Accepted Manuscript

Green ultrasound-assisted extraction of carotenoids from pomegranate wastes

using vegetable oils

Athanasia M. Goula, Maria Ververi, Anna Adamopoulou, Kyriakos Kaderides

PII: S1350-4177(16)30261-9
DOI: http://dx.doi.org/10.1016/j.ultsonch.2016.07.022
Reference: ULTSON 3320

To appear in: Ultrasonics Sonochemistry

Received Date: 4 April 2016

Revised Date: 12 July 2016
Accepted Date: 26 July 2016

Please cite this article as: A.M. Goula, M. Ververi, A. Adamopoulou, K. Kaderides, Green ultrasound-assisted
extraction of carotenoids from pomegranate wastes using vegetable oils, Ultrasonics Sonochemistry (2016), doi:
http://dx.doi.org/10.1016/j.ultsonch.2016.07.022

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 Green ultrasound-assisted extraction of carotenoids from pomegranate
wastes using vegetable oils

Athanasia M. Goula*, Maria Ververi, Anna Adamopoulou, Kyriakos Kaderides

Department of Food Science and Technology, School of Agriculture, Forestry and Natural
Environment, Aristotle University, 541 24 Thessaloniki, Greece
Abstract
The objective of this work was to develop a new process for pomegranate peels application in
food industries based on ultrasound-assisted extraction of carotenoids using different
vegetable oils as solvents. In this way, an oil enriched with antioxidants is produced.
Sunflower oil and soy oil were used as alternative solvents and the effects of various
parameters on extraction yield were studied. Extraction temperature, solid/oil ratio, amplitude
level, and extraction time were the factors investigated with respect to extraction yield.
Comparative studies between ultrasound-assisted and conventional solvent extraction were
carried out in terms of processing procedure and total carotenoids content. The efficient
extraction period for achieving maximum yield of pomegranate peel carotenoids was about 30
min. The optimum operating conditions were found to be: extraction temperature, 51.5oC;
peels/solvent ratio, 0.10; amplitude level, 58.8%; solvent, sunflower oil. A second-order
kinetic model was successfully developed for describing the mechanism of ultrasound
extraction under different processing parameters.

Keywords: Carotenoids; Green extraction; Green solvent; Pomegranate peel; Ultrasound

extraction

1. Introduction
Carotenoids are natural pigments that are present mostly in all plants, algae, some
bacteria, and fungi. Generally, it is believed that carotenoids pigments are responsible for the
color in fruits and flowers, which plays an important role in attracting animals to act as

*
Corresponding author. Tel.: +30 2310 991658; fax: +30 2310 991658.
E-mail address: athgou@agro.auth.gr (A.M. Goula).

1
pollinators and seed dispersion vehicles (Hornero-Méndez & Mínguez-Mosquera, 2000).
More than 650 carotenoids have been described and isolated from natural sources, however,
only about 60 are regularly present in the human diet, and about 20 carotenoids were reported
to be in human plasma and tissues (During & Harrison, 2004). It was found that the
consumption of carotenoids-rich foods, such as fruits and vegetables, decreases the risk of
developing certain types of cancer (Flagg, Coates, & Greenberg, 1995) and other degenerative
and chronic diseases (Cho et al., 2004).
Pomegranate (Punica granatum L.) has been well documented for its potential health
benefits such as its high antioxidant, anti-mutagenic, anti-hypertension, anti-inflammatory,
and anti-atherosclerotic activity against osteoarthritis, prostate cancer, heart disease, and HIV-
1 (Caleb et al., 2013). Since the juice yield of pomegranates is less half of the fruit weight,
very large amounts of by-product wastes, such as peels, are formed every year. Pomegranate
by-product wastes have been traditionally valorised as animal feed. Recently, a number of
studies have proposed that some fruit or vegetable by-products could be a source of natural
antioxidants (Llorach et al., 2003; Wolfe, Wu, & Liu, 2003). Kim et al. (2002) introduced the
peels of pomegranate as a rich source of antioxidants, especially phenolic compounds and
carotenoids. Several studies have been published in the past to extract phenolics from
pomegranate peels with various extraction methods, such as normal stirring, microwave-
assisted extraction, pressurized liquid extraction, and ultrasounds (Cheng et al., 2011; Wang
et al., 2011; Zheng et al., 2011; Pan et al., 2012; Tabaraki, Heidarizadi, & Benvidi, 2012;
Veggi, Martinez, & Meireles, 2013; Kaderides, Goula, & Adamopoulos, 2015). However, in
literature, to the best of our knowledge, no studies have been presented on the extraction of
carotenoids from pomegranate peels.
Traditionally, conventional methods have been used to recover nutritionally valuable
compounds from plant food matrices. However, nowadays, the need for obtaining greener,
sustainable, and viable processes has led both food industries and food scientists to develop
new processes in full correspondence with the green extraction concept. Compared to the
conventional methods, such as Soxhlet extraction, high-energy techniques are gaining a
considerable interest in the field of extraction of bioactive compounds, due to the fast
extraction rate, the high extraction yield, the minimum detrimental effect on the extracted
compounds, the enhancement of the extraction of thermosensitive nutritional components, the
lower processing temperatures, and the use of “recognized as safe” organic solvents.
One promising alternative to traditional methods for carotenoids extraction is the use
of edible oils as solvents, which offer attractive advantages, due to the oil solubility of

