Ingeniería e Investigación vol. 36 n.

° 3, december - 2016 (82-88)

DOI: http://dx.doi.org/10.15446/ing.investig.v36n3.55632

Optimization of castor seed oil extraction process

using response surface methodology
Optimización del proceso de extracción de aceite
de semillas de ricino utilizando la metodología
de superficie de respuesta
J.D. Mosquera-Artamonov1, J.F. Vasco-Leal2, A.A. Acosta-Osorio3, I. Hernandez-Rios4,
E.Ventura-Ramos5, E. Gutiérrez-Cortez6, and M.E. Rodríguez-Garcia7

ABSTRACT
This work focuses on the study of the oil extraction yield from castor seed using three different seed conditions: whole, minced and
bare endosperm. Taguchi design was used to determine the contribution of the following parameters: seed condition, seed load
in the extractor, temperature, and pressure. It was proved that it is necessary to introduce the whole seed and that the presence of
the pericarp increases the extraction yield. The contribution of the control factors has an extraction yield limit. After determining
which factors contributed to the process, these were left at their optimum levels aiming to reduce the control factors to only two. The
complete analysis was done using a surface response methodology giving the best parameter for temperature and pressure that allows
a better yielding mechanical extraction. The oil extraction yield can be kept up to 35 % of the seed.
Keywords: Experiment design, mechanical extraction, Ricinus communis L.

RESUMEN
Este trabajo se centra en el estudio de la extracción y la producción de aceite de semillas de ricino usando tres diferentes estados
de semillas: entera, partida y sin testa. Se utilizó un diseño Taguchi para determinar la contribución de los siguientes parámetros:
estado de las semillas, cantidad de semilla en el extractor, temperatura y presión. Se demostró que es necesario introducir la semilla
entera para aumentar el rendimiento de la extracción. La contribución de los factores de control tiene un límite de extracción en
la variable respuesta (rendimiento). Después de determinar cuáles factores contribuían altamente al proceso de extracción y cuáles
no, se procedió a dejarlos en los niveles de mayor contribución con la finalidad de reducir el número de factores de control a dos.
Posteriormente se utilizó la metodología de superficie de respuesta para la optimización del proceso, dando como resultado los
niveles óptimos para los factores de control temperatura y presión. De acuerdo con las pruebas, el rendimiento de la extracción del
aceite se puede mantener en un 35 % de la semilla.
Palabras clave: Diseño de experimentos, extracción mecánica, Ricinus communis L.
Received: January 1st 2016
Accepted: August 31st 2016

Introduction
1
M.Sc in System Engineering, Universidad Autónoma de Nuevo León. M.I in
Castor bean (Ricinus communis L.) is a shrub that originates Quality Engineering from Universidad Autónoma de Querétaro, México. In-
dustrial Engineer by the Universidad Tecnológica de Pereira, Colombia. Affi-
from Africa. Its seed is commonly known as ‘higuerilla’, liation: Ph.D Student in System Engineering, Universidad Autónoma de Nuevo
‘ricine’ or ‘mamona’ (Ali et al., 2008;Scholz & Da Silva 2008). León. México. Email: [email protected]
The optimal growth conditions are loamy to sandy loam 2
M.Sc Sustainable Production of Bioenergy, Colegio de Postgraduados, Agroin-
dustrial Engineer from the Universidad La Gran Colombia, Colombia. Affilia-
soils and temperatures ranging from 20 to 30 °C. In addition, tion: Ph.D Student in Technology and Innovation Management, Universidad
the annual rainfall should be between 700 and 1500 mm Autónoma de Querétaro, México. E-mail: [email protected]
(Valderrama et al., 1994; Ogunniyi, 2006) for the optimal 3
Ph.D in Engineering and M.Sc. in Instrumentation and Automatic Control, for
the Universidad Autónoma de Querétaro, México. B.Sc in Mechanical Engi-
development of the plant. Nevertheless, it adapts to tropical,
neer by the Universidad Tecnológica de Pereira, Colombia. Affiliation: Co-
subtropical, and semiarid conditions, tolerating extreme nacyt Research fellow Instituto Tecnológico de Veracruz, Veracruz, México.
environmental stresses, including high temperatures and low E-mail: [email protected]
water availability. At present, the oil extracted from its seeds
How to cite: Mosquera-Artamonov, J. D., Vasco-Leal, J. F., Acosta-Osorio,
has many different applications, which include but are not A. A., Hernandez-Rios, I., Ventura-Ramos, E., Gutiérrez-Cortez, E., & Rodrí-
limited to: hydraulic oil, paint thinner, emulsifier, varnishes, guez-Garcia, M. E. (2016). Optimization of castor seed oil extraction pro-
pharmaceutical applications, organic fertilizers, biological cess using response surface methodology. Ingeniería e Investigación, 36(3),
82−88. DOI: 10.15446/ing.investig.v36n3.55632
pest control, manufacture of polymers, and dyes (Ogunniyi,
2006; Lorestani et al. 2012), with biodiesel production being
another well-known use (Kılıç et al., 2013). Attribution 4.0 International (CC BY 4.0) Share - Adapt

