C. R.

Chimie 17 (2014) 268–283

Contents lists available at ScienceDirect

Comptes Rendus Chimie

www.sciencedirect.com

Full paper/Mémoire

Bixin extraction from defatted annatto seeds

Liara M. Rodrigues a, Sylvia C. Alcázar-Alay a, Ademir J. Petenate b,
Maria Angela A. Meireles a,*
a
LASEFI/DEA/FEA (School of Food Engineering), UNICAMP (University of Campinas), Rua Monteiro Lobato, 80,
13083-862 Campinas, SP, Brazil
b
EDTI–Process Improvement, Rua José Ponchio Vizzari, 312, 13085-170 Campinas, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: Bixin is the major carotenoid in the seed of the Annatto plant (Bixa orellana L.). The aim of
Received 8 August 2013 this study was to obtain extracts containing bixin from seeds that had been partially
Accepted after revision 17 October 2013 defatted by supercritical fluid extraction. Pressurized liquid extraction (PLE) and low-
Available online 13 January 2014
pressure solvent extraction (LPSE) methods were used, and the effects of the solvent,
temperature, pressure, solvent mass to feed mass (S/F) ratio and ultrasonication were
Keywords: evaluated for the global yield (X0(%)) and the bixin yield (BY(%)). Extraction conditions
Annatto producing high yields of bixin were established for both the PLE and LPSE methods.
Bixa orellana
Analysis of variance was used to examine the influence of the individual extraction
Bixin
variables in LPSE and PLE. For LPSE; significant effects were found for solvent, temperature,
Pressurized liquid extraction
Low-pressure solvent extraction
and the interactions of temperature with solvent and temperature with S/F. Solvent was
the only variable that significantly affected X0(%) and BY(%), for PLE. While ultrasonication
did not significantly affect X0(%) or BY(%), scanning electron microscopy analysis revealed
structural changes in the vegetal matrix following this treatment.
ß 2013 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction other minor constituents, such as trans-bixin, cis-norbixin

and trans-norbixin. Annatto is almost unique among the
The use of additives to make food and other products sources of carotenoids, as its pigment takes on a number of
more visually appealing is very common in the food and different chemical structures; the range of intense colors
chemical industries, and so the interest in and demand for its compounds take include shades of red, orange and
natural products have increased significantly in recent yellow. Annatto can be obtained from hydrophilic and
years. Due to certain requirements and consumer pre- hydrophobic extracts, and its pigments are very stable due
ferences for natural compounds, a global trend has arisen to their interactions with protein compounds. Thus, it is an
aiming to increase the use of natural products over excellent candidate for a natural pigment to be used in
synthetic versions. The growing demand for natural dyes cosmetics, pharmaceuticals and the food industry [1–3].
is justified by their minimal or absent toxicity. Unlike b-carotene, which is widely distributed in vege-
Annatto is a shrub native to the South American tropics, tables and fruit, bixin can only be found in annatto and
the natural reddish-yellow color of which is obtained from comprises more than 80% of the total carotenoid content of
the outer coating of its seeds. The major pigments present its seeds [3].
are carotenoids, including a large amount of cis-bixin and Various techniques have been studied to develop clean
extraction technologies with environmental benefits (so-
called ‘‘green technologies’’). Innovative technologies, such
as ultrasound-assisted extraction [4–8], microwave-
* Corresponding author.
E-mail addresses: meireles@fea.unicamp.br, maameireles@gmail.com assisted extraction [9], supercritical fluid extraction [10–
(M.A.A. Meireles). 15] and accelerated solvent extraction [7,16,17], can be

1631-0748/$ – see front matter ß 2013 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.10.010
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 269

