Industrial Crops and Products 95 (2017) 733–741

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Industrial Crops and Products

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

Evaluation and mathematical modeling of processing variables for a

supercritical fluid extraction of aromatic compounds from Anethum
graveolens
José Jacques Garcez a , Fernando Barros b , Aline Machado Lucas a , Vanessa Barbieri Xavier a ,
Ana Luisa Fianco a , Eduardo Cassel a , Rubem Mário Figueiró Vargas a,∗
a
Materials Engineering and Technology Program, Engineering College, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, Pr.30, CEP:
90619-900, Porto Alegre, Brazil
b
Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, Pr.30, CEP: 90619-900, Porto Alegre, Brazil

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

Article history: The aim of this study is to identify the extraction conditions, using supercritical carbon dioxide as solvent,
Received 20 June 2016 that maximize the yield of volatile extract from the seeds of Anethum graveolens L. by the use of the
Received in revised form 24 October 2016 response surface method, where the variables pressure, temperature and flow rate of supercritical CO2
Accepted 21 November 2016
were investigated. A 23 factorial design combined with response surface methodology was used in order
Available online 28 November 2016
to achieve the goal. The best set of experimental conditions was determined to be P = 100 bar, T = 35 ◦ C and
solvent flow rate = 1000 g/h. The mathematical modeling of the extraction curve was performed under the
Keywords:
best experimental conditions, and the olfactory profile of the extract and its antimicrobial activity were
Dill
Separation techniques
compared with the oil obtained from hydrodistillation. The gas chromatography analysis (GC) identified
Mass transfer dillapiole, carvone and its isomers to be major compounds. The olfactory profile of A. graveolens extracts
Sensory analysis obtained was determined by gas chromatography-olfactometry (GC-O), and the most often identified
Essential oil aroma was cheese/bacon for the hydrodistillation oil, whereas in the supercritical extract, the most often
identified aroma was burning bush.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction regard to chemical composition, olfactory profile, and antimicrobial

The use of natural products in the pharmaceutical and food
industries has been intensified due to market regulations seeking
to appease a public that requires minimal or no use of artifi-
cial products (Gurib-Fakim, 2006; Lahlou, 2013; Rates, 2001). The
food industry seeks compounds that help preserve food by inhibit-
ing the growth of undesirable microorganisms, as well as natural
compounds that promote the perception of flavors and aromas
(Lanciotti et al., 2004; Ayala-Zavala et al., 2009). Thus, the search
of alternative technologies that meet these demands using raw
materials with an industrial potential is an important step toward
industrial development (Herrero et al., 2006), justifying the pur-
pose of this work: the optimization of supercritical fluid extraction
of volatile extracts from dill seeds and a comparison to the essen-
tial oil obtained by hydrodistillation, a traditional process, with
∗ Corresponding author.
E-mail address: rvargas@pucrs.br (R.M.F. Vargas).
activity.
Anethum graveolens L., also known as dill, is a plant native to
the Mediterranean area. Used mainly for its aromatic properties, A.
graveolens oil contains dillapiole, carvone, and limonene as major
components (Singh et al., 2005; Stanojević et al., 2015; Sintim et al.,
2015; Weisany et al., 2015). The pharmacological properties of A.
graveolens oil, such as its antibacterial activity, have been recently
reported (Rafii and Shahverdi, 2007; Tian et al., 2011; Chen et al.,
2014; Orhan et al., 2013). Other studies have also demonstrated
that the extract of A. graveolens seeds has good antioxidant and
antimicrobial activities that favor the preservation of food and pro-
tection against food pathogens, as well as antifungal activity that
suppresses or deteriorates some types of fungi (Singh et al., 2005;
Prakash et al., 2015). These combined properties give a very wide
range of applications for A. graveolens oil (Fatope et al., 2006; Singh
et al., 2005; Kazemi, 2015). These various properties are the reason
why A. graveolens, that is widely used in traditional medicine and
in culinary, has attracted the attention of the pharmaceutical and
food industry for the development and manufacture of drugs and
food enhancers (Tian et al., 2011).

