...

1. Improved extraction of vegetable oils under high-intensity
ultrasound and/or microwaves
Giancarlo Cravotto a,*, Luisa Boffa a
, Stefano Mantegna a
, Patrizia Perego b
,
Milvio Avogadro b
, Pedro Cintas c,*
a
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` di Torino, Via P. Giuria 9, 10125 Torino, Italy
b
Dipartimento di Ingegneria Chimica e di Processo, Universita` di Genova, Via Pia 15, I-16145 Genova, Italy
c
Departamento de Quı´mica Orga´nica e Inorga´nica, Universidad de Extremadura, Facultad de Ciencias-UEX, E-06071 Badajoz, Spain
Received 19 October 2007; accepted 30 October 2007
Available online 9 November 2007
Abstract
Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) techniques have been employed as complementary
techniques to extract oils from vegetable sources, viz, soybean germ and a cultivated marine microalga rich in docosahexaenoic acid
(DHA). Ultrasound (US) devices developed by ourselves, working at several frequencies (19, 25, 40 and 300 kHz), were used for US-
based protocols, while a multimode microwave (MW) oven (operating with both open and closed vessels) was used for MAE. Combined
treatments were also studied, such as simultaneous double sonication (at 19 and 25 kHz) and simultaneous US/MW irradiation, achieved
by inserting a non-metallic horn in a MW oven. Extraction times and yields were compared with those resulting from conventional pro-
cedures. With soybean germ the best yield was obtained with a ‘cavitating tube’ prototype (19 kHz, 80 W), featuring a thin titanium cyl-
inder instead of a conventional horn. Double sonication, carried out by inserting an immersion horn (25 kHz) in the same tube, improved
the yield only slightly but halved the extraction time. Almost comparable yields were achieved by closed-vessel MAE and simultaneous
US/MW irradiation. Compared with conventional methods, extraction times were reduced by up to 10-fold and yields increased by 50–
500%. In the case of marine microalgae, UAE worked best, as the disruption by US of the tough algal cell wall considerably improved the
extraction yield from 4.8% in soxhlet to 25.9%. Our results indicate that US and MW, either alone or combined, can greatly improve the
extraction of bioactive substances, achieving higher efficiency and shorter reaction times at low or moderate costs, with minimal added
toxicity.
Ó 2007 Elsevier B.V. All rights reserved.
Keywords: Ultrasound-assisted extraction; Microwave-assisted extraction; Soybean germ; Seaweed; Docosahexaenoic acid
1. Introduction and background
Extraction techniques are widely employed for the isola-
tion of bioactive substances from natural sources [1]. How-
ever, they are usually time-consuming and, unless carefully
controlled, are liable to cause degradation or unwanted
chemical changes in the products. Current techniques of
solid–liquid extraction are essentially based on diffusion
and osmosis. To shorten extraction times and improve
yields treatments may be carried out at a higher tempera-
ture, and/or be repeated several times with fresh solvent.
The simplest and cheapest technique is maceration; at
any rate stirring is indispensable if local saturation at the
matrix surface is to be avoided, so that diffusion may work
more efficiently. In the extraction of essential oils and, gen-
erally, of volatile fractions, steam distillation often proves
very useful, although exposure to heat may cause degrada-
tion of thermally labile compounds. The same drawback
affects Soxhlet extraction, the usual preliminary step in
the processing of solid samples according to many official
1350-4177/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2007.10.009
*
Corresponding authors. Tel./fax: +39 0 11 6707684.
E-mail address: giancarlo.cravotto@unito.it (G. Cravotto).
www.elsevier.com/locate/ultsonch
Available online at www.sciencedirect.com
Ultrasonics Sonochemistry 15 (2008) 898–902

2. methods of analysis. Industrial plants, that must produce
large amounts of extract in a short time, resort to percola-
tion: the solvent drips through large cylinders packed with
up to several cubic meters of solid matrix. As a single pass
would yield but a limited amount of extract, the effluent is
enriched by returning it several times to the top of the
column. Because efficiency limits are ultimately set by
diffusion rates that increase with temperature, the above-
mentioned strictures also apply to the possibility of work-
ing with hot solvents.
