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Contents lists available at ScienceDirect

Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Enzyme and high pressure assisted extraction of

carotenoids from tomato waste
Irini F. Strati , Eleni Gogou, Vassiliki Oreopoulou
Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of
Athens, Iroon Polytechniou 5, 15780 Zografou, Athens, Greece

a b s t r a c t
Enzyme (EA) and high pressure (HP) assisted extraction of carotenoids, especially lycopene, from tomato waste
using various organic solvents was examined. Total carotenoid and lycopene extraction yields were increased by
the use of pectinase and cellulase enzymes, when compared to the non enzyme treated solvent extraction process. The increase of extraction yield depended on the solvent. Maximum total carotenoid (127 mg/kg d.w.) and
lycopene (89.4 mg/kg d.w.) extraction yields were obtained in enzyme treated samples extracted with ethyl lactate
(solvent:solid = 10:1 mL:g), corresponding to almost 6-fold and 10-fold increase, respectively, with respect to non
enzyme treated samples. HP assisted extraction led to higher extraction yields (from 2 to 64% increase depending on
the solvent used) compared to conventional solvent extraction process performed at ambient pressure for 30 min.
HP assisted solvent extraction was successfully performed at 700 MPa by using signicantly (P < 0.05) lower ratios of
solvent:solid (6:1 and 4:1 mL:g) and reduced processing time (10 min), compared to solvent extraction performed at
ambient pressure, solvent:solid ratio 10:1 mL:g and 30 min extraction time.
2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Tomato waste; Carotenoids; Lycopene; Solvent extraction; Enzyme-assisted extraction; High hydrostatic
pressure

1.

Introduction

By-products of plant food processing represent a major disposal problem for the industry concerned, but they are also a
promising source of valuable components. Tomato processing
industry generates signicant amounts of waste, consisting
mainly of tomato skins and seeds, in a proportion depending on the specic process from which it is generated (Del
Valle et al., 2006). Tomatoes are a rich source of carotenoids,
particularly lycopene. The global market for carotenoids was
estimated to US$1.07 billion and is projected to top US$1.2
billion in 2015 (Global Industry Analysts Inc., 2011).
According to Sharma and Le Maguer (1996), at the end of
ripening stage, tomato skins contain up to ve times more
lycopene than the pulp. Conventional food grade solvents,
such as hexane, ethanol, and ethyl acetate, have been proposed for the extraction of carotenoids from tomato waste.

However, the yield in most cases is low possibly due to the

difculty for the solvent molecules to penetrate the tomato
peel tissue and solubilize the pigment, while oxidative degradation of carotenoids is also possible (Lavecchia and Zuorro,
2008).
In that aspect, enzyme-assisted extraction methods are
gaining more attention because hydrolytic enzymes break
down the structural integrity of cell walls rendering the
intracellular materials to be more exposed for extraction.
Cellulase and pectinase enzymes were employed by several
researchers as a pretreatment step of tomato based products
prior to solvent extraction for the recovery of carotenoids, and
especially lycopene (Choudhari and Ananthanarayan, 2007;
Lavecchia and Zuorro, 2008; Zuorro et al., 2011; Papaioannou
and Karabelas, 2012; Ranveer et al., 2013). The enzymatic treatment was followed by extraction with hexane, ethyl acetate,
acetone or mixtures of solvents. In all cases, there was an