82
MOSQUERA-ARTAMONOV, VASCO-LEAL, ACOSTA-OSORIO, HERNANDEZ-RIOS, VENTURA-RAMOS, GUTIÉRREZ-CORTEZ, AND RODRÍGUEZ-GARCÍA

Oil from castor bean seeds may undergo changes according The expeller-pressing procedure is widely used in the food
to the extraction procedure (Ortiz et al., 2003). A particularly industry; raw materials are squeezed under high pressure,
noticeable change is the modification in the free fatty acid either in one step or in batches (Pradhan et al., 2011). The
content when heat is used during extraction. Heating extraction is based on a screw that presses the raw materials
around 100 °C generates triricinoleine decomposition of against the walls of a metallic cylinder; oil is recovered
ricinoleic acid and diricinoleine, resulting in increased oil through a mesh that do not allow the passage of solids
acidity (Salimon et al. 2010). (Evangelista 2009). The system is commonly equipped with
a temperature control device to avoid any damage to the oil
The transesterified biodiesel obtained from Ricinus that may affect its properties.
communis L. oil is a potential alternative for solving
problems associated with biofuels produced from other Hydraulic pressing is somehow similar to the expeller
sources (first-generation oil, used cooking oils, waste pressing method, but it yields higher quality oil, and it is
animal fats, etc.), which have shown difficulties in their more economical at an industry level (Sriti et al., 2011);
cold flow properties and oxidative stability, generating however, it works only for seed batch extraction. In this case,
storage problems (Perdomo et al., 2013). a hydraulic piston is used to press raw materials against a
rigid surface with small openings to separate oil and solid
Hincapié et al. (2011) used castor oil to produce biodiesel residues. Extraction may be done at room temperature or
by transesterification, obtaining a conversion rate of 74,9 % under increased seed temperature; in the second case,
when potassium carbonate was used as a catalyst, and oil yield is enhanced, but oil chemical properties may be
66,2 % when the catalyst was hemimorphite. On the other altered (Perdomo et al. 2013).
hand, Montoya et al. (2010) used the response surface
method to optimize the ethanolysis process, achieving a Solvent-extraction methods are based on the addition of
transesterification rate of 93,63 %. These authors have focused organic liquid chemicals to ground seeds, and the mixture
their research mainly on the transesterification process, but is then filtered and subsequently heated to around 150 °C
have not considered the oil extraction procedure from seeds. to remove solvents by evaporation (Qian et al., 2010). Due
to the use of solvents and high temperature, this is a high-
Rios et al. (2007) compared three procedures for oil extraction cost method, and it is used mostly at a laboratory level.
from cardamom seeds. Fontal (2007) worked on oil extraction
by extrusion from coffee seeds, carrying out sensorial tests The aim of this research was to optimize the extraction
and the analysis of the volatile compounds in the oil, without process of oil from Ricinus communis L. using a hydraulic
considering the oil extraction process. Perdomo et al., (2013) press, and the techniques of experimental designs.
characterized physically and chemically seeds of seven
castor bean accessions from central Mexico; also, seed oil
was extracted by three different methods (chemical solvent, Experimental procedure Material
cold pressing, and warm pressing).
The seed accession used in this study was collected in the
State of Queretaro, Mexico, and named VQ-4 (Perdomo et
Methods for vegetable al., 2013). In terms of experiment design, Figure 1a shows a
oil extraction from seeds complete seed (‘whole’) that defines one of the conditions
used for the oil extraction; Figure 1b presents the second
Several processes can be used to obtain oil. The most common seed condition, defined as ‘minced’; and Figure 1c defines
oil extraction procedures are classified as: expeller pressing, the third condition, designated as ‘bare endosperm’. These
hydraulic pressing, and solvent-dependent extraction. three conditions of the seed were used to feed the oil
extractor.
4
Ph.D in Plant Physiology and Weed Science by New Mexico State University,
USA; M.Sc. in Plant Nutrition by Colegio de Postgraduados, México; B.Sc. in
Agronomy by Universidad Autónoma Chapingo, México. Current affiliation:
Associate Professor-Researcher at Colegio de Postgraduados, Campus San Luis
Potosí. Salinas de Hgo, SLP, México. E-mail: [email protected]
5
Ph.D in Soil Water Erosion in the Purdue University. USA. M.Sc. in Soil Phy-
sics for the Colegio de Postgraduados, México. Agronomic Engineering for the
Universidad Autónoma Chapingo. México. Affiliation: Professor-Researcher at
Universidad Autónoma de Querétaro, Querétaro, México.
E-mail: [email protected]
6
Ph.D in Engineering for the Universidad Autónoma de Querétaro. M.Sc. in
Chemical Food Engineering for the Universidad Nacional Autónoma de Mé-
xico. México. Affiliation: Professor-Researcher at Universidad Nacional Autó-
noma de México, México. E-mail: [email protected]
7
Ph.D in Physics in the Centro de Investigación y Estudios Avanzados of Institu- (a) (b) (c)
to Politécnico Nacional, México. M.Sc. in Physics of the Universidad Autóno-
ma de San Luís Potosí, México. B.Sc in Physics of the Universidad del Quin-
dío, Colombia, Affiliation: Researcher at Universidad Nacional Autónoma de Figure 1. a) Whole seed, b) broken, and c) bare endosperm Ricinus
México, Querétaro. México. E-mail: [email protected] communis L.