Ultrasound-assisted extraction (UAE) and microwave-
assisted extraction (MAE) are now recognized as efficient
extraction techniques that dramatically cut down working
times, increasing yields and often the quality of the extract.
Although chiefly exploited on the laboratory scale, both
have already found industrial applications in this field, par-
ticularly the latter [2]. This employ of ultrasound (US) to
enhance extraction yields began in the fifties [3]; more
recently we showed that it dramatically improves the extrac-
tion of oils from plant materials [4], mainly through the phe-
nomenon of cavitation. The mechanical effect of US
promotes the release of soluble compounds from the plant
body by disrupting cell walls, enhancing mass transfer
and facilitating solvent access to the cell content. This effect
is much stronger at low frequencies (18–40 kHz) and prac-
tically negligible at 400–800 kHz. If the matrix has been pre-
viously dried, US accelerates its rehydration and swelling.
Vinatoru et al. [5] showed that it greatly enhanced the
hydration process which takes place simultaneously with
matrix fragmentation, without any appreciable chemical
degradation. UAE has proved to be a versatile technique
that can be scaled up to the benefit of industrial production.
The use of microwave (MW) dielectric heating in analyt-
ical laboratories began in the late 1970s and was first seized
upon by the food industry. Dielectric heating depends on
the ability of materials to absorb MW energy and convert
it to heat. Unlike conductive heating, MW heats the whole
sample volume simultaneously. It disrupts weak hydrogen
bonds by promoting the rotation of molecular dipoles, an
effect that is opposed by the viscosity of the medium. Fur-
thermore, the movements of dissolved ions increase solvent
penetration into the matrix and thus facilitate analyte sol-
vation. The effect is strongly dependent on the nature of
both solvent and matrix. Sometimes MW affects mainly
the latter, while the surrounding liquid, having a low
dielectric constant, remains relatively cold. This set-up,
that presents obvious advantages when dealing with ther-
mosensitive compounds, has been successfully used for
the extraction of natural oils [6]. MW heating of fresh plant
material is also a convenient way to achieve direct distilla-
tion of essential oils [7]. Liquid-phase extraction (often
used to isolate essential oils from plants) is based on the
different absorbance for MW of materials having different
dielectric constants. The matrix to be extracted (usually
water-rich) is mixed with a solvent having a low dielectric
constant, so that most of the heating effect will be concen-
trated on the plant material. Ganzler and associates [8]
were the first to report the advantages of this method over
conventional ones for the extraction of natural com-
pounds. MAE requires less solvent and takes less time,
affording better products at lower costs. Some biologically
active compounds have been so extracted, e.g. taxanes
from Taxus brevifolia [9], glycyrrhizic acid from Glycyr-
rhiza glabra [10], and artemisinin from Artemisia annua
[11].
In a previous paper, we compared yields and times of
rice bran extractions using both UAE and conventional
methods [4]. These results prompted us to extend our study
to related techniques, introducing novel apparatuses as
well. After widely experimenting with the design, construc-
tion and testing of new reactors using US [12] and MW
alone or combined, we report here the first instances of
extraction employing simultaneous US and MW irradia-
tion [13] as well as double sonication combining two trans-
ducers working at different frequencies (19 and 25 kHz).
We applied these environmentally benign methods to: (1)
soybean germ, an important source of tocopherols, carote-
noids, and polyunsaturated fatty acids (PUFAs); (2) a cul-
tivated seaweed that is rich in docosahexaenoic acid
(DHA) (Fig. 1). Gas-chromatographic analyses of methyl
ester derivatives, revealed little or no oxidative degradation
of PUFAs, which was not the case for parallel extraction
runs carried out with conventional methods. In this con-
text, it is worth mentioning a recent study in which Zhao
and coworkers investigated the effects of MW and US on
the stability of a keto-carotenoid, (all-E)-astaxanthin, that
is present in marine animals and algae; they found that
MW induced isomerization to its Z forms, while US pro-
moted degradation to unidentified compounds [14].
