Ultrasonics Sonochemistry 18 (2011) 243–249

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultsonch

Effects of different factors of ultrasound treatment on the extraction yield

of the all-trans-b-carotene from citrus peels
Yujing Sun, Donghong Liu, Jianchu Chen, Xingqian Ye *, Dong Yu
Department of Food Science and Nutrition, School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310029, PR China

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

Article history: The effects of different factors, including the material’s particle size, the extraction solvent, solid/solvent
Received 20 October 2009 ratio, temperature, extraction time, the electrical acoustic intensity, liquid height and duty cycle of ultra-
Received in revised form 28 April 2010 sound exposure on the extraction yield of all-trans-b-carotene from citrus peels by ultrasound-assisted
Accepted 27 May 2010
extraction (UAE) were investigated. The extraction yield was significantly affected by particle size.
Available online 30 June 2010
Dichloromethane caused the degradation of all-trans-b-carotene extracted during UAE. Ethanol showed
a pronounced higher extraction yield during UAE in comparison with classical extraction (CE). The extrac-
Keywords:
tion yield of UAE had a peak value at 25 °C. In comparison with classical extraction, the extraction yield of
Ultrasound-assisted extraction
Pulsed ultrasound
UAE did not easily arrive at equilibrium. The extraction yield increased first, then decreased, then slightly
All-trans-b-carotene increased with an increase in electrical acoustic intensity. The extraction yield of UAE decreased with
Citrus peel increased liquid height. The extraction yield increased with increased duty cycle until equilibrium was
Extraction yield achieved.
Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction polysaccharides [22–24]). Compared with traditional solvent

Carotenoids are the main dietary sources of Vitamin A and are
associated with the reduction in the incidence of cancer [1], age-
related macular degeneration (AMD) [2], and cardiovascular dis-
eases [3]. Carotenoids are mainly distributed in plant-derived
foods. The largest number of carotenoids found in any fruit are
those of citrus fruits, more than 115 different compounds [4].
The carotenoid content in mandarin (Citrus reticulata Blanco) is
the highest among all the varieties of citrus fruits in China [5].
The carotenoid content in the peels is higher than the pulp. Bendi-
zao mandarin (Citrus succosa Hort) has the largest carotenoids con-
tent among mandarins [5]. Bendizao mandarin (Citrus succosa
Hort) and satsuma mandarin (Citrus unshiu Marc) are the main
varieties of canned citrus fruits in China [6]. The by-products
(peels) of canned citrus fruit occupy 30–40% of the total fruit
weight [7]. The peels are an abundant source of natural carote-
noids. However, most citrus peels in conventional food processing
as by-products are wasted, resulting in certain environmental pol-
lution. It is consequently necessary to effectively extract carote-
noids from the citrus peels.
Ultrasound-assisted extraction (UAE) has been widely used for
the extraction of nutritional material, such as lipids [8–10], pro-
teins [11–13], flavoring, essential oils [14,15] and bioactive com-
pounds (e.g., flavonoids [16–18], carotenoids [19–21], and
* Corresponding author. Tel./fax: +86 571 86971165.
E-mail addresses: psu@zju.edu.cn, yjsun666@sina.com (X. Ye).
extraction methods, ultrasound extraction can improve extraction
efficiency and extraction rate, reduce extraction temperature, and
increase the selection ranges of the solvents [25]. In comparison
with other extraction methods such as supercritical fluid extrac-
tion and microwave-assisted extraction, ultrasound equipment is
simpler and economically cheaper.
Ultrasonic cavitation is cited as the major factor leading to the
enhancement of extraction. Cavitation can cause locally high tem-
peratures, locally high pressures [26], and free radicals [25,27],
which may accelerate or trigger chemical reactions of extracted
compounds. In previous studies, researchers mostly focused on
the higher extraction efficiency of UAE. The structure and property
change of extracted compounds during extraction using ultra-
sound have been paid more attention in recent years, especially
for some polymers such as protein and polysaccharides. Yang et
al. [11] found that the extraction yield of soybean protein isolated
from soybean flakes was improved, and simultaneously the prop-
erties of isolated soy protein were changed by ultrasound treat-
ment. For instance, the 7S isolated soybean protein aggregated,
their emulsification was improved, and the viscosity was de-
creased. Gülseren et al. [12] found that bovine serum albumin
aggregated and the number of free sulfhydryl groups decreased
after treatment by high intensity ultrasound. The surface activity,
surface hydrophobicity and surface charge were also found to be
increased. High intensity ultrasound also caused polysaccharide
depolymerization [28], aggregation [24], a viscosity decrease, and
an improvement in immunity [24].

