Food Chemistry 124 (2011) 16521659

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Microwave-assisted extraction of phenolic compounds from wine lees and

spray-drying of the extract
J.A. Prez-Serradilla, M.D. Luque de Castro *
Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Crdoba, E-14071 Crdoba, Spain

a r t i c l e

i n f o

Article history:
Received 22 September 2009
Received in revised form 4 May 2010
Accepted 18 July 2010

Keywords:
Wine lees
Dregs
Wine by-products
Phenolic compounds
Antioxidants
Microwave-assisted extraction
Leaching

a b s t r a c t
Most research on extraction of phenol compounds from wine by-products and commercial exploitation of
extracts use grape seeds and/or skins as raw materials. Looking for alternative antioxidants sources,
obtaining antioxidant extracts from wine lees (also known as dregs), a sub-exploited by-product of winemaking process, is here presented. Microwave-assisted extraction (MAE) of phenolic compounds from
wine lees has been optimized using the total phenols index, the ORAC values and yield of the extraction
as response variables. Under the optimal working conditions, the proposed MAE method provides better
extraction efciency in a much shorter time (17 min) than the conventional extraction method for phenolic compounds (24 h). The liquid extract obtained by MAE was spray-dried. The type and amount of
excipients used, as well as the spray-drying temperature, were optimized in order to minimize the oxidation of phenolic compounds and maximize the yield of the spray-drying process. The total phenols
index in the dried extract thus obtained was 36.8% (expressed as gallic acid), showing an ORAC value
of 3930 lmol TE/g. Additionally, Mv3G, Cm-Mv3G, myricetin, quercetin, quercetin-3-b-glucoside, caffeic
acid and p-coumaric acid were quantied in the dry extract by HPLCDAD. The results indicate that wine
lees antioxidant extracts can be a suitable and cheap alternative to those obtained from grape seeds or
skins.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Phenolics antioxidant activity led Stanner, Hughes, Kelly, and
Buttris (2004) to establish that as antioxidants can prevent oxidative damages, increased intakes from the diet will reduce the risks
of chronic diseases. Their use, mainly in nutraceuticals, has led to
obtaining antioxidants from wine by-products, the exploitation of
which is of great importance, not only because of their benecial
properties, but also from an environmental point of view as several
millions tons of industry wastes are generated every year by the
winemaking industry (Louli, Ragoussis, & Magoulas, 2004). Research in this eld has been mainly focused on the extraction of
phenolic compounds from grape seed or skin (Bleve et al., 2008;
Luque Rodrguez, Luque de Castro, & Prez-Juan, 2007; Prez-Serradilla, Priego-Capote & Luque de Castro, 2007; Schieber, Stintzing,
& Carle, 2001); in fact, all commercial grape extracts seems to be
obtained only from these two by-products (Yilmaz & Toledo,
2006). A very opportunistic business has emerged around the market of skins and seed from red grape (Shrikhande, 2000), making
advisable looking for alternatives for these raw materials. In this
context, some interest in taking prot of wine lees has been observed in the last few years (Alonso, Guilln, Barroso, Puertas, &
* Corresponding author. Tel./fax: +34 957 218615.
E-mail address: qa1lucam@uco.es (M.D. Luque de Castro).
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.07.046

Garca, 2002; Prez-Serradilla & Luque de Castro, 2008). Lees, also

known as dregs, is dened by EEC regulation no. 337/79 as the residue formed at the bottom of recipients containing wine, after fermentation, during storage or after authorized treatments, as well
as the residue obtained following ltration or centrifugation. Wine
lees is mainly composed by microorganisms (mainly yeasts), tartaric acid, inorganic matter and phenolic compounds (Prez-Serradilla & Luque de Castro, 2008). The scarce literature in this eld has
reported the presence of anthocyanins (Morata et al., 2003) and
other phenolics (Alonso et al., 2002) in wine lees. Although Alonso
et al. (2002) studied the composition of wine lees, as far as the
authors know there is no published research on optimization of
the extraction of phenolic compounds from wine lees to obtain
an antioxidant extract.
Extraction of phenolic compounds from solid samples is usually
carried out by stirring (Luthria, Mukhopadhyay, & Kwansa, 2006;
Nepote, Grosso, & Guzman, 2005), although the use of auxiliary
energies has demonstrated to accelerate the process (Japn-Lujn,
Luque-Rodrguez, & Luque de Castro, 2006; Prez-Serradilla, JapnLujn & Luque de Castro, 2007). Microwave-assisted extraction
(MAE) is the process by which microwave energy is used to heat
polar solvents in contact with solid samples and to partition compounds of interest between the sample and the solvent (Luque de
Castro & Luque Garca, 2002), reducing both extraction time and
solvent consumption.