Ingeniería e Investigación vol. 36 n.° 3, december - 2016 (82-88) 83

Optimization of castor seed oil extraction process us-ing response surface methodology

Oil extraction equipment The response surface methodology proposed for the analysis,
is a central composite design (CCD) consisting of two factors
Oil extraction was carried out with a temperature-controlled with two blocks, three points central, three points axial cube,
stainless steel prototype, with a maximum load capacity of with two replications, run randomly (Montgomery, 2013).
400 g. The extraction device has a cylinder to enclose the The central point is replicated several times to provide an
seeds and a plunger for pressure application. The prototype independent estimate of the experimental error; α used for
is assembled with a hydraulic press (Mikel, México), with a this design was 1,414. Temperature factor ranged from 40 °C
maximum pressure capacity of 68,65 MPa and a 150-mm to 60 °C, and pressure from 22,85 MPa to 45,80 MPa.
piston. The system has a thermal casing with a commercial
strength of 1000 W at 127 V, and a data logger connected
to a k-type thermocouple for temperature-controlled oil Results and discussion
extraction.
Taguchi design
Oil extraction performance Table 1 shows the results for Taguchi design for turbidity,
viscosity, color, and extraction yield. Regardless of the
Immediately after extractions, oil was centrifuged twice at combination between pressure and temperature, no oil is
1300 rpm for 16 min to remove debris. extracted at all when bare endosperm is used, suggesting
that seed coat is required for oil percolation to the collector
within the extraction prototype.
Kinetic Viscosity
Table 1. Results obtained from Taguchi design.
Determination of the oil rheological behavior was done with
a Rheomat viscometer (Mettler Toledo, USA). Samples were

Extraction
weight (g)

condition

yield (%)
Turbidity

Viscosity
Pressure

Sample

(NTU)
(MPa)

Color
Temp

(Pa.s)
Seed
(°C)
Run

heated to 40 °C, and measurements were run in triplicate

(c )
using the standard procedure D445 of the American Society
of Testing and Materials (ASTM), designed to determine 8 20 22,85 100 1 188,250 0,330 9,55 20,44
kinematic viscosity of transparent and opaque liquids. 4 20 34,32 200 2 212,500 0,360 10,37 13,62

1 20 45,8 300 3 0,000 0,000 0,00 0,00

9 40 22,85 200 3 0,000 0,000 0,00 0,00

Color 3 40 34,32 300 1 194,400 0,340 12,26 30,69

5 40 45,8 100 2 204,500 0,350 11,70 30,65

Oil color was measured with a Minolta colorimeter Model
7 60 22,85 300 2 228,000 0,370 16,25 31,95
CR300 (Konica Minolta, Japan), according to ASTM
2 60 34,32 100 3 0,000 0,000 0,00 0,00
D1500-12.
6 60 45,8 200 1 191,600 0,330 14,52 35,55

Seed condition (1) Whole, (2) Minced, (3) Bare endosperm.

