food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203

Contents lists available at ScienceDirect

Food and Bioproducts Processing

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

Natural deep eutectic solvents and

ultrasound-assisted extraction: Green approaches
for extraction of wine lees anthocyanins

Tomislav Bosiljkov a, Filip Dujmić a, Marina Cvjetko Bubalo a, Janez Hribar b,

Rajko Vidrih b , Mladen Brnčić a , Emil Zlatic b ,
Ivana Radojčić Redovniković a,∗ , Stela Jokić c
a Faculty of Food Technology and Biotechnology, University of Zagreb, Pierrotijeva 6, 10000 Zagreb, Croatia
b Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
c Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, 31000 Osijek, Croatia

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

Article history: To establish environmentally friendly extraction methods for the anthocyanins in wine lees,
Received 28 June 2016 natural deep eutectic solvents (NADES) were investigated as a green alternative to conven-
Received in revised form 1 tional solvents, coupled with high-efficiency ultrasound-assisted extraction. Screening for
December 2016 the optimal NADES for this extraction was initially performed. A choline–chloride-based
Accepted 9 December 2016 NADES with malic acid as the hydrogen bond donor was selected as the most promising,
Available online 29 December 2016 which provided more effective extraction of wine lees anthocyanins compared to a conven-
tional solvent. To optimise this extraction of wine lees anthocyanins using the NADES and
Keywords: ultrasound-assisted extraction, response surface methodology was successfully applied.
Anthocyanins Considering the maximum amount of extracted compounds, the optimal conditions were:
Natural deep eutectic solvent extraction time, 30.6 min; ultrasound power, 341.5 W; and water content in NADES, 35.4%
Response surface methodology (w/w). This approach using NADES as a green solvent and ultrasound as an alternative
Ultrasound-assisted extraction energy source represents a good choice for designing eco-friendly extraction methods for
Wine less phenolic compounds from various sources.
Eco-friendly extraction © 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction
The winemaking industry produces large volumes of waste and by-
products, including grape pomace, stalks and lees. Only small amounts
of these by-products are up-graded or recycled, frequently used for
composting, or even discarded in open areas, thus potentially result-
ing in environmental problems (Teixeira et al., 2014). Over the last
years, the evaluation of these by-products as sources of biologically
active compounds and the recovery of these into value-added prod-
ucts have attracted great interest. The most representative of the grape
secondary metabolites are the phenolic compounds, due to their valu-
able activities in terms of the prevention of oxidative reactions and
the benefits seen against cardiovascular disease, inflammatory pro-
cesses, and degenerative pathophysiological states that can develop
in adults (Vislocky and Fernandez, 2010). Research in this field has
been mainly focused on the extraction of the phenolic compounds from
grape pomace (Casazza et al., 2010; Deng et al., 2011; Luque-Rodríguez
et al., 2007; Rajha et al., 2014), using the available literature based on
the exploration of wine lees (Delgado de la Torre et al., 2013; Pérez-
Serradilla and Luque de Castro, 2011; Tao et al., 2014; Teixeira Barcia
et al., 2014; Barbaa et al., 2016). However, some data have indicated that
phenolic extracts from wine lees can be suitable as a cheaper alterna-
tive to those obtained from grape seeds and skins (Pérez-Serradilla and
Luque de Castro, 2011).
Among these phenolic compounds, wine lees from red grapes is
a good source of anthocyanins (Pérez-Serradilla and Luque de Castro,
2011; Tao et al., 2014; Teixeira Barcia et al., 2014). Anthocyanins are nat-
ural colorants with an extensive range of colours and beneficial health