J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

Most research on extraction methods reports the optimization

and understanding of the extraction process, as well as characterization of the liquid extract obtained. However, dried extracts are
endowed with advantages over liquid forms such as lower storage
costs and higher concentration and stability of active substances
(Oliveira, Bott, & Souza, 2006). For this reason, the vast majority of
commercial grape-related extracts are presented in dried form.
One of the drying techniques most widely used in the food industry
is spray-drying (Wang & Langrish, 2009)in fact, it was estimated
that more than 15,000 industrial spray-dryers were in operation over
the world in 1996 (Masters, 1996). In order to improve both the
spray-drying yield and the physicalchemical characteristics of the
powder obtained, the use of some type of additive as maltodextrins,
colloidal silicon dioxides or mixtures of them is often necessary
(Wang and Langrish, 2009; Roustapour, Hosseinalipour, Ghobadian,
Mohaghegh, & Azad, 2009; Tewa-Tagne, Brianon, & Fessi, 2006; Ztola et al., 2002). As far as the authors know, no spray-drying process
of wine by-products extracts has been reported.
Usually, the information given in commercial grape-related
preparations is the total phenol index, which generally is determined by the FolinCiocalteu method (Shrikhande, 2000; Singleton
& Rossi, 1965). Despite the correlation between the content of phenolic compounds and antioxidant activity has been reported
(Fernndez-Pachn, Villao, Garca-Parrilla, & Troncoso, 2004; Yi
et al., 2009), the evaluation of this activity is of major interest as
this is the main quality attributed to these compounds. In this context, the oxygen radical absorbance capacity (ORAC) assay is one of
the few methods that combines both inhibition percentage and
inhibition time of the reactive species action by antioxidants in a
single datum (Bleve et al., 2008). Additionally, liquid chromatography with diode array detection (HPLCDAD) is able to provide
valuable information about the phenolic composition of grape-related extracts (Luque Rodrguez et al., 2007).
In order to study the potential industrial relevance of wine lees
as a source of phenols, an MAE method for obtaining phenolic compounds from this by-product has been developed and the results it
provides have been compared with those from a conventional
method. A spray-drying method to remove the extractant, thus
obtaining a powder from the liquid extract provided by the MAE
method, has also been developed. In both methods, multifactorial
optimization designs have been used in order to establish the best
working conditions. Yield of both the MAE and spray-drying processes, total phenols index, percentage of phenolic compounds oxidation and ORAC determinations have been used as response
variables in the optimization studies. The total phenols index,
ORAC and HPLCDAD determinations of individual components
have been used to characterize the spray-dried extract.
2. Materials and methods

1653

mixtures. Acetonitrile and formic acid (both HPLC grade and supplied by Panreac) were used to prepare the HPLC mobile phases.
Deionized water (18 MX cm) was obtained from a Millipore (Bedfore, MA, USA) Milli-Q plus system.
Malvidin-3-glucoside (Mv3G) was purchased from Extrasynthese (Genay, France). Myricetin, quercetin, quercetin-3-b-glucoside and caffeic and p-coumaric acids were from Sigma (St. Louis,
MO, USA). These analytes have been reported as major low-molecular weight phenolic compounds in grape by-products (Alonso
et al., 2002; Luque Rodrguez et al., 2007; Morata et al., 2003).
Fluorescein (30 ,60 -dihydroxyspiro[isobenzofuran-1[3H],90 [9H]xanthen]-3-one) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Fluka (Buches, Switzerland). FolinCiocalteu reagent, sodium carbonate, gallic acid
and AAPH (2,20 -azobis-2-methyl-propanimidamide dihydrochloride) were provided by Sigma.
Maltodextrin Glucidex (a potato starch conversion product,
containing 20% amylose and 80% amylopectin) was provide by Roquette (Lestrem, France). Aerosil 200 was supplied by Degussa
(Frankfurt, Germany).