Corresponding author. Tel.: +30 210 7723166; fax: +30 210 7723163.
E-mail addresses: irinistrati@yahoo.gr, strati@chemeng.ntua.gr (I.F. Strati).
Received 13 January 2014; Received in revised form 22 August 2014; Accepted 28 September 2014
http://dx.doi.org/10.1016/j.fbp.2014.09.012
0960-3085/ 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Strati, I.F., et al., Enzyme and high pressure assisted extraction of carotenoids from tomato waste. Food
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increase in extraction yield compared to the untreated samples, which varied from 2- to 18-fold. These differences may
be attributed to different raw material, enzyme preparation,
experimental conditions and solvents used for the subsequent
extraction. The effect of solvent on the extraction yield of
carotenoids from enzymatically treated tomato waste might
be very important, as different solvents show different penetration abilities and solubilizing effect on carotenoids (Strati
and Oreopoulou, 2011).
On the other hand, HP processing can cause some structural changes, such as cell deformation or cell membrane
damage that increase cell permeability as well as secondary
metabolite diffusion and consequently mass transfer rates,
while it has very little effect on low-molecular-weight compounds such as avour compounds, vitamins and pigments
(Knorr, 1993; Corrales et al., 2008). In the literature, earlier studies have been published with regards to the effect of HP on total
carotenoid content and carotenoid availability of commonly
consumed vegetables (McInerney et al., 2007; Plaza et al., 2012).
As an extraction method, this technology was rst studied
in 2004, and it was found to effectively shorten the extraction time and increase the process efciency (Zhang et al.,
2004). Recently, HP has been implemented to extract bioactive compounds from natural sources, such as anthocyanins
from grape by-products (Corrales et al., 2008), or phenolic compounds from Maclura pomifera fruits (Altuner et al., 2012), while
Xi (2006) applied HP on tomato paste waste and obtained
higher yields of lycopene compared to conventional solvent
extraction performed at ambient pressure for 30 min.
Several solvents have been examined in a previous work
(Strati and Oreopoulou, 2011) for the extraction of carotenoids,
and especially lycopene, from tomato waste and the results
were highly dependent on the solvent. In the present study
we further investigated the possibility to enhance carotenoid
extractability by enzyme pre-treatment or HP assisted extraction. Tomato waste was treated with pectinase and cellulase
enzymes prior to extraction with various solvents, from the
non-polar hexane to the highly polar ethanol so as to
dene the combined effect on extraction yield. Additionally,
HP processing, in the pressure range of 100800 MPa with
the initial (prior to pressurization) temperature of 25 C was
explored in order to obtain the maximum extraction yields of
carotenoids and lycopene from tomato waste.

2.

Materials and methods

2.1.

Chemicals and enzymes

Hexane and ethyl acetate, analytical grade, were purchased

from Thermo Fisher Scientic (Fair Lawn, NJ). Acetone and
ethanol, p.a., were purchased from Sigma Chemical Co.
(SigmaAldrich Company, St. Louis, MO). (-)-Ethyl L-lactate,
p.a., was purchased from Fluka Analytical (SigmaAldrich
Chemie Gmbh, Munich, Germany). All solvents used for HPLC
analysis (acetonitrile, 1-butanol and methylene chloride) were
of HPLC grade and were obtained from Merck (Darmstadt,
Germany). REDIVIVO Lycopene 10% FS (10% microcrystalline
lycopene in corn oil containing a-tocopherol as antioxidant)
from DSM Nutritional Products (Kaiseraugst, Switzerland)
was used for the preparation of standard solutions for spectrophotometric measurements. All-trans lycopene standard
was purchased from Sigma Chemical Co. (SigmaAldrich Company, St. Louis, MO).

Cellulyve AN 3500, a powder enzyme preparation of

Aspergillus niger cellulase, was obtained from Lyven S.A.
(Colombelles, France), with enzyme activity of 3500 200 U/g
and pH 46 (concentration 1%). Pectinex Ultra AFP, a liquid
enzyme preparation produced from Aspergillus aculeatus and A.
niger, containing pectin lyase and polygalacturonase activities
of 10,000 Units/mL, was obtained from Novozymes (Bagsvaerd,
Denmark). Both products were stored at 4 C and prior to use,
calculated amounts of enzyme preparations were diluted with
acetate buffer solution of appropriate pH (4.8 for cellulase and
5.0 for pectinase) to obtain the desired enzyme concentration.

2.2.

Plant material

Tomato processing waste, composed of skin and seeds of

tomato cultivar Red Sea, was collected from a Greek tomato
processing plant (NOMIKOS S.A., Aliartos, Viotia, Greece).
Moisture content was determined at fresh tomato processing
waste upon arrival at the laboratory and found to be
80.48 0.35%. Tomato waste material was air dried at 25 C,
homogenized in a domestic blender and nally ground in a
laboratory mill (Cutting Mill Pulverisette 15, Fritsch Gmbh,
Idar-Oberstein, Germany) equipped with a 0.5 mm sieve. Moisture content of ground dry tomato waste was 7.65 0.21%.
The dry ground material was kept in glass jars wrapped with
aluminium foil at 20 C before further processing.
The critical steps involved in the EA and HP assisted extraction of carotenoids from tomato waste are described in Fig. 1.

2.3.

