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Journal of Food Quality ISSN 1745-4557

ULTRASONIC-ASSISTED SOLVENT EXTRACTION OF

CAROTENOIDS FROM RAPESEED MEAL: OPTIMIZATION USING
RESPONSE SURFACE METHODOLOGY
FENGWEI YAN1,2,3, KAI FAN1, JIE HE1 and MENGXIANG GAO1
1
Department of Food Science and Technology, College of Life Science,
2
Jingchu Food Research and Development Center, Yangtze University, Jingzhou, Hubei Province 434025, China

3
Corresponding author. ABSTRACT
TEL: +86-716-8066712;
FAX: +86-716-8066257; Ultrasound-assisted solvent extraction of carotenoids from rapeseed meal was
EMAIL: yanfw001@yangtzeu.edu.cn investigated and evaluated by response surface methodology. We have compared
the yields of carotenoids by using five different solvent systems, and found that the
Received for Publication May 27, 2014
highest yield can be achieved under petroleum ether/acetone (v/v = 1/1). We have
Accepted for Publication May 19, 2015
further tested the effect of ultrasonic power, modulation frequency, duration, tem-
10.1111/jfq.12154 perature, liquid to material ratio and extraction times on the yield of carotenoids
by adopting Plackett–Burman and Box–Behnken experimental design methods.
Optimal conditions for obtaining high yield of carotenoids were as follows: tem-
perature 49.6C, liquid to material ratio 41.4 mL/g, duration 48.5 min, ultrasonic
power 252.9 W (240 W was used in the control experiments). Under the above
conditions, average carotenoid yield was 0.1577 ± 0.0014 mg/g, and the extraction
percentage was 79.61 ± 0.71%. The results indicated that rapeseed meal was a
promising alternative source for carotenoid extraction.

PRACTICAL APPLICATIONS
Carotenoids are one of the most common natural food colorants and exist widely
in plants, bacteria, fungi and animals. Ultrasound has been utilized to assist the
solvent extraction of bioactive compounds from vegetal materials, and led to high
yield of desired products. In the present study, we have investigated ultrasound-
assisted extraction of carotenoids from rapeseed meal using the response surface
methodology. In order to obtain the highest yield of carotenoids, we have exam-
ined various operational parameters, such as extraction temperature, liquid to
material ratio, solvent composition, extraction time and modulation frequency,
and have established optimal conditions to extract carotenoids from rapeseed
meal. Our results provide basic knowledge on the extraction of carotenoids from
rapeseed meal, which serves as a promising bioresource.

tion (Rutkowska and Stolyhwo 2009). The industrial

INTRODUCTION
Carotenoids are one of the most common natural food
colorants and exist widely in plants, bacteria, fungi and
animals (Sachindra and Mahendrakar 2005). Dietary
intake of adequate carotenoids from fruits and vegetables
(Kim and Kim 2011) and even therapeutic doses of carot-
enoids such as β-carotene (Wang et al. 1999) have been
associated with a lower risk of cancer, such as lung cancer,
due to their antioxidant activity and provitamin A func-
demands for carotenoids in food, pharmaceuticals, cosmet-
ics, nutraceuticals and textiles are continuing to rise. Yet,
the majority of commercially available carotenoid products
are chemically synthesized, instead of being derived from
natural sources that consumers prefer (Gu et al. 2008).
Interest is growing to extract natural carotenoids from
microorganism, microalgae and various agricultural
by-products, e.g., plants and microalgae (Gu et al. 2008;
Jaime et al. 2010).

Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc. 377
EXTRACTION OF CAROTENOIDS
Rapeseed (Brassica napus L.) is one of the most impor-
tant oilseeds in the world, and it ranks as the fourth largest
production followed by soybean, palm and cottonseed (Pan
et al. 2011). In China, the production of rapeseed exceeded
1.2 × 107 tons in 2008 (Pan et al. 2011). Conventional indus-
trial processing of rapeseed involves pressing and hexane
extraction, which produce two products – the oil and a low-
value meal that is mainly used as animal feed or fertilizer.
The total production of rapeseed meal is 4.0 × 106 tons in
2008 in China (Mailer et al. 2008). It is technologically and
economically valuable to recover biologically active ingredi-
ents from rapeseed meal and improve the use of these
bioresources (Thiyam et al. 2009). The current oilseed
industry has focused on deriving valuable ingredients from
rapeseed meal, such as protein isolates, bioactive peptides
and antioxidative compounds (Thiyam et al. 2009), but has
paid no attention to carotenoids. Kull and Pfander (1995)
reported that there are more than 80 different carotenoids
present in the petals of rape, in which (9′Z)-neoxanthin,
(8′S)-luteoxanthin, (8′R)-luteoxanthin, (all-E)-violaxanthin
and (9Z)-violaxanthin are the major components. Studies
have been reported on extraction of carotenoids from
various materials, but there is no known report on extrac-
tion of carotenoids from rapeseed meal that contains abun-
dant carotenoids (Sachindra and Mahendrakar 2010; Mai
et al. 2013; Shi et al. 2013; Hiranvarachat and Devahastin
2014). The most widely accepted extraction methods
involve the extraction of carotenoids with one or more
organic solvents. Methanol, acetone and petroleum ether
were used almost at every turn (Howe and Tanumihardjo
2006).
Ultrasound has been utilized to assist the solvent extrac-
tion of bioactive compounds from vegetal materials, and led
to high yield of desired products. The application of
ultrasound-assisted extraction offers many advantages,
including the reduction of solvent use, lower extraction
temperatures and shorter extraction times, all of which are
critical factors in extracting thermolabile and unstable com-
pounds (Vilkhu et al. 2008). Most ultrasound-assisted
extraction experiments were conducted at laboratory scale,
and it is very challenging to scale up to an industry level.
Recently, industrial ultrasound equipment has advanced to
provide industrially robust processing capability, which lays
a strong foundation for the scaling up of ultrasound-
assisted extraction. A few reports on pilot-plant scale have
been published (Vilkhu et al. 2008; Boonkird et al. 2009;
Achat et al. 2012).
In the present study, we have investigated ultrasound-
assisted extraction of carotenoids from rapeseed meal using
the response surface methodology (RSM). In order to
obtain the highest yield of carotenoids, we have examined
various operational parameters, such as extraction tempera-
ture, liquid to material ratio, solvent composition, extrac-
378
F. YAN ET AL.
tion time and modulation frequency, and have established
optimal conditions to extract carotenoids from rapeseed
meal. Our results provide basic knowledge on the extraction
of carotenoids from rapeseed meal, which serves as a prom-
ising bioresource.
MATERIALS AND METHODS
Materials
Rapeseed meal processed from double-low rapeseed (the
rapeseed meal with low contents of glucosinolates and
erucic acid) was acquired from Tianyi Oil Industrial Co.,
Ltd. (Hubei, China). The rapeseed was harvested in 2009.
The meal processed within 10 h of harvest was packed in
black plastic bags (polyethylene) and immediately brought
to our laboratory. It contained 7.5 ± 0.8% moisture,
43.2 ± 0.7% protein (N × 6.25) and 9.7 ± 0.7% fat.
Rapeseed meal was ground into fine powder using a
mortar and pestle, and passed through a 100 mesh screen
(150-mm aperture), mixed thoroughly, kept in closed dry
clean polyethylene bags and stored at 4C until use.
β-Carotene standard sample was purchased from Sigma-
Aldrich Chemical Co. (St. Louis, MO). All other chemicals
used in the experiments were of analytical grade.
Conventional Extraction Procedure
Conventional extraction was carried out in a water bath
(Model HH-4, Hangzhou Limai Instrument Co., Ltd.,
Hangzhou, China). Five grams of rapeseed meal powder
was used per extraction. Rapeseed meal powder was added
into a 250-mL flask, the extraction solvent was added and
reflux condenser refluxed to the extracts. The flask was
partially immersed into the water bath. The bottom of the
flask was approximately 2 cm above bath.
Carotenoid extraction with petroleum ether, acetone,
chloroform, methanol and petroleum ether/acetone
(v/v = 1/1) was used respectively to choose an appropriate
solvent by water bath at the extraction temperature of 40C,
duration of 60 min, liquid to material ratio of 20 mL/g and
one time extraction. Petroleum ether/acetone was a better
extraction solvent. The extraction process by petroleum
ether/acetone (v/v = 1/1) was repeated until the solvent lost
its color. The supernatant was combined and evaporated.
The total carotenoid content measured by colorimetric
assay was 0.1981 ± 0.0031 mg/g.
To compare with the ultrasound-assisted extraction
method, water bath extraction method was carried out at
the temperature of 49.6C, liquid to material ratio of
41.4 mL/g and duration of 48.5 min. Petroleum ether/