http://dx.doi.org/10.1016/j.indcrop.2016.11.042
0926-6690/© 2016 Elsevier B.V. All rights reserved.
734 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741

make the extraction with supercritical CO2 a prominent technology

Nomenclature in the pharmaceutical and food industries (Perrut, 2003).
Supercritical extraction depends on such variables as flow, pres-
e Mass of extract relative to N sure and temperature of the fluid. Since there are several factors
F Frequency of detection of an aroma that affect an experiment, the use of an experimental design is
FM Modified frequency used in olfactometry assays required in order to evaluate the interactions between these fac-
I Average intensity of an aromatic attribute tors and the overall effect generated by them. The combined use of
k First order rate constant (min−1 ) an experimental design with response surface methodology per-
kf Mass transfer coefficient in fluid phase (m/s) mits the evaluation of the influence of several variables on one or
k Mass transfer coefficient in solid phase (m/s) more responses with a reduced number of experiments, therefore
m Refers to the start of extraction from inside the par- reducing time and cost (Anderson-Cook et al., 2009), permitting the
ticles identification of the optimal process conditions in a fast and effec-
me Mass collected at the end of the extraction process tive way. This methodology has been widely used in engineering
(g) research/experiments as a way of optimizing processes (Liyana-
mp Milled seeds mass used in the extraction (g) Pathirana and Shahidi, 2005; Wang et al., 2007; Domingues et al.,
N Mass of the solute-free solid phase (g) 2013).
n Refers to the end of extraction of easily accessible Mathematical models of the extraction process are important
solute for generating predictions about process efficiency to simulate the
P Pressure condition (bar) process on different scales and to evaluate the influence of the oper-
q Specific amount of solvent ating parameters on the technical and economic feasibility of an
qm Specific amount of solvent for start of the extraction industrial process (Meireles et al., 2009). According to Cassel et al.
from the inside of particles (2009), mathematical modeling of the extraction is an extremely
qn Specific amount of solvent for end of the extraction important step in the development process, since mathematical
of easily accessible solute models can be used to design industrial plants, greatly reducing the
q̇ Mass flow rate of solvent related to N, (s−1 ) number of laboratory experiments. Many models are available in
Q Flow rate (g/h) the literature, and they are needed to evaluate parameters associ-
St Mass of extract obtained over the extraction time ated with the type of operation employed for specific raw material.
(g) Models based on principles of desorption and mass transfer (Tan
S0 Total mass extracted in process (g) and Liou, 1989; Cocero and Garcia, 2001; Reverchon and Marrone,
T Temperature (◦ C) 2001; Sovová, 1994) are often used to represent the extraction of
t Extraction time (s) natural products by different separation techniques (Cerpa et al.,
W Parameter of the slow-extraction period 2008; Vargas et al., 2013; Rahimi et al., 2011; Ferreira and Meireles,
x Molar fraction of the liquid phase 2002; Cassel et al., 2010). The choice of the model to use for the
x0 Overall initial concentration related to the solute- mathematical simulation of a process is dependent on the extrac-
free solid phase tion conditions, plant material used, product type, solvent used and
xk Inaccessible oil concentration inside the solid phase geometry of the extractor. It is important to emphasize that a model
particles with more variables implies a better and more accurate representa-
Y % Global yield of extract tion of the process but also increases the complexity of the model. A
y Molar fraction of the vapor phase complex model often leads to time-consuming simulations which
yr Solubility can be unattractive for industrial use, where a quick response is
Zm Parameter of fast extraction period often needed.
zw Dimensionless coordinate of the boundary between
fast and slow extraction
2. Materials and methods
Greek symbols
␧ Bed porosity This study seeks to determine the best extraction conditions in
␳ Density of solvent (g/cm3 ) terms of volatile extract yield from A. graveolens seeds using extrac-
␳s Density of solid phase (g/cm3 ) tions with supercritical carbon dioxide, a technology not employed
to date for obtaining A. graveolens oil, using a response surface
method where the variables: pressure, temperature and flow rate of
supercritical CO2 were investigated. Factorial design 23 was used
The composition and characteristics of an essential oil is depen-
to determine the number of experiments necessary. For the best
dent on several factors (Retta et al., 2009; Gil et al., 2002) including
conditions identified, the mathematical modeling of the extraction
the type of extraction technique (Flamini et al., 2002; Pereira
curve was performed.
et al., 2008; Bailer et al., 2001) Specifically with regard to A. grave-
The chromatographic analysis of the volatile extract was also
olens essential oil, the soil treatment conditions (Zheljazkov et al.,
conducted in order to compare the composition of the extract
2006; Kapoor et al., 2002), crop maturity, plant density (Callan
obtained by supercritical fluid extraction with the A. graveolens
et al., 2007), extraction time (Sintim et al., 2015), cropping system
essential oil obtained with the use of traditional technique as the
(Weisany et al., 2015) has been investigated.
hydrodistillation (Sintim et al., 2015; Stanojević et al., 2015; Bailer
Supercritical extraction is a method that derives from the tra-
et al., 2001). The global extract was also submitted to olfactometry
ditional extraction principles, which consists of a fluid passing
analysis and to an assay for antimicrobial activity.
through a bed constituted by the material to be extracted. This
process is considered a clean technology, offering advantages over
traditional methods, such as extraction by steam distillation and 2.1. Plant material
hydrodistillation (Reverchon, 1996), when using CO2 as solvent.
The properties associated with the non-toxicity, non-flammability, The A. graveolens seeds used were purchased in a retail store in
and low critical temperature and pressure of the carbon dioxide the city of Porto Alegre (Brazil) in 1 kg packages of dry product. The
 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741 735