2
carotenoids (Sachindra & Mahendrakar, 2005; Higuera-Ciapara, Felix-Valenzuela, &
Goycoolea, 2006; Li et al., 2013; Li et al., 2014). Anderson (1975) developed a process for
extraction of carotenoids from shrimp processing wastes wherein soybean oil was added to
the wastes, mixed, heated and the oil fraction recovered by centrifugation. Chen and Meyers
(1982) applied enzymatic hydrolysis of homogenized crawfish wastes with protease and
subsequent extraction with soy oil for recovery of carotenoids. Acid ensilaging of crawfish
wastes was found to stabilize the astaxanthin in the wastes and also increase the recovery of
astaxanthin in soy oil (Chen & Meyers, 1983). Carotenoids in homogenized crawfish waste
were recovered by acidifying and heating with soybean oil to recover pigment-enriched oil
(Meyers & Chen, 1985). Omara-Alwala et al. (1985) reported that the use of propionic acid
enhances the recovery of astaxanthin from crawfish wastes by 35% during vegetable oil
extraction. Cod liver oil also has been used to extract pigments from processing discards of
snow crab and shrimp wastes (Shahidi & Synowiecki, 1991). A method based on silica gel
column chromatography has been developed for the concentration of carotenoids in Krill oil
(Hara et al., 2001). Sachindra and Mahendrakar (2005) studied the extractability of shrimp
wastes carotenoids in different vegetable oils (sunflower oil, groundnut oil, gingelly oil,
mustard oil, soy oil, coconut oil, and rice bran oil) and optimized the conditions for oil
extraction, such as oil to waste ratio, time, and heating temperature. Vasapollo et al. (2004),
Sun and Temelli (2006), and Krichnavaruk et al. (2008) investigated the potential of using
different vegetable oils as co-solvents with continuous addition into SC-CO2 for the extraction
of carotenoids from carrots, tomatoes, and H. pluvialis. Handayani, Indraswati, and Ismadji
(2008) extracted astaxanthin from giant tiger (Panaeus monodon) shrimp waste using palm
oil. Li et al. (2013) developed a method for ultrasound-assisted extraction of carotenoids from
fresh carrots, in which sunflower oil was applied as a substitute to organic solvents.
Parjikolaei et al. (2015) introduced sunflower oil and methyl ester of sunflower oil as two
green solvents for extracting astaxanthin from shrimp processing waste.
Extraction using edible oils can fulfill almost all the principles as defined by Chemat,
Vian, and Cravotto (2012) to be considered as a green process e.g. applying environmentally
friendly solvents, reducing the energy consumption, and producing the non-denatured extract
without contaminants. Further, the oil plays a barrier role against oxygen and consequently
retards the oxidation time and degradation rate of the carotenoids extract (Pu, Bechtel, &
Sathivel, 2010). However, the high oil viscosity is a major problem, which results in low
diffusivity and consequently low extraction yield even at high temperatures. In recent years,

3
ultrasound-assisted extraction (UAE) of compounds with antioxidant activities has been
widely applied to alleviate this problem (Achat et al., 2012; Li et al., 2013).
Extraction enhancement by ultrasound has been attributed to the propagation of
ultrasound pressure waves and resulting cavitation forces, where bubbles can explosively
collapse and generate localized pressure causing plant tissue rupture and improving the
release of intracellular substances into the solvent (Knorr, Ade-Omowaye, & Heinz, 2002). In
recent years, several studies have compared the effects of UAE and conventional solvent
extraction on carotenoids recovery from different plant food materials and their by-products,
such as tomato (Lianfu & Xiaolin, 2001; Lianfu & Zelong, 2008; Eh & Teoh, 2012), tomato
wastes (Kumcuoglu, Yilmaz, & Tavman, 2013), carrot (Almahy, Ali, & Band-Ali, 2013; Li et
al., 2013), corn (Ye et al., 2011), grapefruit (Xu & Pan, 2013), auyama, cabbage, and lettuce
wastes (Alzate, González, & Londoño-Londoño, 2013). However, no information on the
ultrasound-assisted extraction of carotenoids from pomegranate peels is available in the
literature.
The objective of this work is to investigate the extractability of pomegranate peels
carotenoids using ultrasounds and different vegetable oils as alternative solvents. In this way,
a new process for pomegranate peels application in food industries was developed based on
an innovative, fast, and green method for producing an oil naturally enriched with
antioxidants, recovering carotenoids from pomegranate wastes. Comparative studies between
ultrasound-assisted and conventional solvent extraction were carried out in terms of
processing procedure and total carotenoids content.

2. Materials and methods

2.1. Materials
Pomegranate peels as a by-product of fruit juice industry were provided from a local
producer (Rodi Hellas, Greece). The peels were dried at 40 °C for 48 h and kept at −30 °C
until use. The peels were ground in a laboratory mill (Type A10, Janke and Kunkel, IKA
Labortechnik, Germany) immediately prior to extraction. The particle size distribution of the
milled peels is presented in Fig. 1.

