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

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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 in adults (Vislocky and Fernandez, 2010). Research in this field has
been mainly focused on the extraction of the phenolic compounds from
The winemaking industry produces large volumes of waste and by- grape pomace (Casazza et al., 2010; Deng et al., 2011; Luque-Rodríguez
products, including grape pomace, stalks and lees. Only small amounts et al., 2007; Rajha et al., 2014), using the available literature based on
of these by-products are up-graded or recycled, frequently used for the exploration of wine lees (Delgado de la Torre et al., 2013; Pérez-
composting, or even discarded in open areas, thus potentially result- Serradilla and Luque de Castro, 2011; Tao et al., 2014; Teixeira Barcia
ing in environmental problems (Teixeira et al., 2014). Over the last et al., 2014; Barbaa et al., 2016). However, some data have indicated that
years, the evaluation of these by-products as sources of biologically phenolic extracts from wine lees can be suitable as a cheaper alterna-
active compounds and the recovery of these into value-added prod- tive to those obtained from grape seeds and skins (Pérez-Serradilla and
ucts have attracted great interest. The most representative of the grape Luque de Castro, 2011).
secondary metabolites are the phenolic compounds, due to their valu- Among these phenolic compounds, wine lees from red grapes is
able activities in terms of the prevention of oxidative reactions and a good source of anthocyanins (Pérez-Serradilla and Luque de Castro,
the benefits seen against cardiovascular disease, inflammatory pro- 2011; Tao et al., 2014; Teixeira Barcia et al., 2014). Anthocyanins are nat-
cesses, and degenerative pathophysiological states that can develop 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- Wet wine lees were obtained from the Agrolaguna Com-
metics and pharmaceutical industries (Castañeda-Ovando et al., 2009). pany winemakers (Porec, Croatia). The lees were obtained as
Extraction techniques for such by-products for their evaluation and the semi-solid residue decanted after the alcoholic fermenta-
utilisation should be based on green and sustainable technologies, by tion of the grape juice from Merlot grapes. After the wine lees
following the principles of green extraction. In general, green extrac-
were collected, they were lyophilised and then frozen under
tion is based on the discovery and design of extraction processes with
vacuum until further use.
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, 2.2. Preparation of the deep eutectic solvents
which is devoted to the design of new, environmentally friendly and
tunable solvents that can meet both the technological and economic All of the chemicals used in these preparations were dried
demands (Cvjetko Bubalo et al., 2015). before use in a vacuum concentrator (Savant SPD131DDA
Over the last few years, the use of natural deep eutectic solvents
SpeedVac Concentrator), at 60 ◦ C for 24 h. The NADES were
(NADES) as a green alternative to conventional solvents has been
used at specific ratios of choline chloride to hydrogen donor
dramatically expanded (Smith et al., 2014). NADES present a new
(i.e., citric acid, malic acid, oxalic acid, glucose, fructose,
generation of liquid salts, and they are generally based on mixtures
of relatively cheap and readily available components, such as non-
xylose, glycerol), to obtain liquids at room temperature,
toxic quaternary ammonium salts (e.g., choline chloride) with naturally choline chloride:citric acid (ChCit), choline chloride:oxalic
derived uncharged hydrogen-bond donors (e.g., vitamins, amines, sug- acid (ChOa), choline chloride:malic acid (ChMa), choline chlo-
ars, alcohols, carboxylic acids), which provide a ‘green profile’ and have ride:glucose (ChGlc), choline chloride:fructose (ChFru), choline
good prospects for wider use in the field of green technologies (Abbott chloride:xylose (ChXyl), choline chloride:glycerol, (ChGly)
et al., 2004). Since their emergence, NADES have attracted attention (Radošević et al., 2016). The mixtures of choline chloride
as potential solvents in various industrial fields, including the extrac- with the hydrogen donor were stirred in a flask at 80 ◦ C for
tion of biologically active compounds from plant materials (Paiva et al., 2 h–6 h, until a homogeneous transparent colourless liquid
2014). Some studies have reported NADES applications for the extrac-
was formed. These NADES samples were vacuum dried prior
tion of phenolic compounds (Radošević et al., 2016; Cvjetko Bubalo
to further use.
