Biomass and Bioenergy 106 (2017) 1e7

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Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Research paper

Optimization of carotenoids and antioxidant activity of oils obtained

from a co-extraction of citrus (Yuzu ichandrin) by-products using
supercritical carbon dioxide
John Ndayishimiye, Byung Soo Chun*
Department of Food Science and Technology, Pukyong National University, 45 Yongso-ro, Namgu, Busan 48513, Republic of Korea

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

Article history: The processing of citrus fruits leaves massive by-products. Those by-products which are considered as
Received 2 November 2016 wastes contain a wide range of healthy bioactive compounds where carotenoids are among of them. This
Received in revised form work aimed to study the optimum supercritical carbon dioxide (SC-CO2) extraction conditions for
31 July 2017
maximization of carotenoid content and antioxidant activity for citrus by-products, using response
Accepted 9 August 2017
Available online 17 August 2017
surface methodology so that those by-products can not only be valorized effectively but also to reduce
disposal problems. The effects of pressure, temperature, and mixing ratio (citrus peel-to-citrus seed mass
ratio) on the recovery of carotenoids and antioxidant activity were studied. A second-order polynomial
Keywords:
Carotenoids
model showed a suitable tting of the experimental values regarding the carotenoid content (R2
Antioxidant activity 0.9974, p < 0.05) and antioxidant activity (DPPH (R2 0.9919, p < 0.05) and ABTS assay (R2 0.9885, p <
Citrus by-products 0.05)). An optimization and validation study was performed and the optimum extraction conditions were
Supercritical CO2 extraction 25.196 MPa, 44.88  C and 1.91 for carotenoid content, 29.960 MPa, 41.08  C and 1.52 for DPPH assay and
Response surface methodology 29.983 MPa, 43.14  C and 1.50 for ABTS assay, respectively, for pressure, temperature and mixing ratio.
The corresponding predicted values were 1.983 mg g1 oil for carotenoid content, 0.762 mg cm3 for
DPPH, and 1.220 mg cm3 for ABTS assay. The predicted and the experimental values were well in
agreement, thus afrming the adequacy and validity of the predicted models. Overall, the combination of
citrus by-products could yield the oils with good bio-activity and this could be a new valorization
method.
2017 Elsevier Ltd. All rights reserved.

1. Introduction processing industries. In fact, those CP considered as wastes contain

The consumption of citrus fruit either as fresh produce or as the
juice is common due to its dietary benets and particular avor. It is
broadly grown around the world with the yearly production of
about 102 Tg [1]. Because of the production of juice and other
products from citrus fruits, the big amounts of citrus by-products
are generated every year. As consequence, this not only wastes
useful materials, but also may pose some pollution, disposal, and
other related environmental problems due to microbial spoilage
[2]. These citrus by-products have the potential to be valorized
since they contain a wide range of healthy bioactive compounds
[3,4].
The citrus peels (CP) are the primary by-products of citrus
* Corresponding author.
E-mail address: bschun@pknu.ac.kr (B.S. Chun).
bioactive compounds which can contribute to health. The carot-
enoids are among those bioactive compounds which have been
shown to be in CP [5e7]. Carotenoids are the primary compounds in
ripe citrus fruit peels which contribute to their red, yellow and
golden color [8]. A number of studies have shown the importance of
carotenoids as anti-oxidant compounds and precursors of vitamin
A and other health related benets including regulation of the
immune system and anticancer activity [9,10].
The traditional solvent extraction has been used for extracting
the bioactive compounds from plant matrices. However, due to the
instability of carotenoids to light, heat and/or oxygen, the possi-
bility of oxidation and/or thermal degradation of carotenoids in the
extracts have always been a concern [10].
Supercritical carbon dioxide (SC-CO2) extraction has been used
to remove compounds from plant matrices. Particularly, SC-CO2
extraction can provide the mild conditions of extraction together
with low energy requirements to recover the solvent. The higher