2.2. Process for pomegranate peels utilization

Fig. 2 presents the proposed integrated process for pomegranate peels application in
food industries. This process is based on the ultrasound-assisted extraction of carotenoids

4
using a vegetable oil as an alternative solvent. In this way, an oil enriched with antioxidants is
produced. Fig. 2 also presents the conventional method for producing a carotenoids enriched
oil, which is based on the classic extraction using an organic solvent. In both methods, several
pretreatment procedures (washing, drying, grinding) are employed, whereas the conventional
method consists of several additional operation steps, such as solvent evaporation and
recycling and molecular distillation or vacuum drying to eliminate residual traces of
undesirable solvents according to the regulations in force. These additional steps would result
in high input cost, energy consumption, and generation of hazardous solvent waste.

2.3. Ultrasound extraction using vegetable oils

Ultrasonication can be applied in two ways: directly to the sample or indirectly through
the walls of the sample container using a water bath. In the direct method, the ultrasonic probe
is immersed into the solution and provides an ultrasonic power that is at least up to 100 times
greater than that supplied by the bath, with sonication time usually 5 min or less. In this work,
a 130 W of maximum nominal output power, 20 kHz of frequency VCX-130 Sonics and
Materials (Danbury, CT, USA) sonicator equipped with a Ti-Al-V sonoprobe (13 mm) was
used for ultrasound-assisted extraction in pulsed mode. The pulse duration and pulse interval
refer to ‘‘on’’ time and ‘‘off’’ time of the sonicator. The amplitude control of the processor
allowed the ultrasonic vibrations at the probe to be set at any desired level in the 10–100%
range of the nominal power.
Sunflower oil and soy oil were used as alternative solvents. A sample of powdered
pomegranate peels was mixed with 200 mL solvent to produce different peels/solvent ratios.
During the extraction process, the sample container was held in a thermostat-controlled water
bath. The extracts were collected at 10, 20, 30, 40, 50, and 60 min and filtered through glass
microfiber paper to remove particulate residues before carotenoids analysis.
The extraction temperature (T), the peels/solvent ratio (S/L), and the amplitude level (A)
were varied between 20 and 60oC, 1/10 and 3/10 (g/mL), and 20 and 60%, respectively. A
central composite design was applied to determine the effects of the above parameters on
extraction yield. The effects were studied at five experimental levels –a, –1, 0, +1, and +a,
where a = 2n/4 and n is the number of variables.
Extraction yield, Y, was defined as the percent ratio of the total weight of carotenoids
extracted to the dry peels sample weight.

2.4. Conventional solvent extraction

5
 A traditional solvent extraction was applied based on the method optimized by
Sachindra, Bhaskar, and Mahendrakar (2006) and used by Parjikolaei et al. (2015). Five
grams of peels sample was weighed and repeatedly extracted using 25 mL of mixed hexane
and isopropanol (Hex: IPA, 60:40 v/v) until no further pigment was extracted by the solvent
(four times). In order to separate the phases and remove traces of IPA in each step, the extract
was washed with an equal amount (100 mL in total) of 0.1% NaCl solution. The supernatant
was collected and evaporated under vacuum at 35oC using a rotary evaporator R-210
(Rotovapor R114, Waterbath B480, Büchi, Flawil, Switzerland). The resulting carotenoids
concentrate was re-dissolved in 4 mL of acetone before carotenoids analysis. All experiments
were performed in dim light.

2.5. Carotenoids analysis

Three grams of oil were accurately weighed and dissolved in cyclohexane up to a final
volume of 10 mL. Carotenoids content was calculated from the absorption value, A, of the oil
solution at 470 nm and the specific coefficient for lutein, Eo = 2000, using the Eq. (1)
(Karabagias et al., 2013):

A  10 6
C (1)
2000  100  d
where d is the thickness of the spectrophotometer cell (1 cm) and C is the content of
carotenoids in mg/kg oil.

2.6. Oil quality

To examine the effect of ultrasound on oil quality, the untreated sunflower oil and the
oil treated with ultrasound at the optimum conditions were analyzed for acid value (AOCS,
2015a), peroxide value (PV) (AOCS, 2015b), and conjugated dienes (AOCS, 2015c).
In order to estimate the stability of carotenoids during ultrasound-assisted extraction,
identification of the carotenoids in the extract was carried out by means of high-performance
liquid chromatography (HPLC) as described by Mendes et al. (1999). A SupelcosilTM LC-18
column (Supelco Inc., Bellefonte, PA) was used to separate individual carotenoids at ambient
temperature (25oC). Samples (50 μL) were dissolved in methanol/dichloromethane (1:1, v/v)
and the mobile phase, methanol with 10% (v/v) acetonitrile, was delivered at a flow rate of 1
mL/min under isocratic conditions. Peak assignment was performed by comparing the
retention times and line spectral properties obtained from photodiode array detection with α-
carotene, β-carotene and lutein standards.
6
 In addition, the DPPH radical (1,1-diphenyl-2-picrylhydrazyl) scavenging activity of
the extracts was measured according to the method described by Blois (1958).