Enzyme aided extraction of carotenoids

Dry, ground and homogenized tomato waste was distributed

(1.0 g each) in tightly-closed glass test tubes, covered with
aluminium foil. To each test tube, 7.0 mL of enzyme solution of the appropriate enzyme concentration were added
and the mixture was vortexed for 3 min. The samples were
then incubated under agitation in a Memmert shaking waterbath WB 14 with drive SV 1422 (Memmert GmbH + Co. KG,
Schwabach, Germany). The incubation temperature for both
enzymes (pectinase and cellulase) was chosen according to
that reported by the supplier and literature review as optimal
for enzyme activity and was 45 C and 55 C for Pectinex Ultra
AFP and Cellulyve AN 3500, respectively.
At the end of the incubation period the enzymes were
inactivated by immersing the test tubes in boiling water for
3 min. After ltration, the tomato waste solid residue was
placed in screw-top conical asks in a thermostated water
bath (25 0.1 C) and was subjected to solvent extraction,
using 10 mL of each solvent. The whole system was kept under
agitation for 30 min. On completion of the extraction procedure, the mixture was centrifuged at 1000 g for 10 min
(Centrifuge Thermo scientic Heraeus Megafuge 16R, DJB Labcare Limited, UK), to separate the supernatant.
Blank (enzyme free) samples were prepared for each solvent extraction process, by adding buffer solution in the same
amount as the enzyme solution, prior to solvent extraction.

2.4.

High pressure treatment

Tomato waste samples (2.5 g each) were weighed, mixed

with the appropriate volume of different solvents or solvent mixtures and packed into polypropylene pouches for
HP experiments. The pouch was sealed after eliminating air
from the inside and placed into the HP vessel. HP samples

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equilibrated at the process temperature (25 C). Pressure and

temperature were monitored and recorded (1s intervals) during the process by temperature and pressure sensors installed
within the available HP vessels equipment; temperature and
pressure sensors are positioned inside the 45 mL vessels in
contact with the pressure transmitting uid (polyglycol). Pressure build up (15 MPa/s) resulted in compression heating of the
sample by approximately 3 C/100 MPa (i.e. HP processing of
300 MPa resulted in an adiabatic temperature increase from
25 C to approximately 34 C) followed by cooling to 25 C
within 10 min during pressure holding time (HP decompression was instantaneous). On completion of the HP processing,
the mixture was centrifuged 1000 g for 10 min (Centrifuge
Thermo scientic Heraeus Megafuge 16R, DJB Labcare Limited,
United Kingdom), to separate the supernatant.
Conventional extractions were performed with the same
solvents as those used in HP extractions, to compare the
results. The procedure described by Strati and Oreopoulou
(2011) was followed. Briey, 10.0 g of dry, ground raw material
were mixed with solvent at a liquid to solid ratio of 10:1 in an
agitated vessel, kept at 25 C for 30 min. Subsequently the mixture was centrifuged at 1000 g for 10 min (Centrifuge Thermo
scientic Heraeus Megafuge 16R, DJB Labcare Limited, United
Kingdom), to separate the supernatant, which was used for
subsequent carotenoid analyses.

2.5.

Fig. 1 Enzyme and high pressure assisted extraction

scheme of carotenoids from tomato waste.

preparation was performed 5 min prior to compression in

order to avoid any further carotenoids extraction before HP
treatment. Solvent extract was discharged from the solid
tomato waste immediately after decompression and transferred in tubes before further use in order to determine the
total carotenoid content. HP treatments were conducted at the
pressure range of 100800 MPa for 130 min. Temperature prior
to pressurization was 25 C and was elevated during compression, due to adiabatic heating, approximately 3 C/100 MPa.
Solvent to solid ratios of 10:1 mL:g, 6:1 mL:g and 4:1 mL:g were
examined.
HP unit used (Food Pressure Unit FPU 1.01, Resato International BV, Roden, Holland) comprised a six vessel system of
45 mL capacity each, with a maximum operating pressure of
1000 MPa and temperature of 90 C. The pressure transmitting
uid was polyglycol ISO viscosity class VG 15 (Resato International BV, Roden, Holland). Process temperature in the vessels
was achieved by water circulation in the outer jacket controlled by a heating-cooling system. Before introduction of the
samples in the pressure unit, pressure vessels were already

