 to 30 °C and above all malaxa-
tion time ranging from 30 min to 90 min.
The total concentration of C6 volatiles increased with extended
times of olive paste kneading for both oils regardless of tempera-
ture. For C5 volatiles, this behaviour was maintained in the case
of Manzanilla de Sevilla oils but not in the case of Morisca oils. Con-
cerning temperature conditions, the total concentration of Morisca
C6 volatiles decreased with 30 °C regardless of paste kneading time.
Nevertheless, for C5 volatiles, when malaxation time was set to
30 min an increase in the total amount of C5 compounds was ob-
served, and when time was set to 90 min C5/LnA compounds
diminished and C5/LA compounds increased slightly.
A clear difference was found between oils processed at 30 °C/
30 min and 30 °C/90 min for both varieties in terms of odorant
series:
 The oils from Morisca obtained at 30 °C and 30 min were char-
acterised mainly by bitter-like, pungent-like, leaf, apple and
grass odorant series, while for Manzanilla de Sevilla oils, fruity
(banana, olive), grass and wood notes predominated.
 The sensory profile of oils from Morisca obtained at 30 °C and
90 min changed with respect to shorter times, thus olive and
banana fruits were the characteristic aromatic series instead.
For Manzanilla de Sevilla the sensory profile changed to apple,
sweet-like, bitter-like and leaf notes.
Acknowledgements
This work was funded by EU FEDER, and also under contracts
09TAL045E (Xunta de Galicia and Aceites Abril S.L.) and 2009/
060 (Xunta de Galicia). The authors are grateful to Aceites Abril
S. L., and especially indebted to J. M. Pérez-Canal and F. Osorio-
Rodríguez.
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