2.7. Kinetic model

The solid-liquid extraction process can be considered as the reverse of an adsorption
operation, therefore the second-order law was applied to experimentally evaluate the
extraction rate (Rakotondramasy-Rabesiaka et al., 2009). The general second-order model can
be written as (Pan et al., 2012):
dCt
 k  Ce  Ct 2 (2)
dt
where k is the second-order extraction rate constant (L/g min), Ce is the equilibrium
concentration of carotenoids in the liquid extract (g/L) (extraction capacity), and Ct is the
carotenoids concentration (g/L) in the liquid extract at a given extraction time t.
The integrated rate law for a second-order extraction under the boundary conditions t =
0 to t and Ct = 0 to Ct, can be written as an Eq. (3) or a linearized equation (4) (Qu, Pan, &
Ma, 2010):

k  t  Ce 2
Ct  (3)
1  k  t  Ce
t 1 t
  (4)
Ct k  C e 2 Ce

The initial extraction rate, h (g/L min), when t approaches 0, can be defined as:

h  k  Ce 2 (5)

2.8. Statistical analysis

The data were analyzed using the statistical software MINITAB (Release 13.32).
Regression analysis was used to fit a full second order polynomial, reduced second order
polynomials, and linear models to the data of the response variable. F values for all reduced
and linear models with an R2 > 0.70 were calculated to determine if the models could be used
in place of full second order polynomials to predict the response of a variable to the
independent variables.

3. Results and discussion

7
3.1. Extraction yield
In overall, the highest concentrations obtained depending on the applied extraction
solvent were 0.6134 and 0.6715 mg carotenoids/100 g of dry peels using sunflower oil and
soy oil, respectively (Fig. 3). During an extraction process, a low solvent viscosity is usually
associated with an improved solvent migration through the matrix to increase extraction
efficiency (Pierre et al., 2002). Fasina and Colley (2008) and Diamante and Lan (2014)
reported viscosity values of 0.0234-0.0250 and 0.0232 Pa s (at 38oC) for sunflower oil and
soy oil, respectively. Thus, the higher extraction yield obtained for soy oil may be attributed
to its lower viscosity. However, the effect of solvent type on extraction yield was not
statistically significant (p  0.05). A similar trend was reported by Sachindra and
Mahendrakar (2005), who studied the extractability of shrimp wastes carotenoids in different
vegetable oils. Highest carotenoids yield of 26.3 μg/g waste was obtained by extraction with
sunflower oil. However, the extraction yield of carotenoids in soy oil, coconut oil, and rice
bran oil was similar to that in sunflower oil.
Extracting four times with a fresh mixture of Hex:IPA extracted nearly all the
carotenoids present in the waste material. Based on this, extracting once with the green
solvents and using ultrasound successfully extracted about 85.7 and 93.8% of the total
carotenoids present in the waste material. As carotenoids are degraded to some extent during
extraction, reducing the extraction time is important both from a process time and a yield
point of view. Improved extraction yields may be explained in terms of a) oil-soluble property
of carotenoids and b) cavitational effects caused by the application of high-intensity
ultrasound. As large amplitude ultrasound waves travel through a mass medium, they cause
compression and shearing of solvent molecules resulting in localized changes in density and
elastic modulus. As a consequence, the initially sinusoidal compression and shear waves will
at a finite distance from the ultrasonic transducer be distorted into shock waves. The abrupt
decrease in pressure at the edge of the saw tooth shaped ultrasonic wave in the negative
pressure cycle generates small bubbles. These bubbles collapse in the positive pressure cycle
and produce turbulent flow conditions associated with high pressures and temperatures (Price,
White, & Clifton, 1995; Li, Pordesimo, & Weiss, 2004). In addition, ultrasound facilitates
swelling and hydration and causes enlargement of the pores in the cell wall (Vinatoru, 2001).
Diffusion through the plant cell walls, disruption, and washing out of the cell contents were
also attributed to improved extraction performance, whereas the reduction in the particles size