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 2.3. Selection of optimal deep eutectic solvent
environmental benefits of using NADES as solvents, such as their low
vapour pressures and non-flammability, NADES represent excellent About 33.3 mg of lyophilised wine lees were extracted per mL
candidates for green extractions (Cvjetko Bubalo et al., 2015). Another of the NADES of interest (i.e., solvent-to-solid ratio, 60:1), in
principle of green extraction is reduced energy consumption through
a shaker for 3 h at room temperature (Tao et al., 2014). The
the use of innovative technologies, such as ultrasound. Ultrasound-
extracts were then centrifuged at 5000 × g for 15 min, and the
assistant extraction (UAE) is recognized as providing an outstanding
supernatants obtained were filtered and further analysed. To
energy source to promote extraction, to cut down on extraction times,
to increase yields, and often to improve the quality of the required
compare the extraction efficiencies of these NADES with those
extract (Esclapez et al., 2011). The enhanced heat and mass transfer of extractions performed using conventional solvents, an acid-
provided by ultrasound is mainly attributed to the acoustic effects that ified aqueous solution of ethanol (ethanol/water/formic acid,
are accompanied by ultrasound cavitation phenomena, which makes 50:48.5:1.5, v/v/v; pH 2.7) was carried out under the same con-
ultrasound one of the most promising of the many technological pro- ditions. All of the extraction procedures and the analysis of
cesses considered (Bosiljkov et al., 2011; Brnčić et al., 2010; Dujmić et al., the total anthocyanins were performed in triplicate (Carrera
2013; Herceg et al., 2010; Karlović et al., 2014). UAE has been widely et al., 2012; Sarneckis et al., 2006).
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.,
2.4. Ultrasound-assisted extraction procedure
2008; Tao et al., 2014). Also, UAE methods have been used in conjunc-
tion with different NADES for the extraction of phenolic compounds An ultrasonic bath system was used for the UAE (Elmasonic
(Cvjetko Bubalo et al., 2016; Radošević et al., 2016; Woo Nam et al., 2015; P 300 H; Elma Hans Schmidbauer GmbH & Co. KG, Sin-
Bi et al., 2013; Dai et al., 2013b). gen, Germany), which was equipped with a digital controlled
The aim of the present study was to establish a highly efficient and system for temperature, time and power (ultrasonic power
truly eco-friendly extraction method for the anthocyanins in wine lees effective is 380 W). All of the extractions were performed
using NADES and UAE. Initial screening for the optimal NADES for these at constant frequency (37 kHz) and temperature (35 ◦ C). The
anthocyanins was performed, followed by the statistical optimisation
solid-to-liquid ratio of 0.1 g dried wine lees per mL solvent was
of the UAE operational conditions using response surface methodology
used for these extractions, which were performed according to
(RSM).
the experimental design. The extracts were then centrifuged
at 5000 × g for 15 min, and the supernatants obtained were
filtered and further analysed.
