Analytical Methods

Ultrasound assisted maceration: An original procedure for direct aromatisation

of olive oil with basil
Sbastien Veillet, Valrie Tomao, Farid Chemat
*
UMR408, Scurit et Qualit des Produits dOrigine Vgtale, INRA, Universit Avignon et Pays de Vaucluse, F-84000 Avignon, France
a r t i c l e i n f o
Article history:
Received 9 September 2009
Received in revised form 7 February 2010
Accepted 2 May 2010
Keywords:
Aromatisation
Basil
Olive oil
Ultrasounds
a b s t r a c t
Aromatisation of olive oil is a new trend in the Mediterranean area, both for sensory and for nutritional
improvement. This work presents the development of a green enrichment of an olive oil with basil. In fact
instead of a solvent extraction of the aromas, purify them and add them to the olive oil or instead of doing
steam distillation prior to add the essential oil into the olive oil, basil leaves were directly put into the
olive oil. Ultrasounds were then applied to the mixture in order to accelerate diffusion of the basil volatile
compounds into the olive oil. The processing time is reduced from hours or days to few minutes when
comparing traditional maceration and ultrasound assisted aromatisation. GC/MS chromatographs have
similar proles between macerated and ultrasounds assisted macerated oils. Concentration of linalool
and eugenol were calculated into the aromatised oils and used as indicators of the aromatisation.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Virgin olive oil is widely consumed in the Mediterranean basin.
It is well known for its nutritional properties due to its high con-
tent in volatile and non-volatile compounds of interest. The rst
group of interesting compounds is the fatty acid composition: olive
oil is rich in monounsaturated fatty acids (oleic acid) and polyun-
saturated fatty acids (linoleic, and linolenic acid) that are known to
show protective effects against cardiovascular diseases by prevent-
ing oxidation of low density lipoproteins (LDL) (Kratz et al., 2002;
Ramirez-Tortosa et al., 1999). Other compounds of interest are the
phenolic compounds, which are secondary plant metabolites that
show antioxidant properties and thus have been reported to
induce lower rate of cancers and Alzheimers disease (Owen
et al., 2000; Panza et al., 2007; Scarmeas, Ster, Tang, Mayeux, &
Luchsinger, 2006). The protective role of olive oil also comes from
its content in vitamins, especially vitamin E (Gimeno et al., 2000)
which has been reported to be one of the most effective antioxi-
dant for reducing lipid peroxydation by donating hydrogen to fatty
peroxyl radicals (Ravindra, 2000).
Like olive oil, aromatic plants are known to show antioxidant
activity. Common examples are rosemary (Erkan, Ayranci, &
Ayranci, 2008; Genena, Hense, Smania Junior, & De Souza, 2008)
or thyme (Youdim & Deans, 1999) and at lower level, some
compounds of basil essential oil have also demonstrated antioxi-
dant and preservative capacities (Bagamboula, Uyttendaele, &
Debevere, 2004; Beric, Nikolic, Stanojevic, Vukovic-Gacic, &
Knezevic-Vukcevic, 2008; Lee, Umano, Shibamoto, & Lee, 2005;
Suppakul, Miltz, Sonneveld, & Bigger, 2003). Nevertheless, basil
(Ocinum basilicum L.) is mostly used as culinary herb for its charac-
teristic aroma or in perfumery. Both antioxidant activity and
aromas mostly come from the essential oil contained in these aro-
matic plants. Distillation of this plants leads to extracts very rich in
antioxidants so various extracting methods were developed in
order to preserve the antioxidant molecules from degradation
(Araus, Uquiche, & Del Valle, 2009; Bayramoglu, Sahin, & Sumnu,
2008; Bousbia et al., 2009; Farhat, Ginies, Romdhanea, & Chemat,
2009). One of these new processes is the ultrasound assisted
extraction (UAE).
Ultrasounds are mechanic waves able to propagate through an
elastic medium. The sound wave will temporarily dislodge the
molecules of the medium from their original location, which will
create compression and rarefaction areas into the medium corre-
sponding to the compression and rarefaction cycles of the sound
wave. The higher the power of ultrasounds is, the higher the
depression (or rarefaction) areas are. If the rarefaction cycles are
strong enough, the distance between two molecules that links
the medium might be above the critical molecular distance of
the medium. This medium will then break and voids also call
cavitation bubbles are generated. During rarefaction cycles the
cavitating bubbles will grow up and decrease in size during com-
pression cycles. When the size of these bubbles reaches a critical
size, they will collapse during a compression cycle and this col-
lapse generates hot spot with very high temperature and high
pressure. When this bubble collapse next to the surface of a solid
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.05.005
* Corresponding author.