8
by ultrasound disintegration will increase the number of cells directly exposed to extraction
by solvent and ultrasonic cavitation (Vinatoru, 2001).
In all experiments, the extraction yield (Y) was time dependent and increased with
extended ultrasonic times, especially from 10 to 30 min, but slowly from 30 to 60 min (Fig.
4). Thus, the efficient extraction period for achieving maximum yield of pomegranate peel
carotenoids was about 30 min. This can be attributed to the fact that extraction presents two
stages; the first stage, which is characterized by a rapid rate, involves the penetration of the
solvent into the cellular structure followed by the dissolution of soluble constituents in the
solvent, whereas the second one involves the external diffusion of soluble constituents
through the porous structure of the residual solids and its transfer from the solution in contact
with the particles to the bulk of the solution (Goula, 2013). A similar trend was reported by
Zhang et al. (2008), Pan et al. (2012), Li et al. (2013), Kaderides et al. (2015), and Kalamara,
Goula, and Adamopoulos (2015), who extracted oil from flaxseeds, antioxidants from dry
pomegranate marc, carotenoids from carrots, phenolic compounds from pomegranate peels,
and oil from pomegranate seeds, respectively. They attributed this observation to the fact that
ultrasonic wave could disrupt the cell walls, so larger contact area between solvent and
material was created and more oil appeared on the surface. However, this effect would be
increasingly weak on the inner cell walls as the distance is increased. Thus, the ultrasonic
waves affect the mass transfer rate mainly in the solvent penetration stage. In addition, as the
pomegranate peel cell walls ruptured, impurities such as insoluble substances are suspended
in the extract, lowering the solvent permeability into cell structures (Liu et al., 2009; Tian et
al., 2013). Furthermore, target components also re-adsorb into the ruptured tissue particles,
due to their relatively large specific surface areas, lowering yields of peel phenolics (Dong et
al., 2010). Ahmad-Qasem et al. (2013) reported that, during phenolic extraction from olive
leaves, the ultrasound application led to an immediate leaching of polyphenols into the
solvent; thus, 84% of total phenolics content was extracted during the first 5 min of
ultrasound treatment. Therefore, ultrasound effects accelerated the solubilization of accessible
antioxidant compounds (washing effect) and contributed to the extraction of the non-
accessible compounds. A review of the literature also brings opposite results to light, thus,
Jerman, Trebše, and Vodopivec (2010) determined that the extraction efficiency of
polyphenols from olive fruit was low for the first 4 min of ultrasound application, indicating
that longer times were needed for wall disruption. This mild effect could be linked to the level
of ultrasonic power applied, since these authors carried out the experiments in an ultrasonic

9
bath, which actually supplies lower ultrasonic intensities than probe systems like the one used
in the current study.
As shown in Fig. 5 and Table 1, the effect of temperature on the carotenoids content
extracted by vegetable oils was significant. Increasing extraction temperature from 20 to 40
°C, extraction yield increased, whereas an opposite trend was observed when T increased
from 40 to 60 °C. The first effect may be attributed to the fact that an increase in temperature
increases solubility and diffusion coefficients of the compounds to be extracted and decreases
the viscosity of the solvent, thus facilitating its passage through the solid substrate mass.
However, at temperatures higher than 40 °C, the tendency of impurities dissolution will also
increase and some thermal labile constituents will decompose (Dong et al., 2010). Chemat et
al. (2004a), who compared conventional and ultrasound-assisted extraction of carvone and
limonene from caraway seeds, reported that the activation energies for the ultrasound
procedure are much lower compared with those obtained by the control procedure, which
explains the enhancement of extraction in ultrasound experiments at low temperatures.
Comparing the effect of temperature on the carotenoids content in soy oil with that in
sunflower oil, the lower effect observed may be associated with the lower influence of
temperature on the viscosity of soy oil compared to that of sunflower oil. Fasina and Colley
(2008) and Diamante and Lan (2014) reported activation energy values of 23.40–24.64 and
21.58–22.95 kJ/mol for sunflower oil and soy oil, respectively.
The extraction yield improved with increased amplitude level (A) up to values around
40% (Fig. 5). This effect may be due to the improved cavitation and mechanical effect of
ultrasound, which increased the contact surface area between solid and liquid surfaces and
caused greater penetration of the solvent into the peel matrix. At high amplitude levels,
cavitational bubble collapse is more violent (Suslick & Price, 1999). According to Zhang et
al. (2008), since the temperature and pressure were very high inside the bubbles and the
collapse of bubbles occurred over a very short time, a violent shock wave and a high-speed jet
were generated, which could enhance the penetration of the solvent into the cell tissues and
accelerate the intracellular product release into the solvent by disrupting the cell walls.
However, for amplitude levels above 40%, the increase in A led to a decrease in yield,
possibly due to degradation of the plant material.
As far as the ratio of peel/solvent (S/L) is concerned, the extraction yield increased
with decreasing the ratio up to values around 0.2, a trend that is consistent with the mass
transfer principles. Higher solvent/solid ratio resulted in larger concentration gradient during
the diffusion from the solid into the solution and, thus, in higher carotenoids concentration in

