J.

of Supercritical Fluids 51 (2010) 319–324

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Extraction of phenolic fraction from guava seeds (Psidium guajava L.) using
supercritical carbon dioxide and co-solvents
Henry I. Castro-Vargas a , Luis I. Rodríguez-Varela b , Sandra R.S. Ferreira c , Fabián Parada-Alfonso a,∗
a
Chemistry Department, Universidad Nacional de Colombia, 30 Avenue and 45 street, Bogotá D.C., Colombia
b
Chemical Engineering Department, Universidad Nacional de Colombia, Bogotá D.C., Colombia
c
Chemical and Food Engineering Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

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

Article history: In this work the supercritical fluid extraction (SFE) with carbon dioxide (CO2 ) and with ethyl acetate (EtAc)
Received 21 May 2009 and ethanol (EtOH) as co-solvents was applied to obtain the phenolic fraction from guava seeds (Psidium
Received in revised form 19 October 2009 guajava L.). The extraction was explored at various operating conditions, using 10, 20 and 30 MPa and 40,
Accepted 28 October 2009
50 and 60 ◦ C. The use of EtAc and EtOH as co-solvents in SFE was also studied. The supercritical process
was compared with traditional techniques such as Soxhlet extraction using EtAc and EtOH as solvents.
Keywords:
The quality of the different extracts, obtained using SFE and Soxhlet methods and different solvents, was
Guava seeds
evaluated through the antioxidant activity, obtained by the collection methods of scavenging DPPH and
Psidium guajava L.
Supercritical fluids
bleaching of ␤-carotene, and also through the total phenolic content (TPC) of the samples, by the Folin-
Total phenolic content Ciocalteu method. The antioxidant potential indicates the use of ethanol as co-solvent as the best modifier
Antioxidant activity in SFE, used in concentration of 10% (w/w) at 50 ◦ C and 30 MPa. The quality of the extracts obtained by SFE
with EtOH varied with the operating conditions of temperature and pressure, with higher values obtained
at 10 and 20 MPa for TPC results and also antioxidant methods. The process yield of the phenolic fraction
was also evaluated for all the extraction procedures studied (SFE and Soxhlet), with results varying from
0.380 to 1.738% (w/w).
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Guava (Psidium guajava L.) is a fruit widely distributed in tropical
America, from Mexico to Brazil, as well as in Asia and Africa (South
Africa, India, Algeria and Tunisia), mainly in zones of warm cli-
mate. The guava fruit represents an important economical product
in Colombia, with annual production of 110,000 tonnes in 2007 [1].
This fruit is desired by its high nutritional value [2,3] and important
medicinal properties; some investigations established characteris-
tics such as anti-inflammatory, analgesic, antipyretic, spasmolitic
and anti-bacterial activities from guava [4–6]. Related to guava
seeds, only few studies were performed about its use. Recently,
the presence of compounds with cytotoxic activity in guava seeds
was demonstrated [7]. To the best of our knowledge, the use of
supercritical fluid extraction to obtain guava seeds oil was only
performed by our research group [8].
The supercritical fluid extraction (SFE) is a relatively recent tech-
nique which presents various advantages over traditional methods,
like the use of low temperatures and reduced energy consumption
and high product quality due to the absence of solvent in solute
phase [9,10]. Supercritical carbon dioxide (SC-CO2 ) is the most used
solvent for SFE due to its particular characteristics such as moderate
critical conditions (31.1 ◦ C and 73.8 MPa) and of easy availability. It
is also non-toxic, inflammable and chemically stable. In the areas
of cosmetics, essences, foods and agricultural products, the appli-
cations of SFE are multiple, from decaffeination of coffee and tea,
to extraction of flavors, aromas, pigments, antioxidants and spices,
among others [11].
In the present work, new possibilities of enhance the productiv-
ity chain of guava were studied, especially due to the economical
importance of guava for Colombia. The supercritical technology
was applied for the extraction of phenolic fractions with antiox-
idant activity from guava seeds, using SC-CO2 and SC-CO2 with
co-solvents. This alternative method was compared to traditional
extraction methods such as Soxhlet, in terms of process yield and
product quality evaluated by antioxidant activity of the extracts.
2. Materials and methods