acetone (v/v = 1/1) was used as solvent.
Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc.
F. YAN ET AL.
Ultrasound-Assisted Extraction
Ultrasound-assisted extraction was carried out in an ultra-
sonic cleaning bath (Model JPCT 0328, Wuhan Jiapeng
Electronics Co., Ltd., Wuhan, China) with a frequency of
28 kHz and a modulation frequency of 10–160 Hz. The
extraction of carotenoids was performed by adding 5 g of
rapeseed meal in a 250-mL flask. The flask was partially
immersed into the ultrasonic bath, which contained 20 L of
water (the internal dimensions: 50 × 30 × 20 cm). The
bottom of the flask was approximately 2 cm above bath and
the liquid level in the flask was about 5 mm below the water
surface in the bath. Petroleum ether/acetone was used as the
extraction solvent. The bath water was circulated and regu-
lated at constant temperature to avoid overheating. The
parameters during each extraction – temperature, material
to liquid ratio, duration, extraction times, ultrasonic power
and modulation frequency – were chosen upon experimen-
tal design (Tables 2, 4, and 5).
The extract was filtered with filter paper (Whatman No.
1, Shanghai Mosu Science Equipment Co., Ltd., Shanghai,
China) and the filtrate was centrifuged (Beckman J6HC,
Shanghai ZhaoMing Electronic Technology Co., Ltd.,
Shanghai, China) at 2,072 × g for 20 min to discard the
remainder of the precipitates. The pigmented solvent layer
from the supernatant was collected and evaporated using a
rotary evaporator (Rotavapor R-200, Buchi Labortechnik
A, Flawil, Switzerland) at 35C. The residue was dissolved
and washed three times with petroleum ether and trans-
ferred to a 50-mL volumetric flask, and the carotenoid
content was determined.
At the condition of repeated extraction, the extract was
filtered with filter paper (Whatman No.1), and the filtrates
were collected and the solids were extracted repeatedly.
The rapeseed meal and extractive parameters were the
same for all stages; only the solvent was renewed in each
new extraction.
Determination of Carotenoids
Spectrophotometric analysis was carried out to quantify the
total carotenoid content based on the method of Sachindra
and Mahendrakar (2005). The extract was monitored at
450 nm using a Shimadzu (Shanghai, China) UV-1619
spectrophotometer. Quantification of the total carotenoid
content was carried out based on the absorption coefficients
calculated for β-carotene in petroleum ether at a given
wavelength (450 nm) of the utilized spectrophotometer.
Plackett–Burman Experimental Design
Petroleum ether/acetone was identified as the better extrac-
tion solvent. The important factors were efficiently evalu-
Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc.
EXTRACTION OF CAROTENOIDS
ated using a small fraction of the experiments required for a
full factorial design (Yang et al. 2007). According to our pre-
liminary experimental results, the yield of carotenoids was
significantly influenced by temperature, material to liquid
ratio, duration, extraction times, ultrasonic power and
modulation frequency (data not shown), and their ranges
between the model levels described as −1 and 1 were
selected and evaluated for optimization of carotenoid
extraction: ultrasonic power (A, 120–180 W); modulation
frequency (B, 40–100 Hz); duration (C, 10–45 min); tem-
perature (D, 20–50C), to minimize the solvent loss, and the
ground flask was accompanied with reflux condense); liquid
to material ratio (mL/g), E (15–30) and extraction times, F
(1–3) were evaluated. The following experiments were
designed with Plackett–Burman (PBD) method (Zhou et al.
2009; El Aty et al. 2014), in which six factors (temperature,
material to liquid ratio, duration, extraction times, ultra-
sonic power and modulation frequency) were included and
evaluated using the SAS version 8.2 software (SAS Institute,
Inc., Cary, NC). Each factor (independent variable) was
tested at two levels: high (+1) and low (−1). A total of 16
experiments were carried out in this experimental series,
and the response value was measured in terms of carotenoid
yield (dependent variables), as shown in Table 2. This set of
experiments allows us to determine which factors were
important for carotenoid extraction.
Path of Steepest Ascent
Experimental Design
According to the first-order model and the four important
effect factors above, we concentrated on four most impor-
tant effect factors. The path of steepest ascent was first
determined, so that we can decide the proper direction to
change the variables according to the sign of the main
effects on improving carotenoid yield. The path of steepest
ascent experiments was shown in Table 4.
Box–Behnken Design and RSM
According to the principle of Box–Behnken design (BBD)
(Zhou et al. 2009; Zhu and Liu 2013), a total number of 29
experiments were conducted for four factors and at three
levels, as shown in Table 5. Four independent parameters –
temperature, liquid to material ratio, duration and ultra-
sonic power – were employed at three different levels,
respectively. The parameters and their levels were based on
steepest accent experiments. The results were analyzed using
SAS 8.2 software, in which models were built to plot the
four-dimensional response surface curves. The fitting
quality of the second-order model was expressed by the
coefficient of determination R2, and its statistical signifi-
cance was evaluated by an F-test. The significance of the
379
EXTRACTION OF CAROTENOIDS F. YAN ET AL.