2.3. Apparatus and instruments

Centrifugation of the lees was carried out by a Mixtasel centrifuge from Selecta (Barcelona, Spain).
A Microdigest 301 digestor of 200 W maximum power, furnished with a microprocessor programmer to control the microwave unit and used to accelerate solidliquid extraction, was
provided by Prolabo (Paris, France).
An R-220 rotary evaporator from Bchi (Flawil, Switzerland)
working with a 20 L balloon ask was used to concentrate the
liquid extracts before to be spray-dried, and a B-290 Mini Spray
Dryer provided by Bchi was used for drying the liquid
extracts.
The absorbance of the extracts was monitored by an Agilent
8453E UVvisible spectrometer (Waldbronn, Germany). Fluorimetric monitoring of ORAC assay was performed by an F-2500 Hitachi
uorimeter equipped with a microcuvette (1 cm pathlength) and
connected to a device to keep the temperature at 37 C.
An Agilent 1100 liquid chromatograph consisting of a G1322A
vacuum degasser, a G1315A diode array detector, and a Rheodyne
7725 high-pressure manual injection valve (20-lL) injection loop
was used to analyse the extracts by HPLC. The reversed-phase column used was a Nova-pack C-18 (250  3.9 mm) purchased from
Anlisis Vnicos (Ciudad Real, Spain). A Kromasil 5 C-18 precolumn
(15  4.6 mm) protected with a steel holder, provided by Scharlab
(Barcelona, Spain), was used to clean up the extracts.
Operational variables were optimized by using the software
Statgraphics plus v.5.1 for Windows.

2.1. Samples
Wine lees obtained by raking after alcoholic fermentation of
syrah grapes was provided by Cooperativa Agrcola La Unin
(Montilla, Crdoda, Spain). This grape variety was previously selected by Alonso et al. (2002) both to study major phenolic compounds in lees and show a higher antioxidant capacity as
compared with lees from other grape varieties. The lees was centrifuged at 2100g and the liquid phase was discarded. The solid phase
was dried at 40 C for 48 h in an oven, milled in a mortar, sieved to
a 0.5-mm particle size and stored at 4 C until use.
2.2. Chemicals
Ethanol (96% v/v) PA from Panreac (Barcelona, Spain) and distilled water were used for preparing the different ethanolwater

2.4. Proposed extraction and drying methods

The dried lees (prepared as explained in Section 2.1) is mixed
with the extractant (ethanol 75%, hydrochloric acid 1% in water)
in an 1:10 (w/v) ratio and subjected to MAE at 200 W irradiation
power for 17 min. The extractant is evaporated to a nal volume
containing 20 g dry residue per 100 mL extract (data calculated
as in Section 2.6). Two grams of 75:25 (w/w) Aerosil-200maltodextrin are added to the concentrated extract and the resulting
mixture is homogenized at 50 C before charging it in the spraydryer. The concentrated extract containing the excipients is
spray-dried under the following working conditions: airow
through the system 600 L/h; feed rate 250 mL/h; aspiration suction
velocity, 40 mbar; inlet temperature 128 C.

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J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