origin of seeds is Rajasthan (India) as provider information. Grind- Table 1

Factorial design uncoded randomized.
ing was performed in a knife mill and then, the milled seeds were
divided into 100 g portions for use in extractive processes. Seeds Standard order Run order P (bar) T (◦ C) Q (g/h)
were ground using a knife mill to obtain a powder with a particle 1 1 80 35 600
size of 0.51 mm, and they were later subjected to the extraction 7 2 80 55 1000
process. 6 3 100 35 1000
2 4 100 35 600
5 5 80 35 1000
2.2. Supercritical fluid extraction (SFE) 4 6 100 55 600
3 7 80 55 600
The acquisition of the volatile A. graveolens extract was carried 11 8 90 45 800
out in a pilot plant as described in detail in Scopel et al. (2013). The 9 9 90 45 800
8 10 100 55 1000
equipment has two cylinders for storage of carbon dioxide with
10 11 90 45 800
99.9% purity; a high pressure pump; a condenser; two pre-heaters;
three extraction vessels (100, 500 and 1000 mL); two collector ves-
sels; one mass flow measurement system; two monitoring software with condenser and an oil/water separation system. It used 500 mL
and an automated control system (Scopel et al., 2013). of water together with 100 g of ground seeds and time used for
The experiments were conducted in the 500 mL vessel loaded the extraction was 1.5 h. Two hydrodistillation experiments were
with 100 g of milled A. graveolens seeds at different extraction con- performed.
ditions. The final yield was calculated according to Eq. (1):
me
%Yield = ∗ 100 (1) 2.5. Gas chromatography
mp
where me is the extract mass collected at the end of the extrac- The chemical composition of the A. graveolens oils was
tion process (g); mp is the milled seeds mass initially added to the determined using a gas chromatograph equipped with a mass
extractor vessel (g) and %Yield is the yield of the extraction. The spectrometer (Hewlett Packard – Agilent system GC/MS model
plant and extract masses were obtained with a digital balance with 7890A and mass detector 5975C). The carrier gas was helium
precision (±10−4 ). (0.8 mL/min), injector temperature was 250◦ C, volume injected
The experiment was repeated in duplicate under the condition was 0.2 ␮L, and split mode with split ratio of 1:55 was used.
of best performance and the extraction curve was constructed for The capillary column was HP-5MS (Hewlett Packard – Agilent, 5%
mathematical modeling of the process. The curves were made by fenil metil silox, 30 m × 250 ␮m × 0.25 ␮m). The temperature pro-
measuring the extract mass obtained every 10 min of extraction, gramming was 60 ◦ C (8 min), 60 ◦ C–180 ◦ C, 3 ◦ C/min, 180 ◦ C (1 min),
and these measurements were made from the beginning of the 180 ◦ C–250 ◦ C, 20 ◦ C/min, 250 ◦ C (10 min). MS mode EI, EI voltage
extraction until there was no more variation in the mass of extract 70 eV and an acquisition mass range of m/z 40–450.
between two successive collections. The components of the A. graveolens oils were identified by com-
parison of their Retention Index (RIs) on the GC column determined
2.3. Experimental design and response surface methodology in relation to a homologous series of n-alkanes, with those from
pure standards or reported in literature (Jennings and Shibamoto,
Response surface methodology was applied to identify the best 1980; Davies, 1990). Comparison of fragmentation patterns in the
extraction conditions of A. graveolens oil using supercritical CO2 mass spectra with those stored on the GC–MS databases (Adams,
over a certain range of process variables. Three variables were cho- 2007) was also performed.
sen to be evaluated: flow rate, pressure and temperature of the
supercritical fluid. The minimum pressure was 80 bar and maxi- 2.6. Gas chromatography-olfactometry
mum 100 bar, while the temperature varied between 35 and 55 ◦ C
and the flow rate ranged from 600 to 1000 g/h. The relationship The analysis of A. graveolens volatile extract (SFE) and essential
between the variables and the response was modeled by adjusting oil (hydrodistillation) was performed with a gas chromatograph
the experimental data to a polynomial equation. The identifica- Perkin Elmer Auto System equipped with a detection system flame
tion of the best extraction condition was performed in terms of ionization detector (FID) and a compartment for sensory evalua-
the highest yield. tion (sniffing port) with a humidification system. The column used
The factorial design method 23 with a central point was applied was a HP-5MS (Hewlett Packard – Agilent, 5% phenyl methyl Silox,
to obtain the trend toward the best process conditions. The com- 30 m × 250 ␮m internal diameter, film thickness of 0.25 ␮m). The
pletion of the factorial design was accomplished using Minitab system was operated under programmed temperature conditions.
software, with which the experiments were randomized. This pro- The program starts with temperature at 60◦ C which is main-
cedure is important as it distributes variable errors uncontrollable tained for 4 min, increased at 5 ◦ C/min to 180◦ C after increasing
throughout the experiment allowing the statistical analysis. The 20 ◦ C/min to 250 ◦ C and maintaining that temperature for 5 min.
experimental design matrix, the levels of each factor and their com- The carrier gas used was nitrogen with a flow of 1 mL/min, injector
binations designated by factorial design are shown in Table 1 with temperature of 250 ◦ C, olfactometer temperature of 250 ◦ C, detec-
the values of variables and levels randomized. tor temperature of 250 ◦ C, injection volume equal to 1 ␮L, in the
split mode with split ratio 1:55 (Xavier et al., 2013).
2.4. Hydrodistillation The analysis were performed using a panel with six assessors
(due to availability), where each evaluator should report every time
The extraction of the A. graveolens seeds was also performed that an odor was perceived and provided the following information:
by a hydrodistillation technique. This extraction procedure was (a) time, relative to the beginning of the analysis, (b) description
performed to compare the characteristic of the volatile extract of the odor; (c) odor intensity on an integer scale from 1 to 5 (1
obtained by supercritical extraction with essential oil obtained by being the lowest intensity and largest intensity 5). With the data
a conventional technique. The seeds were introduced in a round obtained by the evaluators it was possible to make a comparison
bottom flask coupled to vertical tube (type Clevenger) combined between the results obtained individually and generate a new vari-
736 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741