2. Experimental (materials and methods)
2.1. Chemicals
All reagents and solvents were purchased from Carlo
Erba Reagenti and Acros Organics. The extraction course
was followed by thin layer chromatography (TLC) on
Fluka F254 (0.25 mm) plates, which were visualized by
DHA (22:6 n-3)
COOH
COOH
α- linolenic acid (18:3 n-3)
linoleic acid (18:2 n-6)
COOH
Fig. 1. Chemical structures of main fatty acids from soybean-germ oil and
docosahexaenoic acid (DHA) from seaweed.
G. Cravotto et al. / Ultrasonics Sonochemistry 15 (2008) 898–902 899

3. heating after a molybdic acid spray. Milled pure soybean
germ (150 mesh) and milled seaweed (150 mesh) were
kindly provided by BF Pharma Spa Italia (Fossano, Italy).
Both were extracted with hexane (Carlo-Erba, Italy): fil-
tered extracts were evaporated under vacuum and weighed.
2.2. Microwave and ultrasonic equipment
All MW runs were carried out with a professional multi-
mode oven operating at 2.45 GHz (Microsynth, Milestone,
Italy). US treatments were conducted with the following
devices developed in our laboratories in collaboration with
Danacamerini sas (Torino, Italy), working at different fre-
quencies (19, 25, 40, and 300 kHz): (1) high-power probe
systems with immersion titanium horns, working at 25
and 40 kHz, (2) a titanium cup-horn working at 300 kHz.
The transducer can work without overheating at high
power (up to 500 W) thanks to an efficient cooling system
that employs a chiller and an oil circulation circuit; (3) the
high-power cavitating tube (Fig. 2), an innovative cup-
horn-like system, consisting of a thin hollow titanium cylin-
der whose base is fixed on a booster. Measurements with a
small hydrophone showed that acoustic energy was effi-
ciently transferred to the liquid both from its bottom and
wall. A thermostatting fluid was circulated through the
outer polytetrafluoroethylene (PTFE) jacket. Inserting an
immersion horn in the cavitating tube made it possible to
combine two transducers working at different frequencies
(Fig. 2); (4) simultaneous US/MW irradiation in a single
reaction vessel was achieved by inserting in a MW oven a
horn made of PEEKÒ
(polyether ether ketone). Fig. 3
shows a professional oven currently employed in our labo-
ratory for such combined US/MW irradiations.
2.3. Chromatographic analyses
A Shimadzu GC-14 B (FID detector) with a Shimadzu
C-R6A Chromatopac integrator was used for analyses by
gas chromatography (GC). All data presented below are
mean values from three extractions. The profile of fatty
acids (derivatized as methyl esters) was determined accord-
ing to the Official Method of Analysis AOAC [15] by gas
chromatography using an OV1 apolar column (length
8 m; i.d. 0.25 mm; film thickness 0.25 lm). GC conditions
were: injection split 1:20, injector temperature 250 °C,
detector temperature 300 °C; temperature program: from
50 °C (2 min) to 100 °C at 3 °C/min, then to 280 °C
(5 min) at 5 °C/min, then the temperature was kept constant
at 280 °C for 20 min; hydrogen as carrier gas at 25 kPa.
3. Results and discussion
The great potential of US- and MW-assisted extractions
has not been adequately exploited as yet; the combined use
of both energy sources has hardly been investigated at all
[16]. High-intensity US generate cavitational bubbles and
streaming. Bubble collapse in the vicinity of plant tegu-
ments may cause microfractures in these tissues [17].
MAE generally works better than conventional methods,
as it saves both time and solvent volume, has a high extrac-
tion efficiency and a low environmental impact [18]. While
the Soxhlet method usually takes hours (up to 15 or more),
MAE only takes a few minutes, using (at most) 10 times
less solvent. In the present MAE treatments samples were
suspended in an apolar solvent such as n-hexane, so that
MW heated the fresh plant material almost exclusively
owing to its high moisture content. The resulting disrup-
tion of cell membranes released the oil to the solvent.
As previously mentioned, we tested several US devices
working at different frequencies and different MW pro-
grams, as well as US/MW combinations. Table 1 compares
results obtained for hexane extraction of soybean germ
under different conditions, including conventional tech-
niques (static extraction at room temperature and refluxing
Fig. 2. Immersion horn (25 kHz) inserted in the cavitating tube (19 kHz);
the inset shows the latter as seen from the top.