2.5. Reference method for extraction of phenolic compounds (PrezSerradilla et al., 2007)
The dried lees (prepared as explained in Section 2.1) and 75%
ethanol in water are stirred at 40 C for 24 h in an 1:10 ratio, after
which the solid residue is removed by centrifugation prior to analysis of the extract.
2.6. Determination of the percentage of dry residue (%DR) in the liquid
extracts
The %DR in the liquid extracts is determined gravimetrically:
25 mL extract is dried in an oven at 105 C for 6 h. The results
are expressed as g of dry residue per 100 mL sample.
2.7. Determination of total phenols index (%PI)
The amount of total phenolics was determined by the Folin
Ciocalteu method using gallic acid (GA) as standard. Briey: a calibration curve is run for GA. When analyzing liquid extracts, 1-mL
aliquots of dilute extracts (dilution with distilled water to adjust
the absorbance within the calibration limits), 10 mL of distilled
water, 1 mL FolinCiocalteu reagent and 3 mL Na2CO3 (20% w/v)
are mixed in this order, made to 25 mL with distilled water and
heated at 50 C for 5 min. After 30 min in the dark, the absorbance
is monitored at 765 nm against a blank similarly prepared, but
containing distilled water instead of extract. The total phenols index is expressed as mg of GA per 100 mg of dry matter contained in
the liquid extract (after calculating the %DR).
When analyzing dry extracts, 200 mg of solid extract is exactly
weighed, added to 25 mL 1:1 (v/v) ethanolwater and sonicated for
10 min. The resulting solution was centrifuged, diluted if necessary, and processed in the same way as the liquid extracts. In this
case, the %PI was expressed as mg of GA per 100 mg of dry extract.
2.8. Determination of the antioxidant capacity of the extracts by the
ORAC assay
The ORAC procedure reported by Bleve et al. (2008) was followed. AAPH as peroxyl radicals generator, uorescein as uorescent probe (uorescein acts as target for the peroxyl radicals
generated by AAPH, which quench the uorescein uorescence)
and Trolox as antioxidant standard are used. Excitation and emission wavelengths are set at 535 and 560 nm, respectively. Briey,
the reaction is carried out in 75 mM phosphate buffer (pH 7.4)
and the nal reaction mixture was 2 mL. Twenty microlitres of diluted extract, blank, or Trolox calibration solution (1, 2, 4 and 8 lM
nal concentrations) are mixed with uorescein solution (1.2 mL,
120 nM) for 15 min at 37 C. Then, AAPH solution (0.6 mL,
40 mM) is added to the mixture. The uorescence was monitored
every minute for 80 min. The nal ORAC values are calculated
using the net area under the decay curves, and expressed as lmol
of Trolox equivalents (TE) per gram of dry matter contained in the
liquid extract (its %DR is previously determined). When analyzing
dry extracts, the method is preceded by dissolution as in the previous section, and the results expressed as TE per gram of dry extract.
2.9. Quantication of the spray-drying yield and phenolic compounds
oxidation (%PCO) during this process
The yield of the spray-drying process is quantied as the percentage of powder obtained after sieving the spray-dried product
at 0.5-mm particle size (to reject sticky powder, if present), with
respect to the dry matter contained in the original liquid sample
(knowing its %DR and amount of excipients, if added). The powder
stuck to the spray-drier walls was rejected.

The percent of phenolic compounds which undergoes oxidation

during the drying step (%PCO) is calculated as the difference between the amount of phenolic compounds in the liquid to be dried
(knowing its %PI and amount of excipients, if added) and that in the
powder obtained after sieving the spray-dried product at 0.5-mm
(knowing its %PI).
2.10. HPLCDAD analysis
A binary gradient from a mobile phase A consisting of 10% (v/v)
formic acid aqueous solution and a mobile phase B consisting of
10% (v/v) formic acid in acetonitrile is used with the following elution program: linear gradient from 10% to 27% B in 15 min and
from 27% to 100% B in 30 min. The ow-rate is set at 1 mL/min.
The analytes are identied by comparing their retention times
and UV spectra with those of the corresponding standards, and
quantied at the following wavelengths: 530 nm for anthocyanins,
360 nm for quercetin, quercetin-3-b-glucoside and myricetin, and
320 nm for hydroxycinnamic acids.
3. Results and discussion
3.1. Preliminary study of the sample
Wine lees is a semisolid residue. The samples used in the present study showed 21.3%DR. After centrifugation, the %PI of the liquid fraction was 3.6%. After checking that the %PI of the extract
from the liquid fraction is not signicant as compared with that
of the solid fraction, the former was discarded.
3.2. Optimization of the microwave-assisted extraction
The inuence of the main factors involved in the extraction process was studied by using a multifactor experimental design.
Experimental designs have never been applied to optimize phenolic compounds extraction from wine lees, from which the amount
of phenolic compounds extracted does not correspond to the total
dry matter extracted. Therefore, in order to maximize the amount
and quality of the extract obtained, three response variables were
examined, namely: the %DR, the %PI (referred to the dry matter
contained in the liquid extract), and the ORAC values (also referred
to the dry matter contained in the liquid extract). The optimization
study (consisted of a complete two-level factorial design allowing
ve degrees of freedom and involving 16 randomized runs plus
three central points) was used to screen the four factors with potential inuence on the extraction process, namely: irradiation
power (tested range 100200 W), irradiation time (tested range
525 min), percent of ethanol (tested range 50100%) and percent
of HCl (tested range 0.21%). The upper and the lower values given
to each factor were selected from the available data and the experience gathered in preliminary experiments. Two grams of sample
were extracted with 20 mL extractant. Ethanol was preferred to
methanol taking in mind a potential use of the product in the food
industry.
The results of this experimental design are shown in Fig. 1 as
Pareto charts corresponding to %PI, ORAC, and %DR. The main conclusion from Pareto charts A and B in Fig. 1 is that the sole factor
signicantly affecting the %PI and ORAC value of the resulting extract was the percentage of ethanol, as it had a positive and significant inuenceat 95% condence level, its inuence is out of the
experimental errorin the two response variables. No signicant
interactions between factors were observed. As regards the effects
of other factors, the percentage of hydrochloric acid shows a positive inuence on both response variables, although no signicant
at 95% condence level; while the inuence of irradiation power