∗
Corresponding author. Fax: +385 1 4605078.
E-mail address: iradojci@pbf.hr (I. Radojčić Redovniković).
http://dx.doi.org/10.1016/j.fbp.2016.12.005
0960-3085/© 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
196 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203
effects, which makes them interesting for applications in the food, cos-
metics and pharmaceutical industries (Castañeda-Ovando et al., 2009).
Extraction techniques for such by-products for their evaluation and
utilisation should be based on green and sustainable technologies, by
following the principles of green extraction. In general, green extrac-
tion is based on the discovery and design of extraction processes with
optimal consumption of raw materials, solvents and energy (Roselló-
Soto et al., 2015; Barbaa et al., 2016; Zhu et al., 2017). Additionally, there
is a growing area of research for the development of green extractions,
which is devoted to the design of new, environmentally friendly and
tunable solvents that can meet both the technological and economic
demands (Cvjetko Bubalo et al., 2015).
Over the last few years, the use of natural deep eutectic solvents
(NADES) as a green alternative to conventional solvents has been
dramatically expanded (Smith et al., 2014). NADES present a new
generation of liquid salts, and they are generally based on mixtures
of relatively cheap and readily available components, such as non-
toxic quaternary ammonium salts (e.g., choline chloride) with naturally
derived uncharged hydrogen-bond donors (e.g., vitamins, amines, sug-
ars, alcohols, carboxylic acids), which provide a ‘green profile’ and have
good prospects for wider use in the field of green technologies (Abbott
et al., 2004). Since their emergence, NADES have attracted attention
as potential solvents in various industrial fields, including the extrac-
tion of biologically active compounds from plant materials (Paiva et al.,
2014). Some studies have reported NADES applications for the extrac-
tion of phenolic compounds (Radošević et al., 2016; Cvjetko Bubalo
et al., 2016; Bi et al., 2013; Dai et al., 2013a,b, 2014; Woo Nam et al., 2015).
NADES have unique physicochemical properties, with the possibility
for their design for particular purposes, and thus along with the other
environmental benefits of using NADES as solvents, such as their low
vapour pressures and non-flammability, NADES represent excellent
candidates for green extractions (Cvjetko Bubalo et al., 2015). Another
principle of green extraction is reduced energy consumption through
the use of innovative technologies, such as ultrasound. Ultrasound-
assistant extraction (UAE) is recognized as providing an outstanding
energy source to promote extraction, to cut down on extraction times,
to increase yields, and often to improve the quality of the required
extract (Esclapez et al., 2011). The enhanced heat and mass transfer
provided by ultrasound is mainly attributed to the acoustic effects that
are accompanied by ultrasound cavitation phenomena, which makes
ultrasound one of the most promising of the many technological pro-
cesses considered (Bosiljkov et al., 2011; Brnčić et al., 2010; Dujmić et al.,
2013; Herceg et al., 2010; Karlović et al., 2014). UAE has been widely
used for extraction of phenolic compounds from different plant mate-
rials, including grapes and winemaking by-products such as pomace
and wine lees (Carrera et al., 2012; Casazza et al., 2010; Novak et al.,
2008; Tao et al., 2014). Also, UAE methods have been used in conjunc-
tion with different NADES for the extraction of phenolic compounds
(Cvjetko Bubalo et al., 2016; Radošević et al., 2016; Woo Nam et al., 2015;
Bi et al., 2013; Dai et al., 2013b).
The aim of the present study was to establish a highly efficient and
truly eco-friendly extraction method for the anthocyanins in wine lees