1350-4177/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2010.05.014
244 Y. Sun et al. / Ultrasonics Sonochemistry 18 (2011) 243–249
Fig. 1. The structure of all-trans-b-carotene.
Little information is available involving sonochemical studies of
the structure and property changes of small molecules such as
carotenoids. Zhao et al. [29] found that (all-E)-astaxanthin in a
model system degraded to unidentified colorless compounds un-
der ultrasound treatment. The degradation increased by increasing
both treatment time and ultrasonic power. Sun et al. [20] extracted
lutein from chicken liver by UAE, and found that while the extrac-
tion rate of lutein was improved under conditions that led to non-
saponification, lutein degradation occurred under conditions
where saponification occurred. The literature mentioned above
only explored the stability of carotenoids under special conditions
of ultrasound treatment. There has been, to our knowledge, no
comprehensive study on the stability of any one kind of carote-
noids under ultrasound treatment in the literature.
The goal of this study is to determine the effects of different
factors of ultrasound treatment on the extraction yield of all-
trans-b-carotene (Fig. 1) from citrus peel. This study may help us
to effectively control the extraction outcome, reduce degradation,
and may establish a better understanding of the mechanism of
ultrasound-assisted extraction of carotenoids.
2. Materials and methods
2.1. Plant materials
Fresh Bendizao mandarin (Citrus succosa Hort) fruits were
kindly offered by the institute of citrus, Huangyan, Zhejiang, China,
in December, 2008. The fruits were peeled by hands. The peels
were dried for 48 h in an oven with air circulation at 45 °C, and
then ground finely in the laboratory with a blade mixer (Wenling
linda machine Co., Wenling, Zhejiang, China). The peels powder
was separated into five groups according to their particle size
through 2, 0.45, 0.36, 0.28, and 0.074 mm sieves (Huakang experi-
ment instrument Co., Shangyu, Zhejiang, China), respectively, then
stored at 25 °C until use.
2.2. Chemicals
HPLC-grade solvents, including dichloromethane and methanol,
were purchased from Tedia Company, Inc. (USA). Hexane and
dichloromethane (analytical grade) were purchased from Sinop-
harm chemical reagent Co., Ltd. (Shanghai, China). Ethyl acetate
and ethanol (analytical grade) were purchased from Dafang chem-
ical reagent Co. (Hangzhou, China). Tetrahydrofuran (analytical
grade) was purchased from Shuanlin chemical reagent Co. (Hangz-
hou, China). The standard all-trans-b-carotene (purity P97%) was
purchased from Sigma chemical Co. (St. Louis, MO, USA).
2.3. Ultrasound-assisted extraction
Ultrasound-assisted extraction was carried out with a probe
ultrasonic processor (Scientz-IID, Ningbo scientz biotechnology
Co., Ningbo, China). Some parameters were set as followed: the
highest power is 950 W, frequency is 21–25 kHz, and diameter of
the horn microtip is 10 mm. The grounded powder was added into
the extraction solvent in brown glass tubes (3 cm diameter  10–
20 cm height), then the tubes with solution were immersed in a
low-temperature thermostatic ethanol (T 6 0 °C) or water
(T > 0 °C) bath (DC-1006, Safe Corporation, Ningbo, China) to main-
tain the same temperature. The probe was placed 1 mm from the
top surface of the extraction cell. The solution was treated by
ultrasound. The treated solutions were filtrated by 0.22 lm polyvi-
nylidene fluoride microfiltration membranes (Shanghai xingya
purification material Co., Shanghai, China) before HPLC analysis.
The Classical extraction (maceration) of all-trans-b-carotene from
citrus peels was used as a control to compare with ultrasonic ex-
tracted samples.
2.4. Calculation of electrical acoustic intensity
The electrical acoustic intensity dissipated from the probe
microtip was calculated according to the following formula [10]:
P
I¼ ð1Þ
pr 2
Where r is the radius of the probe microtip, and P is the input
power. In the present study, the input power levels were adjusted
to 5%, 25%, 45%, 65%, and 85% of total input power (950 W) which
were equal to 47.5, 237.5, 427.5, 617.5, and 807.5 W, respectively.
The corresponding ultrasonic intensities were 60.51, 302.55,
544.59, 786.62, and 1028.86 W/cm2, respectively.
2.5. Experimental design
The main effects of different factors of ultrasound treatment on
the extraction yield of all-trans-b-carotene were tested by single-
factor experiment design. The following variables were examined
for their effect on the extractions yields: particle size (<0.074,
0.074–0.28, 0.28–0.36, 0.36–0.45, 0.45–2 mm), solution (dichloro-
methane, ethanol, ethyl acetate, hexane, tetrahydrafuran), solid/
solvent ratios (1:30, 2:30, 3:30, 4:30, and 5:30), temperature (5,
5, 15, 25, 35, and 45 °C), time (20, 40, 60, 80, 100, and 120 min),
electrical acoustic intensity (ranging from 0 to1028.86 W/cm2), li-
quid height (2, 4, 6, 8, 10, and 12 cm, measured from the horn