2. Materials and methods
2.5. Experimental design and statistical analysis
2.1. Chemicals and materials
Response surface methodology was applied to evaluate the
Choline chloride, citric acid monohydrate, malic acid, oxalic effects of the extraction parameters and to optimise the con-
acid, glucose, fructose, xylose and glycerol were from Sigma ditions according to various responses. The extractions were
(St. Louis, MO, USA). The analytical standards of delphinidin- planned through the application of central composite rotat-
3-O-monoglucoside, petunidin-3-O-monoglucoside, peonidin- able design (Baş and Boyacı, 2007). Five different levels were
3-O-monoglucoside, and malvidin-3-O-monoglucoside were used for each of the following factors: water content in the
from Polyphenols AS (Sandnes, Norway). 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 3.1. Selection of deep eutectic solvent for extraction of
(Agilent 1260; Agilent Technologies) with a diode array detec- wine lees anthocyanins
tor, and with the wavelength set at 520 nm. Separation of the
anthocyanins was carried out on a C18 column (Poroshell Seven different cholinium-based NADES were tested for
120 EC-C18; 100 × 2.1 mm i.d. 2.7 ␮m; Agilent Technologies, extraction of total anthocyanins from wine less, which con-
USA), at a flow rate of 0.2 mL min−1 . The mobile phases were: tained xylose, glucose, fructose, glycerol, malic acid, citric
(A) 1% (v/v) aqueous formic acid; and (B) methanol. The 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 ple, in the less polar NADES (e.g., alcohol and the sugar-based
from 10% to 50% (w/w) were also performed, as this water con- NADES), the optimal extraction efficiency was achieved at
tent has been reported to dramatically influence the extraction about 50% water content, which can be explained in terms
efficiency (Dai et al., 2013a,b, 2014). In general, the results that the addition of the water increased the polarity to reach
obtained indicated that the type of NADES used did indeed similar values to water (Dai et al., 2013a). In contrast, for
strongly influence the anthocyanin extraction (Fig. 1). The the organic-acid-based NADES, which are more polar sol-
best extraction efficiency was obtained with ChMa followed vents than water, the best NADES performances were obtained
by ChOa > ChCit > ChGly > ChFru > ChXyl > ChGlc. These data with the lower water content of 25% probably also due to
indicated that the NADES with organic acids as the hydrogen polarity changing since the addition of the water decreased
bond donor were the best solvents in these test extractions. the polarity to reach similar values to water. However, the
These will also be more acidic (pH < 3) by nature than those yields decrease when lower amounts of water (10%) are used
with sugars or polyalcohols as the hydrogen bond donor probably due to high viscosity. Addition of water reduces
(pH > 6) (Dai et al., 2014; Radošević et al., 2015). This is not sur- the viscosity of these solvents, and therefore allows better
prising as it is well known that an acidic environment favours mass-transfer rates (Dai et al., 2013a).
anthocyanin extraction and stability, as the flavylium ion is To evaluate the NADES as possible replacements for con-
stable under acidic conditions, while when the pH is raised, ventional solvents, the extraction efficiencies were compared
the structures of the bonds around the flavylium ion change with that obtained for the acidified aqueous solution of
to form the blue quinoidal species (between pH 2 and 4). At ethanol, and encouraging results were obtained (Delgado de
pHs between 5 and 6, the colourless carbinol pseudobase and la Torre et al., 2013; Pérez-Serradilla and Luque de Castro,
chalcone are predominant, while at pHs > 7 the anthocyanins 2011; Tao et al., 2014). Also, data were comparable to the
are degraded (Castañeda-Ovando et al., 2009). results obtained with other conventional methods (Teixeira
Furthermore, the NADES polarity should also be considered Barcia et al., 2014; Barbaa et al., 2016). Due to the stability of
when discussing the differences observed in the extraction the red flavylium cation in highly acid medium, the extrac-
efficiencies, given that anthocyanins are highly polar com- tion of anthocyanins is commonly performed with acidified
pounds that are solubilised better in water then in non-polar organic solvents and methanol, with those containing HCl
solvents. The results obtained here are in agreement with this, as the most effective. Nevertheless, HCl is corrosive, while
as among the NADES tested, the organic acid-based NADES methanol produces toxic effects after human exposure, and
were more polar that the sugar- and polyalcohol-based NADES consequently food scientists would prefer the use ethanol,
(Dai et al., 2013b). which can be classified as a bio-solvent due to its environ-
For all of the NADES tested, the addition of water to the mental safety and sustainable production (Chemat et al.,
NADES system at certain levels improved the extraction effi- 2012; Castañeda-Ovando et al., 2009). In the present study,
ciencies, as this reduces the viscosity of these solvents, and among three types of NADES tested, alcohol- and sugar-based
therefore allows better mass-transfer rates. However, at the NADES showed similar contents of total anthocyanins as acid-
higher water contents (>50%) this became unfavourable, pos- ified aqueous solutions of ethanol, while organic-acid-based
sibly because of the weakening of the interactions between the NADES that contained 25% water showed significant improve-
NADES and the target compounds (Bi et al., 2013). Additionally, ments (p < 0.05). Based on the aforementioned, among the
interesting patterns of extraction efficiencies were observed NADES tested, ChMa was the most promising solvent for this
with this addition of water to the NADES. The water con- wine lees anthocyanin extraction, and was thus selected for
tent that provided the optimal extraction of the anthocyanins further extraction optimisation.