using NADES and UAE. Initial screening for the optimal NADES for these
anthocyanins was performed, followed by the statistical optimisation
of the UAE operational conditions using response surface methodology
(RSM).
2. Materials and methods
2.1. Chemicals and materials
Choline chloride, citric acid monohydrate, malic acid, oxalic
acid, glucose, fructose, xylose and glycerol were from Sigma
(St. Louis, MO, USA). The analytical standards of delphinidin-
3-O-monoglucoside, petunidin-3-O-monoglucoside, peonidin-
3-O-monoglucoside, and malvidin-3-O-monoglucoside were
from Polyphenols AS (Sandnes, Norway).
Wet wine lees were obtained from the Agrolaguna Com-
pany winemakers (Porec, Croatia). The lees were obtained as
the semi-solid residue decanted after the alcoholic fermenta-
tion of the grape juice from Merlot grapes. After the wine lees
were collected, they were lyophilised and then frozen under
vacuum until further use.
2.2. Preparation of the deep eutectic solvents
All of the chemicals used in these preparations were dried
before use in a vacuum concentrator (Savant SPD131DDA
SpeedVac Concentrator), at 60 ◦ C for 24 h. The NADES were
used at specific ratios of choline chloride to hydrogen donor
(i.e., citric acid, malic acid, oxalic acid, glucose, fructose,
xylose, glycerol), to obtain liquids at room temperature,
choline chloride:citric acid (ChCit), choline chloride:oxalic
acid (ChOa), choline chloride:malic acid (ChMa), choline chlo-
ride:glucose (ChGlc), choline chloride:fructose (ChFru), choline
chloride:xylose (ChXyl), choline chloride:glycerol, (ChGly)
(Radošević et al., 2016). The mixtures of choline chloride
with the hydrogen donor were stirred in a flask at 80 ◦ C for
2 h–6 h, until a homogeneous transparent colourless liquid
was formed. These NADES samples were vacuum dried prior
to further use.
2.3. Selection of optimal deep eutectic solvent
About 33.3 mg of lyophilised wine lees were extracted per mL
of the NADES of interest (i.e., solvent-to-solid ratio, 60:1), in
a shaker for 3 h at room temperature (Tao et al., 2014). The
extracts were then centrifuged at 5000 × g for 15 min, and the
supernatants obtained were filtered and further analysed. To
compare the extraction efficiencies of these NADES with those
of extractions performed using conventional solvents, an acid-
ified aqueous solution of ethanol (ethanol/water/formic acid,
50:48.5:1.5, v/v/v; pH 2.7) was carried out under the same con-
ditions. All of the extraction procedures and the analysis of
the total anthocyanins were performed in triplicate (Carrera
et al., 2012; Sarneckis et al., 2006).
2.4. Ultrasound-assisted extraction procedure
An ultrasonic bath system was used for the UAE (Elmasonic
P 300 H; Elma Hans Schmidbauer GmbH & Co. KG, Sin-
gen, Germany), which was equipped with a digital controlled
system for temperature, time and power (ultrasonic power
effective is 380 W). All of the extractions were performed
at constant frequency (37 kHz) and temperature (35 ◦ C). The
solid-to-liquid ratio of 0.1 g dried wine lees per mL solvent was
used for these extractions, which were performed according to
the experimental design. The extracts were then centrifuged
at 5000 × g for 15 min, and the supernatants obtained were
filtered and further analysed.
2.5. Experimental design and statistical analysis
Response surface methodology was applied to evaluate the
effects of the extraction parameters and to optimise the con-
ditions according to various responses. The extractions were
planned through the application of central composite rotat-
able design (Baş and Boyacı, 2007). Five different levels were
used for each of the following factors: water content in the
NADES (w/w) (X1 ), time (X2 ), and ultrasonic power (X3 ). The
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203 197