combined to develop processes that are free of the residues of the solid matrix pretreatment, solvent, and ultrasound
of organic solvents. These techniques are used to extract power.
bioactive substances to shorten the processing time, The major part of bixin in annatto seeds is located in the
reduce solvent consumption, increase the extraction yield outside layer of the seed. Nonetheless, there is a lipid layer
and improve the quality of the extracts. These studies are strongly associated to the bixin in the seed; the removal of
needed because of the general trend of the market to the lipidic layer during the extraction of bixin from annatto
identify products that generate economic, social and is the most difficult step. Bixin has a very low solubility in
environmental advantages [11,18]. Extraction with carbon dioxide while that of the lipids is very high.
organic solvents is limited by the need for a solvent that Therefore, we envisioned a process in two steps: (i)
is compatible with the end use of the product. In food, the removal of the lipids using supercritical carbon dioxide
dye will be subject to serious technical restrictions on the followed (ii) by removal of bixin. The removal of the lipids
amount of residues from potentially toxic solvents [15]. using supercritical carbon dioxide has proven to be a very
Solid-liquid extraction or solvent extraction occurs efficient process [11]. Additionally, as reported in litera-
through the selective dissolution of one or more solutes ture [23] supercritical fluid extraction is an interesting tool
from a solid matrix by a liquid solvent. This unit operation for solid matrix pretreatment. Therefore, in this work, we
is also called leaching, decoction, elution or low-pressure are seeking for the best process to remove bixin from
solvent extraction (LPSE). Regardless of the name, this defatted annatto seeds. Therefore, two process were
technique is one of the most widely used operations in the investigated PLE and LPSE. The effects of solvent, ratio of
chemical industry [18]. mass of solvent to mass of solvent, temperature, pressure
For instance, a certain liquid can be pressurized and and ultrasound assistance were evaluated on the total
heated at pressures and temperatures below its critical yields of extract and of bixin.
point and then employed in the extraction of several
compounds, based on the potentially increased solubility 2. Material and methods
of the compounds to be extracted and on the acceleration
of the desorption kinetics of these compounds from the 2.1. Material
vegetable matrix. The liquid extraction process at pres-
sures higher than ambient pressure and moderate to high Annatto seeds were partially defatted as described by
temperatures has various names; in this paper, we use the Albuquerque [11]. Briefly, annatto seeds of the Piave
name ‘‘pressurized liquid extraction’’ (PLE). variety were defatted at 313 K and 20 MPa using super-
The extraction methods used for the production of dyes critical CO2. The extraction was carried out in a commercial
from annatto seeds may produce bixin or, by aqueous SFE unit (Thar Technologies, SFE-2  5LF-2-FMC, Pitts-
hydrolysis, the simultaneous extraction of norbixin [1,19]. burgh, Pennsylvania, USA).
The most commonly used methods to extract the
pigments from annatto seeds are alkaline extraction 2.2. Characterization of the raw materials
(norbixin salt), extraction with oil (bixin) and extraction
with solvents, such as ethyl acetate, ethanol, chloroform The real density (rr) of the seeds was determined
and acetone, to yield products with higher purity. These following the method of helium pycnometry using a gaseous
dyes differ in solubility and pigmentation [1]. pycnometer (Quantachrome, UltrapycTM 1200e, Boynton
The great demand for annatto extracts with high quality Beach, Florida, USA) in the Analytical Central/Institute of
characteristics has accentuated the deficiencies of the Chemistry/Unicamp. Apparent density (ra) was calculated
commonly used processes to produce dyes. Typically, these as the ratio of the mass of raw material used to fill the
techniques require high extraction times and provide a low extraction cell to its volume. The porosity of the bed and the
efficiency, even while including the risk of the thermal particles (e) was determined as (1  (ra/rr)). The average
degradation or oxidation of the pigment extracts, which diameter (dp) of the seeds was determined according to the
requires the use of extraction techniques at milder FAO/WHO report [1], using the geometric means of the
conditions to avoid degradation. height, width and thickness of twenty randomly selected
Ultrasonic energy has been identified as an efficient tool seeds, as measured by a universal pachymeter. For the
to improve performance in different applications of analysis of their chemical composition, the samples were
analytical chemistry, such as the extraction of organic milled (Tecnal, model TE-631, Piracicaba, São Paulo, Brazil).
and inorganic compounds, homogenization, and disper- The moisture [24], ash [25], lipid [26] and protein [27]
sion of suspensions, among other applications [4–6,18]. contents were determined. Scanning electron microscopy
The improvement of the extraction efficiency for organic analysis was performed at the Analytical Laboratory of
compounds by the use of ultrasonication is based on the Resources and Calibration (LRAC) at the School of Chemical
phenomenon of cavitation, which is produced in the Engineering (FEQ)/UNICAMP, São Paulo, Brazil. The aim was
solvent by the passage of sound waves [20–22]. In general, to analyze the seed surface, evaluating the effects of the
for ultrasound techniques to be efficient, an ultrasonic extraction conditions used in this study on the structure of
probe should be used, as described by Veggi et al. [4]; the pericarps of the seeds, where bixin is most commonly
however, there have been reports in the literature of located. A coating of gold with a thickness of 92 Å was
improvements in the processing performance even with applied by metallic sputter coating (Polaron SC7620 sputter
ultrasonic bath-assisted extractions [8]. Nonetheless, it is coater, VG Microtech, Uckfield, UK) in the presence of an
import to observe that the extraction efficiency is a result inert gas, such as argon. To obtain the micrographs, a
270 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

scanning electron microscope with an energy dispersive 395 K) and pressure (2, 4 and 20 MPa); the assays were
detector X-ray (Leo 440i, 6070, LEO Electron Microscopy/ performed in a semi-continuous apparatus (Fig. 1) at flow
Oxford, Cambridge, England) was used; the accelerating rates of approximately 1.14, 1.31 and 1.45 g/min, respec-
voltage was equal to 20 kV, and the beam current used was tively, for ethanol, ethyl acetate and sodium hydroxide
100 pA. solutions. LPSE were performed simultaneously with
periodic agitation (every 1 minute). The extraction time
2.3. Chemical characterization was approximately 20 minutes, and three ratios of S/F for
both methods were used: S/F = 6, 7 and 7.5 g (solvent)/g
The bixin contents of the seeds and extracts were (seed) for the ethanol, ethyl acetate and sodium hydroxide
determined according to the methodology described in the solutions, respectively. The extractions were performed in
FAO/WHO report [1] and adapted by Albuquerque and batches at ambient conditions (approximately 0.1 MPa and
Meireles [11]. Bixin was exhaustively extracted from the 300 K).
seeds with acetone PA ACS (Exodus scientific Lot:
A8979RA, Hortolândia, Sao Paulo, Brazil) until complete 2.5. Procedure for pressurized liquid extraction (PLE)
discoloration; the absorbance of this solution was read
[11,19]. The obtained extracts by PLE and LPSE were Fig. 1 illustrates the extraction equipment using
diluted in acetone to the concentrations appropriate for pressurized liquid. All connections within the system are
analysis. The absorbances of the diluted samples were made of stainless steel tubing (1/16’’ and 1/8’’). The
measured at 487 nm with a UV-vis spectrophotometer extraction cell (Thar Designs, CL 1373, Pittsburg, USA) of
(Hach DR/4000 U Loveland, Colorado, USA), and the 5 cm3 (inner diameter of 19.6 mm and height of 22.6 cm)
concentration of bixin was calculated according to the was packed with approximately 3 g of the sample. After-
Lambert-Beer law, using E1%
1 cm ¼ 3090 [1]. wards, the cell was attached to the heating jacket, which
was already at the extraction temperature. The sample was
2.4. Preliminary tests heated over 5 minutes to ensure that the extraction cell
was at the desired temperature for the subsequent
Preliminary tests were performed to evaluate the procedures of filling and initially pressurizing the system
efficiency of the extraction with pre-selected solvents with the solvent. After reaching these conditions, the
under different conditions of temperature and pressure. blocking valve was opened, and the solvent was pumped
These tests aimed to evaluate the extraction of bixin from by a HPLC pump (Thermoseparation Products, Model 3200
the defatted seeds in extreme conditions (temperature and ConstaMetric P/F, Fremont, California, USA) into the cell,
pressure), and the results obtained using both methods increasing the pressure to the desired value before a static
allowed for the selection of the best conditions for further extraction period of 10 minutes began. After this period,
studies. For the PLE method, the use of water as the solvent the block and back-pressure valves were opened, and the
in conjunction with high pressures had a negative effect on solvent was again pumped into the cell. During this step,
the global yield and bixin recovery. Therefore, we opted to the pressure was controlled by the back-pressure valve.
use other solvents that had been reported in the literature The extract was collected in pre-weighed glass bottles up
for the extraction of bixin from annatto, as follows: ethanol to a predetermined volume. Alcoholic extracts were
(PA, dynamics, 52990, Diadema, SP, Brazil), ethyl acetate evaporated in a rota-evaporator (Marconi, model MA120/
(Merck KGaA, K40235423, Darmstadt, Germany) and a TH, Piracicaba, São Paulo, Brazil) under vacuum (Marconi,
mixture of water + NaOH (pH 10 and 14). The PLE model MA057/1, Piracicaba, São Paulo, Brazil) at 310 K for
conditions tested were temperature (305, 335, 355 and the removal of the solvent (the same procedure was used in

Fig. 1. Equipment used for PLE [8].