E-mail address: farid.chemat@univ-avignon.fr (F. Chemat).
Food Chemistry 123 (2010) 905911
Contents lists available at ScienceDirect
Food Chemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
material (like a plant matrix), a micro-jet is directed toward the
plant material and is strong enough to break the cell walls. As
essential oil glands are usually present at the surface of the aro-
matic plant, the collapse of the cavitation bubbles will destroy
the glands that will release the essential oil or the plant extract
into the surrounding medium. Thus ultrasounds have been used
in the past few years for some aromas extractions (Da Porto, Decor-
ti, & Kikic, 2009; Jadhav, Rekha, Gogate, & Rathod, 2009; Kimbaris
et al., 2006; Shotipruk, Kaufman, & Wang, 2001). All the results ob-
tained showed either better yields, better quality of the extract
and/or shorter times of extraction. These results were very prom-
ising but as they were all using solvents (either petroleum solvents
or ethanol), the extracts have to be puried prior to be used in the
industry. Alternative solvents that would allow good extraction
yields have to be found in order to facilitate the use of extracts
in the food industry or in cosmetics.
Conventional extraction of antioxidants or aromas can be per-
formed in two different ways: the rst one is to do a solvent
extraction before to add the extract into the food matrix of interest.
This rst technique raises different problems such as the use of
petroleum solvents, the long time of extraction (23 h), the tem-
perature of extraction (often performed in a reuxing system),
the evaporation of the solvent and the purication of the extract.
The other conventional technique is the steam distillation of the
essential oil contained in some plants and herbs. This process also
raises some problems such as the use of big amounts of water (to
generate the steam and to cool the system), the plant matrix is
boiled so there could be thermal damages and it is a long proce-
dure (24 h).
Some problems due to the use of ultrasonic systems have been
raised by different teams (Chemat, Grondin, Shum Cheong Sing, &
Smadja, 2004; Patrick, Blindt, & Janssen, 2004; Schneider, Zahn,
Hofmann, Wecks, & Rohm, 2006). Off avours and oil degradation
were observed because of the too high intensity of the probe and
the horn systems. In fact these two systems are very powerful be-
cause ultrasounds are delivered on a very small surface. Thus the
intensity of ultrasounds at the tip of the probe is very high (about
50200 W/cm
2
). The new device developed by REUS does not con-
sist of a probe system, but the power is delivered on the whole
base of the system which corresponds to a power of about 1 W/
cm
2
. At this power very little oxidation could be observed and this
system is more applicable to food processing.
Hamed (2006) proposed a good idea, the use edible oil as an
alternative solvent for extraction of antioxidant components from
natural herbs. Japon-Lujan et al. (2008) used olive leaves for a di-
rect enrichment of edible oils. This work presents a combination
of the method proposed by Hamed and the ultrasound technology
experimented by Luque de Castro and collaborators. Basil leaves
were directly put into the olive oil and ultrasounds were applied
to the mixture in order to enhance the speed of the maceration.
This resulted in a greener procedure using olive oil as solvent (no
petroleum solvent), run out at room temperature (to prevent ther-
mal degradation) and in a dramatically reduced time (so reduced
energy costs and CO
2
emissions). The main idea was the use of a
direct aromatisation procedure in order to make it more conve-
nient and to reduce the time of aromatisation.
2. Materials and methods
2.1. Materials
Fresh basil leaves (Ocimum basilicum L.) were taken from basil
plants purchased at Saint-Rmy Basilic (St. Rmy de Provence, Bou-
che du Rhne, France). Olive oil was purchased from a local
supermarket.
2.2. Isolation of basil essential oil by steam distillation
Basil leaves (1 kg) were placed in a 20 L distillation tank with
2 L of deionised water. The mixture was heated by induction plate
and the steam distillation lasted for 3 h. The essential oil was col-
lected in a Clevenger system and this oil was stored at 4 C in the
dark before GC/MS analysis.
2.3. Conventional aromatisation
Freshly cut basil leaves (150 g) were put in 1 L of olive oil and
the mixture was allowed to stand at room temperature for several
hours with intermediate samplings for following the kinetic of
aromatisation.