able, that relates intensity and frequency detection, called modified

frequency (FM), Eq. (2), proposal by Dravnieks (1985):

FM (%) = F (%) .I (%) (2)

where F (%) is the frequency of detection of an aroma between the

universe of evaluators in percentage terms, and I (%) is the average
intensity of an aromatic attribute in relation to the scale, with 100%
corresponding to average 5. If an aroma has FM equal to 100%, it
means that the flavoring was detected by all evaluators and with
maximum intensity.

2.7. Antimicrobial activity

An antimicrobial activity analysis was performed by determin-

ing the minimum inhibitory concentration for each microorganism.
The minimum inhibitory concentration was determined using a
dilution method on microplates (Falcão et al., 2012). The inocu-
lum of the organism was prepared by adding the colonies in saline
solution after being added to Mueller-Hinton broth, resulting in a
final concentration of 1.0 × 104 CFU/mL. In each well of microplate
was added 100 ␮L of Mueller-Hinton broth containing inoculum
and then were added 100 mL of extract, obtained by supercrit-
ical extraction or hydrodistillation, solubilized in Tween 20 and
water (at final concentrations of 25, 30, 35, 40, 45 and 50 mg/mL).
The microplates were incubated for 24 h at 37 ◦ C and after the
growth phase, the results were evaluated by the presence/absence
of turbidity. Organisms tested were Staphylococcus aureus (ATCC
25922), Escherichia coli (ATCC 25923) and Enterococcus faecalis
(ATCC 29212). The positive control used was amoxicillin 0.1 mg/mL
and the negative proof was only Mueller Hinton broth inoculated.