 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 271

the preliminary tests for the removal of ethanol and ethyl BY(%), kinetic experiments were performed in duplicate at
acetate). The solvent-free extracts were kept at 265 K to these conditions. During the extraction process, the bottles
determine the overall yields and for quantification of the for the collection of the extract were replaced by clean, pre-
total carotenoids, expressed as the bixin percentage (BY(%)). weighed flasks every 5 min.
The overall extraction curves (OECs) were constructed
2.6. Extraction procedure for LPSE, with and without with the purpose of determining the quantity of extracted
ultrasound assistance soluble material and process parameters as a function of
time. For the determination of kinetic parameters, these
The LPSE equipment with or without ultrasound (US) included tCER (duration of the constant extraction rate
assistance is illustrated in Fig. 2. The solvent was pumped period, [min]); MCER (mass transfer rate during the CER
through a peristaltic pump (Cole-Parmer Instrument Co, period, [g/s]); YCER (mass ratio of extract at the bed outlet,
catalog number 7554-30, Chicago, IL, USA) into the cell [g-ext/100 g solvent]) and RCER (yield during the CER
extraction, which was immersed in an ultrasonic bath period, [%]). These calculations were performed with the
(frequency 40 kHz, power 135 W) (Unique, Clean Max assistance of SAS1 9.3 software (SAS Institute Inc., version
model 1400, Indaiatuba, SP, Brazil) and maintained at the 9.2, Cary, USA), using the procedure PROC REG and PROC
process temperature by a thermostatic bath (Marconi, NLIN, as described in the literature [28].
model MA 127/BO, Piracicaba, SP, Brazil). The extraction
cell, a glass column of 3.04 cm3 (inner diameter of 16.3 mm 2.9. Calculations
and height of 14.6 cm), was sealed with silicone stoppers at
both ends. The assays with US assistance were performed 2.9.1. Determinations of overall yield (X0)
in the same apparatus. The ethanol was removed as Global yield in dry basis, X0(%), was calculated
previously described; the water was removed by freeze- according to Eq. (1) as the ratio of the total mass of the
drying (LIOTOP, L101, São Carlos, SP, Brazil). The solvent- extract (mextract) and the initial mass of the sample
free extracts were kept at 265 K to determine the overall (msample) on a dry basis.
yields and quantifications of the total carotenoids, mextract
expressed as the bixin percentage (BY(%)). X 0 ð%Þ ¼  100 (1)
msample

2.7. Experimental design

2.9.2. Extraction yield bixin BY(%)
For PLE, based on the preliminary results, ethanol was Bixin yield in dry basis, BY(%), was calculated according
chosen as the solvent. The independent variables studied to Eq. (2), which involves X0(%), bixin in the extract,
were S/F (5 e 10 g/g), temperature (313, 323 e 333 K) and Bixinextract(%), and bixin in the seeds, Bixinseeds(%).
pressure (2, 6 e 10 MPa). The response variables were X0(%)
X 0 ð%Þ  Bixinextract ð%Þ
and BY(%). The experimental design was a full factorial BY ð%Þ ¼ (2)
Bixinseeds ð%Þ
design (2  3  3) that was totally randomized without
replication.
For LPSE, we studied the following independent 2.9.3. Statistical analysis
variables: S/F (approximately 4 and 8 g/g), temperature For both methods (PLE and LPSE), an analysis of
(313, 323 and 333 K), ultrasound (without, intermittent variance (ANOVA) was performed using Minitab1 16
and continuous use) and solvent (water and ethanol). The software and the general linear model (GLM) with a
response variables were X0(%) and BY(%). The experimental confidence interval of 95% (P-value  0.05).
design was a full factorial design (2  3  3  2) that was
totally randomized without replication. 3. Results and discussion

2.8. Study of the kinetics of extraction 3.1. Characterization of the raw material

After carrying out the extractions and determining the The chemical profile of the defatted seed (variety Piave)
conditions that produce optimal (maximum) X0(%) and was (12.3  0.7)% moisture, (5.8  0.2)% ash, (9.7  0.0)%

Fig. 2. Equipment for the low pressure extraction assisted by an ultrasound.

272 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

protein, (2.7  0.01)% lipids, (68.9)% carbohydrates and

(2.5  0.2)% bixin. The values obtained in this study are
similar to those found in the literature for annatto seeds. Silva
et al. [15] found approximately 2.8% bixin, 3.1% lipids, and
11.3% moisture. Albuquerque and Meireles [11] found similar
values for annatto seeds of the Piave variety: (4.9  0.2)%
bixin, (12.3  0.1)% moisture, (6.2  0.1)% ash, (3.7  0.0)%
lipids, (12.1  0.2)% protein and 65.7% carbohydrates. The
mean diameter of the nondefatted seeds was 3.50 mm, and
their real density, 1220 kg.m3.
Fig. 3. Comparison of the extract colors from extractions using water as
the solvent at different temperatures and pressures.
3.2. Preliminary assays