The study of the guava seeds extraction was performed in order

∗ Corresponding author. Tel.: +57 1 3165000x14448; fax: +57 1 3165220. to obtain the phenolic fraction and the total extract. The assays
E-mail address: fparadaa@unal.edu.co (F. Parada-Alfonso). were divided in three groups:

0896-8446/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2009.10.012
320 H.I. Castro-Vargas et al. / J. of Supercritical Fluids 51 (2010) 319–324

(1) Soxhlet extraction (SE) using ethyl acetate (EtAc) and ethanol
(EtOH) as solvents.
(2) SFE at 50 ◦ C and 30 MPa, performed with pure CO2 , and with
CO2 added with EtAc and EtOH as co-solvent at concentra-
tion of 10% (w/w). The operating condition of temperature and
pressure used in this assays was defined based on yield data
reported in literature (for guava seeds with pure SC-CO2 [8])
indicating 30 MPa and 50 ◦ C as the best conditions to obtain the
phenolic fraction. The concentration of co-solvent in SFE was
defined based on literature data for tamarind seeds [12,13] and
the assays were performed at fixed condition of 10% (w/w) of
co-solvent.
(3) The effect of temperature and pressure in SFE was evaluated
considering the best extraction agent (solvent) found in assay
group (2). The solvent agent was based on process yield and
extract (phenolic fraction or total extract) quality, evaluated in
terms of antioxidant activity and total phenolic content. Then,
based on results session, this assay group was performed in SFE
with CO2 and EtOH as co-solvent, at concentration of 10% (w/w),
in different extracting conditions (40, 50 and 60 ◦ C and 10, 20
and 30 MPa).

Each extraction was carried out in triplicate. In SE and SFE with

co-solvent the total extract obtained was in two phases: lipid phase
(oil) and solvent phase, the phenolic fraction. The phases were
separated and the high polarity phase was submitted to further
investigation concerning the antioxidant analysis. In SFE with pure Fig. 1. Supercritical fluid extraction unit.

CO2 the extract was submitted to liquid–liquid extraction obtained

the phenolic fraction. All extracts were concentrated by rotaevapo-
rator at 40 ◦ C for posterior reconstitution to a final volume of 25 mL
in ethanol.
The results from all assays were evaluated considering the
extraction yield (total and phenolic fraction) for guava seeds and
also the extract quality (antioxidant activity and total phenolic con-
tent), performed by the methods described as follows.
2.1. Materials and sample preparation
The guava seeds were obtained from the residue material from
different industries of guava jelly located in the municipality of
Barbosa (Santander, Colombia). The guava seeds were washed and
dried at room temperature for 24 h until the final moisture con-
tent 11.8%. The samples were cold-crushed using solid CO2 and
then the guava seeds particles were separated and the fraction
selected for extractions presented particle size from 0.2 to 0.5 mm.
The grounded samples were conditioned in plastic bags at 5 ◦ C until
the extraction was performed.
The CO2 used was 99.9% of purity (Ingegas, Colombia). Ethanol,
ethyl acetate, chloroform, 2,2-diphenyl-1-picryl-hydrazyl (DPPH),
␤-caroteno and methyl linoleate were purchased in analytical
degree from Biocol Ltda (Colombia). Ethanol, ethyl acetate and chlo-
roform were distillated previous from use.
2.2. Supercritical fluid extraction unit and experimental
procedure
For the development of this research a SFE unit was assembled
and the extraction equipment is shown in Fig. 1. This unit allows
the extraction in static mode using SC-CO2 and SC-CO2 with co-
solvents. The unit was manufactured using stainless steel 316 and
is formed by the following parts: (1) CO2 reservoir; (2) pneumatic
pump (HASKEL® model AGT-7/30, CA, USA); (3) mixture cell for
CO2 and co-solvent, consisted of a stainless steel tube with 0.8-in.
internal diameter with sealed extremities gathered by thermo
dilatation/contraction (TDC) technique [14]; (4) extraction cell
with 50 mL; (5) 150 mL separator vessel; (6) thermocouple type K
(WATLOW serie SD 31, MI, USA, ±1 ◦ C) connected to extraction cell
which was heated by a 300 W resistance; (7) sample flask collec-
tors. The flow was controlled by valves (WHITEY series SS-1VS4 y
SS-1VR4, OH, USA) (a–g) and the pressure system was monitored
by analogical manometers (BOURDON HAENNI, Vendôme cedex,
France, 700 bar) (h–j).
The extraction procedure consisted of placing the sample
(grounded guava seeds) inside the extraction cell (30 g for each
assay) and submitted to the extraction. Then, the unit was pres-
surized and the solute was kept in contact with SC-CO2 or SC-CO2
with co-solvent at pre-established conditions of temperature and
pressure for 30 min in static mode. Then the valve e (Fig. 1) was
open for complete depressurization of the extraction cell (4), CO2
get out of separator vessel (5) when upper valve (f) was open. The
extract was obtained in the sample flask collector (7). The extrac-
tion/depressurization process was repeated four times for each
sample inside the extractor, until completing a total extracting time
of 2 h. The time interval and the total extraction time were defined
based on preliminary assays.
2.3. Soxhlet extraction (SE)
Extractions with Soxhlet method were performed using ethyl
acetate (EtAc) and ethanol (EtOH) as solvents [15,16], in descend-
ing order of polarity. The soxhlet method consisted of 30 g ground
guava seeds placed inside a thimble made by thick filter paper and
loaded into the main chamber of the soxhlet extractor, composed
by an extracting tube, a glass balloon and a condenser. The total
extracting time was 10 h and 250 mL of solvent were used for each
extraction, with solvent continuously refluxing over the sample.
After the extraction the solvent was removed by rotaevaporator
at 40 ◦ C, for further reconstitution of the extracts, as previously
mentioned.
2.4. Extracting conditions
As stated previously, the assay group (3) was based on quality
results of the extracts collected in assay (2), which indicate the
 H.I. Castro-Vargas et al. / J. of Supercritical Fluids 51 (2010) 319–324 321