2.8. Mathematical modeling

The curve of the supercritical extraction yield versus time was

obtained for the best condition provided by the response surface
method. Two models were fit to the experimental data. One model
is based on desorption principles, being faster and simpler to imple-
ment, and the other model is based on mass transfer principles that
take into account more parameters to fit the data.
Model 1: This model can be used in processes that are controlled
by intra-particle diffusion. The model considers one type of extract
being removed during the extraction with a fast rate of removal,
and assumes that the extract is uniformly distributed within the
matrix (Anekpankul et al., 2007). The model is represented by Eq.
(3):
St
= 1 − e−kt (3)
S0
where St is the mass (g) of extract obtained in the extraction time, t
(min); S0 is the total mass extracted in process (g); k is the first order
rate constant to be estimated and which describes the efficiency
of extraction (min−1 ). This constant k can be taken as the desorp-
tion constant of the extract and assumes the extract composition
is constant throughout the process.
Model 2: The second model is a classical mathematical model Fig. 1. Response surfaces for effects of two independent variables on the global yield
of extract obtained by supercritical extraction: (a) process pressure and temperature
proposed by Sovová (1994). This model is based on the presence of
at a constant CO2 flow rate of the 800 g/h; (b) pressure and flow rate of CO2 at a
easily accessible solute and inaccessible solute in the bed particles. constant temperature of the 45 ◦ C; (c) flow rate of CO2 and temperature at a constant
This feature allows us to assume the existence of two regions in pressure of the 90 bar.
the extractor, one where solute is removed from broken particles
and another where solute difficult is extracted from intact particles.
The model is constructed by mean of differential mass balance in concentrations xk , x0 and yr , the void fraction ␧, the density of sol-
terms of the solute in the fluid and solid phases. The solution was vent ␳, the density of solid phase ␳s , the mass flow rate of solvent
obtained by Sovová (1994) in analytical form and the summarized related to mass of solute-free solid phase q̇, the mobile coordinate
results are shown as follow in terms of amount of extract as a func- that represents the border between the easy and the difficult access
tion of the specific amount of solvent q, specific interfacial area a0 , solute regions zw and parameters Zm and W (Scopel et al., 2014).
 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741
Table 2
Yield of supercritical extraction of dill seeds for each studied condition.
Run order P (bar) T (◦ C) Q (g/h)
1 - - -
2 - + +
3 + - +
4 + - -
5 - - +
6 + + -
7 - + -
8 0 0 0
9 0 0 0
10 + + + 1.34
11 0 0 0 1.40
− and +: Minimun and maximum values of the variables investigated; 0: values of
the center point.
The parameters Zm and W are directly related to the solid and fluid
mass transfer coefficients, ks and kf , respectively, as described in
the literature (Sovová, 1994).
Aiming to conduct the mathematical modeling of the process,
it was necessary to perform the calculation of the following input
parameters: specific interfacial area (a0 ), porosity (ε), seed density
(s ), CO2 flow rate (QCO2 ) and specific mass of CO2 under different
extraction conditions.
The unknown quantities xk , yr , ks and kf were estimated by min-
imization of the sum of squares of errors between the experimental
data and the prediction using the model (Hornbeck, 1975).
3. Results and discussion
3.1. Extraction process and experimental design
The yields (w/w) ranged from 0.31% for the lowest pressure,
lowest temperature and highest flow rate conditions to 3.16% for
the highest pressure, highest flow rate and lowest temperature
conditions.
Table 2 shows the results of the experiments carried out accord-
ing to the factorial design presented in Table 1. The uncoded
variables and yields are presented in Table 2. The analysis of vari-
ance was performed to determine which process variables were
significant. For this purpose, ANOVA was used and the results are
shown in Table 3.
The data presented in Table 3 indicated that all variables and
their interactions are statistically significant for a significance level
equal to 95%, i.e., p < 0.05. The results presented in Table 3 permit
us to state that, for the conditions investigated, the linear terms
have a high influence on the yield of volatile extract of A. graveolens
seeds. Among all the significant variables, the temperature was the
parameter with the largest effect on the yield due to the highest F
value associated with it. In decreasing order of strength, the most
important variables were temperature, pressure and flow rate.
The data in Table 3 allowed the development of a response
surface and the generation of an equation to describe the experi-
mental results. The adjusted model represents the data adequately
with a coefficient of determination R2 equal to 0.9994 and adjusted
coefficient of determination of 0.9972. Equation (4) represents the
adjusted model for the uncoded variables valid for all the intervals
investigated.
Y = 24.4185 − 0.277881P − 0.54T − 0.03531Q + 0.00618875
+0.000450344PQ + 0.0007055TQ − 8.75625E −06 PTQ
where Y is the % global yield of extract obtained by supercritical
extraction; P is the pressure (bar); T is the processing temperature
(◦ C) and Q is the flow rate of CO2 (g/h). Fig. 1 shows the response
737
surfaces in terms of the% yield of extract (R) in relation to PxT, and
PxQ and QxT, respectively.
Extract global Upon observation of Fig. 1, it is easy to observe that the best per-
yield (% w/w) formance is directed towards the extremes of the variable ranges.
1.15 This procedure allows observing that the highest yield (3.16%) is
0.71 obtained for a pressure of 100 bar, a temperature of 35 ◦ C and a
3.16 CO2 flow rate of 1000 g/h. It is also noticeable that the approach of
1.65
the range limits extreme of intervals leads to a higher value for the
1.51
1.19 yield, but transcending the limits for the range investigated there
0.31 is no guarantee of obtaining the extract with the characteristics
1.40 observed in this study.
1.47
3.2. Extraction at the best condition identified
The mathematical simulation was performed using the best con-
ditions identified by the mean statistical method. The experimental
procedure was conducted using particles with specific area equal
to 1.178 × 10−4 m−1 in a bed with porosity equal to 0.68 and the
extraction condition with best yield, 100 bar, 35 ◦ C and 1000 g/h
was repeated in duplicate for mathematical simulation. The extrac-
tion yield versus time curves were plotted and the results with their
standard deviations are shown in Fig. 2.
For the hydrodistillation, extraction curves were not generated
because the comparison among the extracts obtained by supercriti-
cal fluid extraction and hydrodistillation was the only goal to which
this extraction technique was performed. For this technique it was
obtained approximately 1.5 mL essential oil from out of 100 g of
grinded seeds. The total extraction time was 130 min. Sintim et al.
(2015) employed hydrodistillation for extract A. graveolens essen-
tial oil from seeds, getting similar yield (1.31%) the one found in this
study. Moreover, Bailer et al. (2001) obtained yields among 2.6 to
4.6% to 26 dill varieties. In this case the variable that influences
the results was the extraction time (12 h). A differential aspect
of supercritical fluid extraction in relation to traditional methods,
hydrodistillation and steam distillation, is the higher process vari-
able number that is possible to manipulate for maximize an answer
such as the extract yield or target compound composition in the
extract.
3.3. Mathematical modeling
The mathematical models fit the experimental data very
well. Model 1 required the adjustment of a single parameter,
k = 0.0273 min−1 , and the extraction curve can be observed in Fig. 2
where the experimental data and standard deviation are presented.
The coefficient of determination for model 1 is equal to 0.9963. The
magnitude order of the parameter shows similarity with the others
studies (Moura et al., 2005; Almeida et al., 2013).
Model 2 was adjusted from the determination of four parame-
ters and the coefficient of determination (R2 = 0.9996) adjust as well
as model 1. Model 2 requires more information on the extraction
and its adjustment process takes longer because the technique is
sensitive to the initial choice of values for the unknown parameters.
The adjustment technique provided the value of 5.508 × 10−8 m/s
for kf , 1.2117 × 10−8 m/s for ks , 0.0166 for xk , and 0.0122 for yr . The
magnitude order of the parameters was consistent with the work of
Mezzomo et al. (2009), Scopel et al. (2014), and Vargas et al. (2006).
3.4. Chromatographic analysis
The major compounds of the A. graveolens essential oil and
(4)
volatile extracts obtained by hydrodistillation and supercritical
fluid extraction, respectively, were identified and quantified by gas
chromatography coupled to a mass spectrometer (GC/MS) and they
are shown in Table 4. Note that all extracts obtained by supercrit-
ical fluid extraction to perform the statistical analysis showed no
738 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741