TABLE 1. PRELIMINARY SCREENING FOR THE SOLVENT OF CAROTENOID EXTRACTION

Solvent Acetone Petroleum ether Methanol Chloroform Petroleum ether/acetone (v/v = 1/1)
Carotenoid yield (mg/g) 0.0970 ± 0.0007c 0.1057 ± 0.0034b,c 0.1177 ± 0.0049a 0.1303 ± 0.0094a 0.1230 ± 0.0049a

All data presented are means ± standard deviation (n = 3). Values with different letters (a, b, c) differ significantly (P < 0.05, Duncan’s multiple range
tests). Carotenoids were extracted by water bath at the extraction temperature of 40C, duration of 60 min, liquid to material ratio of 20 mL/g and
one time extraction.

regression coefficients was tested by a t-test (Tables 6 and 7).
The experiments were run in a random order to minimize
the effects of unexpected variability in the observed
responses due to extraneous factors. Finally, the model vali-
dation was carried out.
Statistical Analysis
All tests were carried out in triplicate and the averages of
carotenoid yields were taken as responsive value. All data
shown in the tables and figures were expressed as
mean ± standard deviation of the three replicates. A second-
order polynomial regressed equation was established on the
basis of analysis of Box–Behnken experimental data, and the
optimal conditions for extraction were determined by SAS
8.2 analysis. When mean values of compared sets are differ-
ent at P < 0.05, the trends were considered as significant and
as very significant at P < 0.01.
RESULTS AND DISCUSSION
the results are shown in Tables 2 and 3. The carotenoid
yields varied considerably within the tested conditions in
the range of 0.0890∼0.1970 mg/g. The adequacy of the
model was tested, and the parameters with statistically sig-
nificant effects were identified using the Fisher’s test for
analysis of variance (ANOVA). The ANOVA for the selected
factorial model showed that the model was significant at the
1% level of significance with a model P value <0.0001. The
coefficient of determination (R2) of the model was calcu-
lated to be at 0.9740 (adjusted R2 = 0.9566), indicating that
the model could explain 97.40% of the variability in the
response.
From the data shown in Table 3, four factors had a very
significant (P < 0.001) effect and one factor had significant
(P < 0.05) effect on the response. The very significant
factors in a descending order were D (temperature,
P < 0.001), F (extraction times, P < 0.001), E (liquid to
material ratio, P = 0.001) and C (duration, P = 0.0020). The
significant factor was A (ultrasonic power, P = 0.0182). The
operation would become disadvantageous when the extrac-

Preliminary Screening for the Solvent of TABLE 2. FRACTIONAL FACTORIAL SCREENING DESIGN IN CODED
Carotenoid Extraction UNITS AND EXPERIMENTAL RESULTS

We evaluated five solvent mixtures and their extraction Carotenoids yield

yields were compared. Chloroform, petroleum ether/ Run A B C D E F (mg/g)
acetone (v/v = 1/1) and methanol were shown as the most 1 −1 −1 −1 −1 −1 −1 0.0890 ± 0.0018
effective solvents, with carotenoid yields of 0.1303, 0.1230 2 +1 −1 −1 −1 +1 −1 0.1050 ± 0.0040
and 0.1177 mg/g, respectively, and have no significant dif- 3 −1 +1 −1 −1 +1 +1 0.1313 ± 0.0023
ference (Table 1). Considering the potential toxicity and 4 +1 +1 −1 −1 −1 +1 0.1228 ± 0.0033
5 −1 −1 +1 −1 +1 +1 0.1394 ± 0.0018
vapor cost of chloroform and methanol, petroleum ether/
6 +1 −1 +1 −1 −1 +1 0.1248 ± 0.0019
acetone (v/v = 1/1) was chosen as the solvent for extracting 7 −1 +1 +1 −1 −1 −1 0.1040 ± 0.0001
carotenoids. According to the theory of “similarity and 8 +1 +1 +1 −1 +1 −1 0.1238 ± 0.0019
intermiscibility” (Tian et al. 2009), a high molecular affinity 9 −1 −1 −1 +1 −1 +1 0.1471 ± 0.0108
between the solvent and solute is highly preferred in the 10 +1 −1 −1 +1 +1 +1 0.1708 ± 0.0090
solvent selection. The difference in carotenoid yields might 11 −1 +1 −1 +1 +1 −1 0.1356 ± 0.0028
be due to the polarity difference between the solvent and 12 +1 +1 −1 +1 −1 −1 0.1315 ± 0.0030
13 −1 −1 +1 +1 +1 −1 0.1468 ± 0.0046
carotenoid in rapeseed meal (Remeteiová et al. 2008; Jaime
14 +1 −1 +1 +1 −1 −1 0.1353 ± 0.0005
et al. 2010). 15 −1 +1 +1 +1 −1 +1 0.1552 ± 0.0021
16 +1 +1 +1 +1 +1 +1 0.1970 ± 0.0034

Screening Principal Factors for
Carotenoid Extraction
A total of 16 groups of extraction experiments were con-
ducted using different combinations of these six factors and
All data presented are means ± standard deviation (n = 3).
A, ultrasonic power (W, −1: 120, +1: 180); B, modulation frequency
(Hz, −1: 100, +1: 40); C, duration (min,−1: 10, +1: 45); D, temperature
(C, −1: 20, +1: 50); E, liquid to material ratio (mL/g, −1: 15, +1: 30); F,
extraction times (−1: 1, +1: 3).