10
the extract. Li et al. (2005), Zhang et al. (2008), and Qu et al. (2010) also reported this effect
of solvent/sample ratio on the extraction yield of chlorogenic acid from Eucommia ulmodies,
oil from flaxseed, and antioxidants from pomegranate marc, respectively. However, in the
case of sunflower oil, where the effect of S/L is significant (Table 1), with further decrease in
solid-to-liquid ratio, a decrease in carotenoids content was observed. A similar observation
was reported by Prasad et al. (2011) and Kaderides et al. (2015), who found the maximum
extraction yield of total phenolics from Mangifera pajang and pomegranate peels at a
solid/solvent ratio of about 0.03. Sachindra and Mahendrakar (2005) found a marginal
decrease in carotenoids extraction yield by further increasing the solvent(oil)/solids ratio from
2 to 3 and hence there is no additional advantage of increasing the ratio above 2. Regarding
soy oil as a solvent, no statistically significant effect of peels/solvent ratio was observed
(Table 1).
Using the central composite design, the effect of the examined variables on extraction
yield was analyzed. The variables evidencing statistically significant effects were screened via
Student's t-test for ANOVA (Table 1). According to the ANOVA analysis of the optimization
study, temperature, peels/solvent ratio, and amplitude level influence significantly the
extraction yield using sunflower oil (p < 0.05) (Table 1). These parameters also showed a
significant quadratic effect. Regarding soy oil as a solvent, the extraction temperature, with a
probability value of 0.022, was determined to be the only significant factor. The desirability
profile for optimum carotenoids yield indicates that the maximum desirability level of 1.0 (on
a scale of 0–1) can be achieved with a temperature of 51.5oC, an amplitude level of 58.8%, a
peels/solvent ratio of 0.10 g/mL, and sunflower oil as solvent (Fig. 6). Under these optimized
conditions, the predicted value for extraction yield was 0.3291 mg carotenoids/100 g of dry
peels, whereas the observed experimental value was found 0.3255 mg carotenoids/100 g of
dry peels.
The process proposed in this work was based on the ultrasound-assisted extraction of
carotenoids using a vegetable oil as an alternative solvent. In this way, an oil enriched with
antioxidants was produced. On the contrary, the conventional method for producing a
carotenoids enriched oil is based on the classic extraction using an organic solvent. This
conventional method consists of several additional operation steps, such as solvent
evaporation and recycling and molecular distillation or vacuum drying to eliminate residual
traces of undesirable solvents according to the regulations in force. These additional steps
result in high input cost, energy consumption, and generation of hazardous solvent waste.

11
Generally, reducing the number of steps in a process chain leads to a reduction in costs and
better use of energy.
According to Kaderides et al. (2015), while conventional extraction procedures are
often time and/or energy consuming, ultrasound-assisted extraction provides numerous
advantages from an industrial perspective. Ultrasound as a food processing technology has
shown large commercial large scale application, with high returns on capital investment.
Improvements in product efficiency, process enhancement, and low maintenance cost are
achievable on a commercial scale. Also, depending on the application, the required energy is
comparable to those of other operation units currently utilized in the industry (Pingret et al.,
2012).
In addition, vegetable oils as substitutes to organic solvents, besides being
biodegradable and non-toxic, do not emit VOCs (volatile organic compounds), they have
technical performances comparable with those of petrochemical solvents. One significant
advantage of using vegetable oils is that there is no need for subsequent separation of oil and
carotenoids since the pigmented oil can find use as carotenoids source in different products,
such as aquaculture feeds (Krichnavaruk et al., 2008). According to Spinelli and Mahnken
(1978), in aquaculture feed preparations, vegetable oil or fish oil is commonly used as a
source of energy. The use of pigmented oil in feeds thus serves the dual purpose of pigment
carrier as well a source of lipid energy. Sowmya and Sachindra (2014) extracted carotenoids
using vegetable oils and found that the enriched oil has a protective effect against ammonia-
induced stress in common carp (Cyprinus carpio) and prevents lipid peroxidation in the
tissues exposed to ammonia. Sachindra and Mahendrakar (2010) used carotenoids pigmented
oil as a colorant in fish sausage and reported that addition of carotenoids enhanced the
sensory color, flavor, and overall quality score of sausage, whereas the added carotenoids
were stable during processing.
However, the flavor and composition of some edible oils may be deteriorated by
ultrasound treatment (Chemat at al., 2004b; Patrick, Blindt, & Janssen, 2004). According to
Chemat et al. (2004c), the ultrasonic oxidation of edible oils is probably attributed to
cavitation that influences structural and functional components up to the point of lipid
oxidation and deterioration. Metals such as copper, which occur naturally in edible oils, in
combination with ultrasound cavitation, were also suspected as possibly responsible for the
formation of oxyradical species. A comparison of untreated sunflower oil and oil treated with
ultrasounds at the optimum conditions showed that there was no major difference between the
two oils. The acid value (0.13 and 0.12 mg KOH/g oil for the treated and the untreated oil,

12
respectively) and the percentage of conjugated dienes (0.064 and 0.066% for the treated and
the untreated oil, respectively) were about the same and only a slight increase in peroxide
value was observed. PV increased from 5.68 meq O2/kg oil for the untreated oil to 6.22 meq
O2/kg oil measured immediately after sonication of sunflower oil. However, the Codex
Alimentarius standard for the quality of oils states a maximum acid value of 4.0 mg KOH/g
oil and a maximum peroxide value of 15 meq O2/kg oil, thus, the treated oil is below the limit
despite the increase in PV value observed after the ultrasounds-assisted extraction.
In addition, the chromatograms correspond to carotenoids extracted by ultrasound at the
optimum conditions and organic solvent showed that there was no carotenoids degradation in
sonicated sunflower oil. β-carotene was the main carotenoid detected in pomegranate peels
extracts obtained by both ultrasound and conventional techniques, made up 93% of the total
carotenoids content, while the other carotenoids identified, such as a-carotene and lutein, were
only present in small amounts. A similar observation was reported by Sun et al. (2010) and Li
et al. (2013), who found that the ultrasonic degradation of β-carotene is typically slow in
comparison with that of more volatile aromatics that diffuse more readily into the cavitation
bubble in an aqueous system. As far as the antioxidant activity is concerned, carotenoids
extracted by ultrasound at the optimum conditions and organic solvent showed about the same
antioxidant activity (13.69 and 14.08% inhibition for the ultrasound and the conventional
method, respectively). Similar results were reported by Roidaki, Zoumpoulakis and Proestos
(2015), who concluded that UAE was proven to be effective for leaves extraction for Leikora
variety, concerning the antioxidant activity measured by DPPH and ABTS.