Table 3
Analysis of variance (ANOVA).

Factor DF Seq SS Adj SS Adj MS F p

Linear 3 4.319 4.319 1.439 1038.01 0.001

P 1 1.668 1.668 1.668 1202.63 0.001
T 1 1.925 1.925 1.925 1388.39 0.001
Q 1 0.725 0.725 0.725 523.01 0.002
Two cross product 3 0.372 0.372 0.124 89.62 0.011
P*T 1 0.053 0.053 0.053 38.43 0.025
P*Q 1 0.101 0.101 0.101 73.16 0.013
T*Q 1 0.218 0.218 0.218 157.27 0.006
Three cross product 1 0.245 0.245 0.245 176.89 0.006
P*T*Q 1 0.245 0.245 0.245 176.89 0.006
Lack of fit 1 0.004 0.004 0.004 3.03 0.224
Residual 2 0.002 0.002 0.001 - -
Pure error 2 0.002 0.002 0.001 - -
Total 10 4.944 - - - -

DF: Degrees of freedom; Seq SS: sequential sum of squares; Adj SS: adjusted sum of squeres; F: F-statistics; p: p value.

Fig. 2. Extraction curve for volatile extract obtained by supercritical fluid extraction at 100 bar and 35 ◦ C with flow rate equal to 1000 g/h in terms of yield versus time:
experimental data and results from mathematical modeling.