Table 1 – The coded and real levels of the independent variables for the experimental design.
Independent variable Symbol Levels

−˛ −1 0 +1 +˛

Water content in DES (%) X1 10 18.11 30 41.89 50

Time (min) X2 15 21.08 30 38.91 45
Power (W) X3 190 228.5 285 341.5 380

coded and uncoded levels of the independent variables and following elution gradient was used, according to solvent
the experimental design are given in Table 1. B: 0–20 min, 14%–23%; 20–40 min, 23%–35%; 40–50 min, 40%;
The responses evaluated were the total anthocyanin 50–60 min, 60%; 60–65 min 95%. The column temperature was
content (Y1 ), and the total contents of the anthocyanin- kept at 30 ◦ C and the autosampler at 4 ◦ C. The individual
3-O-monoglucosides (Y2 ) and the anthocyanin-3-(6-O-p- anthocyanins were identified by comparing their retention
coumaroyl)monoglucosides (Y3 ). times with their respective standard. The quantification
The second-order polynomial given in Eq. (1) was used of the different phenolic compounds was carried out at
to express the responses investigated (Y) as functions of the 520 nm, applying each calibration line constructed using the
coded independent variables: corresponding standard (Supplementary Fig. S1 in the online
version at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005).

k 
k 
k−1 
k For the compounds lacking of standards, the quantification
Y = ˇ0 + ˇi Xi + ˇii Xi2 + ˇij Xi Xj (1) was achieved using similar compounds. Thus peonidin-
i=1 i=1 j=2 3-(6-O-p-coumaroyl)monoglucoside was quantified in
i=1
equivalents of peonidin 3-O glucoside, malvidin-3-(6-O-
i<j
p-coumaroyl)monoglucoside was quantified in equivalents
of malvidin 3-O glucoside. All anthocyanins were also
where X1 , X2 ,. . ., Xk are the independent variables that affect
confirmed using mass spectrometer (Agilent 6460, Agilent
the Y responses, ˇ0 , ˇj (i = 1, 2,. . ., k), ˇii (i = 1, 2,. . ., k), and ˇij
Technologies). The system was equipped with an electrospray
(i = 1, 2,. . ., k; j = 1, 2,. . ., k) are the regression coefficients for the
interface (ESI) that was used in positive ionisation mode.
intercept, linear, quadratic and interaction terms, respectively,
These data are presented in mg g−1 dw for each individual
and k is the number of variables.
anthocyanin group: the anthocyanin-3-O-monoglucosides
The statistical analysis was performed using the RSM soft-
® (AMon): as the sum of delphinidin 3-O glucoside, petunidin
ware Design-Expert , v.7 (Stat Ease, Minneapolis, USA). The
3-O glucoside plus malvidin 3-O glucoside; the anthocyanin-
results were tested statistically using analysis of variance
3-(6-O-p-coumaroyl)monoglucosides (ACm): as the sum of
(ANOVA) at the significance level of p = 0.05. The adequacy
peonidin-3-(6-O-p-coumaroyl)monoglucoside plus malvidin-
of the model was evaluated by the coefficient of determina-
3-(6-O-p-coumaroyl)monoglucoside. The data acquisition and
tion (R2 ) and the model p value. Mathematical models were
analysis were carried out using the Agilent ChemStation
established to describe the influences of the single process
software. The HPLC analyses were conducted in triplicate.
parameters and/or the interactions of multiple parameters
HPLC method was validated in the terms of linearity of cal-
on each response investigated. Response surface plots were
ibration graphs, limits of detection (LOD), limits of quantifica-
generated with the same software, and were drawn using the
tion (LOQ) and precision (Supplementary Table S1 in the online
function of two factors, and keeping the other factor constant.
version at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005).
Linearity was evaluated on the basis of eight point calibration
2.6. Determination of total anthocyanin content
curves for each standard. Linear least-square regression anal-
ysis was employed to calculate slope, intercept and correlation
The total anthocyanins extracted from the wine lees was
coefficient of the calibration curve. Correlation coefficients of
determined by the bisulphite bleaching procedure (Ribéreau-
calibration curves for all standards were higher then 0.99, indi-
Gayon and Stonestreet, 1965), as anthocyanins form colourless
cating very good linearity. Limit of detection was determined
compounds with the bisulphite ion, thus transferring from
form the amount of standard required to give signal-to-noise
the red-coloured flavylium cation to a non-coloured form.
ratio of 3, and limit of quantification was determined as the
The differences obtained between the non-bleached and
lowest concentration giving signal-to-noise ratio of 10. The
the bisulphite-bleached samples for their absorbance at
precision of method was determined in terms of intraday
520 nm are proportional to the total anthocyanin con-
repeatability of peak area for all standards, with relative stan-
tents. The total anthocyanin contents are expressed as mg
dard deviation (RSD%) being lower than 0.96% and indicating
malvidin-3-glucoside equivalents g−1 dw (mg g−1 dw). The
adequate degree of precision.
spectrophotometric analyses were conducted in triplicate.

2.7. HPLC analysis of anthocyanins 3. Results and discussion

The anthocyanin analysis was performed on an HPLC system
(Agilent 1260; Agilent Technologies) with a diode array detec-
tor, and with the wavelength set at 520 nm. Separation of the
anthocyanins was carried out on a C18 column (Poroshell
120 EC-C18; 100 × 2.1 mm i.d. 2.7 ␮m; Agilent Technologies,
USA), at a flow rate of 0.2 mL min−1 . The mobile phases were:
(A) 1% (v/v) aqueous formic acid; and (B) methanol. The
3.1. Selection of deep eutectic solvent for extraction of
wine lees anthocyanins
Seven different cholinium-based NADES were tested for
extraction of total anthocyanins from wine less, which con-
tained xylose, glucose, fructose, glycerol, malic acid, citric
acid, and oxalic acid as the hydrogen bond donors. Extrac-
198 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203

Fig. 1 – Total anthocyanin extraction yields for acidified ethanol (EtOH) in comparison with the deep eutectic solvent
combinations initially tested. Mean values ± SD (n = 3) in each column followed by different lower-case letters are not
significantly different (p < 0.05) as measured by Tukey’s HSD test. The deep eutectic solvent combinations comprised choline
chloride (Ch) plus xylose (Xyl), fructose (Fru), glucose (Glc), glycerol (Gly), malic acid (Ma), citric acid (Cit), and oxalic acid (Oa),
the numbers on the graph (50–90) indicate DES content of aqueous solution of DES (%).