Preliminary tests were performed with the aim of was 6.48, and the BY(%), 22.62. Therefore, for PLE, feasible
evaluating the efficiency of certain pre-selected solvents, alternative solvents include ethanol and ethyl acetate. In
using different temperatures and pressures. The prelimin- spite of the larger X0(%) and BY(%) for ethyl acetate, ethanol
ary experiments were performed without a formal has obvious advantages, such as its status as a benign
experimental design. Conditions were selected based in solvent.
results for other raw materials worked at our laboratory. In Considering again PLE using water, another point worth
industrial practice, bixin is extracted from annatto seeds noting is that, the color of the extracts changed substan-
using water at pH = 14; the main reason to use this highly tially depending on the pressures and temperatures used
alkaline solution is due to the presence of the lipid layer in (Fig. 3). At 393 K/20 MPa, the extract color was dark brown,
the in natura seeds. Afterwards, to obtain bixin of high most likely indicating the degradation of the annatto
purity several steps are required. In the preliminary assays, pigments, while at 303 K/2 MPa, the extract had the
we were testing the hypothesis that the use of defatted yellow-reddish color characteristic of bixin. The yellow-
seeds would allow the extraction of bixin using pressur- reddish color seen at 303 K/0.1 MPa was even more
ized water as the solvent. Nonetheless, for comparison characteristic of annatto pigments. Consequently, the
purposes, we included the use of alkaline water at two pHs following methods were selected for further study: (i)
10 and 14 and the use of two other solvents ethanol and PLE using ethanol as a solvent and (ii) LPSE, assisted or not
ethyl acetate. Even so, the results shown (Table 1) that by ultrasound, using water and ethanol as solvents.
using water as solvent the bixin recovery was very low at
all PLE conditions used in spite of the high total yields 3.3. Pressurized liquid extraction–PLE
(X0(%)) at certain conditions. For instance, at 333 K and
2 MPa the bixin recovery was approximately 0.9%. The Table 2 presents the results for the variables studied.
conclusion was that for PLE water was not a good solvent We observed that the X0 had maximum and minimum
while the solvents of choice are ethyl acetate and ethanol.
At 333 K and 2 MPa, the X0(%) was equal to 2.33, and the
BY(%), 5.85 for ethanol. However, in ethyl acetate, the X0(%) Table 2
X0 overall extraction yield (%, d.b.), the amount of bixin extract (%, d.b.),
Table 1 and extraction yield bixin BY (%, d.b.) in PLE using ethanol as a solvent.
Results of preliminary tests of extraction using defatted annatto seeds.
Conditions of extraction
Conditions of extraction
Temperature Pressure X0(%) Bixinextract BY(%)
Temperature Pressure Solvent X0(%) Bixinextract BY(%) (K) (MPa) (%)
(K) (MPa) (%)
S/F = 4
Method: PLE 323 6 3.17 3.19 3.60
393 20 Water 14.45 0.03 0.23 313 6 4.40 2.34 3.67
393 2 Water 13.61 0.06 0.42 333 10 4.02 6.08 8.69
353 2 Water 6.20 0.08 0.26 323 10 2.95 4.39 4.61
333 2 Water 19.62 0.1 0.92 333 2 2.67 1.84 1.75
303 2 Water 3.00 0.06 0.16 323 2 3.94 2.7 3.78
303 4 Water 3.00 0.1 0.09 333 6 3.54 3.76 4.74
353 2 Water (pH = 10) 6.48 0.2 0.67 313 2 2.71 2.37 2.29
353 2 Water (pH = 14) 17.90 0.05 0.47 313 10 4.92 2.33 4.08
333 2 Ethanol 2.33 4.82 5.85
S/F = 8
333 2 Ethyl acetate 6.48 6.71 22.62
333 10 7.22 3.53 9.07
Method: manual agitation using a glass rod 333 2 4.06 3.47 5.02
Ambienta LPSEb Water 3.32 13.89 24.02 323 6 3.25 6.38 7.38
Ambient LPSE Water (pH = 10) 2.61 8.95 12.17 323 10 4.27 2.99 4.54
Ambient LPSE Water (pH = 14) 15.20 0.43 3.40 313 6 4.64 2.15 3.55
Ambient LPSE Ethanol 3.61 5.83 10.94 323 2 3.69 2.83 3.72
Ambient LPSE Ethyl acetate 4.56 6.96 16.50 333 6 3.62 4.48 5.77
a
313 10 4.77 2.69 4.57
Approximately 300 K.
b
313 2 3.86 3.46 4.76
Approximately 0.097 MPa.
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 273

values, respectively, of 7.22% and 2.67%; the maximum and Table 3

Overall extraction yield, X0(%), the amount of bixin in the extract,
minimum values of BY were 9.07% and 1.75 (%).
Bixinextract (%), and bixin yield BY(%) obtained by LPSE.
Analyses of variance for X0(%) and BY(%) did not show
any significant influences of the parameters studied; in Conditions of extraction
other words, these two response variables were not Temperature (K) Method X0(%) Bixinextract (%) BY(%)
significantly affected by variations in S/F or pressure. Solvent: water; S/F = 4
Pineiro and Palma [26] compared different types of 333 LPSE-C 7.97 1.93 5.48
extraction: extraction by magnetic agitation, ultrasound- 313 LPSE-P 0.77 4.32 1.19
assisted extraction (UAE) and PLE. In the three extraction 333 LPSE 0.70 0.80 0.20
333 LPSE-P 1.36 1.65 0.80
systems, four different pure solvents were used, as follows:
313 LPSE 1.30 5.02 2.31
water, methanol, ethanol and ethyl acetate. PLE with 323 LPSE-C 2.62 1.57 1.46
methanol as the solvent resulted in better yields in terms 323 LPSE-P 7.24 1.09 2.81
of the recovery of catechin and epicatechin, notably higher 323 LPSE 8.44 1.08 3.24
than any of the other extraction conditions tested. Plaza 313 LPSE-C 7.03 0.32 0.80