380 Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc.
F. YAN ET AL. EXTRACTION OF CAROTENOIDS

TABLE 3. TESTS OF BETWEEN-SUBJECT

Parameter Standard
EFFECTS DEPENDENT VARIABLE: CAROTENOID
Variable DF estimate Error t value Pr > |t|
YIELD (MG/G)
Intercept 1 0.13496 0.00136 99.29 <0.0001
Ultrasonic power 1 0.00391 0.00136 2.88 0.0182
Modulation frequency 1 0.00269 0.00136 1.98 0.0794
Duration 1 0.00582 0.00136 4.29 0.0020
Temperature 1 0.01745 0.00136 12.84 <0.0001
Liquid to material ratio 1 0.00875 0.00136 6.44 0.0001
Extraction times 1 0.01359 0.00136 10.00 <0.0001

DF, degree of freedom; Pr, P value.

tion time was increased, and multiple extraction could be liquid to material ratio, duration and ultrasonic power all
done under the optimizing conditions, so rapeseed meal increased. The design and results of the path of steepest
was extracted once in the latter experiment, and B (modula- ascent experiments were shown in Table 4. The highest
tion frequency) was kept at its +1 level (40 Hz). D, E, C and response was 0.1504 mg/g, at the parameters of temperature
A were selected for further studies. 51C, liquid to material ratio 35 mL/g, duration 50 min, and
Table 3 also shows that the carotenoid yield increased ultrasonic power 210 W. Clearly, the point was near the
with increasing levels of all factors. A higher temperature region of maximum extraction response.
results in a higher diffusion velocity of extractants and a
lower solvent viscosity, so the extraction yield increases.
Under the experimental conditions specified in Table 2, the BBD and Response Surface Analysis
carotenoids cannot be extracted thoroughly in a single
The BBD was conducted to evaluate the optimum param-
extraction, the carotenoid yields from the second and third
eters of temperature (C, X1), liquid to material ratio (X2),
extraction are not negligible, so the extraction time had a
duration (min, X3) and ultrasonic power (W, X4) (indepen-
very significant effect on the carotenoid yield. Both raising
dent variables) for carotenoid yields (dependent variables).
the liquid to material ratio and extending duration can lead
The levels of the variables for the BBD experiments were
to an enhanced mass transfer process, which results in a
based on the results of previous experiments. The design
higher extraction yield. It has been suggested that acoustic
parameters and the corresponding experimental data were
cavitation, mechanical function and thermal function have
shown in Table 5, where x1 = (X1 − 51)/6, x2 = (X2 − 35)/
a direct effect on the efficiency of ultrasonic extraction,
10, x3 = (X3 − 50)/10 and x4 = (X4 − 210)/60. x1–x4 are the
where cavitation is the most significant factor. When a
dimensionless values of independent variables.
liquid is irradiated by ultrasound, microbubbles will appear,
Tables 6 and 7 show the ANOVA results of the response
grow, oscillate extremely fast and even collapse violently if
surface design for carotenoid yields and the significance of
the acoustic pressure is high enough. The occurrence of
the regression coefficients of the model, respectively. The P
these collapses near a solid surface will generate micro jets
value serves as a tool for checking the significance
and shock waves. Moreover, in the liquid phase surrounding
(P < 0.001) of each term. The responses taken from Table 7
the particles, high micromixing will increase the heat and
reveal that linear model term (x1, coded temperature and
mass transfer and even the diffusion of species inside the
x2, coded liquid to material ratio), quadratic model terms
pores of the solid (Sun and Temelli 2006). Meanwhile, the
(x12) and interactive model terms (x2x4, x4: coded ultra-
mass transfer can also be improved by the mechanical
sonic power) are very significant (P < 0.01).
function-induced solvent penetration into solid and
thermal function-induced solvent viscosity decrease (Vilkhu
et al. 2008). These factors, taken together, enhance the effi-
ciency of ultrasonic extraction. We note that the modula- TABLE 4. EXPERIMENTAL DESIGN OF STEEPEST ASCENT AND
CORRESPONDING RESPONSE
tion frequency of ultrasonic bath has an effect on the size
and intensity of microbubbles, so low modulation fre- Liquid to
quency is advantageous for the extraction, which is consis- Temperature material Duration Ultrasonic Carotenoids yield
Run (C) ratio (mL/g) (min) power (W) (mg/g)
tent with the above explanation.
1 45 25 40 150 0.1301 ± 0.0058
2 48 30 45 180 0.1413 ± 0.0017
Path of Steepest Ascent 3 51 35 50 210 0.1504 ± 0.0018
The path of steepest ascent started from the center of the 4 54 40 55 240 0.1410 ± 0.0023

PBD and moved along the path in which the temperature, All data presented are means ± standard deviation (n = 3).

Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc. 381
EXTRACTION OF CAROTENOIDS F. YAN ET AL.

TABLE 5. EXPERIMENTAL DESIGN AND RESULTS OF THE which reveals that this regression was statistically significant
SECOND-ORDER REGRESSION FOR BOX–BEHNKEN DESIGN at 93.53% of confidence level. The value of the adjusted
Carotenoid yield determination coefficient (Adj. R2 = 0.8706) was also very
Run x1 x2 x3 x4 (mg/g) high to advocate for a high significance of the model.
1 −1 −1 0 0 0.1297 ± 0.0011 According to our statistical analysis, the carotenoid yield
2 1 −1 0 0 0.1100 ± 0.0043 follows the second-order polynomial equation:second-order
3 −1 1 0 0 0.1406 ± 0.0009 polynomial equation:
4 1 1 0 0 0.1226 ± 0.0007
5 0 0 −1 −1 0.1422 ± 0.0027 y = 0.14522 − 0.01150 x1 + 0.00586 x 2
−1 0.1575 ± 0.0031
(1)
6 0 0 1 − 0.01599 x12 + 0.01125 x 2 x 4
7 0 0 −1 1 0.1466 ± 0.0016
8 0 0 1 1 0.1475 ± 0.0011 where y is the carotenoid yield (mg/g).
9 −1 0 0 −1 0.1422 ± 0.0008 It is apparent that higher liquid to material ratio and
10 1 0 0 −1 0.1190 ± 0.0004
higher ultrasonic power lead to higher carotenoid yield. In
11 −1 0 0 1 0.1444 ± 0.0010
12 1 0 0 1 0.1200 ± 0.0012
general, response surface equation only fitted a local area
13 0 −1 −1 0 0.1438 ± 0.0007 data, so it is difficult to explain theoretically response
14 0 1 −1 0 0.1547 ± 0.0009 surface equation. In contrast to the results of Table 3, lower
15 0 −1 1 0 0.1344 ± 0.0007 temperature is beneficial to the extraction of carotenoids.
16 0 1 1 0 0.1491 ± 0.0010 There are several possible explanations. The temperature
17 −1 0 −1 0 0.1444 ± 0.0005 used in BBD experiments is higher than that in PBD. As we
18 1 0 −1 0 0.1222 ± 0.0011
know, temperature can affect the solvent polarity, so the
19 −1 0 1 0 0.1465 ± 0.0007
20 1 0 1 0 0.1160 ± 0.0005
polarity of petroleum ether/acetone (v/v = 1/1) and rape-
21 0 −1 0 −1 0.1469 ± 0.0014 seed meal carotenoids might be closer in BBD experiments
22 0 1 0 −1 0.1350 ± 0.0053 as the temperature is lower. It has been reported that cavita-
23 0 −1 0 1 0.1328 ± 0.0007 tion intensity decreases as vapor pressure increases, and the
24 0 1 0 1 0.1659 ± 0.0009 vapor pressure increases at higher temperature, which
25 0 0 0 0 0.1379 ± 0.0011 results in higher oxidation rate of carotenoids. It is likely
26 0 0 0 0 0.1444 ± 0.0015
that these effects can be ignored in PBD experiments, but
27 0 0 0 0 0.1466 ± 0.0019
28 0 0 0 0 0.1501 ± 0.0021
must be included in BBD experiments.
29 0 0 0 0 0.1471 ± 0.0007 The two-dimensional contour and surface plots are
shown in Fig. 1, which are based on the regression equation.
Data are expressed as means ± standard deviations (n = 3).
We kept two variables constant at the level of zero while
x1 = (X1 − 51)/6, x2 = (X2 − 35)/10, x3 = (X3 − 50)/10, x4 = (X4 − 210)/
60.
varying the other two within their experimental range.
X1, temperature (C); X2, liquid to material ratio (mL/g); X3, duration The plot depicting the interaction of temperature level
(min); X4, ultrasonic power (W). and liquid to material ratio level is given in Fig. 1a.
Maximum carotenoid yield can be obtained at lower tem-
perature and higher liquid to material ratio. Figure 1b
The fitting of the model was calculated to be 0.9353 by illustrates the interactive effect of liquid to material ratio
the coefficient of determination R2, which indicates that level and ultrasonic power level on carotenoid yield, where
93.53% of the variability in the response can be explained the maximum carotenoid yield can be obtained at high
by the model. The statistical significance (P < 0.0001) of the levels of ultrasonic power. Figure 1c shows the interaction
second-order equation was evaluated by the F-test ANOVA, between temperature level and ultrasonic power level. The