http://dx.doi.org/10.1016/j.biombioe.2017.08.014
0961-9534/ 2017 Elsevier Ltd. All rights reserved.
2 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7
selective extraction process and the low possibility of extracts
oxidation make SC-CO2 extraction a suitable technique for extrac-
tion of plant bioactive compounds such as carotenoids [10e12].
Even though carotenoids are considered being non polar, their
solubility in SC-CO2 is usually inhibited due to their large molecular
weight. To overcome this, different modiers have been used to
facilitate the solubility of carotenoids in SC-CO2 where ethanol and
other organic solvents have been the modiers of choice [10].
Nonetheless, the need for heat to separate the extracts from the
solvents (used as modiers) and the possible remaining of some
traces of those solvents in extracts oppose the key advantages of
SC-CO2 extraction [13]. Vegetable oils have been also used instead
of organic solvents to improve SC-CO2 extraction of carotenoids
from plant matrices. The usage of vegetable oils has benets since
not only it enhances the extraction of carotenoids but also there is
no need of separating the oil from the extract [14].
Although many studies have been dedicated to the extraction of
carotenoids by SC-CO2 using vegetable oils as co-solvent [13,14], no
such study on the carotenoid content of oils from a co-extraction of
CP and citrus seeds (CS) using SC-CO2 where CS oil portion (since CS
contain about 30% of oil) in the mixture may act as a co-solvent to
enhance the carotenoids extraction from CP matrix has emerged.
More importantly, this mixing of CP and CS may not only increase
the availability of carotenoids content but also the bio-potentiality
of resulting oils may be increased because of the synergistic effect
between their compounds [13,15].
In this study, the effects of the independent variables i.e.,
pressure, temperature, and mixing ratio (CP-to-CS ratio) on the SC-
CO2 extraction of carotenoids from CP matrix were studied. In
addition, the effects of those independent variables on the anti-
oxidant activity of extracted oils were investigated, and the optimal
extraction conditions were studied.
2. Materials and methods
2.1. Chemicals
Carotenoids standards, 2,20 -azinobis-(3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt (ABTS), 1,1 diphenyl-2-picryl-
hydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis,
USA). Ethanol, acetonitrile, chloroform, dichloromethane, meth-
anol were purchased from Samchun Company (Busan, South Ko-
rea). Carbon dioxide (with 99.99% purity) was bought from KOSEM
Company (Busan, South Korea). All the reagents and chemicals
were of analytical grade.
2.2. Plant material
The citrus fruits (Citrus ichangensis  C. reticulate) at the mature
stage were harvested and collected from Namhae-gu, Gyeong-
sangnam-do Province, South Korea (35 150 000 N, 128 150 000 E) in
December 2015. The collected fruits were of eating quality and
without blemishes or damage. After collection, the samples were
transferred to the laboratory at the same day to keep the freshness
of the fruits.
2.3. Sample preparation
Citrus fruits were cleaned, peeled off and the peels and seeds
were collected. The percentage weight of CP and CS compared with
the weight of the fresh fruit was about 43 1.52% and 9 1.1%,
respectively (on a fresh weight basis). The CP were freeze dried
(50  C for 4 days) while the CS were oven dried at 103  C. The
average weight of CP and CS were 106.21 0.91 g kg1 and 80.82
0.85 g kg1, respectively, of the fruit (on dry weight basis) and the
moisture content was 75.3 0.73% and 10.2 0.38% for CP and CS,
respectively. The sample materials were then crushed, sieved (us-
ing a 710 mm metal sieve) to get powder for extraction.
2.4. Supercritical carbon dioxide extraction
The SC-CO2 extraction diagram used in the study is shown in Fig.
1. The extraction setup and procedures were the same as those
reported in our previous work [16]. The SC-CO2 extraction condi-
tions (pressure, temperature and mixing ratio (CP-to-CS mass ra-
tio)) are depicted in Supplementary Table 1. The extraction time
was 2 h and the CO2 ow rate was 27 g min1.
2.5. Analysis of carotenoid composition by HPLC
Carotenoids identication and quantication were performed
by normal-phase HPLC using a Hitachi chromatography system.
Approximately 20 mg of extracted oil were dissolved in 1 cm3 HPLC
grade chloroform, ltered through 0.45 mm lters and 20 mm3 were
injected directly into an XDB-C18 column (5 mm, 4.6 mm  150
mm) (Agilent Technologies, Hewlett-Packard, CA, USA). The mobile
phase was acetonitrile/methanol/dichloromethane (60:35:5 v/v),
the ow rate was 1.0 cm3 min1 and the detection wavelength was
450 nm by a Hitachi L-2420 UV-Vis detector. The individual ca-
rotenoids standards were diluted with chloroform at different
concentrations to construct an external standard curve which was
used to identify and quantify the carotenoids in the extracted oils.
2.6. Antioxidant activity determination
2.6.1. DPPH radical scavenging assay
The DPPH assay of extracted oils was carried out in accordance
with a method reported by Zhang et al. [17] with some modica-
tions. Exactly, 1.5 cm3 of oil with different concentrations in ethanol
was added to 1.5 cm3 of 100 mmol m3 ethanol DPPH radical so-
lution. The absorbance was read at 492 nm using a microplate
reader (Synergy HTX BioTek Instruments, Winooski, VT, USA) after
30 min of incubation at ambient temperature (in dark). Ethanol was
used as a control. A percent inhibition against concentration curve
was plotted and the concentration of oil required for inhibiting 50%
was determined and expressed as IC50 values.
2.6.2. ABTS radical scavenging assay
The ABTS radical scavenging assay of extracted oils was
determined by a method reported by Xu et al. [18] with some
modications. In brief, the pre-formed ABTS radical was obtained
by reacting 2.45 mmol m3 potassium persulfate with ABTS solu-
tion (0.007 mol m3) for fourteen hours (in the dark at room
temperature). Thereafter, the solution was diluted (with ethanol) to
get the absorbance of 0.7 0.2 at 734 nm. The extracted oils were
dissolved in ethanol at different concentrations and an aliquot of 1
cm3 of each concentration was added to 3 cm3 of already prepared
ABTS radical solution and shaken vigorously. After one hour in
the dark, the absorbance was measured at 734 nm using the same
microplate reader. A percent inhibition against concentration curve
was plotted and the required concentration of oil for 50% inhibition
was determined and expressed as IC50 values.
2.7. Experimental design and statistical analysis
The effect of independent variables (pressure: 20e30 MPa,
temperature: 40e50  C and mixing ratio: 1.5e3 g g1) on responses
(carotenoid content and antioxidant activity) of extracted oils
was investigated by applying the response surface methodology
(RSM). BoxeBehnken experimental design with three numeric
 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7 3