et al. [7] evaluated the extraction of bioactive compounds Solvent: water; S/F = 8
from Chlorella vulgaris (carotenoids and fatty acids) using 313 LPSE-P 0.63 2.01 0.45
PLE and UAE (analytical scale). The authors found no 333 LPSE 8.75 0.97 3.02
323 LPSE 8.49 1.30 3.93
significant differences among the extraction techniques; 333 LPSE-C 6.96 0.91 2.25
however, the PLE method resulted in higher yields for 323 LPSE-P 8.15 1.46 4.23
similar bioactive compositions. 323 LPSE-C 7.68 3.53 9.65
313 LPSE-C 0.30 2.09 0.22
313 LPSE 1.08 0.66 0.25
3.4. Extractions at low pressures, assisted or not by
333 LPSE-P 8.64 1.7 5.23
ultrasound (LPSE and USA-LPSE)
Solvent: ethanol; S/F = 4
313 LPSE 10.58 3.21 12.09
Table 3 shows the experimental results obtained for the
313 LPSE-P 9.55 4.7 15.97
studied variables. We can observe that X0(%) has maximum 333 LPSE-P 11.31 5.96 23.99
and minimum values of 13.06 (ethanol; 333 K, S/F = 8 and 333 LPSE-C 9.73 5.56 19.25
LPSE-C) and 0.30 (water, 313 K, S/F = 8 and LPSE-C), 323 LPSE-P 10.19 6.49 23.54
corresponding to a BY(%) of 22.89 and 0.22, respectively. 313 LPSE-C 11.98 7.42 31.62
323 LPSE 12.54 2.57 11.46
Nonetheless, the highest BY(%) was equal to 31.62 and was 323 LPSE-C 10.01 1.41 5.02
obtained with ethanol, 313 K, S/F = 313, LPSE-C; under 333 LPSE 9.07 3.87 12.50
these conditions, the X0(%) was 11.98. The minimum BY(%)
Solvent: ethanol; S/F = 8
was equal to 0.20 and was determined with water, 333 K, 313 LPSE-P 10.50 4.16 15.54
S/F = 4, LPSE; the X0(%) was 0.70. The analyses of variance 313 LPSE-C 9.51 5.76 19.49
showed that, for these extraction conditions, significant 333 LPSE-P 9.53 5.65 19.17
differences were observed in X0(%) for the variables solvent 323 LPSE-P 10.33 4.13 15.18
323 LPSE-C 11.05 5.40 21.23
(P-value = 0.000) and temperature (P-value = 0.023) and
323 LPSE 10.11 4.48 16.12
the interaction between solvent and temperature (P- 313 LPSE 10.77 3.54 13.57
value = 0.034); although smaller, the interaction between 333 LPSE-C 13.06 5.14 23.89
S/F and temperature (P-value = 0.092) was also detected. 333 LPSE 10.56 7.58 28.49
Nonetheless, for BY(%) significant differences were LPSE: low-pressure solvent extraction; LPSE-P: low-pressure solvent
observed only for the variable solvent (P-value = 0.000). extraction assisted by ultrasound pulses; LPSE-C: low-pressure solvent
Ethanol was the best solvent, most likely because bixin is extraction assisted by ultrasound along the extraction.

lipid soluble. This behavior was expected, because, bixin is

a carotenoid, therefore, insoluble in water. Ethanol is less
polar than water, but more polar than acetone, a solvent in ultrasound on the surface of the seeds, where the bixin
which bixin is also soluble. The values of X0(%) and BY(%) is primarily located, was expected to improve the
obtained by PLE were lower than those obtained by LPSE extraction. In this work, an ultrasonic bath was used,
using the same solvent under the same conditions of and therefore, energy dispersion occurred. We then
temperature and S/F. For ethanol, a possible explanation determined that the assistance of an ultrasound as it
for this is that the ethanol dielectric constant increases was used was insufficient to significantly increase X0(%) or
with pressure; thus, its polarity also increases, which BY(%). Most likely, the energy consumption of this
reduces bixin solubility. For water, the effects of pressure technology would be justified if employing an ultrasound
were similar, as were observed in the preliminary assays probe; however proving this fact would require new
(Table 1). Thus, in spite of the very strong beliefs reported experimental data and an analysis of the economics of the
in the literature that PLE performs better than LPSE, a entire process. Additionally, as will be discussed later, the
careful analysis of the raw material shows opposite use of the ultrasound bath was sufficient to alter the
behaviors, as was observed for annatto. characteristics of the defatted annatto seeds. Therefore, it
The analysis of variance showed that the use of is possible that assistance of ultrasound can prove to be of
ultrasound (LPSE-C and LPSE-P) did not significantly affect importance for process setup employing large ultrasound
the response variables. The cavitation effect of the energy.
274 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

Veggi et al. [4] studied the viability of using suitability of ultrasounds for the preparation of anti-
ultrasound to assist with obtaining extracts rich in oxidant-rich plant extracts.
polyphenols from jatoba bark (Hymenaea courbaril L. var Pingret et al. [5] used apple pomace (Malus domestica
stilbocarpa); these results were compared with those Borkh) for an ultrasound-assisted extraction (UAE) to
from a conventional agitation extraction. A statically produce extracts rich in antioxidants. The optimized
significant (P-value < 0.05) effect was observed for the conditions obtained by response surface methodology
use of the ultrasound (an approximately 15% increase in for extracting polyphenols using water were 315 K, 40 min
total phenolic compounds content) when using the and 0.764 W/cm2 (555 and 420 mg of catechin equivalents
maximum power (60 W). Moreover, the three-stage and per 100 g dry weight, for UAE and conventional agitated
scaled-up stages of the ultrasound experiments demon- extraction, respectively). Both of the tested methods had
strated the efficiency of this process. DPPH analysis the same extraction kinetics. In addition, the extracts
confirmed the higher antioxidant activity of the ultra- obtained by UAE showed a higher antioxidant activity,
sound extracts, and HPLC analysis demonstrated that, which was confirmed by HPLC analysis; this confirmed
besides the increase in yield, ultrasound irradiation that the polyphenols were not degraded under the
modified the composition of the extracts, supporting the conditions applied.

30 Kinecs III (333K/in natura) Kinecs II (333K/defaed)

Kinecs I (323K/defaed) CER Stage
FER Stage DC Stage
25
tFER
20
Yield, % (d.b.)

15 tCER

10

0
0 15 30 45 60 75 90 105 120
S/F
Kinecs III (333K/in natura) Kinecs II (333K/defaed)
30 Kinecs I (323K/defaed) CER Stage
FER Stage DC Stage
25

20
Yield % (d. b.)

15

10

0
0 10 20 30 40 50 60 70 80 90 100
Time, min.
Fig. 4. a: overall extraction curves and splines fitted to the experimental data of Kinetics II (defatted) and III (in natura) as a function of S/F; b: overall
extraction curve and splines fitted to the experimental data of Kinetics of II (defatted) and III (nondefatted) as a function of time.
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 275