Turbidity
The effects of temperature (a), pressure (b), sample weight
Turbidity was determined with a Hanna Instruments (c), and seed conditions (d) on extraction yield are presented
turbidimeter (Hanna, Italia) set with an infrared mode (Lau in Figure 2. There is a slight increase in oil extraction (Figure
et al., 2000). 2a) when the temperature increases from 20 °C to 60 °C
because temperature facilitates the breaking of fatty acids in
the seed, allowing the exudation of oil. Figure 2b presents
Statistical Analysis the extraction yield as a function of the pressure, showing
an increase in the extraction yield (from 34,32 MPa to 45,80
Statistical analysis was performed from a Taguchi MPa). Figure 2c shows that increasing the sample weight
experimental design, with an orthogonal array of L9, had just a little contribution to the extraction yield (6 %
with two replications (Taguchi, 1987; Miranda, 2009). -100g to 300g). It can also be noticed a significant decrease
Temperature values were: 20 °C, 40 °C, and 60 °C; pressure in the extraction performance affected by the condition of
levels were 22,85 MPa, 34,32 MPa, and 45,80 MPa; seed the seed, with no oil extraction at all from bare endosperms
materials used were 100 g, 200 g, and 300 g, for the three Figure 2d; this leads to the inference that the seed coat is
seed conditions: whole, minced, and bare endosperm required for oil percolation from the top of the collector
(Figure 1). Analysis of variance (α = 0,05) was used to cylinder to the filter. As it happened with bare endosperm,
determine statistical significance of response variables. minced seed (broken) also had a decrease in oil extraction
With the information collected through the Taguchi design, yield, compared to the whole seed, which presented the
correlation and principal components analyses were higher extraction values. In terms of the industry, these
performed (Esbense & Geladi, 2009). results show that it is necessary to use the whole seed in

84 Ingeniería e Investigación vol. 36 n.° 3, december - 2016 (82-88)

MOSQUERA-ARTAMONOV, VASCO-LEAL, ACOSTA-OSORIO, HERNANDEZ-RIOS, VENTURA-RAMOS, GUTIÉRREZ-CORTEZ, AND RODRÍGUEZ-GARCÍA

order to increase the extraction yield, suggesting that the The contribution of each factor to oil extraction yield was
paths formed by the pericarp facilitates oil extraction. as follows: seed condition (82 %), temperature (12 %),
pressure (4 %) and seed amount in the container (2 %). The
extraction of oil increases when the process involves the
whole seed and also when the container temperature is
increased.

Correlation and principal component analysis

The percentages of correlation between the response
variables were: very high, with four iterations over 95 %
(viscosity-turbidity, color, turbidity, color-performance, and
color-viscosity), and two iterations being less than 90 % (oil
extraction performance-turbidity, viscosity-yield) although
(a) over 85 %. Thus, raising the level of either variable will
have a positive effect on the remaining three variables.

Table 2 shows the principal components for analysis; for the

first component (PC1), variation is explained by 95,7 %, and
the correlations of the variables are positive, although these
are low. In the second component, variation is explained
by 99,4 %, and the correlations changed significantly. Then,
the experiment contains two major components.

Table 2. Principal component analysis.

Eigenvalue 3,827 0,1500 0,022 0,000

Proportion 0,957 0,0380 0,006 0,000

(b)
Accumulated 0,957 0,9940 1,000 1,000

Variable PC1 PC2 PC3 PC4

Oil extraction 0,487 0,765 -0,410 -0,093

Turbidity 0,503 -0,456 -0,088 -0,729

Viscosity 0,503 -0,430 -0,355 0,660

Color 0,506 0,145 0,835 0,158

When considering two principal components, the

correlation matrix shows that increasing the system
performance could decrease oil turbidity and viscosity.
This type of relationship has also been observed using a
supercritical fluid process (Sheibani & Ghaziaskar 2008).
(c) Those decreased oil properties could affect the use of castor
bean oil for biodiesel production, since low viscosity and
acidity are recommended for that purpose (ASTM D6751).