microtip to the tube bottom) at the same ratio of solid/liquid
(1.2:18, 2:30, 2.8:42, 3.6:54, 4.4:66, and 5.2:78), and duty cycle
(0, 33.3%, 40%, 50%, 66.7%, and 100%), at a width of pulse of 2 s.
The general ultrasound conditions of all the treatments were as
follows: an extraction temperature of 5 °C, an electrical acoustic
intensity of 60.51 w/cm2, a solid/liquid ratio of 2:30, an extraction
solvent of ethyl acetate, an extraction time of 20 min, and pulse
mode (2 s on and 2 s off). The reason why we chose a very low tem-
perature was that we presumed that the extraction yield of all-
trans-b-carotene would be the highest at 5 °C, as the intensity
of cavitation decreases with increasing temperature [31–33].
2.6. Analytical method of all-trans-b-carotene
All-trans-b-carotene extracts from bendizao mandarin peel
were analyzed by using an HPLC analytical method. The HPLC sys-
tem included a Waters1525 pump, a Waters2487 Dual k Absor-
bance Detector and a 250  4.6 mm diameter 5-lm C30 YMC™
carotenoid column (Wilmington, NC, USA). The mobile phases
were methanol (100%) (A) and dichloromethane (100%) (B) with
the gradient elution: 78% A and 22% B in the beginning, maintained
for 5 min, then decreased to 62% A in 20 min, 58% A in 32 min. The
flow rate was 1.0 ml/min. The temperature was maintained at
35 °C, and the injection volume was 10 ll. The all-trans-b-carotene
peak was monitored at 455 and 480 nm. All-trans-b-carotene con-
centration was expressed as micrograms per gram of dry weight
(lg/g of DW), and was calculated using an external standard cali-
bration curve prepared with a pure all-trans-b-carotene standard.
The detection limit was determined based on a method described
 Y. Sun et al. / Ultrasonics Sonochemistry 18 (2011) 243–249
by Lee [30]. The lowest detection limit (DL) was 0.57 lg/ml, and
the lowest quantization limit (QL) was 1.7 lg/ml.
2.7. Statistical analysis
Each treatment was replicated three times. The results were ex-
pressed as the mean value ± the standard deviation. All the data
were subjected to statistical analyses using SPSS 16.0. The main ef-
fect of each factor of ultrasound treatment was subjected to anal-
ysis of variance and Duncan’s multiple range test using a One-Way
ANOVA procedure. Mean values were considered significantly dif-
ferent at p < 0.05.
3. Results and discussion
3.1. Effect of particle size
The extraction yield increased with decreasing of particle size
both by UAE and by CE (p < 0.05) as shown in Fig. 2. The ANOVA
statistical analysis results indicated that there were no significant
differences between UAE and CE (p > 0.05) on the extraction yields
when the particle size was less than or equal to 0.28 mm. However,
significant differences in extraction yields between UAE and CE
were found when the particle sizes were greater than 0.28 mm.
For example, the extraction yield of UAE was 2.99 times that of
CE (p < 0.05) at 0.45–2 mm particle size. The results indicate that
UAE did not have a pronounced advantage in comparison to CE if
the particle size of the treated material were too small.
The increase in extraction yield with a decrease in the particle
size by UAE and CE may be explained by the natural distribution
differences of all-trans-b-carotene in different particle sizes
[31,32] or by the decreasing areas of the particles, which is inver-
sely proportion to the extraction yields. The relationship of the
extraction yields and particle area can be expressed by the follow-
ing equation [33]:
K ¼ pr 2 E ð2Þ
Where r is the radius of the particles, E is extraction yield, and K
is a constant.
Fig. 2. Effect of particle size on the extraction yield of all-trans-b-carotene under
UAE and CE. Different letters on bars indicate significant differences (p < 0.05).
245
Ultrasound can improve extraction yield mainly because ultra-
sound can penetrate the matrix material, rupturing the cell walls
[34], resulting in all-trans-b-carotene being more easily released
from the matrix into the extraction medium. Also, ultrasound can
enhance the extracting power of the solvent by driving solvent into
the matrix to extract the targeted components. However, when the
material was first reduced to a very small particle size, the use of
ultrasound had less effect. That is presumably because the cell
walls were already somewhat ruptured and there was a greater
surface area, permitting closer contact of the extracting solvent
with the matrix [35]. No significant differences in extraction yield
were found between the UAE and CE methods when the material
particle size was relatively small. Therefore, this indicates that
ultrasound has less pronounced effects on extraction yield as the
particle size is reduced to a certain level.
3.2. Effect of solvent
Fig. 3 shows that the highest extraction yield was obtained
using dichloromethane, and the lowest yield was obtained using
ethanol in the CE extraction method (p < 0.05). In contrast, the
dichloromethane hardly had any extractability in the UAE. The eth-
anol had a higher extraction yield, and was at a level that was sim-