solvent mixture formed by SC-CO2 and EtOH as the best extraction Table 1
Yield obtained in different extraction processes.
agent for the phenolic fraction. Therefore, the supercritical fluid
extraction was performed with CO2 and 10% (w/w) of EtOH as co- Assay group Solvent ◦
C/MPa PFa TEb
solvent using different extracting conditions: temperatures of 40, c
Yield (%) SD Yield (%) SD
50 and 60 ◦ C and pressures of 10, 20 and 30 MPa. A factorial design
1 EtAc 0.380 0.010 4.603 0.900
of 32 was used, with nine experiments for the conditions studied,
1 EtOH 6.730 0.500 10.74 0.900
all of then performed in triplicate. 2 CO2 50/30 0.389 0.003 1.392 0.020
2 CO2 /EtOH 50/30 1.182 0.020 16.367 0.200
2 CO2 /EtAc 50/30 0.451 0.003 14.536 0.200
2.5. Antioxidant activity DPPH scavenging method
3 40/10 1.254 0.020 15.530 0.200
3 40/20 1.513 0.010 17.064 0.050
The 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical method
3 40/30 1.738 0.005 19.033 0.300
was based on procedure proposed by Brand-Williams [17] and 3 50/10 0.822 0.010 13.567 0.100
considered the modifications presented for analysis of sample 3 CO2 /EtOH 50/20 0.958 0.090 14.571 0.300
composed by fruit extracts [18,19]. Briefly, 2 mL of DPPH solu- 3 50/30 1.182 0.020 16.367 0.200
3 60/10 0.767 0.010 12.281 0.200
tion 0.1 M in ethanol, with absorbance measured at 517 nm (A0 ).
3 60/20 0.901 0.008 13.311 0.010
Then, 50 ␮L of extract was added and the absorbance measured 3 60/30 1.265 0.030 16.197 0.100
after 60 min (Af ). The inhibition percentage was determined as a
PF: phenolic fraction.
%inhibition = (A0 − Af )/A0 . The results were compared with TROLOX b
TE: total extract.
solutions (0.08 a 1.24 mM), as antioxidant references. Results are c
SD: standard deviation.
expressed in Trolox equivalent antioxidant capacity (TEAC in mmol
of Trolox/100 g of guava seeds) [18,20–22].
3. Results and discussion