1655

J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

A
+
-

0.5

1.5

2.5

B
C:%EtOH
D:%HCl
BD
AB
BC
B:Irr time
A:Irr power
CD
AD
AC

+
-

3.3. Comparison of the liquid extracts obtained by the proposed MAE

and conventional methods

C
C:%EtOH
AD
B:Irr time
A:Irr power
BD
AC
D:%HCl
CD
BC
AB

+
-

The percentage of ethanol in the extractant of the conventional

method was 75 (the same as that selected in the proposed method)
for proper comparison of the effect of the microwave energy on the
quality and quantity of the extract. The %DR, %PI and the ORAC

Total phenols index

C:%EtOH
D:%HCl
BD
AB
CD
BC
AC
A:Irr power
AD
B:Irr time

optimum valuesselected in the rst optimization study. As in

the rst optimization study, the inuence of the studied factors
on the %PT and ORAC value was similar, only the %PT and %DR were
used as response variables in this second optimization study. The
range of ethanol percentage in the extractant was reduced in this
study to 6090%, as in the rst design the extreme values of this
factor showed a very negative inuence on one or other of the response variables. The range within which the irradiation time was
studied coincided with that of the rst design (525 min). The response surfaces obtained in this second design are shown in Fig. 2
(different axis scales were used in the two surfaces in order to
facilitate visualization). It can be observed as the effect of the irradiation time on the %DR is more important than on the %PI. It can
also be seen as increased ethanol percentage decreases the %DR;
meanwhile the %PI is improved up to a certain percentage of ethanol, after which the %PI decreases. So, as the behaviour observed in
the two surfaces is opposite when using the highest percentage of
ethanol, a compromise must be adopted: 17 min extraction time
and 75% ethanol in the extractant were selected as with these values acceptable %DR and %PI can be obtained.

Fig. 1. Standardized Pareto charts obtained by the rst experimental design for
optimization of the MAE process. The vertical line indicates the limit at which the
inuence of the factor is signicant at 95% condence level. (A) % of total phenolics;
(B) ORAC value; (C) % of dried residue.

Irradiation time

% Ethanol

% Dry residue

and irradiation time on the %PI and ORAC of the resulting extract
was even less important. It can also be observed as both %PI and
ORAC Pareto charts are very similar, which could be ascribed to
the previously described correlation between %PT and antioxidant
capacity in winemaking products, (Fernndez-Pachn et al., 2004;
Yilmaz & Toledo, 2006; Alonso et al., 2002). Concerning the %DR
Pareto chart (Fig. 1C), as in the case of %PT and ORAC response variables, the sole signicant factor on the percentage of dry residue is
the ethanolwater ratio, but in this case the effect of this factor is
the opposite: a negative inuence. With respect to the other three
factors, the inuence of hydrochloric acid in the %DR of the extracts
is negligible, meanwhile the inuence of both the irradiation
power and irradiation time is positive, although no signicant.
From the foregoing, 1% HCl was selected for further experiments
as it increases the %PI and ORAC values, and does not affect the
%DR. The maximum irradiation power (200 W) provided by the
digestor used was selected as, in more or less extent, it improves
the values of the three response variables.
As from the results of the rst optimization study, the optimum
values of extraction time and percentage of ethanol could not be
established, a second experimental design was required. Thus,
two response-surface central-composite 22 + star designs allowing
ten degrees of freedom and involving eight centre points were constructed for a better understanding of both the extraction kinetics
and inuence of ethanol percentage in the extractant on the quantity and quality of the extracts obtained. In this study, the percentage of hydrochloric acid and irradiation power were xed at their