Fig. 1. Schematic diagram of SC-CO2 extraction process.

independent variables on three levels was used. The design con-
sisted of fteen runs in random and three replicates were put at the
central point to reduce the error. Each independent variable was
coded in dimensionless numbers from 1 to 1 according to Equa-
tion (1) [19,20].
of independent variables used are displayed in Supplementary
Table 1. The relationship between the independent variables and
the responses was examined by tting the responses to the second-
order polynomial model (Equation(2)) [21].

X
3 X
3 X
2 X
3
X  X0 Y b0 bi Xi bii Xi 2 bij Xi Xj (2)
xi i i 1; 2; 3; :::; k (1)
DX i1 i1 i1 j2
i<j
Where xi : coded value, Xi : corresponding actual value, X0 : actual
value in the center of the domain, and DX: increment of Xi corre- Where Y: response variable, Xi and Xj represent the independent
sponding to a variation of 1 unit of X. The uncoded and coded values variables which affect the response, and b0, bi, bii, and bij represent

Table 1
Analysis of variance for the quadratic model for the carotenoid content and antioxidant activity.

Variation Source Sum of Squares df Mean Square F Value P-value

Carotenoid content (mg g1 oil)

Model 1.78 9 0.20 209.46 <0.0001 signicant
Residual 0.004725 5 0.000945
Lack of Fit 0.003925 3 0.001308 3.27 0.2429 not signicant
Pure Error 0.0008 2 0.0004
Total 1.79 14
Antioxidant activity(mg cm3)
DPPH assay
Model 0.22 9 0.024 68.09 0.0001 signicant
Residual 0.001792 5 0.0003583
Lack of Fit 0.001525 3 0.0005083 3.81 0.2147 not signicant
Pure Error 0.0002667 2 0.0001333
Total 0.22 14
ABTS assay
Model 0.20 9 0.022 47.89 0.0003 signicant
Residual 0.002325 5 0.0004650
Lack of Fit 0.002125 3 0.0007083 7.08 0.1262 not signicant
Pure Error 0.0002 2 0.0001
Total 0.20 14

Values of p-value less than 0.05 indicate models are signicant.