tions with NADES that contained different water contents
from 10% to 50% (w/w) were also performed, as this water con-
tent has been reported to dramatically influence the extraction
efficiency (Dai et al., 2013a,b, 2014). In general, the results
obtained indicated that the type of NADES used did indeed
strongly influence the anthocyanin extraction (Fig. 1). The
best extraction efficiency was obtained with ChMa followed
by ChOa > ChCit > ChGly > ChFru > ChXyl > ChGlc. These data
indicated that the NADES with organic acids as the hydrogen
bond donor were the best solvents in these test extractions.
These will also be more acidic (pH < 3) by nature than those
with sugars or polyalcohols as the hydrogen bond donor
(pH > 6) (Dai et al., 2014; Radošević et al., 2015). This is not sur-
prising as it is well known that an acidic environment favours
anthocyanin extraction and stability, as the flavylium ion is
stable under acidic conditions, while when the pH is raised,
the structures of the bonds around the flavylium ion change
to form the blue quinoidal species (between pH 2 and 4). At
pHs between 5 and 6, the colourless carbinol pseudobase and
chalcone are predominant, while at pHs > 7 the anthocyanins
are degraded (Castañeda-Ovando et al., 2009).
Furthermore, the NADES polarity should also be considered
when discussing the differences observed in the extraction
efficiencies, given that anthocyanins are highly polar com-
pounds that are solubilised better in water then in non-polar
solvents. The results obtained here are in agreement with this,
as among the NADES tested, the organic acid-based NADES
were more polar that the sugar- and polyalcohol-based NADES
(Dai et al., 2013b).
For all of the NADES tested, the addition of water to the
NADES system at certain levels improved the extraction effi-
ciencies, as this reduces the viscosity of these solvents, and
therefore allows better mass-transfer rates. However, at the
higher water contents (>50%) this became unfavourable, pos-
sibly because of the weakening of the interactions between the
NADES and the target compounds (Bi et al., 2013). Additionally,
interesting patterns of extraction efficiencies were observed
with this addition of water to the NADES. The water con-
tent that provided the optimal extraction of the anthocyanins
was significantly different for each NADES type. For exam-
ple, in the less polar NADES (e.g., alcohol and the sugar-based
NADES), the optimal extraction efficiency was achieved at
about 50% water content, which can be explained in terms
that the addition of the water increased the polarity to reach
similar values to water (Dai et al., 2013a). In contrast, for
the organic-acid-based NADES, which are more polar sol-
vents than water, the best NADES performances were obtained
with the lower water content of 25% probably also due to
polarity changing since the addition of the water decreased
the polarity to reach similar values to water. However, the
yields decrease when lower amounts of water (10%) are used
probably due to high viscosity. Addition of water reduces
the viscosity of these solvents, and therefore allows better
mass-transfer rates (Dai et al., 2013a).
To evaluate the NADES as possible replacements for con-
ventional solvents, the extraction efficiencies were compared
with that obtained for the acidified aqueous solution of
ethanol, and encouraging results were obtained (Delgado de
la Torre et al., 2013; Pérez-Serradilla and Luque de Castro,
2011; Tao et al., 2014). Also, data were comparable to the
results obtained with other conventional methods (Teixeira
Barcia et al., 2014; Barbaa et al., 2016). Due to the stability of
the red flavylium cation in highly acid medium, the extrac-
tion of anthocyanins is commonly performed with acidified
organic solvents and methanol, with those containing HCl
as the most effective. Nevertheless, HCl is corrosive, while
methanol produces toxic effects after human exposure, and
consequently food scientists would prefer the use ethanol,
which can be classified as a bio-solvent due to its environ-
mental safety and sustainable production (Chemat et al.,
2012; Castañeda-Ovando et al., 2009). In the present study,
among three types of NADES tested, alcohol- and sugar-based
NADES showed similar contents of total anthocyanins as acid-
ified aqueous solutions of ethanol, while organic-acid-based
NADES that contained 25% water showed significant improve-
ments (p < 0.05). Based on the aforementioned, among the
NADES tested, ChMa was the most promising solvent for this
wine lees anthocyanin extraction, and was thus selected for
further extraction optimisation.
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203
3.2. Determination of process parameter ranges and
modelling of the ultrasound-assisted extraction process
Many factors can affect the extraction efficiency of UAE, such
as the ultrasonic power, temperature, extraction time, type
of solvent, solvent-to-solid ratio, particle size of sample, and
extraction cycles.
Based on our preliminary data and the current literature,
we selected the following independent variables for RSM in
this study: water content in NADES (50%–90%); extraction time
(15–45 min); and ultrasonic power (190–380 W). The impor-
tance of the water content in NADES for extraction efficiency
has already been discussed here, which indicated that a low
level of added water is essential for easy handling, while on
the other hand, an excess of water in NADES appears to inter-
fere with the halide–hydrogen bond donor supramolecular
complex, with a simple aqueous solution of the individual
components obtained (Gutierrez et al., 2009). Furthermore, the
ultrasonic power and the time of the extraction process are
important parameters to study, not only to maximise the con-
centration of bioactive compounds in the extracts, but also to
minimise the energy costs of the process.
It is also worth mentioning that the extraction tempera-