2.4. Ultrasound assisted maceration procedure
A sono-extraction reactor (from 0.5 to 3 L, Fig. 1) developed by
REUS (www.etsreus.com, France) has been used to perform ultra-
sonic tests. The intensity of ultrasounds is about 1 W/cm
2
with a
frequency of 25 kHz. In order to keep constant temperature into
the mixture, the reactor is made of a double mantle into which
cooling water circulate. Olive oil (1 L) was placed into the reactor
and different amounts of basil leaves were added to the oil. A rotat-
ing pale allowed homogenisation of the mixture during all the
experiment. Ultrasounds were applied for different periods and
the nal aromatised mixture was ltered through a coffee lter
in order to remove traces of leaves.
Fig. 1. Ultrasonic device (www.reus.com).
906 S. Veillet et al. / Food Chemistry 123 (2010) 905911
2.5. GC/MS analysis
The volatiles of the olive oil and basil extracts were estimated
using HS-SPME/GC/MS. Four grams of olive oil was put in a 20 ml
vial and pre-incubated at 40 C for 15 min. The volatile compounds
contained into the headspace of the vials were then absorbed on a
SPME bre for 30 min under stirring of the oil. The bre used for
absorption and desorption was a SPME bre 75 lm carboxen-
10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0
0.5
1.0
1.5
2.0
2.5
3.0
(x100,000)
TIC
(x100,000)
TIC
2
5
6
8
9
1
1
1
2
1
4
1
5
1
6
1
7
1
8
1
9
2
0
2
1
2
2
2
3
2
4
2
5
2
6
2
72
8
2
9
3
0
10 20 30 40 50
2.5
5.0
7.5
1
2
3
Olive oil volatile compounds
a
b
Fig. 2. (a) GC/MS chromatogram of Ocinum basilicim essential oil volatile compounds; (b) chromatogram of an aromatised olive oil (15 min US and 150 g of basil leaves in 1 L
of olive oil). (1) Linalool, (2) methyl-eugenol, and (3) eugenol.
Table 1
Volatile compounds of basil essential oil identied by GC/MS.
Compound Peak number RT RI Area % Total volatiles
Limonene 5 17.158 1201 95,218 0.39
Eucalyptol 6 17.475 1207 323,702 1.32
b-Ocimene 8 19.933 1258 554,976 2.26
Terpinolene 9 21.392 1288 153,452 0.62
Camphor 11 32.317 1523 216,068 0.88
Linalool 12 33.583 1551 61,75,480 25.12
Bornyl acetate 14 35.100 1585 458,114 1.86
a-Guaiene 15 35.550 1595 250,995 1.02
4-Terpineol 16 36.042 1607 413,597 1.68
b-Farnesene 17 38.642 1672 12,21,338 4.97
d-Terpineol 18 38.958 1679 483,403 1.97
a-Terpineol 19 39.942 1704 85,989 0.35
Borneol 20 40.117 1708 225,458 0.92
a-Cubebene 21 40.558 1719 342,587 1.39
d-Guaiene 22 40.792 1725 107,393 0.44
Elixene 24 41.550 1744 223,563 0.91
c-Muurolene 25 42.567 1769 136,158 0.55
Methyl-eugenol 26 51.858 2020 37,29,694 15.17
Eugenol 30 57.142 2177 91,02,251 37.03
Total 98.85
S. Veillet et al. / Food Chemistry 123 (2010) 905911 907
PDMS (polydimethylsiloxane) and the volatile compounds were
separated with GC/MS Shimadzu QP2010 (Kyoto, Japan) equipped
with AOC 5000 auto-injector. The separation occurred on a capil-
lary column UB WAX (30 m 0.25 mm 0.5 lm). Helium was
used as gas carrier with a velocity of 41 cm per second. The sepa-
ration was performed as follows: initial oven temperature 35 C
hold for 3 min, then rise at a rate of 3 C per min until 200 C fol-
lowed by an increase at a rate of 10 C per min until 230 C. The
samples were injected at injector temperature of 250 C. The mass
spectra were recorded at three scan per second from 29 to 300 amu
and the ionisation mode was electronic impact at 70 eV. The vola-
tile compounds were identied by comparison of their retention
index and their mass spectra with those of authentic standards
or using the NIST98 [US National Institute of Standards and Tech-
nology (NIST), Gaithersburg, MD, USA] mass spectral library.
2.6. Scanning electron microscopy (SEM)
Basil leaves were carefully collected from the basil plants and
directly golden plated before being observed by scanning electron
microscopy. No further treatment has been performed in order to
not destroy essential oil glands. Leaves were also collected after
maceration (24H) and ultrasound assisted maceration (15 min)
and directly golden plated.