3.5. Kinetics of extraction We observe that the tCER of Kinetic III (in natura seeds)
was twice as high as that of Kinetics II obtained using
From the results obtained for X0(%) and BY(%), two defatted seeds. The presence of oil in the seeds made it
conditions were selected for the kinetic study. The difficult for the solvent to access the substrate. Comparing
extraction conditions were selected in consideration of Kinetics I and II, significant differences can be noted in the
the levels of the independent variables that maximized the kinetics parameters. The tCER for Kinetics I was smaller, and
response variables, X0(%) (Kinetics I: LPSE/323 K/ethanol) the value of RCER was similar to that of Kinetics II.
and BY(%) (Kinetics II: LPSE/333 K/ethanol). A kinetic assay Therefore, the method that has the lowest energy
(Kinetics III) was established to test the condition BY(%) consumption and processing time should be chosen: LPSE
using nondefatted seeds in order to study the differences in at 323 K. Still, for a more appropriate selection of the
BY(%) in the presence of oil in the seed, considering that the processing conditions, it would be necessary to calculate
presence of oil in the seed can assist in the extraction of the cost of manufacturing (COM), which is beyond the
bixin and affect its solubility in the oil. For all kinetics objectives of this study. In short, COM is directly related to
experiments, the flow rate was standardized at 0.34 g/min. the production rate, the cost of raw material (annatto seeds
Fig. 4a and b show OECs for Kinetics I, II and III. It should and solvents), fixed costs (costs of equipment, installation,
be noted that no significant differences in global yield were depreciation, taxes, insurance and so on) and general
obtained by changing the temperatures in the LPSE method expenses including administrative costs, sales expenses,
with defatted seed. In Fig. 4b, it can be seen that bixin research and development. Therefore, the time of extrac-
extract is still being recovered, even for S/F = 60, while S/ tion will affect directly the productivity of the system.
F = 50 resulted in a substantially reduced extraction rate Then, the choice of extraction process depends on both the
compared to the initial values of the process. At quality of the extract as well its cost.
approximately S/F = 14, which is larger than the maximum
value used in the experiments to determine X0(%) (S/ 3.5.2. Extraction yield of bixin in OECs
F = 10), we were able to recover approximately 18% Upon comparing the kinetics of OECs I and II, significant
(Kinetic I) and 9% (Kinetic II) of the extract. Thus, S/ differences are not observed in the results for the total
F = 10 was insufficient to exhaust the vegetal raw material; yield; however, the kinetic behavior differed, as tCER at
in spite of this, the observed behavior due to increasing S/F 323 K was lower, and both OECs obtained an MCER of 5.01
remained the same, as X0(%) is an intensive variable [29]. and 5.63 g.min1, respectively. Thus, there are advantages
The exhaustive extractions for Kinetics I and II were to using the extraction conditions of Kinetics I (Ethanol/
achieved at approximately S/F = 120 over 85 minutes LPSE/323 K). However, the overall yield cannot be con-
(Fig. 4a); during this time, 94 and 93% of the total weight sidered as a comparative parameter alone because a higher
of extractable compounds was recovered, respectively. yield does not necessarily reflect higher yields of the target
For the macela extract (Achyrocline satureioides), using substance. Instead, higher yields may represent the
fixed-bed percolation and ethanol as a solvent, S/F = 20 was coextraction of compounds other than those of interest.
sufficient to exhaust the raw material, according to Therefore, it is necessary to analyze the yield and chemical
Takeuchi et al. [10]. composition of the extract obtained in order to determine
the best processing conditions to satisfy the conditions of
3.5.1. Kinetic calculations obtaining the compound of interest [29].
The OEC provides the information necessary to The OECs obtained for BY(%) are shown in Fig. 5, which
calculate the kinetic parameters, including the processing shows that a significant decrease in BY(%) can be observed
time. Using straight-lines splines adjusted to the experi- when using nondefatted seeds.
mental data, we obtained the kinetic parameters in Table 4.
The slope of the first line represents the rate of mass 30 Kinecs I (323K/defaed)
transfer of the CER period (MCER), and the time correspond- Kinecs II (333K/defaed)
ing to the intersection between the first and second lines Kinecs III(333K/in natura)
Bixin Yield, % (d.b.)

25
was identified as tCER, which represents the duration of the
CER period and may be considered the minimum time for 20
the extraction process. The concentration of the extract
leaving the bed (YCER) was calculated as the ratio between 15
MCER and the mass flow rate of the solvent. RCER is the yield
obtained during the CER period [29]. 10

Table 4 5
Kinetic parameters.

Kinetic parameters Conditions of processing 0

0 10 20 30 40 50 60 70 80 90 100
Kinetics I Kinetics II Kinetics III Time, min.
tCER (min) 7.35 18.74 44.99
Fig. 5. Yield curves for the extraction of bixin BY(%) from annatto seeds
MCER (g.min1) 0.0208 0.00914 0.00713
(Bixa orellana L.) under different processing conditions. Kinetics I: LPSE,
RCER (%) 5.01 5.63 10.54
323 K, defatted seeds; Kinetics II: LPSE, defatted seeds and Kinetics III:
YCER (g extract.g1 ethanol) 0.0612 0.0269 0.021
LPSE, 333 K, nondefatted seeds.
276 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

Vatai et al. [30] performed extractions of phenolic 333 K. For the purposes of comparison, the authors also
compounds from elderberries and grapes in a single step pretreated the raw materials with supercritical CO2 (with
with ethanol, ethyl acetate and acetone in different or without ethanol as a cosolvent), and the residual
proportions with water, at temperatures of 293, 313 material was re-extracted with a 50% ethanol–water
and 333 K. Extractions in two stages, combining super- mixture to 333 K. Results show that pretreatment with
critical fluid extraction (SFE) and conventional extrac- supercritical carbon dioxide improved the extraction of
tions, have also been used. Conventional extraction was phenolic compounds. The phenolic yield obtained in
more efficient than one-step extractions, in which extractions over two stages was significantly higher
mixtures of organic solvent and water were used at compared to the single-step process.