ture is a critical parameter. For the extractions here, this was
keep at a constant 35 ◦ C, as anthocyanins are very unstable and
are susceptible to degradation at higher temperatures (Carrera
et al., 2012; Castañeda-Ovando et al., 2009; Roobha et al., 2011).
It is well known from the literature data that not only
the extraction conditions, but also the characteristics of the
samples can affect the efficiency of the extraction, either inde-
pendently or interactively. Particle size is one of the important
factors that influence the extraction process. The extraction
yield is higher for smaller particles, due to shorter diffusion
path length and enhancement of the surface area with parti-
cle size reduction. If the particles are too small, however, they
can pose problems of channelling inside the extraction bed,
which can cause efficiency and yield losses.
Also, the solvent-to-solid ratio can have an influence on
the extracted compounds. Indeed, Pinelo et al. (2005) showed
that higher solvent-to-solid ratios provide the highest values
for extracted solids.
The response surface methodology (RSM) study was con-
ducted with the aim of determining the optimal experimental
conditions for the total anthocyanins extracted, for the sum
of the anthocyanin-3-O-monoglucosides (AMon), and for the
sum of the anthocyanin-3-(6-O-p-coumaroyl) monoglucosides
(ACm). These three independent variables were coded at five
levels, which resulted in the experimental design given in
Table 2. The total anthocyanins in the extracts obtained var-
ied from 2.89 mg g−1 dw to 6.42 mg g−1 dw, the AMon varied
from 1.05 mg g−1 dw to 3.63 mg g−1 dw, and the ACm varied
from 0.21 mg g−1 dw to 1.61 mg g−1 dw, depending on the differ-
ent levels of the investigated parameters (Table 2). The lowest
yield for all three of these responses investigated was obtained
using the NADES with 10% water, with an extraction time of
30 min, and an ultrasonic power of 285 W.
The regression coefficients of the model for each response,
and the results of the analysis of variance (ANOVA) are sum-
marised in Supplementary Tables S2 and S3 in the online
version at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005. For
the case model equations for total anthocyanins and AMon,
the linear terms of the water content in the NADES and
the ultrasonic power had significant influences (p < 0.05). For
199
the case model equation for ACm, all three independent
variables had significant influences. For all three of these
responses, the mathematical models were statistically accept-
able due to significant regression (p < 0.05). The regression
equations from Supplementary Table S2 in the online ver-
sion at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005 allowed
the calculation of the predicted values of total anthocyanins,
AMon and ACm to be compared with the experimental val-
ues (Table 2). The predicted values were analysed for the
calculation of the coefficients of determination (R2 ). The coef-
ficients of determination values in this study were higher than
0.80 (see Supplementary Table S3 in the online version at
DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005), which indi-
cated the adequacy of the applied regression models. The best
results were obtained for the AMon (R2 = 0.929: p = 0.0001).
The experimental data acquired were used for the creation
of three-dimensional response–surface plots for each of the
responses (Fig. 2). These response–surface plots show that
the total anthocyanins content of the extracts increased with
increased ultrasonic power. Thus, the power parameter clearly
significantly affected the extraction yield. The extraction time
shows significant effects on the ACm content, where it can be
seen that this increased with longer extraction times, while
for the total anthocyanins and AMon contents, the extrac-
tion time did not have any significant influence. On the other
hand, the water content in the DES had a double effect on
each response. The increase in water content from 10% to 25%
in the DES showed increased anthocyanins contents, while a
further increase led to decreased anthocyanins contents. This
indicates the importance of water content optimisation when
applying DESs as extraction solvents (Bi et al., 2013; Dai et al.,
2013b; Woo Nam et al., 2015).
3.3. Determination of the optimal conditions and
verification of the model
Optimal extraction parameters were obtained for three dif-
ferent responses TA, AMon and ACm. Treatment of multiple
responses and selection of optimal conditions were based on
desirability function D (Cojocaru et al., 2009). In this study, the
desirability was set to show the best conditions that maxi-
mize the TA, AMon and ACm content. By applying desirability
function method the optimum extraction conditions were
obtained at the extraction time of 30.6 min, with ultrasound
power of 341.5 W, using 35.4% water content of the NADES.
Verification and validity of the generated mathematical model
were performed by the extraction at these optimal condi-
tions for solvent concentration, extraction time and ultrasonic
power. Determination of optimal conditions and predicted val-
ues was based on desirability function which was 0.956. A
value of one represents the ideal case, while zero indicates
that one or more responses fall outside the desirable limits.
Experimental and predicted values were further compared.
The predicted total anthocyanins was 6.33 mg g−1 dw, AMon
was 3.54 mg g−1 dw, and ACm 1.50 mg g−1 dw. At the calcu-
lated optimal condition the following experimental data were
obtained: total anthocyanins was 6.55 mg g−1 dw, AMon was
3.66 mg g−1 dw, and ACm 1.57 mg g−1 dw. It can be seen very
close agreement between experimental and predicted values.
3.4. Scale-up perspectives
Use of NADES as green solvents might be a good choice in
the design of eco-friendly extraction methods for plant phe-
200 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203