59
56
53
50
47
44
41
5 0
90 85
80 75
15 10
20
70 65
60 25

1.7
1.4
1.1
0.8
0.5
0.2
-0.1
60 65

70 75

80 85 90 0 5

15
10

20

25

Irradiation time

% Ethanol
Fig. 2. Response surfaces obtained by the second experimental design used to
optimize MAE. Note that axis scales are not the same in both plots.

Table 1
%DR, %TP and ORAC values of the liquid extracts obtained under the optimal working
conditions selected for the MAE and by the conventional method.

Extract

%DRa

%TPb

ORACc

MAE
Conventional method

1.27
1.05

53.2
54.7

6250
6100

Expressed as g of dry residue per 100 mL of liquid extract.

Expressed as mg of gallic acid per 100 mg of dry matter contained in the liquid
extract.
c
Expressed as lmol of Trolox equivalents per gram of dry matter contained in
the liquid extract.
b

J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

As a liquid is not the proper form for industrial exploitation of

compounds extracted from a given solid raw material, a suitable
spray-drying method to convert into powder the liquid extract obtained by MAE was developed.
As commonly dened, the yield of the spray-drying process is
the percentage of theoretical product obtained in a suitable form
after spray-drying (Wang & Langrish, 2009). In order to obtain a
high yield and stable powder, the use of additives in the spray-drying process is generally required.
Taking into account the antioxidant capacity of the extracted
compounds, formation of oxidation products is foreseeable, with
the concomitant decrease in the concentration of active principles;
so oxidation should be avoided or, at least, minimized. This phenomenon has been previously reported as mainly correlated with
the spray-drying temperature (Georgetti, Casagrande, Fernandes
Souza, Pereira Oliveira, & Vieira Fonseca, 2008).
From the foregoing, in order to maximize the yield of the spraydrying process and minimize oxidation of the phenolic compounds
(quantication of these parameters is explained in Section 2.9), the
use of two additives (maltodextrin and Aerosil-200 (Wang & Langrish, 2009; Gonnissen, Remon, & Vervaet, 2008; Roustapour et al.,
2009)) as well as the inuence of the spray-drying temperature
were tested. Other experimental factors were xed in all experiments according to the available data from the literature on the
use of the same spray drier approach (Tajber, Corrigan, & Healy,
2009) and the experience gathered in preliminary experiments:
the airow through the system was xed at 600 L/h, the peristaltic
pump worked at 250 mL/h, the aspiration worked at 40 mbar and
the %DR (taking into account the amount of excipients, if added)
of the liquid to be spray-dried was adjusted to 20% (the extracts
were previously concentrated in a rotary evaporator). The duration
of the experiments was 20 min in all cases.
Due to the dimensions of the microwave-assisted extractor
used in this work, the available amount of extract provided by
MAE under optimal working conditions was not enough for optimization of the spray-drying step, so 30-L extract was obtained
by the reference method (the composition of which was very similar to that of the MAE extract) to be used for this purpose. The
mixture between excipients and concentrated liquid extract was
homogenized at 50 C before being charged in the spray-dryer. In
previous experiments it could be checked that, when no excipients
were added to the extract to be dried, the yield of the process was
highly affected by depositions in the spray-dryer walls. In order to
determine if the use of maltodextrin and/or Aerosil-200 improved
the yield of the process, a preliminary study was carried out keeping the inlet temperature at 150 C and using 10% excipients with
respect to the dry matter contained in the liquid to be spray-dried
(%Exc). Five experiments were carried out in which the excipient
composition varied from 100% maltodextrin to 100% Aerosil-200.
The best yield was achieved when a 75:25 (w/w) Aerosil-200maltodextrin mixture was used as excipient.
Once the composition of the additive to be used was xed, two
response-surface central-composite 22 + star experimental design