Fig. 6. a: nondefatted annatto seeds, magnified 45; b: nondefatted annatto seeds, magnified 1000.
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 277

3.6. Scanning Electron Microscopy (SEM) defatted seeds extracted by LPSE-C (333 K), defatted seeds
extracted in Kinetics I, defatted seeds extracted in Kinetics II,
With the purpose of analyzing the surfaces of annatto and defatted seeds extracted in Kinetics III.
seeds as a means of better understanding the results Fig. 6a and b show the nondefatted seeds with their
obtained for the different extraction methods, SEM analyses homogeneous and intact structures. In Fig. 7a and b, the
were performed in 7 seed samples: nondefatted, defatted modifications caused by the supercritical fluid extraction
seeds, defatted seeds extracted by PLE (333 K and 10 MPa), of the oil can be observed; the surface appears to be more

Fig. 7. a: annatto seeds after extraction with supercritical CO2 (313 K and 20 MPa), magnified 45; b: annatto seeds after extraction with supercritical CO2
(313 K and 20 MPa), magnified 1000.
278 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

porous, which may have helped in the extraction of produced by the ultrasound bath. This shows that bixin is
defatted bixin seeds. indeed present in the surface of annatto seeds. At larger
Fig. 8a and b and Fig. 9a and b show, respectively, magnification (Fig. 8b), the arils remained in the seeds’
defatted seeds after extractions by LPSE-C (333 K) and PLE surfaces. The changes in their structures can be better
(333 K, 10 MPa), both of which were performed with observed through comparison with the corresponding
ethanol. In Fig. 8a, certain highlighted structures were surfaces in Fig. 6b, taken before the extraction was
visualized; this may be due to the effect of the cavitation performed assisted by ultrasound. A visible effect of the

Fig. 8. a: defatted annatto seeds after extraction with LPSE-C at 333 K using a continuous ultrasound, magnified 45; b: defatted annatto seeds after
extraction with LPSE-C at 333 K using a continuous ultrasound, magnified 1000.
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 279

ultrasound appears in the resinous structure, facilitating Fig. 10a and b show micrographs of defatted seeds after
the extraction. Fig. 9a and b show micrographs of the the extraction of bixin for 95 minutes at 323 K/LPSE
defatted seeds after PLE. Note the effect of the pressurized (Kinetic I). Damage to the structure can be observed; the
liquid on the resinous aril, which appears to have a perceived disruption and displacement of the surface may
rounded shape (Fig. 7b). After extraction with pressurized be the result of a longer exposure time of the seeds to the
liquid, this structure was broken. solvent at elevated temperatures.

Fig. 9. a: defatted annatto seeds after extraction with PLE at 333 K and 2 MPa, magnified at 45; b: defatted annatto seeds after extraction with PLE at 333 K
and 2 MPa, magnified 1000.
280 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

Fig. 10. a: defatted annatto seeds after extraction with ethanol at 323 K/LPSE for 95 minutes (Kinetics I), magnified 45; b: defatted annatto seeds after
extraction with ethanol at 323 K/LPSE for 95 minutes (Kinetic I), magnified 1000.

Fig. 11a and b are micrographs of defatted seeds after used. In Fig. 12a and b, the surfaces of the annatto seeds
extraction for 95 minutes at 333 K/LPSE (Kinetic II). were similar to those of nondefatted seeds (Fig. 6a and b),
Compared with Fig. 10a and b, it is clear that temperatures while Fig. 11a and b show a substantial removal of the
influence the displacement of the seeds’ surfaces. surfaces.
We were able to easily observe the differences between Micrographs helped us to understand the results for
the seeds shown in Fig. 12a and b, which had not BY(%). Some authors have claimed that bixin is in the aril of
undergone the pretreatment (defatting step), and those the surface of the annatto seeds. Noble et al. [31,32] stated
shown in Fig. 11a and b, for which defatted seeds were that the yield increased without milling the seeds, possibly
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 281

Fig. 11. a: defatted annatto seeds after extraction with ethanol at 333 K/LPSE for 95 minutes (Kinetics II), magnified 45; b: defatted annatto seeds after
extraction with ethanol at 333 K/LPSE for 95 minutes (Kinetic II), magnified 1000.

due to the localization of these compounds on the surface 1 minute for 20 minutes) had yields of approximately 24%.
of the seed. The highest value of BY(%) was obtained under In the LPSE experiments for the determination of X0(%),
the conditions LPSE-C/333 K/defatted seeds. Observing lower yields were obtained using water, indicating that
Fig. 8a and b, one realizes that these seeds, compared to agitation is an important variable.
other seeds analyzed by SEM, were most affected on their Barrozo et al. [2] studied the mechanical extraction of
surfaces; this finding explains the results of this method. bixin seeds using a spouted bed and observed the effects of
During the preliminary extractions tests (Table 1) using the air flow and seed mass on the bed, as well as the effect
water at ambient conditions and manual agitation (every of inserting a suction tube onto the powder-rich bixin. On
282 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283

Fig. 12. a: defatted annatto seeds after extraction with ethanol at 333 K/LPSE for 95 minutes (Kinetics III), magnified 45; b: defatted annatto seeds after
extraction with ethanol at 333 K/LPSE for 95 minutes (Kinetic III), magnified 1000.

average, the yield of these extractions was about three The results of this study allow us to infer that the use of
times higher when using the suction pipe. The authors seeds previously defatted by supercritical CO2 as a raw
concluded that a 10% increase in air flow caused an average material facilitates the displacement and subsequent
increase of 30% in mass extracted, whereas a 25% increase separation of the dye layer of the seed, providing bixin
in the seed bed caused an average increase of 33% in the extraction yields of approximately 25% (LPSE/333 K/
mass extracted. ethanol). This yield can be obtained without the need
 L.M. Rodrigues et al. / C. R. Chimie 17 (2014) 268–283 283