Table 2 – The experimental matrix and the values of the responses observed.
Run no. Independent variable Extraction response (mg g−1 dw)

X1 X2 X3 Total anthocyanins AMon ACm

1 −1 −1 −1 4.35 2.38 0.88

2 +1 −1 −1 5.92 2.97 1.17
3 −1 +1 −1 3.89 2.58 0.95
4 +1 +1 −1 4.41 2.50 1.36
5 −1 −1 +1 5.27 2.42 1.04
6 +1 −1 +1 6.40 3.63 1.54
7 −1 +1 +1 4.81 2.58 1.20
8 +1 +1 +1 5.62 3.13 1.58
9 −˛ 0 0 2.89 1.05 0.21
10 +˛ 0 0 4.09 2.49 0.90
11 0 −␣ 0 4.09 2.57 0.82
12 0 +˛ 0 4.67 2.93 1.61
13 0 0 −˛ 5.28 2.93 0.99
14 0 0 +˛ 6.42 3.50 1.50
15 0 0 0 5.73 3.04 1.33
16 0 0 0 5.79 3.12 1.43
17 0 0 0 5.95 3.39 1.28
18 0 0 0 5.16 3.20 1.19
19 0 0 0 6.02 3.25 1.14
20 0 0 0 5.86 3.25 1.23

X1 , water content in the DES (w/w); X2 , time; X3 , ultrasonic power.

AMon, sum of the anthocyanin-3-O-monoglucosides; ACm, sum of the anthocyanin-3-(6-O-p-coumaroyl) monoglucosides.