3.2. Ultrasound kinetics

For all the factors studied, the plots of t/Ct versus t resulted in linear functions, in
agreement with the second order leaching model expressed by Eq. (4). The slopes and
intercepts allowed the determination of extraction capacities (Ce) and extraction rate constants
(k) from Eq. (4) and of initial extraction rates (h) from Eq. (5). Figs. 7–8 present the achieved
values of Ce, k, and h.
The kinetic parameters of carotenoids extraction were enhanced with temperature
increase and extraction at 40–50 °C had highest efficiencies. The values of kinetic parameters
declined when the temperature was higher than 50°C. According to Spigno, Tramelli, & De
Faveri (2007), an increase in temperature favors extraction due to the enhancement of both
solute solubility and diffusion coefficient, but beyond a certain temperature level, carotenoids
can be denatured. This fact has already been reported in the literature, where there is a

13
controversy surrounding the influence of temperature in antioxidants extraction processes.
Thus, Jerman et al. (2010) reported an increase in extraction efficiency at temperatures of up
to 45 °C for olive fruit phenolic compounds. The same trend was observed by Zhang et al.
(2009) in the temperature range of 15–45 °C, where high temperatures reduced the extraction
yield. González-Centeno et al. (2015) reported that temperature has a positive effect only
when it is not too high, as most of the bioactive compounds are susceptible to degradation at
higher temperatures, reducing the observed extraction rate. However, Zhang, He, and Hu
(2011) found that extraction yields rose as the temperature increased from 60 to 80 °C, while
Rostagno, Palma, and Barroso (2007) found that phenolics underwent an important
degradation at temperatures of over 60 °C. Therefore, it seems that the temperature influence
may be product-dependent, it being necessary to determine the proper extraction temperature
for a specific commodity.
As it can be seen in Figs. 7–8, the kinetic parameters increased with increased
amplitude level up to values around 40%, due to the improved cavitation and mechanical
effect of ultrasound. Similar results were also found for extraction of antioxidants from
pomegranate peels and seeds (Pan et al., 2012; Goula, 2013; Kaderides et al., 2015).
However, for amplitude levels above 40%, the increase in A led to a small decrease in kinetic
parameters. A similar trend was reported by Abdullah and Koc (2013), who mentioned that
the coefficients of equilibrium concentration for the diffusion stage were higher than the
washing stage, which indicates that ultrasound power had a greater effect during the diffusion
stage than the washing stage. The mass transfer coefficient for washing increased with
increasing ultrasound power, while the mass transfer coefficient for diffusion decreased with
increasing ultrasound power.
The highest Ce, k, and h values were obtained at peels/solvent ratio values around 0.2.
The kinetic parameters increase with the increase of the solid to liquid ratio up to a certain
limit of the tested range. However, further increases in solid to liquid ratio did not translate
into significant improvement in the yield. This observation can be explained by the fact that
the system has become saturated as the solute has entirely dissolved in the fluid. A similar
trend was reported by Prasad et al. (2011), Anuar et al. (2013), Stamatopoulos, Chatzilazarou,
and Katsoyannos (2014), and Kaderides et al. (2015) during extraction of phenolic
compounds from Melastoma malabathricum fruit, olive leaves, and peels of Mangifera
pajang and pomegranate.
Qu et al. (2010) fitted the relationships between kinetic parameters and processing
factors (particle size, water/sample ratio, and temperature) by linear, power or second-order

14
polynomial functions. However, they developed a different model for each factor. In this
work, multiple regression analysis was used to develop equations predicting the effect of all
extraction factors simultaneously (Table 2). Substituting equations presented in Table 2 into
Eq. (4), a kinetic model for predicting carotenoids extraction from pomegranate peels can be
obtained.
Even though the empirical models in Table 2 cannot account for the phenomena
governing extraction processes, they could be used to determine the effects of temperature,
solids/solvent ratio, and amplitude level on the carotenoids extraction capacity during the
extraction process. According to Goula (2013), mechanistic models provide more from a
basic understanding of a given system, a greater basis for extrapolation and a representation
of a response function that is more precise than one attained empirically. In the food industry,
on the other hand, if it is desired to optimize a given unit process, the process of developing
an empirical model would most likely be more economically feasible. Such models may be
more limiting than mechanistic models. However, for the purposes of the manufacturer, the
main concern is narrowing the range of processing variables to produce the best product
possible. Thus, the empirical models in Table 2 should provide the guidance for optimizing
the extraction process decreasing the operating cost and time.