Response surface methodology

For the implementation of the central composite design
with two controllable factors, the ‘whole’ seed condition is
used for the high contribution (82 %), and a load of 100 g
of seeds, to increase oil extraction performance (Table 2).

The controllable factors are: temperature between 40 °C

and 60 °C, and pressure from 22,85 MPa to 45,70 MPa.
(d) Table 3 presents the design used with data obtained for
Figure 2. Effects of extraction factors. each performed run.

Ingeniería e Investigación vol. 36 n.° 3, december - 2016 (82-88) 85

Optimization of castor seed oil extraction process us-ing response surface methodology

Using the average values for extraction showed in Equation (1) represents the variation present in the bearing
Table 3, and the Kolmogorov-Smirnov statistic test with performance, as control variables are temperature and
95 % confidence, it was shown that data collected for pressure:
performance follows a normal distribution with a p-value
of 0,090 (Izraelevitz et al., 2011). For standard deviation, OEY (%) = 30,571 + 3,712P + 3,564T-2,241P2-1,033T 2 + ε (1)
Bonferroni confidence intervals of 95 % were performed
for each level. Using the statistics proposed by Bartlett, Where: OEY is oil extraction yield, P is pressure, T is
values are greater than α = 0,05, stating that it does not have
problems of heteroskedasticity.
temperature, and ε is the absolute error ε ∈N µ ,σ ;
2
( )
regression equation in units unencrypted.

Table 3. Experimental design runs for central composite design. According to the Equation (1) and Table 4, all the terms
proposed are significant (p-value <0,05), opposed to the
Experiment
Temperature Pressure Average Average results obtained in a study conducted by Goswami et al.,
extraction viscosity
(rum) Encrypted
(°C)
Encrypted
(MPa) yield (%) (Pa.s)
(2009). This research seeks to optimize bioconversion of
units units
castor bean oil into ricinoleic acid, finding an equation
14 −1 40 −1 22,9 20,166 0,391
that characterizes that process. However, due to the lack
11 1 60 −1 22,9 27,849 0,481 of significances (p-value > 0,05) in several of the studied
9 −1 40 1 45,8 26408 0,473 variables, these results cannot be conclusive on this
10 1 60 1 45,8 33,611 0,511 pursuit.
12 0 50 0 34,3 31,210 0,479
The normality of the residuals (p-value = 0,109 Anderson
13 0 50 0 34,3 31,210 0,462
Darling test) is supported by the results and the model
8 0 50 0 34,3 31,219 0,466 presented.
7 −1,41 36 0 34,3 24,008 0,484

2 1,41 64 0 34,3 33,611 0,452 Figure 3 shows the contours produced for increasing the
6 0 50 −1,41 18,1 20,166 0,46
yield in oil extraction. Oil yield increases when levels of
both controllable factors are raised.
5 0 50 1,41 50,6 32,65 0,456

4 0 50 0 34,3 29,769 0,468

3 0 50 0 34,3 31,210 0,529

1 0 50 0 34,3 28,809 0,466

Table 4 presents the iteration pressure*temperature, which

had no significant effect on the response variable studied;
therefore, it was excluded from the analysis. The response
variable had a quadratic response (p-value 0,001), in which
both pressure and temperature showed an effect, and had a
quadratic relationship.

Table 4. ANOVA for central composite design.

Source Degree Adjusted sum Adjusted

F ratio P value
of Variations of freedom of squares mean square
Regression 5 253,64 20,067 39,78 0,000 Figure 3. Contour plots for pressure and temperature.
Linear 2 40,13 34,997 15,74 0,002