A
112
92

% Yield

3.4. Optimization of the spray-dried extract

allowing ten degrees of freedom and involving eight centre points

were constructed in order to study the inuence of both %Exc and
inlet temperature on the quantity and quality of the dry extract obtained. The response variables studied were %PI in the powder,
yield of the spray-drying process and %PCO. Although the %PI in
the nal product and spray-drying yield were the response variables taken into account to optimize spray-drying, the %PCO was
also included in the study in order to evaluate the possible effect
of excipients on the oxidation of the phenolic compoundsas the
addition of excipients produces dilution of the active principles, a
potential relationship between the amount of excipients added
and the oxidation of the phenolic compounds could not be observed only with the two responses used in the optimization.
Fig. 3 shows the three response surfaces obtained. From Fig. 3A
and B it can be concluded that the amount of excipients added to
the liquid extract improves the yield and decreases the %PI in the
nal product; but Fig. 3C shows that this decrease is only due to
de dilution effect, as a slightly decrease of the %PCO (especially
at low temperatures) can be observed when high amounts of
excipient are added. On the other hand, Fig. 3B shows as high inlet
temperatures improved the yield but also slightly decreased the
%PI; Fig. 3C proves that increased temperatures promotes the oxidation of phenolic compounds. In view of these effects, a compromise solution was adopted, so 22% of 75:25 (w/w) Aerosil-200
maltodextrin and 128 C inlet temperature were chosen. The extract obtained by MAE was spray-dried under the adopted working
conditions, obtaining a spray-drying yield of 84%. Under vacuum
packing in plastic glass bags, the powder obtained maintains its

72
52
32
150140
130120
110100

Inlet temp

26 30
18 22
90 10 14

% Excipients

B
Total phenols index

values of the extract obtained by the MAE under optimal working

conditions (given in Section 3.2) were compared with those of the
extract provided by the reference method (Section 2.5) as summarized in Table 1. As can be seen, both extracts are similar in terms
of %PI and ORAC values. This behaviour was foreseeable from
Fig. 2A: 75% ethanol in the extractant makes negligible the inuence of the extraction time on the%PI and, consequently, that of
microwave energy. However, 17 min (vs. 24 h extraction time)
are enough in the MAE method to obtain a better extraction efciency (in terms of %DR) than in the conventional method.

41
39
37
35
33
31
29
90 100
110120
14 10
130140
22 18
150 30 26

Inlet temp

% Excipients

C
% PCO

1656

28
25
22
19
16
13
10 14
140 150
18 22
120 130
26 30 90 100110

% Excipients

Inlet temp

Fig. 3. Response surfaces from optimization of the spray-drying step. Note that axis
scales are not the same in both plots.

J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

characteristics during at least 5 months. The results of the chemical analysis of this power are in the section below.
3.5. Analysis of the spray-dried extract
Different analyses of the powder obtained by spray-drying the
extract obtained by MAE were carried out, namely: total phenols
index, ORAC measurements and individual determination of the
most representative phenolics in grape by-products (Alonso
et al., 2002; Luque Rodrguez et al., 2007; Morata et al., 2003).
Fig. 4 shows the chromatograms obtained at the three wavelengths
selected for monitoring and the analytes identied. Peak two was
attributed to p-coumaroyl derivatives (Cm-Mv3G, tentatively identied) taking into account the information provided by Morata
et al. (2003) and Luque Rodrguez et al. (2007).
The results of the %PI and ORAC determinations, as well as the
concentrations of the identied compounds, are summarized in Ta-

1657

ble 2. As in the articles from Morata et al. (2003) and Luque Rodrguez et al. (2007), Cm-Mv3G was the most abundant anthocyan in
the extract (Fig. 4). Concerning avonols such as myricetin, quercetin and quercetin-3-b-glucoside, the concentrations ratio in the
lees extracts is not far from that previously reported in grape seeds
by Luque Rodrguez et al. (2007). Concerning caffeic and p-coumaric acids, Alonso et al. (2002) reported a concentration of caffeic
acid in syrah wine lees about four times higher than that of p-coumaric acid. The difference with the results shown in Table 2 can be
explained by the presence of the liquid fraction of the lees. Adsorption of phenolic compounds from yeasts of wine lees involves a
hydrophobic interaction (Morata et al., 2003), so p-coumaric acid
(less polar) could be more adsorbed than caffeic acid (more polar)
by the solid fraction of the lees (that used in this work). In addition,
comparisons between results obtained by other researchers must
take into account that the phenolic composition of grape is
inuenced by factors such as grape variety, vineyard location,

Fig. 4. HPLCDAD chromatograms of the wine lees spray-dried extract. Identied compounds: 1, Mv3G; 2, Cm-Mv3G (tentatively identied); 3, quercetin-3-b-glucoside; 4,
Myricetin 5, quercetin; 6, caffeic acid; 7, p-coumaric acid.