4. Conclusions
An integrated approach for utilization of pomegranate peels is suggested based on the
ultrasound-assisted extraction of carotenoids using different vegetable oils as solvents. In this
way, an oil enriched with antioxidants is produced. On the contrary, the conventional method
for producing a carotenoids enriched oil is based on the classic extraction using organic
solvents. The proposed method meets the terms of green process definition, since reduces
energy consumption and solvent amount, allows the use of renewable natural products, and
ensures a safe and high-quality extract/product. In addition, there is no need for subsequent
separation of oil and carotenoids, since the pigmented oil can find use as carotenoids source in
different products.
It was found that:
 Extracting once with the green solvents and using ultrasounds successfully extracted
about 85.7 and 93.8% of the total carotenoids present in the waste material.
 The efficient extraction period for achieving maximum yield was about 30 min.
 Extraction yield increased increasing extraction temperature from 20 to 40oC, amplitude

15
 level up to values around 40%, and peels/solvent ratio up to values around 0.2.
 The optimum extraction yield was about 0.3255 mg carotenoids/100 g of dry peels.
 A second-order kinetic model can describe the extraction process under different
ultrasound-assisted extraction parameters.

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Table 1

Table 1. Regression analysis of central composite design.

Sunflower oil Soy oil
Variable Coefficient p-value Coefficient p-value
Constant –1.5841 0.001 –0.5005 0.150
A 0.04085 0.001 0.008812 0.286
T 0.02011 0.011 0.018857 0.022
S/L 6.348 0.005 1.688 0.299
A*A –0.0003254 0.002 –0.0000863 0.242
T*T –0.0002885 0.005 –0.0001316 0.076
S/L*S/L –10.089 0.014 –0.951 0.747
A*T 0.0001004 0.494 0.0000362 0.717
A*S/L –0.08172 0.006 –0.01211 0.544
T*S/L –0.00119 0.968 –0.04397 0.029
Table 2

Table 2. Best model predictions for the kinetic parameters (Ce, h, k) in relation to the
processing factors, amplitude level (A), extraction temperature (T), peels/solvent ratio
(S/L).
Parameter Model R2
Sunflower oil
Ce  2.53  0.0792  A  0.0448  T  4.35  S / L  0.000618  A 2  0.000544  T 2  0.131  A  S / L 0.918

h  0.338  0.0106  A  0.00597  T  0.580  S / L  0.000082  A 2  0.000073  T 2  0.0174  A  S / L 0.892

k (×105)  61.1  2.0  A  1.1 T  98.4  S / L  3.3  A  S / L  0.3  T  S / L 0.938
Soy oil
Ce  1.03  0.0354  T  6.60  S / L  0.000251  T 2  10.5  S / L2  0.0713  T  S / L 0.902

h  0.0562  0.00102  A  0.00269  T  0.111  S / L  0.000011  A 2  0.000017  T 2  0.00588  T  S / L 0.876

k (×105)  6.3  0.2  A  0.4  T  0.1  A 2  0.1  T 2  0.6  T  S / L 0.956

Figure 1

Fig. 1. Particle size distribution of milled pomegranate peels.

Figure 2

Fig. 2. Production of vegetable oil enriched with carotenoids using: a) conventional

organic solvent extraction, b) ultrasound–assisted extraction with a vegetable oil as an
alternative solvent.
Figure 3

Fig. 3. Box plot showing the effect of solvent (vegetable oil) type on extraction yield
(Y).
Figure 4

0.6
Y (mg carotenoids/100 g dry peels) A = 40%, T = 40oC, S/L = 0.2 g/mL
0.5
Sunflower oil
0.4 Soy oil
A = 28%, T = 52oC, S/L = 0.14 g/mL
0.3

0.2

0.1

0
0 10 20 30 40 50 60
t (min)
Fig. 4. Extraction yield (Y) of carotenoids from pomegranate peels as a function of
time (t) for some experiments (continuous lines and closed symbols for
sunflower oil; dotted lines and open symbols for soy oil).
Figure 5

Fig. 5. Main effects of amplitude level (A, %), extraction temperature (T, °C),
peels/solvent ratio (S/L, g/mL) on extraction yield (Y, mg carotenoids/100 g dry
peels) for the two vegetable oils.
Figure 6

Fig. 6. Optimization of green ultrasound-assisted extraction of carotenoids from

pomegranate peels.
Figure 7

Fig. 7. Effect of amplitude level (A, %), extraction temperature (T, °C), and
peels/solvent ratio (S/L, g/mL) on kinetic parameters of extraction using
sunflower oil.
Figure 8

Fig. 8. Effect of amplitude level (A, %), extraction temperature (T, °C), and
peels/solvent ratio (S/L, g/mL) on kinetic parameters of extraction using soy oil.
 Green ultrasound-assisted extraction of carotenoids from
pomegranate wastes using vegetable oils

Athanasia M. Goula*, Maria Ververi, Anna Adamopoulou, Kyriakos Kaderides

Department of Food Science and Technology, School of Agriculture, Forestry and
Natural Environment, Aristotle University, 541 24 Thessaloniki, Greece

Highlights
 A new process for pomegranate peels application in food industries was
developed.
 Carotenoids were ultrasound extracted from pomegranate wastes using
vegetable oils as solvents.
 An oil enriched with antioxidants is produced.
 The effect of different parameters on extraction efficiency was studied.

*
Corresponding author. Tel.: +30 2310 991658; fax: +30 2310 991658.
E-mail address: athgou@agro.auth.gr (A.M. Goula).