4 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7
the regression coefcients for intercept, linear, quadratic and
interaction terms, respectively. The Design-Expert v.7 (Stat-Ease,
Minneapolis, Minnesota, USA) was used.
2.8. Optimization and validation of optimized conditions
The optimization of responses (carotenoid content and antiox-
idant activity (DPPH and ABTS assay)) was performed by a nu-
merical optimization technique using the same statistical software
package (Design-Expert v.7). Optimal conditions for carotenoid
content and antioxidant activity (DPPH and ABTS assay) of extrac-
ted oils dependent up on extraction pressure, temperature and
mixing ratio (CP-to-CS ratio) were obtained and the responses were
evaluated after extraction under those optimal conditions. In order
to validate the models, the predicted and experimental values were
compared.
3. Results and discussion
3.1. Model tting
The quadratic models representing carotenoid content (Y1) and
antioxidant activity (DPPH assay (Y2) and ABTS assay (Y3)) are
presented in Equations (3)e(5), respectively.
Y1 /20.1375 0.03485X10.72725X21.745X30.00079X1X2
0.0026X1X3/0.012X2X3/0.000149X21/0.0102X22/0.48889X23 (3)
Y2 9.97208/0.020808X1/0.2375X2/0.6416X30.0003X1
X20.0026X1X30.0026667X2X3/0.00000183333X21
0.00171667X22/0.00814815X23 (4)
Y3 10.20875/0.020175X1/0.2315X2/0.64833X3
0.00022X1X20.00286667X1X30.00266667X2X30.0000035X21
0.00185X22/0.02X23 (5)
Table 1 shows the results of tting quadratic models to the data.
The statistical signicance of the models was tested by performing
the analysis of variance of the experimental data. The analysis of
variance results show that the quadratic models contributed
signicantly. The signicance was examined using the p-value and
F-test and the F-values of 209.46, 68.09 and 47.89 associated with
p-values of <0.0001, 0.0001, and 0.0003 for carotenoid content,
DPPH assay, and ABTS assay, respectively were obtained, which
indicated that the tness of the models was signicant (p < 0.05)
[22].
The lack of t testing (p > 0.05) was performed to conrm the
Fig. 2. 3-D response surfaces for carotenoid content.
adequacy of the t [23] and the adequacy of the model with respect
to carotenoid content, DPPH assay, and ABTS assay was 0.2429,
0.2147, and 0.1262, respectively, which indicates that the model can
satisfactorily t the experimental data.
In addition, the results in Supplementary Table 2 demonstrated
that the proposed regression model for carotenoid content, DPPH
assay, and ABTS assay was satisfactory, with high determination
coefcients (R2) of 0.9974, 0.9919, and 0.9885, respectively. The R2
adjusted values (0.9926, 0.9773, and 0.8301 for carotenoid content,
DPPH assay, and ABTS assay, respectively) were also high and that
indicates a high signicance of the models. The models showed the
high R2 predicted values of 0.9638, 0.8871, and 0.8301 for carot-
enoid content, DPPH assay, and ABTS assay, respectively, which
indicates the high correlation between the predicted and experi-
mental values. Moreover, the R2 adjusted values and R2 predicted
values showed to be in reasonable agreement (within approxi-
mately 0.20 of each other), which in turn demonstrates a good
agreement between the predicted and experimental values.
Meanwhile, the Adequate precision which indicates the signal-to
-noise ratio was shown to be > 4, which signify an adequate
signal and prove that all predicted models are adequate [24,25].
3.2. Regression coefcients and responses surfaces analysis
3.2.1. Carotenoid content
The regression coefcients of the models for carotenoid content
are demonstrated in Supplementary Table 3. The effect of pressure,
temperature, and mixing ratio was signicant (p < 0.05) for all ef-
fects (linear, quadratic, and interactive) and all effects were posi-
tive. 3-D response surface plots in Fig. 2 shows the change in
carotenoid content as a function of pressure, temperature, and
mixing ratio.
The Fig. 2a and b show the inuence of pressure on the carot-
enoid content. When the temperature and mixing ratio were kept
constant (at center level), the carotenoid content increased to a
value with elevating pressure, thereafter decreased. As shown in
the gure, the pressure range of 22.5 MPae27.5 MPa showed the
high carotenoid content. Below 22.5 MPa, the decrease of pressure
showed to decrease the carotenoid content. Similarly, the further