was significantly different for each NADES type. For exam-
food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 195–203 199

3.2. Determination of process parameter ranges and the case model equation for ACm, all three independent
modelling of the ultrasound-assisted extraction process variables had significant influences. For all three of these
responses, the mathematical models were statistically accept-
Many factors can affect the extraction efficiency of UAE, such able due to significant regression (p < 0.05). The regression
as the ultrasonic power, temperature, extraction time, type equations from Supplementary Table S2 in the online ver-
of solvent, solvent-to-solid ratio, particle size of sample, and sion at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005 allowed
extraction cycles. the calculation of the predicted values of total anthocyanins,
Based on our preliminary data and the current literature, AMon and ACm to be compared with the experimental val-
we selected the following independent variables for RSM in ues (Table 2). The predicted values were analysed for the
this study: water content in NADES (50%–90%); extraction time calculation of the coefficients of determination (R2 ). The coef-
(15–45 min); and ultrasonic power (190–380 W). The impor- ficients of determination values in this study were higher than
tance of the water content in NADES for extraction efficiency 0.80 (see Supplementary Table S3 in the online version at
has already been discussed here, which indicated that a low DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005), which indi-
level of added water is essential for easy handling, while on cated the adequacy of the applied regression models. The best
the other hand, an excess of water in NADES appears to inter- results were obtained for the AMon (R2 = 0.929: p = 0.0001).
fere with the halide–hydrogen bond donor supramolecular The experimental data acquired were used for the creation
complex, with a simple aqueous solution of the individual of three-dimensional response–surface plots for each of the
components obtained (Gutierrez et al., 2009). Furthermore, the responses (Fig. 2). These response–surface plots show that
ultrasonic power and the time of the extraction process are the total anthocyanins content of the extracts increased with
important parameters to study, not only to maximise the con- increased ultrasonic power. Thus, the power parameter clearly
centration of bioactive compounds in the extracts, but also to significantly affected the extraction yield. The extraction time
minimise the energy costs of the process. shows significant effects on the ACm content, where it can be
It is also worth mentioning that the extraction tempera- seen that this increased with longer extraction times, while
ture is a critical parameter. For the extractions here, this was for the total anthocyanins and AMon contents, the extrac-
keep at a constant 35 ◦ C, as anthocyanins are very unstable and tion time did not have any significant influence. On the other
are susceptible to degradation at higher temperatures (Carrera hand, the water content in the DES had a double effect on
et al., 2012; Castañeda-Ovando et al., 2009; Roobha et al., 2011). each response. The increase in water content from 10% to 25%
It is well known from the literature data that not only in the DES showed increased anthocyanins contents, while a
the extraction conditions, but also the characteristics of the further increase led to decreased anthocyanins contents. This
samples can affect the efficiency of the extraction, either inde- indicates the importance of water content optimisation when
pendently or interactively. Particle size is one of the important applying DESs as extraction solvents (Bi et al., 2013; Dai et al.,
factors that influence the extraction process. The extraction 2013b; Woo Nam et al., 2015).