Total carotenoid and lycopene determination

The total carotenoid content of the supernatants obtained

from enzyme- and HP assisted extractions was measured
spectrophotometrically (Helios Alpha & Beta UV-Visible Spectrometer, Unicam Instruments, Cambridge, United Kingdom),
according to the procedure of Strati and Oreopoulou (2011) and
it was expressed as mg of total extracted carotenoids per kg
of dry material weight (mg/kg d.w.).
The lycopene content of the extracts was determined
by high performance liquid chromatography (HPLC), and
quantied using a reference curve. After complete solvent
evaporation in a rotary vacuum evaporator (Rotavapor RE
111, Switzerland), the samples were dissolved in 1 mL methylene chloride and transferred to a vial. The new solution was
ltered through a 0.45 m membrane lter and 20 L were
injected for HPLC analysis (Strati et al., 2012). The HPLC (Agilent 1100 Series, Waldbronn, Germany) system was composed
of a HP 1100 Series Diode Array Detector, a HP 1100 Quaternary Pump, an Agilent 1100 Series Micro Vacuum Degasser
and a Rheodyne model 7010 Sample Injector. The HPLC system was equipped with a YMC (Tokyo, Japan) C30 column
(250 mm 4.6 mm I.D., 5 m particle). A mobile phase of acetonitrile (A), 1-butanol (B), and methylene chloride (C) with
the following gradient elution was used: 69.3% A, 29.7% B and
1.0% C initially, increased to 67.2% A, 28.8% B and 4% C in
10 min, 61.6% A, 26.4% B and 12% C in 20 min, 49% A, 21% B
and 30% C in 40 min and returned to 69.3% A, 29.7% B and 1%
C in 50 min. The ow rate was maintained at 2 mL/min, the
column temperature at 25 C, and detection was carried out at
472 nm. The analysis of the chromatographic data was carried
out on a ChemStation for LC 3D software (Agilent Technologies
19992000, Waldbronn, Germany).

2.6.

Statistical analysis

EA and HP assisted extractions were performed in two separate trials and analyses in triplicate. The average of three

Please cite this article in press as: Strati, I.F., et al., Enzyme and high pressure assisted extraction of carotenoids from tomato waste. Food
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Fig. 2 Effect of enzyme pretreatment time on the extracted

total carotenoid yield from tomato waste.
measurements was calculated for each experiment and the
presented results are the mean values of duplicate experiments. Statistical analysis was carried out using the software
STATISTICA (Stat soft. Inc., 1999). The analysis of variance
technique and Duncans multiple range tests were used to
determine the signicant difference in total carotenoid and
lycopene content between different treatments at 95% condence level (P < 0.05).

3.

Results and discussion

3.1.

Enzyme assisted extraction of carotenoids

3.1.1. Determination of improved enzyme incubation

conditions
Preliminary series of experiments were conducted to determine the effective incubation time and concentration for both
enzymes. Hexane was used as the extraction solvent in each
series. In the rst series, incubation time varied from 30 to
240 min, while the enzyme concentration was kept constant
and equal to 70 U/g and 122.5 U/g for pectinase and cellulase,
respectively (Fig. 2). As indicated from the results the highest
carotenoid yield for both enzymes was observed after 180 min
of incubation. Increasing the incubation time resulted in a progressive reduction in yields. This suggests that the enzymatic
degradation of cell-wall components occurs within 180 min of
incubation. Lycopene and other carotenoids that are released
from the protective chromoplast structures during this time
are further exposed to the conditions of the external environment and can undergo rapid oxidative degradation that
may lead to reduction in extraction yield (Ranveer et al., 2013).
In the second series, the incubation time was kept constant
(180 min) and the concentrations of pectinase and cellulase
enzymes varied from 35 to 140 U/g of tomato waste, and 61.25
to 350 U/g of tomato waste, respectively (Fig. 3). Maximum
carotenoid yield was obtained at pectinase and cellulase concentrations of 70 U/g and 122.5 U/g, respectively.