3. Results and discussion
3.1. Essential oil composition
The yield of the essential oil obtained from basil plants was 0.3%
(w/w). The GC/MS chromatograph corresponding to this essential
oil is presented in Fig. 2(a) and the relative percentages of the dif-
ferent volatile compounds are described in Table 1. Nineteen vola-
tile compounds were identied and they account for 98.85% of the
total volatile fraction. The essential oil is mainly composed of three
different volatile compounds: eugenol (37%), linalool (25%) and
methyl-eugenol (15%). Estragole that is the major compound in
some basil cultivars was not found here but this observation has
already been done in previous works (Klimankova et al., 2008).
Wide ranges of values were found for every compounds depending
on the cultivar (Telci, Bayram, Yilmaz, & Avci, 2006; Vina & Murillo,
2003), the growing conditions (organic or conventional conditions
(Klimankova et al., 2008)), or even depending on the season when
plants were harvested (Hussain, Anwar, Hussain Sherazi, &
Przybylski, 2008). In fact eucalyptol (1,8 cineole) was found from
trace level up to 20% of total volatiles (Klimankova et al., 2008),
eugenol from trace level up to 44% (Politeo, Jukic, & Milos, 2007),
estragole from trace level up to 53% (Chalchat & Ozcan, 2008)
and linalool from trace level up to 60% (Hussain et al., 2008). The
values found in this work were all in the same range than previ-
ously published values.
3.2. Comparison of the conventional maceration and ultrasound
assisted maceration
As described in Table 1, eugenol and linalool were the two major
compounds in the volatile fraction of the essential oil, thus these
two compounds were used as indicators of the aromatisation level
of the olive oil. Standards of linalool and eugenol were analysed by
GC/MS and a calibration curve was performed in order to calculate
their concentrations in the aromatised olive oils (Table 2).
Samples of olive oils were taken at different time of maceration
in order to follow the kinetic of aromatisation (Fig. 3). What was
rst observed is that ultrasounds dramatically increased the ki-
netic of aromatisation. Olive oils were aromatised with 150 g of ba-
sil leaves per litre of olive oil and after only 15 min of ultrasound
assisted maceration, estimated concentrations of linalool and
eugenol reached 3.68 and 1.34 mg/L, respectively. At the same
Table 2
Calibration parameters of linalool and eugenol and calculated concentration into the olive oil after aromatisation.
Linalool Eugenol
Calibration parameters
RT (min) 33.58 57.14
RI 1551 2177
Calibration curve equation + correlation
coefcient (R
2
)
y = 100018543x + 605,525 y = 18759486x + 103,409
R
2
= 0.992 R
2
= 0.967
Concentration in the olive oil (mg/L of olive oil)
12 h maceration + 150 g basil/L olive oil 1.66 0.31
15 min ultrasound + 150 g basil/L olive oil 3.68 1.34
15 min ultrasound + 50 g basil/L olive oil 1.94 0.79
0
1
2
3
4
0 5 10 15
t (min)
0
1
2
3
4
0 100 200 300 400 500 600 700 800
t (min)
C

(
m
g
/
L
)
C

(
m
g
/
L
)
a
b
Fig. 3. (a) Kinetic of aromatisation by ultrasound assisted maceration (150 g basil
leaves in 1 L of olive oil), () linalool, (j) eugenol; (b) kinetics of aromatisation by
traditional maceration or ultrasound assisted extraction (150 g basil leaves in 1 L of
olive oil), (h) eugenol by maceration, (}) linalool by maceration, (j) eugenol by
ultrasound aromatisation, () linalool by ultrasound aromatisation.
908 S. Veillet et al. / Food Chemistry 123 (2010) 905911
experimental conditions but with traditional maceration only 1.66
and 0.31 mg/L of linalool and eugenol were found in the oil after
12 h. Experiments with only 50 g of basil leaves per litre of olive
oil were run out and calculated concentrations of linalool and
eugenol were 1.94 and 0.79 mg/L after 15 min of ultrasound
assisted aromatisation. Concentrations of linalool and eugenol
were still increasing in the olive oil after 15 min of experiment,
but longer ultrasound macerations gave bitterness to the oil, thus
15 min has been chosen as the best maceration time.