TABLE 6. ANOVA RESULTS OF RESPONSE

Source DF Sum of squares Mean square F value Pr > F
SURFACE DESIGN FOR CAROTENOID YIELD
Model 14 0.00457 0.00032640 14.45 <0.0001 DEPENDENT VARIABLE
Error 14 0.00031623 0.00002259
Corrected total 28 0.00489
R2 0.9353
Coeff. var. 3.41150
Root MSE 0.00475
Adj. R2 0.8706

DF, degree of freedom; Pr, P value.

382 Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc.
F. YAN ET AL. EXTRACTION OF CAROTENOIDS

TABLE 7. THE SIGNIFICANCE OF THE

Parameter Standard
REGRESSION COEFFICIENTS OF THE MODEL
Variable DF estimate error t value Pr > |t|
Intercept 1 0.14522 0.00213 68.32 <0.0001
x1 1 −0.01150 0.00137 −8.38 <0.0001
x2 1 0.00586 0.00137 4.27 0.0008
x3 1 −0.00024167 0.00137 −0.18 0.8627
x4 1 0.00120 0.00137 0.87 0.3965
x12 1 −0.01599 0.00187 −8.57 <0.0001
x22 1 −0.00251 0.00187 −1.34 0.2007
x32 1 0.00242 0.00187 1.30 0.2158
x42 1 0.00181 0.00187 0.97 0.3494
x1x2 1 0.00042500 0.00238 0.18 0.8606
x1x3 1 −0.00207 0.00238 −0.87 0.3973
x1x4 1 −0.00030000 0.00238 −0.13 0.9013
x2x3 1 0.00095000 0.00238 0.40 0.6954
x2x4 1 0.01125 0.00238 4.73 0.0003
x3x4 1 −0.00360 0.00238 −1.51 0.1520

x1 = (X1 − 51)/6, x2 = (X2 − 35)/10, x3 = (X3 − 50)/10, x4 = (X4 − 210)/60.

X1, temperature (C); X2, liquid to material ratio (mL/g); X3, duration (min); X4, ultrasonic power
(W).

maximum carotenoid yield can be obtained at low tem-
perature level and high ultrasonic power level, which is
consistent with Fig. 1a,b.
From SAS statistical software, the optimal parameters
were identified as follows: x1: −0.230661 (actual tempera-
ture is 49.6C), x2: 0.642344 (actual liquid to material ratio is
41.4), x3: −0.151262 (actual duration is 48.5 min), x4:
0.715059 (actual ultrasonic power is 252.9 W). The
maximum carotenoid yield under the above conditions was
0.1570 mg/g.
Validation of the Model
Validation of the statistical model and regression equation
was conducted at the temperature of 49.6C, liquid to mate-
rial ratio of 41.4 mL/g, duration of 48.5 min and ultrasonic
power of 240 W (ultrasonic power can be only adjusted at
30 W interval). With these optimized conditions, the pre-
dicted response for carotenoid yield was very close to
0.1570 mg/g, and the observed experimental value was
0.1577 ± 0.0014 mg/g. These results confirmed the validity
of the model, and the experimental values are in agreement
with the predicted values.
Comparison with the Conventional
Extraction
The carotenoids were extracted completely after three times
of conventional water bath extraction. The carotenoid
amounts of the first time and third time water bath extrac-
tion were 0.1145 ± 0.0012 and 0.1544 ± 0.0033 mg/g,
respectively. The percentage of carotenoid extraction at the
first time ultrasound-assisted extraction is 79.61 ± 0.71%,
which implies that one time ultrasound-assisted extraction
is appropriate. One time water bath extraction gives only
57.80% of the total carotenoids, which is about 38% less
than that of one time ultrasound-assisted extraction. There-
fore, the application of ultrasound during the extraction of
carotenoids from rapeseed meal has dramatically increased
the extraction yield. A few researchers have reported that
ultrasonic-assisted extraction of compounds obtained great
extraction yield. For example, Zheng et al. (2014) reported
that ultrasonic-assisted extraction of Trametes orientalis
polysaccharides’ maximal yield (7.49 ± 0.14%) is at ratio of
water to raw material 30.6 mL/g, ultrasonic power 109.8 W,
extraction temperature 40.2C and extraction time 42.2 min.
Teng and Choi (2014) reported that ultrasonic-assisted
extraction of rhizoma coptidis’s maximal total alkaloid
content, berberine content, palmatine content and antioxi-
dant capacity are, respectively, 16.57 g of berberine chloride
equivalent per 100 g of dry rhizoma coptidis, 7.69 g of ber-
berine chloride equivalent per 100 g of dry rhizoma
coptidis, 1.46 g of palmatine chloride equivalent per 100 g
of dry rhizoma coptidis and 3.32 mmol/L of trolox equiva-
lent of rhizoma coptidis extract at ethanol concentration of
59%, extraction time of 46.57 min, and temperature of
66.22C. Chen et al. (2015) reported that ultrasonic-assisted
extraction of sugar beet molasses’ maximal total phenolic
contents, antioxidant activity, and total anthocyanins are,
respectively, 17.36 mg gallic acid equivalent/100 mL,
16.66 mg trolox equivalent/g and 31.81 mg/100 g at HCl
concentration 1.55–1.72 mol/L, ethanol concentration
57–63% (v/v), extraction temperature 41–48C and extrac-
tion time 66–73 min.
Carotenoids can be extracted from many other sources
such as carrot, red paprika, marigold, tomatoes and

Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc. 383
EXTRACTION OF CAROTENOIDS F. YAN ET AL.

FIG. 1. (A) TEMPERATURE LEVEL AND LIQUID TO MATERIAL RATIO LEVEL, (B) LIQUID TO MATERIAL RATIO LEVEL AND ULTRASONIC POWER LEVEL,
AND (C) TEMPERATURE LEVEL AND ULTRASONIC POWER LEVEL. RESPONSE SURFACES AND CONTOUR PLOTS OF THE CAROTENOID YIELD (ML/G)
FROM RAPESEED MEAL AS AFFECTED BY EXTRACTION TEMPERATURE, LIQUID TO MATERIAL RATIO AND ULTRASONIC POWER

specially cultured microorganism (Sun and Temelli 2006;
Topal et al. 2006; Macias-Sánchez et al. 2007; Rutkowska
and Stolyhwo 2009; Jaime et al. 2010), but the carotenoid
yield from rapeseed meal extraction is higher than that
from carrot (Sun and Temelli 2006), tomato skin (Topal
et al. 2006) and some specially cultured microorganism for
carotenoid extraction (e.g., Sporidiobolus salmonicolor and
Synechococcus sp.) (Macias-Sánchez et al. 2007; Valduga
et al. 2009), and it is at the same order of magnitude
as certain cultured microorganism such as Rhodobacter
sphaeroides (Gu et al. 2008). All results above were measured
by colorimetric assay method. In terms of yield, rapeseed
meal is a promising alternative source for the carotenoid
extraction.
Pressing and hexane extraction are two indispensable
steps in the conventional industrial processing of rapeseed.
Even in cold-squeezed process, the temperature is higher
than 60C, the degree of protein denaturation in carotenoid
extraction is unlikely higher than that in pressing. It is pos-
sible that petroleum ether/acetone (v/v = 1/1) and hexane

384 Journal of Food Quality 38 (2015) 377–386 © 2015 Wiley Periodicals, Inc.
F. YAN ET AL.
have approximate effect on protein denaturation. These sol-
vents can be removed through volatilization. So, to a certain
extent, the disadvantageous effect on the utility of the
residual meal by carotenoid extraction is acceptable.
CONCLUSIONS
We have compared the yields of carotenoids by using five
different solvents: acetone, petroleum ether, methanol,
chloroform and petroleum ether/acetone (v/v = 1/1), and
found that the highest yield can be achieved under petro-
leum ether/acetone (v/v = 1/1). The statistical approach
of fractional factorial experimental results showed that
temperature, extraction times, liquid to material ratio,
duration and ultrasonic power can significantly affect
carotenoid yield. The RSM allowed us to develop a poly-
nomial model to optimize the parameters during the
carotenoid extraction. The R2 value of 0.9353 showed a
good fitting of the model with the experimental data, and
the model predicted accurately the maximum point of
carotenoid extraction. The optimal parameters were deter-
mined at the temperature of 49.6C, liquid to material ratio
of 41.4 mL/g, duration of 48.5 min and ultrasonic power
of 252.9 W, and the maximum carotenoid yield was pre-
dicted to be 0.1570 mg/g, which is very close to the experi-
mental value of 0.1577 ± 0.0014 mg/g. Because of its high
yield, compared with the above-mentioned microorgan-
ism, rapeseed meal is a promising alternative source for the
carotenoid extraction.
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