3.1.2. Effect of enzyme treatment combined with different

extraction solvents
The effect of enzyme treatment combined with different
extraction solvents was studied in a subsequent series of
experiments. Pectinase and cellulase enzymes were effective

Fig. 3 Effect of enzyme concentration on the total

carotenoid extraction yield from tomato waste using
hexane as extraction solvent.
in increasing total carotenoid and lycopene yield, while
no statistical (P < 0.05) difference was generally observed in
extraction yield by using either enzyme (Fig. 4a and b). However, the treatment with pectinase increased the extraction
of total carotenoids and especially of lycopene, compared to
cellulase, when hexane, acetone and ethyl lactate were used
as solvents. Pectinase was also reported as more effective
than cellulase for the enzymatic treatment of tomato peel and
waste by Choudhari and Ananthanarayan (2007) and Ranveer
et al. (2013). A preparation consisting of a mixture of the two
enzymes in equal volumes was also assessed; however, a possible synergistic effect of the combined use of two enzymes
was not observed (data not shown).
Tomato peel tissue is a highly structured material containing many different polysaccharide components, such as
cellulose, hemicelluloses, and pectins. Cellulase hydrolyzes
the 1,4--d-glycosidic linkages in cellulose, which is present in
the primary cell wall; therefore cellulase is mainly responsible
for cell opening. Pectinase has pectolytic and hemicellulolytic
activities and therefore breaks down pectic substances and
pectin found in the middle lamella and primary cell walls
(Choudhari and Ananthanarayan, 2007; Gross, 1984). Lycopene
is predominantly found in the tomato peel chromoplasts,
deeply embedded within the membrane structures. By the
degradation of structural components of tomato tissue, solvent molecules penetrate more easily the ruptured peel tissue
and dissolve lycopene (Cuccolini et al., 2013).
Overall, total carotenoid extraction yields were 1.5-fold
(with acetone as solvent) to 6-fold (with ethyl lactate as
solvent) higher when compared to those from the non
enzyme-treated tomato waste sample (Fig. 4a). Lycopene yield
also increased by 2.5-fold (with ethyl acetate as solvent) to 10fold (with ethyl lactate as solvent) when compared to the non
enzyme-treated samples (Fig. 4b). The yields obtained with
ethanol were very low in all cases, in agreement with previous experiments (Strati and Oreopoulou, 2011) and will not
be further discussed. The highest increase was observed with
hexane or ethyl lactate as solvents. A similar increase was
also reported by Lavecchia and Zuorro (2008) and Zuorro et al.
(2011) for lycopene extraction with hexane, from enzymetreated tomato peel or waste. Hexane is an excellent solvent
for lycopene but, as non polar cannot penetrate easily in the

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Fig. 5 Response surface graph for total carotenoid yield

from tomato waste at different HP processing conditions
(solvent: hexane, solvent to solid ratio = 10:1). Graph created
with Sigmaplot 10.0, Systat Software, Inc.
in between the values observed by the individual solvents, as
expected.
These results suggest that sufcient carotenoid yields can
be obtained from the wet industrial tomato waste by enzymatic treatment followed by extraction with ethyl lactate.

3.2.
Fig. 4 (a) Total carotenoid and (b) lycopene yield extracted
from tomato waste samples using different solvents
combined with enzyme pretreatment using pectinase and
cellulase enzymes. Values with different superscripts differ
signicantly (P < 0.05)
(a < b < c < d < e < f < g < h < i < j < k < l < m).

wet material. The rupture of the plant cells by enzymatic

hydrolysis, renders the lycopene much more accessible, and,
therefore increases considerably the extraction yield. Ethyl
lactate on the other hand, which is also an excellent solvent
of tomato carotenoids (Ishida and Chapman, 2009; Strati and
Oreopoulou, 2011), is a water and hydrocarbon miscible solvent that can penetrate much more easily the wet material,
and consequently extract higher amounts of lycopene from
the untreated samples, compared to hexane. Also in this case,
the rupture of the plant cells and the release of lycopene allow
an easier contact of the pigment with the solvent and an
almost quantitative extraction.
The lowest increase of extraction yield was observed with
acetone. Acetone is a strong, basic solvent that causes swelling
of the plant tissue (Mantanis et al., 1995; Obataya and Gril,
2005), therefore further hydrolysis obtained by the enzymes
offered a small increase of recovery. When enzymatic treatment was followed by ethyl acetate extraction, the yield was
approximately 2.4-fold higher compared to the non-treated
material, which is in agreement with the results reported by
Papaioannou and Karabelas (2012), although Lavecchia and
Zuorro (2008) reported a higher increase. Finally, the increase
obtained with the mixture of hexane and ethyl acetate was

High pressure treatment of tomato waste

3.2.1. Determination of improved conditions of HP

treatment
In order to select the most effective HP conditions for the
extraction of total carotenoids, preliminary experiments were
conducted as described in Section 2.4. The results showed that
higher extraction yields were observed at pressures higher
than 400 MPa; increasing pressure up to 700800 MPa led to
a signicant increase in extraction yield when compared to
solvent extraction performed at ambient pressure (0.1 MPa).
The highest total carotenoid extraction yield was observed
at process time treatments of 10 min, while further increase
in processing time did not result to further increase of the
extraction yield. According to the results depicted in Fig. 5,
HP processing at 700 MPa and 10 min were selected as the
processing parameters enabling the highest extraction yield.