significant differences in their compositions. The oil composition
obtained by supercritical fluid extraction presented in Table 4 is
concerning the best condition identified by the statistical analysis.
The extract obtained by hydrodistillation has a very similar com-
position to what is found in the literature (Singh et al., 2005). The
main compounds are the limonene, carvone and their isomers and
dillapiole. The compound encountered in major quantity is carvone.
Although Nautiyal and Tiwari (2011) have investigated a different
kind of dill of that was investigated in this article, the composi-
tion of the extracts obtained by hydrodistillation is quite similar.
Compared to the extracts obtained by supercritical carbon dioxide
extraction, these compounds presented a very similar composition,
but its major compounds are not the same. In these extracts, the
compound with greatest amount was dillapiole, 77.93% and the
identified content of carvone was 12.65%. This high concentration
of dillapiole in the volatile extract obtained using the supercritical
fluid extraction is something of great value since it is a component
with important properties such as antifungal and larvicide (Aciole
et al., 2014; Almeida et al., 2009; Arif et al., 2009). Nautiyal and
Tiwari (2011) conducted a comparative study of extracts obtained
by hydrodistillation and supercritical fluid extraction using seeds
of Anethum sowa as raw matter, the authors reported increased
dillapiole content in the extract obtained by supercritical extrac-
tion compared to hydrodistillation extract. This behavior was also
observed in our research, despite using another kind of dill in our
experiments. The extract does not have a significant amount of
limonene, one of the major compounds of hydrodistillation extract,
only 0.32%, and it has a small but significant amount of an uniden-
tified compound in hydrodistillation, the n-eicosane 2.54 area%.
The difference in the composition of the extracts obtained using
supercritical CO2 in relation to the composition of the ones obtained
by hydrodistillation is explained by the loss of volatiles during with-
drawal of the extract, when CO2 and some light compounds are
released to the atmosphere. This fact justifies obtaining an extract
rich in compounds of higher molecular weight which is also due to
the effect of high-density of the supercritical fluid.
3.5. Olfactometry analysis
The olfactometry analysis has a utility in the identification
of flavorings (Xavier et al., 2013), which can be used in food to
enhance taste during processing or even to confer flavor in foods
that may lose some of their characteristics during the processing
steps. The identification of spectral regions with compounds that
have desirable characteristics in food processing allows submit-
ting the extracts (essential oil and volatile extract) to a subsequent
distillation where fractions rich on compounds with the desired
characteristics are obtained. The calculated modified frequency
(MF) data with values above 30 are shown in Table 5for A. graveolens
essential oil (hydrodistillation) and A. graveolens volatile extract
(SFE), respectively.
Figs. 3 and 4 show the results of the GC-O analysis for A.
graveolens essential oil (hydrodistillation) and A. graveolens volatile
extract (SFE), respectively.
The olfactory profile of the essential oil of A. graveolens is differ-
ent in relation to profile obtained by supercritical extraction. This
finding shows that the type of extraction provides distinct char-
acteristics to the products and this can be further supported by
chromatographic analysis results which indicate a similar compo-
sition but not equal to the oils obtained by the different extraction
techniques. The most intense odors of the oil obtained by super-
critical extraction are burning bush, pepper and sweet, while the
essential oil has aromas more strongly identified as cheese/bacon,
floral/burnt, oil/fat, and rotten potatoes. In function of the loss of
 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741 739

Table 4 oil/fat
Chromatographic analysis (GC/MS) of the dill essential oil obtained by supercritical floral/sweet 100 cheese/bacon
fluid extraction (SFE) and hydrodistillation (H). sweet bush/condiment
80
Compounda LRIb % Ac burnt roen…
60
H SFE %A salty sweet
˛-Pinene 929 0.013 ± 0.001 - 40
Myrcene 988 0.059 ± 0.009 - floral/burnt bad smell/foot…
p-Cymene 1022 0.068 ± 0.011 - 20
Limonene 1028 11.863 ± 0.548 0.321 ± 0.142
burnt 0 bush/cleaner
-Terpinene 1056 0.010 ± 0.001 -
meta-Cymene 1086 0.216 ± 0.002 -
trans-p-Mentha-2,8-dien-1-ol 1118 0.035 ± 0.000 - refreshing herbaceous
cis-Limonene oxide (Me vs IPP) 1131 0.068 ± 0.001 -
trans-Limonene oxide (Me vs IPP) 1135 0.078 ± 0.011 - refreshing sweet
Dill ether 1183 0.086 ± 0.003 -
cis-Dihydro carvone 1197 7.316 ± 0.877 1.516 ± 0.239
sweet floral/sweet
trans-Dihydro carvone 1206 11.826 ± 0.429 2.706 ± 0.048
iso-Dihydro carveol 1213 0.058 ± 0.001 0.122 ± 0.024 land burnt/metal
trans-Carveol 1224 0.285 ± 0.013 0.111 ± 0.025 floral floral/caramel
Carvone 1252 34.801 ± 1.320 12.654 ± 1.365 caramel
trans-Carvone oxide 1277 0.052 ± 0.004 -
E-Anethole 1283 0.141 ± 0.007 0.104 ± 0.001 Fig. 3. Sensory profile of the dill essential oil extracted by hydrodistillation.
Piperitenone 1339 0.012 ± 0.001 -
Eugenol 1355 0.017 ± 0.000 -
sweet/burn
Myristicin 1519 0.543 ± 0.009 0.706 ± 0.077
sweet 40 herbs
Elemicin 1556 0.153 ± 0.021 0.385 ± 0.048
Dodecanoic acid 1566 – 0.236 ± 0.003 burn/grass 35 burning bush
Dill Apiole 1632 31.029 ± 0.859 77.931 ± 0.998 30
Apiole 1680 0.023 ± 0.009 0.096 ± 0.003 fragrant 25 refreshing
n-Eicosane 1967 – 2.544 ± 0.949 20
Ethyl hexadecanoate 1992 – 0.140 ± 0.069
Total identified 98.751 99.571
15
sweet bush/grass
10
a
The identification of peaks is based on comparison between the experimental
5
Linear Retention Index (LRI) data with those from literature (Adams, 2007).
b
Linear retention time calculated in relation to n-hydrocarbons series reported floral/herbs 0 burnt
according to their elution order on HP-5MSseries.
c
The values correspond to relative proportions of the constituents of essential
oils that were expressed as percentages obtained by normalizing the peak area. sweet pepper