elevation of pressure (above 27.5 MPa) showed to decrease the
carotenoid content. This low pressure (below 22.5 MPa) which is
associated with low carotenoid content might be due to that at that
low pressure; the density of SC-CO2 was low, which might cause
the low solubility of carotenoids in SC-CO2 which in turn might
result in the decrease of carotenoid content. On the other hand, the
high carotenoid content obtained in the pressure range of 22.5
MPae27.5 MPa might be attributed to the increase of SC-CO2
 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7
Fig. 3. 3-D response surfaces for antioxidant activity: A&B) DPPH assay and C) ABTS assay.
density and hence the solubility increase of carotenoids [26,27].
However, as can be seen in the gure, the carotenoid content
started to decline as the pressure increased towards 30 MPa. This
might be ascribed to the fact that the increase of pressure above
that pressure might have a great impact on the extraction of other
molecules than carotenoids due to the reduction of selectivity of
SC-CO2 which consequently might reduce the carotenoid content
since it is like diluting effect [28,29].
Regarding the temperature, the pattern was the same as pres-
sure. At low temperature, the carotenoid content was lower, but by
increasing temperature the carotenoid content seemed to increase,
then after decrease (Fig. 2a and c). This might be explained by
considering the two effects of temperature on the solubility of
substances. In fact, the vapor pressure of the solute increases al-
ways with temperature, whereas the density (or solvent power due
to pressure augmentation) of SC-CO2 decreases [30,31]. According
to our results, it seemed like between 42.5  C and 47.5  C, the
density effect was predominating hence the carotenoids might
have be extracted at the high level. On the other hand, above that
range, the vapor pressure effect (due to an increase of temperature)
might hinder signicantly the solubility of carotenoids in SC-CO2
hence the carotenoid content might be reduced.
Moreover, the carotenoid content showed to vary with mixing
ratio (Fig. 2b and c). In general, the mixing ratio (1.5e2.25) resulted
in high carotenoid content up to 1.94 mg g1 oil while a further
increase of the mixing ratio showed to decrease the carotenoid
content. This variation of carotenoid content resulting in the vari-
ation of mixing ratio might be explained by looking into the nature
of carotenoids solubility in SC-CO2 and the possible role of CS in
carotenoids extraction from CP matrix. Structurally, the existence of
long non-saturated aliphatic chains gives the carotenoids their
hydrophobic property. The absence of strong polar moieties in their
molecules would imply that the solubility of carotenoids in SC-CO2
would be reasonably higher. But, regardless of their reasonably
apolar behavior, their solubility in SC-CO2 is inhibited by their high
molecular weight. Many works have used the vegetable oils as co-
solvent for SC-CO2 extraction of carotenoid from plant matrices
[11,14]. In the same concept, instead of using vegetable oil, the CS
which contains about 30% of the oil was used to leverage the
extraction of carotenoids from CP matrix (acting as co-solvent) in
our work. So, it might be assumed that the oil part of CS might have
played a signicant role in the extraction of carotenoids by
increasing the solubility of carotenoids in SC-CO2 in that range of
mixing ratio (1.5e2.25). But, as the gure shows, above the mixing
ratio of 2.25, the carotenoid content started to decline as the mixing
ratio increased, this might be due to that above that ratio, the effect
of CS oil part in the system to leverage the carotenoids extraction
5
diminished. Actually, the increase of mixing ratio means the
reduction of CS proportion and increase the CP proportion in the
mixture. Conversely, the decrease of mixing ratio means the in-
crease of CS proportion and decrease of CP proportion in the
mixture. So, the increase of mixing ratio might result in a dimi-
nution of CS oil to help extract the carotenoids from CP matrix. In
addition, a similar pattern can be seen when CP was extracted alone
(without combining it with CS) at the optimum conditions (Table
3), where the carotenoid content is signicantly low compared to
that of a combination (at the optimum conditions). Here, it seems