3.2.2.

Effect of HP treatment on extraction yield

Total carotenoid and lycopene extraction yields under HP

processing, as compared to extraction at ambient pressure
(reported as conventional solvent extraction) are presented in
Table 1. In general, HP assisted solvent extraction increased
total carotenoid and lycopene extraction yield, compared to
extraction with the same solvents at ambient pressure, while,
statistically signicant (P < 0.05) differences were observed in
a few cases of solvents; ethanol, hexane, hexaneethyl acetate
and hexaneethyl lactate. However, these yields were achieved
at shorter processing time (10 min), compared to the required
processing time of conventional extraction techniques at
ambient pressure, which usually ranges from 30 to 60 min.
The reduced processing times of HP assisted solvent extraction indicate that under HP processes the mass transfer rates

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Table 1 Total carotenoids and lycopene content obtained by conventional and high pressure (HP) assisted extraction.

Total carotenoids
(mg/kg dw)

Lycopene content
(mg/kg dw)

Solvents

Conventional

Ethanol
Hexaneethanol (50:50)
Hexane
Hexaneacetone (50:50)
Acetone
Ethyl acetate
Hexaneethyl acetate (50:50)
Hexaneethyl lactate (50:50)
Ethyl lactate
Ethanol
Hexaneethanol (50:50)
Hexane
Hexaneacetone (50:50)
Acetone
Ethyl acetate
Hexaneethyl acetate (50:50)
Hexaneethyl lactate (50:50)
Ethyl lactate

3.63
19.98
18.15
22.03
21.06
23.68
26.98
57.20
147.69
2.47
13.87
15.14
15.94
14.53
16.62
17.14
46.27
82.48

0.15a
0.50a
0.49a
0.32a
0.90a
0.44a
0.21a
0.40a
2.33a
0.08a
0.09a
0.30a
0.10a
0.31a
0.05a
0.64a
0.04a
1.33a

HP
9.30
17.88
22.34
23.34
23.24
24.78
44.19
64.50
165.27
7.04
14.57
17.84
16.01
15.04
17.70
25.01
50.40
83.59

0.16b
1.48a
0.52b
0.86a
1.24a
0.72a
0.99b
0.60b
2.73b
0.11b
0.05a
0.59b
0.08a
0.16a
0.16a
0.48b
0.73b
4.63a

Values with different superscripts within the same row differ signicantly (P < 0.05).

of bioactive compounds from the tomato waste to the solvent

are increased, conrming the theory that HP can favour the
mass transfer phenomena leading to increased mass transfer
rates during the extraction processes due to changes in the
diffusivity coefcient. Diffusivity coefcient changes can be
mainly attributed to pressure induced changes in the plant cell
membrane leading to an increased cell membranes permeability thus facilitating permeation of the extraction solvent
into the tomato waste cells (Tangwonchai et al., 2000). In addition, denaturation of the carotenoid-binding protein induced
by pressure could be also involved in the cell wall damage of
tomato and, therefore, facilitate the extraction of carotenoids
under HP treatment (Snchez-Moreno et al., 2009). Qiu et al.
(2006) reported an increase in extractable lycopene in tomato
puree after processing at 500 MPa, at 20 C for12 min, which
they attributed to the fact that HP can rupture the tissue and
thereafter favour lycopene release. High pressure impels the
extracting solvent to come into cells to integrate with bioactive
components, the pressurized cells show increased permeability, the solubility of extracts is improved as the pressure
increases, and thus the leaching-out rates of bioactive components are improved (Xi, 2013). This leads to shorter processing
times with HP extraction compared to conventional and consequently to a more time-effective processing technique.
Also, HP deprotonates charged groups and disrupt salt
bridges and hydrophobic bonds making the cellular membranes less and less selective, thereby rendering the
compounds more accessible to extraction up to equilibrium
(Barbosa-Canovas et al., 1998). Furthermore, Smelt (1998) has
reported that HP can affect membranes in vegetable cells
and produce disruption of chromoplasts where carotenoids
are located, inducing a better release of these compounds. Xi
(2006) reported that the yield of lycopene by using 500 MPa of
HP assisted extraction of tomato paste waste with 75% ethanol
concentration in water (1:5 g/mL solid/liquid ratio, at room
temperature) for 1 min was higher (17%) than that obtained
by 30 min ultrasound-assisted extraction (using frequency of
38.5 kHz, power of 810 W, at 25 C). Similarly, during HP extraction of active ingredients from green tea leaves and with the
aid of scanning electron microscopy (SEM) and transmission
electron microscopy (TEM), Xi et al. (2011) indicated that ultrahigh pressure resulted in the disruption of the leaf tissue (cell