nolic compounds. However, applicability of this technology in
commercial processes is still in the initial phases of research
and development. Only a few studies have reported scale-ups
of extraction processes involving NADES (Abbott et al., 2007).
When the scale-up possibilities are deliberated, many factors
need to be discussed and taken into consideration, including
both the economic and environmental issues of NADES, as
well as the exaction methods.
The costs of NADES are comparable with conventional
solvents, while at the same time, the production of NADES
can be classified as sustainable processes (Bi et al., 2013;
Paiva et al., 2014). An economical evaluation of the NADES
showed the average price to be around 2 $/kg and comper-
ing with organic solvent and ionic liquids, the NADES are
slightly expansive that organic solvents but much cheaper that
commercially available ionic liquids (Bogaars, 2015). Further-
more, the physicochemical properties of NADES (e.g., viscosity,
vapour pressure) also have large influences on the integra-
tion of these solvents into industrial processes. For example,
the high viscosities of NADES can be a restrictive factor for
using NADES as extraction solvents not only because of lower
extraction efficiencies, but also due to the energy required for
stirring and pumping. On the other hand, the high viscosi-
ties of NADES might be a desired characteristic, as it has been
reported that the high viscosities of sugar-based NADES with
low water contents allow stable molecular interactions. Such
features have important roles in the stabilities of phenolic
compounds in NADES. Hopefully, the viscosity can be easily
adjusted by addition of the optimal amount of water, to thus
satisfy both demands (Dai et al., 2014).
At the same time, it is well known that NADES have almost
zero vapour pressure, which minimises the risks of air pol-
lution by NADES, in contrast to organic solvents. Then again,
this property of NADES might be a huge problem when the
recovery of the phenolic compounds is needed, particularly
on an industrial scale. Organic solvents can be easily removed
by evaporation, while with NADES this is not possible.
Several approaches have been reported for phenolic com-
pound recoveries, such as solid-phase extraction and the
application of anti-solvents (Woo Nam et al., 2015; Dai et al.,
2013b). This is the major obstacle in NADES applications on
an industrial scale. NADES recovery and/or recycling after
extraction is definitely understudied in the current literature,
so further efforts should be carried out to solve this prob-
lem (Vigier et al., 2015). There is a few studies discussing if
NADES can be implemented in a commercially viable process
(Bi et al., 2013). In one case study an economic evaluation of
the pectin extraction was made and showed that if a recycle
rate of 99% can be achieved that at a solvent to product ratio of
approximately 50 the process becomes commercially interest-
ing (Bogaars, 2015). However, based on current knowledge, the
application of NADES on an industrial scale is possible only
when the extract is directly used without expensive down-
stream purification steps.
Conventional extraction of phenolic compounds is gen-
erally performed by maceration, which can often be time
and/or energy consuming. This is not always interesting from
an industrial point of view, and currently, many innovative
technologies have emerged. Among these novel extraction
methods, the up-scaling of the ultrasonic device has emerged
as an easier and cheaper option than, for example, microwave-
assisted extraction or supercritical fluid extraction (Virot et al.,
2010). When setting up any scale-up of the UAE process, dis-
continuity of the ultrasonic bath is a crucial factor in the
process optimisation, which can result in inefficiency of the
whole process. Here, the continuous flow of an ultrasonic sys-
tem with a directly immersed ultrasonic probe in the flow
cell can produce higher intensities, which would then yield
larger amounts of the extracted compounds. The intensity
of any cavitation mechanism inside the flow cell can also
provide better heat and mass transfer. The optimum flow con-
ditions are controlled by the control unit of the peristaltic
pump. Accordingly, when setting-up the optimal flow param-
eters, it is possible to process large amounts of material.
As a comparison with conventional extraction, theoretically,
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203 201

Fig. 2 – Response surface plots showing combined effects of process variables on contents of (A) total anthocyanins (TA) (B)
anthocyanin-3-O-monoglucosides (Amon) and (C) anthocyanin-3-(6-O-p-coumaroyl)monoglucosides (ACm).

ultrasonic bath extraction uses 40% less energy, while real-
istic expectations are of 10%–20% less energy consumption
using a continuous ultrasonic system (Karlović et al., 2014;
Karman et al., 2012). Furthermore, it is possible to use large-
scale ultrasound extraction reactors of up to 1000 L, within
which about 200 kg dry plant material can be processed each
time. Considering the above-mentioned, ultrasonic extraction
is a promising method for industrial applications (Virot et al.,
2010).
4. Conclusions
Among the NADES tested here, ChMa can be selected as the
most promising solvent, and it has proven to be effective in
the extraction of anthocyanins from wine lees, as compared to
conventional solvents. Considering the maximum amounts of
extracted compounds, the optimal conditions were: extraction
time, 30.6 min; ultrasound power, 341.5 W; and water content
in NADES, 35.4% (v/v). This approach that uses NADES as
green solvents and ultrasound as the alternative energy source
might be a good choice for the design of eco-friendly extraction
202 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203
methods for plant phenolic compounds. Nevertheless, further
studies are needed to scale-up this technology, in terms of
NADES recovery and recycling.
Acknowledgements
This study was supported by the University of Zagreb (Grant
no. 0582261-7002223). The authors are also grateful to the Josip
Juraj Strossmayer University of Osijek, Republic of Croatia, for
financial support.
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