as though the extraction of CP alone by SC-CO2 was similar to its
extraction using neat SC-CO2, whereas the extraction for the
mixture (CS CP) was similar to the extraction using a modied
SC-CO2. This is speculated to be because CS oil can act as a co-
solvent, justifying why the carotenoid content was higher in oil
from a mixture than CP alone.
3.2.2. Antioxidant activity
The results for the effect of each independent variable to the
antioxidant activity, expressed as IC50 values, are depicted in
Supplementary Table 3 and Fig. 3. The low IC50 value corresponds to
a strong antioxidant activity. Regarding the pressure, linear and
interactive effects were demonstrated to be statistically signicant
while the quadratic effect was found to be insignicant
(Supplementary Table 3).
3-D response surface plots for the effect of pressure on the
antioxidant activity are presented in Fig. 3a and b. As can be seen in
the gure, the elevation of pressure reduced the IC50 value (an in-
crease of the antioxidant activity) and at high pressure, the IC50
value was low (strong antioxidant activity). This increase of anti-
oxidant activity due to the increase of pressure might be justied by
correlating density, solubility, and selectivity. As discussed above,
the pressure augmentation causes SC-CO2 density increase, which
thereby increases the solubility of analytes in SC-CO2. Here it is so
important to say that when the density increases, it not only permit
a wide range of components to be dissolved in SC-CO2 but also the
selectivity of SC-CO2 decreases which allows even the extraction of
other dense molecules (i.e., coumarins) and other non saponiable
substances (like phospholipids and phytosterols) [32]. So these
additional substances which might be extracted due to increase in
pressure might contribute to the overall antioxidant activity, which
is the reason why the high pressure might have showed low IC50
value.
For the temperature, a linear effect was detected to be signi-
cant for ABTS assay but insignicant for DPPH assay. The quadratic
and interactive effects were found to be signicant for both assays
(Supplementary Table 3). The Fig. 3a and c show the effect of
6 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7
Table 2
Optimum extraction conditions for the responses.
Responses Independent variables
Pressure (X1)
(MPa)
Carotenoid content (mg g1 oil) 251.96
Antioxidant activity (mg cm3)
DPPH assay 299.60
ABTS assay 299.83
Table 3
Predicted and experimental values of responses under optimum extraction conditions.
Responses Mixturea
Predicted value
Carotenoid content (mg g1 oil) 1.983
Antioxidant activity (mg cm3)
DPPH assay 0.762
ABTS assay 1.22
a
Predicted and experimental values of responses obtained by extracting the mixture (under optimum pressure, temperature and mixing ratio).
b
Experimental values of responses obtained by extracting the citrus peels only (under optimum pressure and temperature).
temperature on the antioxidant activity. The temperature showed
to affect slightly antioxidant activity even though it was insigni-
cant for DPPH assay. Here, the temperature could have had an in-
uence due to interaction with other independent variables by
enhancing the diffusion rate of analytes.
Concerning the mixing ratio, linear effects were signicant, but
the quadratic and interactive effects were not signicant for both
assays (Supplementary Table 3). Fig. 3b and c show the response
surfaces for the effect of mixing ratio on antioxidant activity. The
results show that at lower CP-to-CS ratio, the antioxidant activity
was higher than at higher CP-to-CS ratio. This increase of antioxi-
dant activity resulting to the decrease of mixing ratio might be
justied by looking into the composition. The combination of CP
and CS not only help the extraction of carotenoids (due to the oil
portion of CS which act as co-solvent) but also it might contribute
to the activity due to the other active compounds that CS contain.
Among those compounds, the tocopherols and phytosteols which
can be co-extracted during extraction are the major ones and many
studies have proven that those compounds can exhibit the anti-
oxidant activity [33,34]. Even though the tocopherol and phytos-
terol content was not reported here, it might be understandable
that the reduction of CS proportion in the mixture (an increase of