2.6. Antioxidant activity ˇ-carotene bleaching method 3.1. Extraction yield

The technique proposed by Miller was applied to deter-
mined the antioxidant activity by ␤-carotene bleaching method
[23,24]. This method is based on the reaction of ␤-carotene
with free radicals, reducing its absorbance capacity at 470 nm.
Methyl linoleate/␤-carotene emulsion was prepared using 2 mL
of ␤-carotene solution in chloroform (1 mg/mL) and mixing
with 40 ␮L methyl linoleate and 400 ␮L de Tween 20. In three
test tubes 5 mL of emulsion were added, the first for con-
trol, the second with 50 ␮L of 2,6-di-tert-butyl-4-methyl phenol
(BHT 0.3 mg/mL), as the reference antioxidant compound, and
third with 50 ␮L of phenolic fraction. The tubes were incu-
bated at 50 ◦ C and the absorbance at 470 nm was measured
in 20, 40, 60, 80 and 100 min. The antioxidant activity was
calculated considering the velocity of ␤-carotene bleaching of
control (Vc ) and sample (Va ), according to the relation: %
AA = (Vc − Va )/Vc .
2.7. Determination of total phenol content (TPC)
The total phenols concentration (TPC) in the guava extracts
was determined by the Follin-Ciocalteu method [25,26]. A cal-
ibration curve was prepared with 50 ␮L galic acid solutions
(0.002–0.01 mg/mL) in 0.5 mL of Follin-Ciocalteu reagent and
1.5 mL Na2 CO3 20%, maintained in dark for 90 min. The absorbance
was measured at 760 nm (Thermo ELECTRON CORP., GENESYS
10uv, CA, USA). The TPC values of the extracts were performed
according to calibration curve procedure and expressed as galic
acid equivalent in 100 g of guava seeds (mg GA/100 g seeds)
[16,27].
The results of extraction yield obtained by the three groups of
assays: SE (with EtAc and EtOH) and SFE (with CO2 , CO2 /AcEt and
CO2 /EtOH) are presented in Table 1. The highest extraction yields in
SE were obtained using EtOH as solvent, especially for the phenolic
fraction, a result of the higher polarity of this solvent compared with
EtAc. The yield of the SFE process in terms of phenolic fraction is
also lower than the value obtained by SE-EtOH, although the total
extraction yields for SFE with CO2 /EtOH are mainly higher. This
behavior is explained by the non-polar characteristic of the carbon
dioxide, which increases the extraction of low polarity compounds,
compared with polar ones (particularly found in the phenolic frac-
tion).
In SFE the yield results (phenolic and total) increase directly
with solvent polarity (CO2 , CO2 /EtAc and CO2 /EtOH), particularly,
the use of EtOH as co-solvent is useful to enhance the pheno-
lic fraction yield. The highest yield in SFE with CO2 /EtOH was at
30 MPa and 40 ◦ C (1.738%, w/w phenolic fraction and 19.033%, w/w
total extract). The effect of extracting conditions in SFE is pre-
sented in Fig. 2 by the yield isotherms for phenolic fraction and
total extract. At constant temperature, the increase in pressure
increases the yield due to the density enhancement. At constant
pressure, the phenolic and the total yield decrease with temper-
ature increase due to the solvent density reduction, although, at
30 MPa the variation of temperature from 50 to 60 ◦ C produce an
increase in phenolic yield, probably due to the enhancement in
the solute vapor pressure, the ANOVA show significant differences
between each data. This behavior is an indication of a crossover
pattern, nearly to 25 MPa [10,28].
3.2. Quality data for assays groups 1 and 2