Pressure 1 34,99 7,28 27,44 0,001

Looking at the response surface for oil extraction yield
(Figure 4), the optimization point to get the greatest oil
Temperature 1 7,28 21,14 5,71 0,044
yield performance (35,07 %) was observed at a pressure of
Square 2 42,29 36,75 16,58 0,001 50,31 MPa (0,8403 units unencrypted), and a temperature
Pressure*Pressure 1 36,76 7,81 28,82 0,001
of 64 °C (1,41 units unencrypted). On the other hand, the
control factors had a significant increase in performance
Temperature*-
Temp
1 7,81 1,27 6,12 0,038 (Figure 3), but this significance tends to have a limit,
where the control factors did not increase oil yield (Figure
Residual Error 8 10,20 1,82 − −
4). This outcome could be due to a high compression of
Lack-of-fit 4 7,28 0,73 2,49 0,199 the seed within the extraction device, resulting in a lack of
Pure error 4 2,92 20,07 − − paths for oil flowing to the collector component.
Total 13 263,84 − − −

S= 1,12929 R-sq. = 96,13 % R-sq.(adjusted) = 93,72%

86 Ingeniería e Investigación vol. 36 n.° 3, december - 2016 (82-88)

MOSQUERA-ARTAMONOV, VASCO-LEAL, ACOSTA-OSORIO, HERNANDEZ-RIOS, VENTURA-RAMOS, GUTIÉRREZ-CORTEZ, AND RODRÍGUEZ-GARCÍA

setting values obtained (Table 6). According to these tests,

oil yield can be increased by 35 % or even more just by
controlling those factors that generate higher variability
during the extraction process.

Table 6. Confirmation runs.

Experiment Seed Pressure Temperature Extraction

(run) condition (MPa) (°C) yield (%)

1 Whole 50,31 64 36,01

2 50,31 64 34,81

Figure 5 illustrates the extraction yield percent of both

process, that one carried out in this work (new), and that
performed by Perdomo et al. (2013) in four castor seeds
varieties. The new extraction process showed an oil yield
enhancement in all the seed varieties compared to the
extraction yield obtained by Perdomo et al. (2013). In
Figure 4.
addition, the new process decreases the deviation by
12 % ± 5% to 7 % ±2 %, obtaining an improvement of 54 %
 

Scenario analysis in comparison with Perdomo et al,(2013).

Scenario analysis was developed using optimization

techniques (Derringer & Suich, 1980). Three scenarios
are detected (Table 5) to determine the optimal levels
for the extraction process, taking into account oil yield
and viscosity. Table 5 shows the variation related to the
possibility of changing pressure and temperature for the
extraction process, depending on the importance given to
either oil yield or viscosity.

Table 5. Analysis of scenarios to determine the best combination,

between variables extraction yield and viscosity.

Control Response
Scenario Target Value Desirability
factor variable

Pressure Max 49,380

Temperature Min 40,830

1 0,668
Extraction yield Max 28,161

Viscosity Min 0,471

Pressure Min 30,870

Temperature Max 64,000

2 0,651
Extraction yield Max 30,180 Figure 5.
Viscosity Min 0,477

Pressure Min 50,310

Conclusions
Temperature Max 63,030
3
Extraction yield Max 34,546
0,894 The condition of the seed has a high contribution to the
performance of the oil extraction process (82 %), while the
Viscosity Min 0,470
amount of the material load has no effect on the process.
Extraction yield, turbidity, viscosity, and color are highly
correlated (> 85 %). These variables presented two main
Confirmation runs following components: extraction yield and viscosity, coupled with
the recommended settings turbidity. The contribution of the control factors has a yield
extraction limit. Increasing pressure (> 50,31 MPa) has no
In order to test the results of the analysis developed, two effect on extracting performance, which is due to a high
seed oil extraction runs were performed using the optimal compaction of the seed within the device.

Ingeniería e Investigación vol. 36 n.° 3, december - 2016 (82-88) 87

Optimization of castor seed oil extraction process us-ing response surface methodology

Acknowledgments Montgomery, D.C. (2013.). Design and Analysis of Experi-

ments 8th ed. Wiley: New York.
This work was partially supported by Dirección de Montoya, J., Benjumea, P., & Pashova, V. (2011). Optimization
Vinculación Tecnológica y Proyectos Especiales UAQ. J.D. of the basic ethanolysis of ricin oil using the response sur-
Mosquera-Artamanov and J.F. Vasco-Leal were supported face methodology. DYNA, 78(168), 90-97.
by a scholarship from the CONACYT. Ogunniyi, D.S. (2006). Castor oil: A vital industrial raw mate-
rial. Bioresource Technology, 97, 1086–1091.
DOI: 10.1016/j.biortech.2005.03.028.
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