for agitation or friction, instead using a simple system for References

extraction by percolation.
[1] Joint FAO/WHO Expert Committee on Food Additives, Compendium of
Food Additive Specifications, 67th meeting, 2006.
4. Conclusions [2] M.A.S. Barrozo, K.G. Santos, F.G. Cunha, Ind. Crops Prod. 45 (2013) 279–
282.
[3] H.D. Preston, M.D. Rickard, Food Chem. 5 (1980) 47–56.
The best method to obtain high yields in bixin was [4] P.C. Veggi, D.T. Santos, A.-S. Fabiano-Tixier, C. Le Bourvellec, M.A.A.
determined to be low-pressure solvent extraction using Meireles, F. Chemat, Food Public Health 3 (2013) 119–129.
ethanol as a solvent. While water can be used, ethanol [5] D. Pingret, A.-S. Fabiano-Tixier, C. Le Bourvellec, C.M.G.C. Renard, F.
Chemat, J. Food Eng. 111 (2012) 73–81.
produces higher yields and allows for the process to be [6] M. Virot, V. Tomao, C. Le Bourvellec, C.M.C.G. Renard, F. Chemat,
coupled to a particle formation method, such as SAS Ultrason. Sonochem. 17 (2010) 1066–1074.
(supercritical anti-solvent), to produce solid and micro- [7] M. Plaza, S. Santoyo, L. Jaime, B. Avalo, A. Cifuentes, G. Reglero, G.
Garcia-Blairsy Reina, F. Javier Senorans, E. Ibanez, LWT – Food Sci.
sized particles. According to the results of the OECs and
Technol. 46 (2012) 245–253.
micrographs, the use of pretreatment with supercritical [8] D.T. Santos, P.C. Veggi, M.A.A. Meireles, J. Food Eng. 101 (2010) 23–31.
fluid to extract the oil is able to increase the bixin yield [9] F. Asma, R. Mehrez, C. Farid, in: M. Neffati, A.O. Belgacem, M. El Mourid
through a low-cost methodology (LPSE). It has been (Eds.), International Symposium on Medicinal and Aromatic Plants -
Sipam 2009, 2010, 251–262.
demonstrated that PLE does not improve the yield using [10] T.M. Takeuchi, M.L. Rubano, M.A.A. Meireles, Food Bioprocess Technol.
either water or ethanol. The use of an ultrasound bath 3 (2010) 804–812.
had no effect on the yield, but it should not be ruled out, [11] C.L.C. Albuquerque, M.A.A. Meireles, J. Supercrit. Fluids 66 (2012) 86–95.
[12] M.E.M. Braga, P.A.D. Ehlert, L.C. Ming, M.A.A. Meireles, J. Supercrit.
because effects from the ultrasound bath were observed Fluids 34 (2005) 149–156.
in micrographs, showing that the cavitation surface of [13] T. Hatami, R.N. Cavalcanti, T.M. Takeuchi, M.A.A. Meireles, J. Supercrit.
the ultrasound assisted in the extraction. However, the Fluids 65 (2012) 71–77.
[14] J.M. Prado, I. Dalmolin, N.D.D. Carareto, R.C. Basso, A.J.A. Meirelles, J.V.
use of an ultrasonic probe is recommended. LPSE was Oliveira, E.A.C. Batista, M.A.A. Meireles, J. Food Eng. 109 (2012) 249–257.
found to be more feasible at temperatures of approxi- [15] G.F. Silva, F.M.C. Gamarra, A.L. Oliveira, F.A. Cabral, Braz. J. Chem. Eng.
mately 323 K, as there were small differences in X0(%) 25 (2008) 419–426.
[16] M.E.M. Braga, M. Angela, A. Meireles, J. Food Process Eng. 30 (2007)
when the temperature was increased to 333 K. In spite of
501–521.
this, the literature states that the higher the temperature, [17] D.T. Santos, M.A.A. Meireles, Innovative Food Sci. Emerg. Technol. 12
the higher the yield of bixin extraction. This is because (2011) 398–406.
[18] L.J. Wang, C.L. Weller, Trends Food Sci. Technol. 17 (2006) 300–312.
heated cis-bixin is converted to its more soluble form,
[19] R.C. Chiste, A.Z. Mercadante, A. Gomes, E. Fernandes, J.L. Fontes da Costa
trans-bixin. Still, using higher temperatures should result Lima, N. Bragagnolo, Food Chem. 127 (2011) 419–426.
in a significant increase in the yield; otherwise, the [20] M.A. Rostagno, A. Villares, E. Guillamon, A. Garcia-Lafuente, J.A. Marti-
increase in energy expenditure would not be justified. nez, J. Chromatogr. A 1216 (2009) 2–29.
[21] D. Pingret, A.S. Tixier-Fabiano, F. Chemat, Ultrasound-assisted extrac-
The use of raw materials previously defatted by super- tion, in: M.A. Rostagno, J.M. Prado (Eds.), Natural product extraction:
critical CO2 here is proven to be beneficial to the principles and applications, RSC Publishing, London, 2013, pp. 89–112.
extraction of bixin. [22] M.D. Escaplez, J.V. Garcı́a-Pérez, A. Mulet, J.A. Cárcel, Food Eng. Rev.
(2011) 108–120.
[23] J.Q. Albarelli, R.B. Rabelo, D.T. Santos, M.M. Beppu, M.A.A. Meireles, J.
Supercrit. Fluids 58 (2011) 343–351.
Disclosure of interest [24] A. Sluiter, B. Hames, D. Hyman, C. Payne, R. Ruiz, C. Scarlata, J. Sluiter,
D. Templeton, J. Wolfe, National Renewable Energy Laboratory, Color-
ado (2008).
The authors declare that they have no conflicts of
[25] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton,
interest concerning this article. Laboratory analytical procedure, National Renewable Energy Labora-
tory, Golden, 2008.
[26] N.J. Thiex, S. Anderson, B. Gildemeister, J. AOAC Int. 86 (2003) 888–898.
Acknowledgements [27] N.J. Thiex, H. Manson, S. Anderson, J.A. Persson, J. AOAC Int. 85 (2002)
309–317.
[28] V.M. Rodrigues, E. Sousa, A.R. Monteiro, O. Chiavone-Filho, M.O.M.
The authors acknowledge the financial support from
Marques, M.A.A. Meireles, J. Supercrit. Fluids 22 (2002) 21–36.
CAPES (DEA/FEA/PROEX), CNPq (470916/2012-5) and [29] M.A.A. Meireles, Extraction of bioactive compounds from Latin Amer-
FAPESP (2009/17234-9 and 2012/10685-8). L.M. Rodrigues ican plants, CRC Press–Taylor & Francis Group, USA, 2008.
and S.C. Alcazar-Alay thank CAPES for the MSc and Ph.D. [30] T. Vatai, M. Skerget, Z. Knez, J. Food Eng. 90 (2009) 246–254.
[31] Z. Pineiro, M. Palma, C.G. Barroso, J. Chromatogr. A 1026 (2004) 19–23.
assistantships, respectively. M.A.A. Meireles thanks CNPq for [32] B.P. Nobre, R.L. Mendes, E.M. Queiroz, F.L.P. Pessoa, J.P. Coelho, A.F.
the productivity grant (301301/2010-7). Palavra, Braz. J. Chem. Eng. 23 (2006) 251–258.