2.8. Statistical analysis
An analysis of variances (ANOVA) for each experiment (extrac-
tion yield and quality evaluation) was carried out with the purpose
to determine significant differences between the treatments car-
ried out. The results are reported as standard deviation ±SD
(standard deviation) obtained from the three measurements. The
assays were performed in triplicate for each extracting condition
(Soxhlet with different solvents, election of the extracting agent
and extracting conditions).
The results that support the selection of the mixture CO2 plus
EtOH (10%, w/w) as solvent in SFE are the combination of antioxi-
dant activity performed by DPPH and ␤-carotene methods and TPC
method performed for extract samples obtained in assays from
groups 1 and 2. The results are expressed in Table 2 where it is
possible to observe that the results for DPPH method (in TEAC) and
TPC are higher for samples SE-EtOH, followed closely by SFE with
CO2 added with EtOH, while the ␤-carotene (% AA) assays indicate
the sample obtained by SFE-CO2 /EtOH as the best results among
the samples analyzed. This behavior suggests that the EtOH is a
322 H.I. Castro-Vargas et al. / J. of Supercritical Fluids 51 (2010) 319–324
Fig. 2. Effect of pressure in three isotherm yields (40, 50 and 60 ◦ C) for SFE obtained
with CO2 added with 10% (w/w) EtOH as co-solvent: (a) yield in phenolic fraction;
(b) total yield.
good co-solvent for SFE in order to obtain high valuable compounds.
Therefore, the process SFE (CO2 /EtOH) was selected to evaluate the
pressure and temperature effect on the quality extract.
Comparing the TPC results for different extraction methods pre-
sented in Table 2 (assays groups 1 and 2) we observe that the
best relation between % AA and TPC was obtained by the SFE-
CO2 /EtOH, with higher TPC (131 ± 5 mg GA/100 g seeds) and TEAC
of 130 ± 3 mmol Trolox/100 g seeds and % AA of 63 ± 5. For the Soxh-
let extraction, the solvent EtOH result in higher TPC and antioxidant
activity (172 ± 6 mg GA, 224 ± 10 mmol Trolox/100 g seeds and
44 ± 6% AA), due to the large solvent polarity, compared with EtAc.
Comparing SFE and SE, the phenolic fraction by SE-EtOH presented
higher TPC and % AA by DPPH method (TEAC), while % AA by ␤-
carotene bleaching was lower than for SFE with CO2 /EtOH.
3.3. Antioxidant activity
3.3.1. DPPH scavenging method
Fig. 3 shows the results obtained for antioxidant activity
by DPPH method related to the phenolic fractions extracted
using SFE with CO2 /EtOH at different conditions (40, 50 and
60 ◦ C and 10, 20 and 30 MPa). The results expressed in TEAC
values varied from 81 ± 12 mmol Trolox/100 g seeds (fraction
obtained at 40 ◦ C/10 MPa) to 136 ± 3 mmol Trolox/100 g seeds
(fraction obtained at 60 ◦ C/10 MPa). The conditions 60 ◦ C/20 MPa
and 60 ◦ C/30 MPa also presented high TEAC values.
In Fig. 3 we also observe an increase in TEAC (antioxidant poten-
tial) with extraction temperature, for all pressures studied. The
Table 2
Quality results in terms of antioxidant activity (by DPPH and ␤-carotene methods) and TPC, for SFE (50 ◦ C/30 MPa: CO2 , CO2 with co-solvent) and for SE (EtOH and EtAc).
Analytical method Supercritical fluid extraction
CO2 CO2 /EtAc
% AA (␤-carotene) 41 ± 2 39 ± 2
TEAC (mmol Trolox/100 g seeds) 19 ± 1 36 ± 1
TPC (mg GA/100 g seeds) 72 ± 2 50 ± 1
Fig. 3. DPPH results (TEAC) for the phenolic fraction obtained by SFE with CO2 /EtOH.
Fig. 4. Antioxidant activity percentage for the phenolic fractions obtained for SFE
with CO2 /EtOH.
pressure effect shows that TEAC increases from 10 to 20 MPa, for the
isotherms at 40 and 50 ◦ C, while from 20 to 30 MPa the TEAC values
were nearly constant for all isotherms. The increase in TEAC with
pressure (up to 20 MPa) could be justified by the density effect of
the solvent, which increases with pressure, enhancing the extrac-
tion for more complex substances such as the ones responsible for
the antioxidant behavior (or responsible for the DPPH scavenging).
The ANOVA results show no significant differences at 5% confi-
dence level on TEAC values for the phenolic fractions obtained at 20
and 30 MPa. Also, at 50 and 60 ◦ C, no significant difference in TEAC
values were detected.
Comparing the results from Fig. 3 with literature data we
observe that phenolic fraction obtained from guava seeds using
SFE-CO2 /EtOH are higher then TEAC values detected for: methano-