The proles of the oils aromatised either by maceration or by
ultrasounds shown differences compared to the essential oil vola-
tile prole: linalool was the major compound in the aromatised
oils (Fig. 2(b)) when eugenol was the major compound in the
essential oil. This came from differences of solubility and volatility
of the two compounds in the olive oil.
3.3. Sensory evaluation
To analyse the level of aromatisation of the olive oils and to
detect potential off avours due to ultrasonic treatment, sensory
evaluation of olive oils treated and untreated with ultrasounds
was conducted by 12 panellists (graduated students and staff
members in the laboratory UMR A-408 Avignon University,
France). Randomly coded samples were presented to the panel-
lists who were asked to indicate the presence or absence of off
avours and to evaluate the level of aromatisation. No signicant
difference was found between the 15 min sonicated oil and the
macerated one concerning the off avours caused by oil degrada-
tion. In both 15 min ultrasounds (50 g/L of basil leaves) olive oil
and the macerated olive oil the plant taste was not very devel-
oped because of low concentrations of volatiles but 15 min of
Fig. 4. (1 and 2) SEM scan at 10 kV of a fresh basil leaf, magnication 200 and 800, respectively on the original picture scale, (3) SEM scan at 10 kV of a basil leaf after 6 h
maceration, magnication 800 on the original picture scale, (4) SEM scan at 10 kV of a basil leaf after 12 h maceration, magnication 1600 on the original picture scale, (5)
SEM scan at 10 kV of a basil leaf after ultrasonic extraction, magnication 1000 on the original picture scale, and (6) representation of a cavitation bubble collapsing on the
surface of an essential oil gland. (a) Plant prole with and essential oil gland at the surface of the leaf, (b) generation of a cavitation bubble, (c) collapse of the cavitation bubble
which generates a micro-jet directed toward the essential oil gland, and (d) collapse of the essential oil gland and release of the essential oil into the surrounding medium.
S. Veillet et al. / Food Chemistry 123 (2010) 905911 909
ultrasounds and 150 g/L gave good basil aroma to the olive oil
which was well appreciated by the panellists.
3.4. Mechanism of ultrasound assisted maceration
Basil leaves issued from the two processes of aromatisation
were observed by SEM and compared with a fresh sample
(Fig. 4). In the fresh leaf (Fig. 4(1) and (2)), basil cells were well
lled and basil essential oil glands appeared to be at the surface
of the leaf but push into the inferior epidermis. Stomata of the
plant could easily be seen on the picture, either open or close
which mean that they were working correctly. When a macera-
tion procedure has occurred, cells looked like they have been
dehydrated or emptied of they content (Fig. 4(3) and (4)). Thus
essential oil glands appeared like if they have been pulled out
of the cell layer, but most of the glands remained intact but emp-
tied of part of their content. The passive diffusion process of the
essential oil from the gland toward the surrounding medium
takes time and maceration of 12 h was not sufcient to let this
process fully happen. The ultrasonic maceration is not a diffusion
process: the cavitation bubbles generated by application of ultra-
sounds in an elastic medium can explode at the surface of the
plant and destroy the plant cells. As seen on Fig. 4(1), basil essen-
tial oil glands were located on the surface of the leaves so they
were directly exposed to cavitation bubbles (Fig. 4(6)). This re-
sulted in explosion of the glands and essential oil releasing into
the surrounding medium (Fig. 4(5)). As the surrounding medium
was the olive oil, the explosion of the glands made the aromati-
sation process going much faster than the conventional diffusion
process.
4. Conclusions
This work presented the potential of an ultrasound assisted
aromatisation of an olive oil with fresh basil. The main advantage
of this new technique is that the essential oil contained in the ba-
sil leaves was directly extracted into the olive oil without any
intermediate stage. This process leaded to an aromatised olive
oil in few minutes, which can be opposed to the several hours re-
quired in conventional maceration process. It was the ability of
the ultrasounds and the cavitation bubbles to destroy plant mate-
rial that accelerated dramatically the rate of extraction of the
essential oil from their glands into the olive oil. This new tech-
nique could be applicable to lab work for students because it is
a very educational and easy to handle procedure. Different aro-
matic plants could also be tested in order to get a wide range
of aromatised oils.
Acknowledgements
The authors are very grateful to Isabelle Bornard (INRA domaine
St. Maurice, Le Pontet, France) for her help in scanning electron
microscopy and want to thank Christian Ginies for the GC/MS anal-
yses. Authors are also grateful to the Rgion PACA for its nancial
support.
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