yield is higher for smaller particles, due to shorter diffusion
path length and enhancement of the surface area with parti- 3.3. Determination of the optimal conditions and
cle size reduction. If the particles are too small, however, they verification of the model
can pose problems of channelling inside the extraction bed,
which can cause efficiency and yield losses. Optimal extraction parameters were obtained for three dif-
Also, the solvent-to-solid ratio can have an influence on ferent responses TA, AMon and ACm. Treatment of multiple
the extracted compounds. Indeed, Pinelo et al. (2005) showed responses and selection of optimal conditions were based on
that higher solvent-to-solid ratios provide the highest values desirability function D (Cojocaru et al., 2009). In this study, the
for extracted solids. desirability was set to show the best conditions that maxi-
The response surface methodology (RSM) study was con- mize the TA, AMon and ACm content. By applying desirability
ducted with the aim of determining the optimal experimental function method the optimum extraction conditions were
conditions for the total anthocyanins extracted, for the sum obtained at the extraction time of 30.6 min, with ultrasound
of the anthocyanin-3-O-monoglucosides (AMon), and for the power of 341.5 W, using 35.4% water content of the NADES.
sum of the anthocyanin-3-(6-O-p-coumaroyl) monoglucosides Verification and validity of the generated mathematical model
(ACm). These three independent variables were coded at five were performed by the extraction at these optimal condi-
levels, which resulted in the experimental design given in tions for solvent concentration, extraction time and ultrasonic
Table 2. The total anthocyanins in the extracts obtained var- power. Determination of optimal conditions and predicted val-
ied from 2.89 mg g−1 dw to 6.42 mg g−1 dw, the AMon varied ues was based on desirability function which was 0.956. A
from 1.05 mg g−1 dw to 3.63 mg g−1 dw, and the ACm varied value of one represents the ideal case, while zero indicates
from 0.21 mg g−1 dw to 1.61 mg g−1 dw, depending on the differ- that one or more responses fall outside the desirable limits.
ent levels of the investigated parameters (Table 2). The lowest Experimental and predicted values were further compared.
yield for all three of these responses investigated was obtained The predicted total anthocyanins was 6.33 mg g−1 dw, AMon
using the NADES with 10% water, with an extraction time of was 3.54 mg g−1 dw, and ACm 1.50 mg g−1 dw. At the calcu-
30 min, and an ultrasonic power of 285 W. lated optimal condition the following experimental data were
The regression coefficients of the model for each response, obtained: total anthocyanins was 6.55 mg g−1 dw, AMon was
and the results of the analysis of variance (ANOVA) are sum- 3.66 mg g−1 dw, and ACm 1.57 mg g−1 dw. It can be seen very
marised in Supplementary Tables S2 and S3 in the online close agreement between experimental and predicted values.
version at DOI: http://dx.doi.org/10.1016/j.fbp.2016.12.005. For
the case model equations for total anthocyanins and AMon, 3.4. Scale-up perspectives
the linear terms of the water content in the NADES and
the ultrasonic power had significant influences (p < 0.05). For 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 Several approaches have been reported for phenolic com-
commercial processes is still in the initial phases of research pound recoveries, such as solid-phase extraction and the
and development. Only a few studies have reported scale-ups application of anti-solvents (Woo Nam et al., 2015; Dai et al.,
of extraction processes involving NADES (Abbott et al., 2007). 2013b). This is the major obstacle in NADES applications on
When the scale-up possibilities are deliberated, many factors an industrial scale. NADES recovery and/or recycling after
need to be discussed and taken into consideration, including extraction is definitely understudied in the current literature,
both the economic and environmental issues of NADES, as so further efforts should be carried out to solve this prob-
well as the exaction methods. lem (Vigier et al., 2015). There is a few studies discussing if
The costs of NADES are comparable with conventional NADES can be implemented in a commercially viable process
solvents, while at the same time, the production of NADES (Bi et al., 2013). In one case study an economic evaluation of
can be classified as sustainable processes (Bi et al., 2013; the pectin extraction was made and showed that if a recycle
Paiva et al., 2014). An economical evaluation of the NADES rate of 99% can be achieved that at a solvent to product ratio of
showed the average price to be around 2 $/kg and comper- approximately 50 the process becomes commercially interest-
ing with organic solvent and ionic liquids, the NADES are ing (Bogaars, 2015). However, based on current knowledge, the
slightly expansive that organic solvents but much cheaper that application of NADES on an industrial scale is possible only
commercially available ionic liquids (Bogaars, 2015). Further- when the extract is directly used without expensive down-
more, the physicochemical properties of NADES (e.g., viscosity, stream purification steps.