lic extracts obtained from different guava genotypes (pulp and peel)
[22]; alcoholic extracts obtained from inga (Inga edulis) seeds [29],
methanolic extracts from different spice and herbs (Curcuma longa,
Valeriana officinalis, Origanum vulgare, Melisa officinalis, Artemisia
vulgaris) [30], ethanolic extracts from star fruit residues [31]; and
different pomegranate juice [32].
3.3.2. ˇ-Carotene bleaching method
The results for % AA by ␤-carotene bleaching method are pre-
sented in Fig. 4 for the phenolic fractions obtained by SFE-CO2 /EtOH
at different extracting conditions. The % AA values varied from
Soxhlet extraction
CO2 /EtOH SE-EtAc SE-EtOH
63 ± 5 38 ± 17 45 ± 12
130 ± 3 95 ± 2 224 ± 4
131 ± 5 133 ± 9 176 ± 10
 H.I. Castro-Vargas et al. / J. of Supercritical Fluids 51 (2010) 319–324
Fig. 5. TPC for the phenolic fractions obtained for SFE with CO2 /EtOH.
39 ± 2 (40 ◦ C/10 MPa) to 79 ± 8 (60 ◦ C/20 MPa). The last value was
close to the correspondent standard used for comparison, the BHT
(82 ± 1), the ANOVA data show no significant differences from these
data.
The effects of temperature and pressure on the % AA are com-
plex. While at 40 and 60 ◦ C the % AA increases with pressure from
10 to 20 MPa, the ANOVA data show significant differences from
these data. At 50 ◦ C, no detectable pressure effect was observed by
ANOVA evaluation. Also, the maximum % AA value was detected at
60 ◦ C and 20 MPa. An increase in % AA was observed at 20 MPa with
enhancing temperature, while at 30 MPa the % AA decreased with
temperature, although all treatments are statistically equals.
3.3.3. Total phenol content
The TPC results from phenolic fractions obtained from guava
seeds, expressed in mg gallic acid/100 g seeds (mg GA) are pre-
sented in Fig. 5. The TPC values varied from 79 ± 4 mg GA at
(60 ◦ C/30 MPa) to 153 ± 1 mg GA (at 60 ◦ C/10 MPa). For the pressure
effect it was detected at 40 ◦ C an increase in TPC with enhancement
in pressure, while at 60 ◦ C the TPC decreased with temperature. The
ANOVA data indicate significant difference among all TPC values.
The temperature effect show an increase in TPC from 40 to 60 ◦ C
at 10 and at 20 MPa, while at 30 MPa the TPC reaches a maximum
at 50 ◦ C, although no significant difference was detected between
50 ◦ C/10 MPa and 60 ◦ C/10 MPa. The antioxidant activity of the phe-
nolic fractions can be related to TPC values considering that the
class of phenolic compounds is well known as to present antioxi-
dant activity [13,18,21,27].
Comparing the TPC results found in guava seed with other litera-
ture data we observe that: TPC data from this work were higher than
values obtained from logan, jackfruit, avocado and tamarind [27],
inga seeds [29], mexican chia seeds [33], while inferior to almond
extracts [34] and tamarind [13], being this one obtained by SFE with
CO2 /EtOH.
4. Conclusions
Different methods were applied to obtain extracts and phenolic
fractions from guava seeds. The SFE with CO2 /EtOH was the best
method used, combining extraction yield (total and fraction) and
product quality (antioxidant activity and TPC). The effect of pres-
sure and temperature on the SFE was observed on yield and quality.
It was demonstrated that the more appropriate condition for the
phenolic extraction by TPC data was 60 ◦ C/10 MPa, with value of
153 mg GA. This result is in agreement with TEAC result (DPPH col-
lection capacity) of 136 mmol trolox/100 g of seeds, nevertheless
related to the inhibition of the bleaching of ␤-carotene, the condi-
323
tions associated to the higher activity were 60 ◦ C/20 MPa (79% AA).
Therefore, guava seeds can be considered as a source of antioxidant
potential, with a wide range of applications in nutritional, cosmetic
and medicine industries. The extraction procedures can provide
a high aggregate-value by-product of fruit residues, fortifying the
guava productive chain in Colombia.
Acknowledgments
Authors thank Ministerio de Agricultura y Desarrollo Rural-
MADR and Asociación Hortifruticola de Colombia-ASOHOFRUCOL
(Project: Obtaining of antioxidants and dietary fibre from guava)
and the Dirección Nacional de Investigación-Universidad Nacional
de Colombia (Project: Application of extraction methods with pres-
surized fluids in the advantage of agricultural product) for their
financing support.
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