mixing ratio) might reduce those additional compounds. So given
that CS might play an important role in antioxidant activity by
providing those compounds (tocopherols, phytosterols, etc), it is
plausible to say that the reduction of CS proportion in the mix-
ture(increase of mixing ratio) might reduce those compounds in
the extracted oils which probably could affect negatively the overall
antioxidant activity.
3.3. Optimization and validation
Table 2 shows the optimal extraction conditions for each
response. The optimal extraction conditions for carotenoid content
were pressure of 25.196 MPa, temperature of 44.88  C and mixing
ratio of 1.91. On the other hand, the optimal extraction conditions
for DPPH assay were pressure of 29.96 MPa, temperature of 41.08  C
and mixing ratio of 1.52 while the optimal conditions for ABTS
assay were pressure of 29.983 MPa, temperature of 43.14  C and
mixing ratio of 1.50. These conditions gave predicted values of
1.983 mg g1 oil, 0.762 mg cm3 and 1.22 mg cm3 for carotenoid
Temperature (X2) Mixing ratio (X3)
( C) (g g1)
44.88 1.91
41.08 1.52
43.14 1.50
Citrus peelb
Experimental value Experimental value
(mean SD, n 3) (mean SD, n 3)
1.952 0.01 1.463 0.03
0.75 0.06 0.98 0.01
1.26 0.08 1.51 0.1
content, DPPH assay, and ABTS assay, respectively and the experi-
mental values were reasonably close to the predicted values (p >
0.05) (Table 3), thus justifying the adequacy and validity of the
predicted models.
3.4. Correlation between the carotenoid content and antioxidant
activity
The correlation between carotenoid content and antioxidant
activity was calculated and the antioxidant activity did not well
correlate with carotenoid content (R2 0.6302 for DPPH assay and
R2 0.5871 for ABTS assay). This indicates that the antioxidant
activity of extracted oils could not be entirely attributed to their
carotenoids since there are other several antioxidant compounds
like phytosterols, tocopherols, phenolics and avonoids which
might not only contribute individually to the overall antioxidant
activity but also through the synergistic effects among them [35].
As far as the methods used are concerned, the DPPH assay
showed low IC50 values compared with ABTS assay (Supplementary
Table 1). This difference might be due to ABTS assay is specic to the
antioxidant capacity of hydrophilic compounds [36] while DDPH
assay can perform well in both aqueous-organic extracts of plant
foods and vegetable oils [37,38]. Based on the nature of our
extracted oils, it seems like DPPH assay presented an advantage
over ABTS assay because it might be able to react with both hy-
drophilic and lipophilic antioxidants compounds present in a
sample, which could have contributed to the augmentation of
overall antioxidant activity for DPPH. Contrarily, ABTS is hydro-
philic in nature; its ability to react might sometimes be limited only
to the hydrophilic compounds in the extract and it may not react
with lipophilic compounds, which in turn might contribute to the
diminution of overall antioxidant activity compared with that of
DPPH.
4. Conclusion
The RSM was effectively achieved for optimization of caroten-
oids and antioxidant activity from a mixture of citrus by-products.
The independent variables showed to affect the carotenoid content
and antioxidant activity of extracted oils. More importantly, it
appeared that the oil from a mixture of CS and CP presented higher
 J. Ndayishimiye, B.S. Chun / Biomass and Bioenergy 106 (2017) 1e7
carotenoid content and antioxidant activity than oil from CP only,
which in turn may reect how that mixture oil can be applied in
many areas. Generally, this study showed that the extraction (using
SC-CO2) of a mixture of CS and CP, which are considered as wastes,
might not only yield the oils with high bio-potentiality, but might
also be a promising work in many areas since the SC-CO2 extraction
is an undisputable environmentally-friendly extraction technique,
which could be an advantage over the conventional solvent
extraction.
Acknowledgements
The authors gratefully acknowledge the nancial support for the
research work provided by Business for Cooperative R&D between
Industry, Academy, and Research Institute funded Korea Small and
Medium Business Administration in 2015.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biombioe.2017.08.014.
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