1658

J.A. Prez-Serradilla, M.D. Luque de Castro / Food Chemistry 124 (2011) 16521659

Table 2
Results of the analyses of the lees extract powder.
Concentration in the dry extract
Photometry
Total phenolics
ORAC
HPLCUV
Anthocyans
Mv3G
Cm-Mv3G
Other phenolics
Myricetin
Quercetin
Quercetin-3-b-glucoside
Caffeic acid
p-Coumaric acid
Total
a
b
c
d
e

36,4a
3930b

91c
11729d

uct highly demanded by virtue of their nutraceutical properties).

So, wine lees can be a suitable alternative raw material (nowadays
undervalued) to obtain grape-related antioxidant extracts.
While there are hundreds of papers concerning grape seed and/
or grape skins extraction, only a few studies focused on wine lees
extraction have been reported. In the authors opinion, the potential uses of this poorly exploited grape by-product deserve more
research in this eld.
Acknowledgement

4292c
13656c
8900c
663c
2449c
4.18e

mg of gallic acid per 100 mg of lees extract powder.

lmol of Trolox equivalents per gram of lees extract powder.
lg of the corresponding standard per gram of lees extract powder.
lg of Mv3G per gram of lees extract powder.
mg of phenolics identied by HPLC per 100 mg of lees extract powder.

cultivation system, climate, soil type, wine cultivation practices

and harvesting time (Shahidi & Naczk, 1995).
Sum-up the individual concentration of the identied phenolic
compounds (last line in Table 2), the total concentration obtained
is far from total phenolics determined photometrically. This behaviour, previously reported by Alonso et al. (2002) for wine lees, must
not be only ascribed to non-identied chromatographic peaks, as
their areas are not signicant enough with respect to those of
the identied compounds. One explanation given to this difference
by Luque Rodrguez et al. (2007) was the presence of anthocyanins
linked to other molecules forming higher molecular-weight anthocyanin-derivatives, which are retained in the ltration step previous to HPLC analysis. This predominance of polymeric pigments
is a positive aspect of the extract, as their antioxidant activity
has been reported to be higher than that of low-molecular weight
phenolic compounds (Spranger, Sun, Mateus, de Freitas, & Ricardoda-Silva, 2008).
The %PI (the most usual parameter for standardization of commercial grape seed extracts) and the ORAC values of the spraydried lees extract here presented are comparable to those reported
by Bozan, Tosun, and zcan (2008) in grape seed liquid extracts,
but there is no available information about oxidation of phenolic
compounds and the need of using excipients during drying of grape
seed extracts. A comparison of the quality of the extracts obtained
in the present study with those provided by Alonso et al. (2002) is
unaffordable as these authors express their results with respect to
the volume of lees extracted with no discard of the liquid fraction.
4. Conclusions
Optimization of extraction of phenolic compounds from wine
lees has been reported for the rst time. The proposed MAE method provides better extraction efciency (1.27 vs. 1.05%DR) in a very
shorter time (17 min) as compared to the conventional extraction
method for phenolic compounds (24 h).
In order to facilitate an industrial methodology, a spray-drying
process has also been optimized, thus minimizing oxidation of
phenolic compounds and maximizing the yield of the process.
The effect of both temperature and excipients amount on the oxidation of phenolic compounds during the spray-drying process has
been also studied.
Optimization of both extraction and spray-drying steps has allowed obtaining an antioxidant wine lees extract in powder form
with features comparable to those of the grape seed extracts (prod-

The Spanish Ministerio de Ciencia e Innovacin (MICINN) and

the FEDER program are thanked for nancial support through projects PET2006_0193 and CTQ2009-07430. Central Enolgica
Manchega (LIEC) is also thanked for nancial support.
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