Fig. 3. Apparatus for simultaneous US/MW irradiation.
900 G. Cravotto et al. / Ultrasonics Sonochemistry 15 (2008) 898–902

4. in a Soxhlet). As expected, significant differences are seen in
terms of extraction times and yields. The best result was
achieved by our novel US device we call a cavitating tube,
working at 19 kHz. Inserting in the cavitating tube a tita-
nium immersion horn (25 kHz and same US power)
improved the oil yield only slightly but halved the extrac-
tion time. To avoid solvent splashing, the horn and the
external wall of the tube were joined by an elastomeric
sleeve to seal the system. Good results were likewise
obtained (Table 1, last entry) with closed-vessel MW,
although this required a much higher temperature. Simul-
taneous US/MW irradiation cannot be performed by intro-
ducing conventional metallic horns inside the MW
chamber, as this would cause arcing and explosions. We
resorted to a PEEKÒ
horn (21 kHz, 50 W) inserted in the
MW oven (100 W) (Fig. 3); this setup afforded satisfactory
extraction yields at moderate temperatures.
Although MAE and UAE can be conducted at lower
temperatures than those attained in Soxhlet extraction,
nevertheless superheating and pyrolytic degradation can
occur; therefore much care must be exerted in applying
these techniques to the extraction of bioactive compounds.
Soybean-germ oil is rich in thermolabile unsaturated com-
pounds [19,20] that include PUFAs (both omega-3 and
omega-6 acids), carotenoids, tocopherols, phytosterols
and coenzyme Q10. UAE of the whole soybean has been
reported [21] as well as its application to separating isoflav-
ones from freeze-dried ground soybean [22]. Gas chroma-
tography (GC) analyses generally showed no differences
between the methyl esters profiles of soybean germ oils
extracted under conventional and ultrasonic conditions.
Owing to the very brief sonication times and the low
moisture content (4.5%) of the matrix, we observed but a
very small decrease in the relative percentage of unsatu-
rated fatty acids. Data for oils extracted by Soxhlet and
simultaneous double sonication are shown in Table 2.
Results indicate that only a very slight oxidation occurs,
irrespective of the degree of unsaturation, upon application
of high-intensity US using metallic horns.
These positive, reproducible results encouraged us to
extend our study to other matrices, in particular the axeni-
Table 1
Hexane extraction of soybean germ and seaweed: optimal extraction times and oil yields as % of matrix weight
Entry Extraction method Matrice
weight (g)
Solvent
volume (ml)
Temp.
(°C)
Time
(h)
Yield SGa
(%)
Yield SWa
(%)
1 Separatory funnel (static) 15 50 r.t. 8 3.5 2.0
2 Soxhlet 15 100 rfx 4 8.6 4.8
3 Immersion horn 25.0 kHz, 80 W 8 50 45° 1 12.2 18.7
4 Immersion horn 40.0 kHz, 80 W 8 50 45° 1 8.9 13.6
5 Cup horn 300 kHz, 70 W 8 40 45° 1 6.4 12.0
6 Cavitating tube 19.0 kHz, 80 W 8 50 45° 1 17.7 24.7
7 Cavitating tube 19.0 kHz, 65 W and
immersion horn 25.0 kHz, 60 W
8 50 45° 0.5 17.9 25.9
8 Comb. US/MW (60/100 W)b
8 40 45° 1 14.1 24.0
9 MW, open vesselc
3 40 60° 1 10.0 12.5
10 MW under pressurec
2 35 120° 0.5 16.5 17.8
a
SG, soybean germ; SW, seaweed.
b
US horn made of PEEKÒ
.
c
Magnetic stirrer (P/N 86116) and weflon button for apolar solvents (P/N WO1703).