bad smell bacon

sweet peanut/nuts
volatile compounds during extraction with CO2 supercritical it is bush/Grass floral
possible to observe that essential oil presents compounds with Burn
higher FM, consequently higher aromatic potential.
Fig. 4. Sensory profile of the dill volatile extract obtained by supercritical extraction.
GC-olfactometry refers to the use of human assessors as a sensi-
tive and selective detector for odor-active compounds (Xavier et al.,
3.6. Antimicrobial activity
2013). Many odors were identified by the evaluators however only
those who had FM values higher the 30 were presented in Table 5.
Minimum inhibitory concentration (MIC) for supercritical
extraction (SC) and hydrodistillation (HD) extracts were deter-
Table 5
Olfactometry analysis of the dill essential oil.

Extraction technique MFa LRIb Aroma description Compoundc

hydrodistillation 43 841 oil/fat ndd

82 859 cheese/bacon nd
39 909 bush/condiment nd
41 916 rotten potato/súlfur nd
*
39 943 bush/cleaner
*
34 969 sweet
32 1004 burnt/metal nd
32 1131 sweet cis-limonene oxide (Me vs IPP)
55 1213 floral/burnt iso-dihydro carveol
39 1252 burnt carvone

SFE 39 918 burning bush ndd

37 989 pepper nd
32 1193 sweet cis-Dihydro carvone

SFE refers to supercritical fluid extraction.

a
Modified frequency.
b
Linear retention reported according to their elution order on HP-5MS.
c
Compounds identification based on Table 5.
d
Non-detected.
*
Peak was detected, but cannot be identified by GC/MS.
740 J.J. Garcez et al. / Industrial Crops and Products 95 (2017) 733–741
mined. The HD extracts provided higher antibacterial activity than
the extracts obtained by supercritical extraction, with MIC values
lower than 25 mg/mL to inhibit gram-positive and negative bacte-
ria growth. Supercritical extracts have shown to be less effective to
gram-negative bacteria (E. coli), with a MIC value of 40 mg/mL. Con-
cerning to S. aureus and E. faecalis the MIC value were 25 mg/mL. The
results confirm the effectiveness of the A. graveolens extracts as an
antimicrobial agent against studied bacteria. The results corrobo-
rate previous studies that Singh et al. (2005) reported the significant
antimicrobial activity of A. graveolens seed essential oil against
08 different food pathogenic fungi, and 06 bacteria and Sintim
et al. (2015) that identified antileishmanial activity of A. graveolens
essential oil. Thus, the combined assessment of olfactometric and
antimicrobial analysis could result in new possible applications of
the A. graveolens volatile extracts in the food industry following a
market trend of searching multifunctional products.
4. Conclusions
The method of response surface applied to supercritical fluid
extraction of aromatic compounds of A. graveolens indicated that
pressure, temperature and flow rate are statistically significant
variables. The ideal condition for the ranges investigated in this
work, is P = 100 bar, T = 35 ◦ C and flow = 1000 g/h.
The phenomenological mathematical models fitted particularly
well to the experimental curve, providing values for parameters
that can enable the prediction of other process conditions without
the need for conducting experiments.
Finally, the chromatographic analysis revealed that the extract
obtained using supercritical CO2 has more compounds with higher
molecular weight, since the more volatile components are lost
during the expansion phase due to drag promoted by the carbon
dioxide. The major compounds were dillapiole, n-Eicosane, carvone
together with the isomers of Dihydro carvone, whose yields (w/w)
relative to 100 g of seed were 2462.62 mg, 80.39 mg, 399.86 mg and
133.42 mg, respectively.
Acknowledgment
R.M.F.V and E.C. are gratefully indebted to CNPqfor partial finan-
cial support to this work.
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