wall, membrane and organelles) and consequently improved

the mass transfer of the solvents into the leaves and the
soluble constituents into the solvents. Moreover, HP assisted
extraction is performed at ambient temperature (2530 C)
that do not favour degradation of carotenoids and especially
lycopene (Xianquan et al., 2005).
Additionally, in the present work, the reduction of solvent volume used during HP assisted extraction was studied
in order to investigate the ability of extracting lycopene and
total carotenoids using lower solvent volumes. The study
was performed for selected solvents that were found to
enable higher extraction yields, namely: hexane, hexaneethyl
acetate, hexaneethyl lactate and ethyl lactate. The specic solvents were used in three different ratios of solvent
volume to dry tomato waste mass; 10:1 mL:g, 6:1 mL:g, and
4:1 mL:g. According to the results depicted in Fig. 6, HP assisted
extraction of lycopene and carotenoids can be performed at
lower solvent to solid ratio, without signicantly (P < 0.05)

Fig. 6 HP assisted extraction of carotenoids performed at

different solvent to solid ratio values (4:1 mL:g, 6:1 mL:g and
10:1 mL:g) for selected solvents. Values with different
superscripts differ signicantly (P < 0.05) (a < b < c < d < e).

Please cite this article in press as: Strati, I.F., et al., Enzyme and high pressure assisted extraction of carotenoids from tomato waste. Food
Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012

FBP-544; No. of Pages 7

ARTICLE IN PRESS
food and bioproducts processing x x x ( 2 0 1 4 ) xxxxxx

reducing the extraction yields. These results indicate that HP

can facilitate the extraction process using reduced volumes of
extraction solvents. The ability of performing extraction using
low solvent volumes may be attributed to the increased mass
transfer phenomena observed in HP extraction, compared to
conventional extraction.

4.

Conclusions

The exploitation of enzymes in industry for extracting plant

bioactives for their application in food is a promising eld.
The results of this study indicate that total carotenoid and
lycopene recovery from tomato processing waste can be
increased by the use of enzymes with pectinolytic and cellulolytic activities. The increase of extraction yield by EA
solvent extraction compared to the non enzyme treated solvent extraction process depended on the solvent used. EA
solvent extraction using ethyl lactate presented the best
results with a 10-fold increase in lycopene recovery when compared to the non enzyme treated tomato waste. Thus, enzyme
treatment can be proposed as a pretreatment step to solvent
extraction process in order to improve the extraction yields.
The advantage of EA extraction is that it can be performed on
wet industrial tomato waste, omitting the cost for drying the
material.
Additionally, the current study showed that HP assisted
extraction can be used to obtain high extraction yields
of carotenoids and lycopene, from waste derived by the
tomato processing industry. HP assisted extraction led to
higher extraction yields when compared to solvent extraction process. Moreover, it was demonstrated that HP assisted
extraction can be performed using lower solvent volume at
reduced processing times without affecting the extraction
yields.

Acknowledgements
The authors thank NOMIKOS S.A. (Aliartos, Viotia, Greece)
for kindly providing the tomato waste and Novozymes
(Bagsvaerd, Denmark) and Lyven S.A. (Colombelles, France) for
the enzyme preparations. The authors are also grateful to Dr.
A. Batrinou for her substantial suggestions and to S. Katsou
and Ch. Tsekou for their assistance in the experimental work.

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Please cite this article in press as: Strati, I.F., et al., Enzyme and high pressure assisted extraction of carotenoids from tomato waste. Food
Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012