Table 2
Methyl ester profiles of soybean-germ oil from UAE and Soxhlet
extraction
Fatty acid compositiona
Soxhlet (%) Double sonicationb
(%)
C18:2 linoleic 56.8 56.3
C18:3 linolenic 15.7 15.4
C16:0 palmitic 12.7 12.7
C18:1 oleic 10.6 10.6
C18:0 stearic 3.6 3.7
C20:0 arachidic 0.4 0.4
a
GC analyses of fatty acid methyl esters were replicated twice.
b
Cavitating tube 19.0 kHz, 65 W + immersion horn 25.0 kHz, 60 W.
Table 3
Methyl ester profiles of seaweed oil from UAE and Soxhlet extraction
Fatty acids compositiona
Soxhlet (%) Double sonicationb
(%)
C12:0 lauric 0.2 0.2
C14:0 myristic 3.8 3.9
C15:0 pentadecanoic 2.0 2.0
C18:1 oleic 0.1 0.1
C18:0 stearic 1.5 1.6
C20:0 arachidic 0.1 0.1
C16:0 palmitic 38.0 37.9
C16:1 palmitoleic 0.1 0.1
C17:0 margaric 0.5 0.6
C18:2 linoleic 0.1 0.1
C18:3 c linolenic 0.1 0.1
C18:4 stearidonic 0.3 0.2
C22:0 behenic 1.3 1.2
C20:3 eicosatrienoic n-6 0.2 0.2
C20:4 arachidonic 0.1 0.1
C20:5 eicosapentaenoic 0.4 0.3
C22:5 docosapentaenoic n-6 8.8 8.6
C22:5 docosapentaenoic n-3 0.4 0.4
C22:6 docosahexaenoic 39.5 39.3
C24:0 lignoceric 0.1 0.1
Others 2.4 2.9
a
GC analyses of the fatty acid methyl esters were replicated twice.
b
Cavitating tube 19.0 kHz, 65 W + immersion horn 25.0 kHz, 60 W.
G. Cravotto et al. / Ultrasonics Sonochemistry 15 (2008) 898–902 901

5. cally cultivated marine microalga Crypthecodinium cohnii
[23]. Some varieties of seaweed, long known as traditional
foodstuff, possess high added value due to their content in
biologically active substances [24]. Of particular impor-
tance is DHA [25], an omega-3 fatty acid, present in sea-
weed; this is in fact the source from which fishes get their
DHA, also serving as a natural antifreeze. Health benefits
of DHA and other PUFAs contained in marine plants have
been reported in the literature; they include prevention of
cardiovascular disease, obesity, besides their well-known
roles in inflammation and the immune response [26–30].
Results for MAE and UAE of the microalga Crypthe-
codinium cohnii are shown in Table 1. As was found with
soybean germ oil, the methyl ester profiles of seaweed oil
obtained under conventional and ultrasonic conditions
were comparable, showing that no significative PUFAs
degradation had occurred (Table 3).
4. Conclusions
UAE of soybean-germ oil using several types of US
apparatuses (cup horn, immersion horn, cavitating tube)
working at different frequencies (19, 25, 40 and 300 kHz)
has been evaluated. In a newly developed apparatus simul-
taneous US/MW irradiation was achieved by inserting a
PEEKÒ
horn in a multimode oven. Optimum extraction
times were determined and yields were compared with
those obtained by MAE (open- and closed-vessel) and con-
ventional methods. The best oil yield was obtained with the
cavitating tube (19 kHz) and double sonication employing
an additional immersion horn (25 kHz). Compared with
conventional methods much higher yields were also
achieved with closed-vessel MW irradiation at 120 °C and
simultaneous US/MW irradiation. Results were even more
striking in the case of seaweed extraction, as the cell wall of
the microalga is very tough. Extraction times were reduced
up to 10-fold and yields increased by 50–500% in compar-
ison with conventional methods. GC analyses showed only
slight or negligible differences in methyl esters profiles of
oils extracted under high-intensity US and in Soxhlet. It
should also be pointed out that techniques using US or
combined US/MW irradiation should be well suited for
other processes, such as two-step extraction and transeste-
rification for the production of biofuels.
Acknowledgements
The present work was supported by Regione Piemonte –
CIPE bando 2004, BF Pharma (Fossano, Italy), and the
Spanish Ministry of Education and Science (CTQ2005-
07676). The authors thank Danacamerini sas for designing
and assembling the US apparatuses.
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