vapour pressure) also have large influences on the integra- Conventional extraction of phenolic compounds is gen-
tion of these solvents into industrial processes. For example, erally performed by maceration, which can often be time
the high viscosities of NADES can be a restrictive factor for and/or energy consuming. This is not always interesting from
using NADES as extraction solvents not only because of lower an industrial point of view, and currently, many innovative
extraction efficiencies, but also due to the energy required for technologies have emerged. Among these novel extraction
stirring and pumping. On the other hand, the high viscosi- methods, the up-scaling of the ultrasonic device has emerged
ties of NADES might be a desired characteristic, as it has been as an easier and cheaper option than, for example, microwave-
reported that the high viscosities of sugar-based NADES with assisted extraction or supercritical fluid extraction (Virot et al.,
low water contents allow stable molecular interactions. Such 2010). When setting up any scale-up of the UAE process, dis-
features have important roles in the stabilities of phenolic continuity of the ultrasonic bath is a crucial factor in the
compounds in NADES. Hopefully, the viscosity can be easily process optimisation, which can result in inefficiency of the
adjusted by addition of the optimal amount of water, to thus whole process. Here, the continuous flow of an ultrasonic sys-
satisfy both demands (Dai et al., 2014). tem with a directly immersed ultrasonic probe in the flow
At the same time, it is well known that NADES have almost cell can produce higher intensities, which would then yield
zero vapour pressure, which minimises the risks of air pol- larger amounts of the extracted compounds. The intensity
lution by NADES, in contrast to organic solvents. Then again, of any cavitation mechanism inside the flow cell can also
this property of NADES might be a huge problem when the provide better heat and mass transfer. The optimum flow con-
recovery of the phenolic compounds is needed, particularly ditions are controlled by the control unit of the peristaltic
on an industrial scale. Organic solvents can be easily removed pump. Accordingly, when setting-up the optimal flow param-
by evaporation, while with NADES this is not possible. 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- 4. Conclusions
istic expectations are of 10%–20% less energy consumption
using a continuous ultrasonic system (Karlović et al., 2014; Among the NADES tested here, ChMa can be selected as the
Karman et al., 2012). Furthermore, it is possible to use large- most promising solvent, and it has proven to be effective in
scale ultrasound extraction reactors of up to 1000 L, within the extraction of anthocyanins from wine lees, as compared to
which about 200 kg dry plant material can be processed each conventional solvents. Considering the maximum amounts of
time. Considering the above-mentioned, ultrasonic extraction extracted compounds, the optimal conditions were: extraction
is a promising method for industrial applications (Virot et al., time, 30.6 min; ultrasound power, 341.5 W; and water content
2010). 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 Dai, Y., Witkamp, G.J., Verpoorte, R., Choi, Y.H., 2013b. Natural
studies are needed to scale-up this technology, in terms of deep-eutectic solvents as a new extraction media for phenolic
NADES recovery and recycling. metabolites in Carthamus tinctorius L. Anal. Chem 85,
6272–6278.
Delgado de la Torre, M.P., Ferreiro-Vera, C., Priego-Capote, F.,
Acknowledgements Luque de Castro, M.D., 2013. Anthocyanidins,
proanthocyanidins, and anthocyanins profiling in wine lees
by solid-phase extraction–liquid chromatography coupled to
This study was supported by the University of Zagreb (Grant
electrospray ionization tandem mass spectrometry with
no. 0582261-7002223). The authors are also grateful to the Josip dana-dependent methods. J. Agric. Food Chem. 61,
Juraj Strossmayer University of Osijek, Republic of Croatia, for 12539–12548.
financial support. Deng, Q., Penner, M.H., Zhao, Y., 2011. Chemical composition of
dietary fiber and polyphenols of five different varieties of
wine grape pomace skins. Food Res. Int. 44, 2712–2720.
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