ilar to hexane and tetrahydrofuran in the UAE (p < 0.05).
To investigate the reasons for the low extraction yield of dichlo-
romethane under UAE process, the stability of the standard all-
trans-b-carotene in dichloromethane was investigated under the
same ultrasound treatment conditions. The result is represented
in Fig. 4. The all-trans-b-carotene degraded significantly over time
(p < 0.05). The results suggest that the lower extraction yield in
dichloromethane under UAE may be attributed to the degradation
of the all-trans-b-carotene extracted.
The high extraction yield of ethanol under UAE in comparison of
CE may be due to its physical properties. The physical properties
(surface tension, viscosity, vapour pressure) of the solvent are the
main factors affecting extraction yield under UAE. The vapour pres-
sure of the solvent is the most important factor among these prop-
erties [36]. Moreover, the vapour pressure is in proportion to the
boiling point. The boiling point of ethanol is the highest of all the
solvents that were used for this study. Thus, the relatively high
Fig. 3. Effect of solvents on the extraction yield of all-trans-b-carotene under UAE
and CE. Different letters on bars indicate significant differences (p < 0.05).
246 Y. Sun et al. / Ultrasonics Sonochemistry 18 (2011) 243–249
Fig. 4. The stability of standard all-trans-b-carotene in dichloromethane under UAE
at 5 °C.
boiling point, combined with its molecular affinity with b-carotene
make ethanol extractability similar to hexane and tetrahydrofuran
in UAE, while molecular affinity of solvent is the main factor affect-
ing extraction yield under CE. Ethanol had the weakest extraction
yield because of the weak affinity between ethanol and all-trans-
b-carotene.
The four nonpolar solvents (hexane, tetrahydrofuran, ethyl ace-
tate and dichloromethane) are most often used for the extraction
of b-carotene, but they have a certain toxicity. Thus, ethanol, which
is generally recognized as a safe (GRAS) solvent, may be an alterna-
tive solvent for extraction of all-trans-b-carotene under UAE.
3.3. Effect of solid/solvent ratio
Fig. 5 shows the highest extraction yield was both obtained for
the solid/solvent ratio of 3:30 under UAE and CE. The extraction
yield increased as the solid/solvent ratio increased from 1:30 to
3:30, and decreased as solid/solvent ratio increased from 3:30 to
Fig. 5. Effect of solid/solvent ratio on the extraction yield of all-trans-b-carotene
under UAE and CE. Different letters on bars indicate significant differences
(p < 0.05).
5:30 under UAE (p < 0.05). The decrease in the extraction yield as
the solid/solvent ratio was increased from 3:30 to 5:30 was due
to the fact that the concentration of solvent was not high enough
to allow for extract transfer from the material matrix. The reason
for the increase in the extraction yield as the solid/solvent ratio in-
creased from 1:30 to 3:30 remains unclear. The phenomenon of
lower extraction yield at lower solid/solvent ratio was also ob-
served by Vongsangnak [37], who found that a larger solvent vol-
ume did not lead to a higher saponin yield from cultured cells of
Panax notoginseng by microwave extraction.
3.4. Effect of temperature
The extraction yield of all-trans-b-carotene under CE was not
significantly different as the temperature ranged from 5 to
25 °C (p > 0.05), but it increased significantly with increasing of
temperature from 25 to 45 °C (p < 0.05) (Fig. 6). In comparison with
CE, the extraction yield of all-trans-b-carotene under UAE had a
peak value at 25 °C (p < 0.05). The variation of extraction yield with
temperature under CE could be explained in that the softening and
swelling of materials accelerated and the solubility and diffusivity
of all-trans-b-carotene increased with increasing temperature from
25 to 45 °C. The variation of extraction yield with temperature un-
der UAE may be attributed to the combination of the cavitation ef-
fect and thermal effect. From the cavitation effect point of view,
increasing temperature had a negative effect because the cavita-
tion intensity decreases with increasing temperature [38–40].
From the thermal effect point of view, the increase in temperature
had a positive influence because of the same reason as under CE.
There is a temperature when the combination of cavitation effects
and thermal effects was the best. This result was not in agreement
with our original presumption.
3.5. Effect of extraction time
Fig. 7 shows that the extraction yield increased with time rang-
ing from 20 to 40 min (p < 0.05) and did not vary significantly with
time ranging from 40 to120 min under CE (p > 0.05). However, the
extraction yield increased significantly with prolonged time from
20 to 120 min (p < 0.05) under UAE. The results indicate that the
Fig. 6. Effect of temperature on the extraction yield of all-trans-b-carotene under
UAE and CE. Different letters on bars indicate significant difference (p < 0.05).
 Y. Sun et al. / Ultrasonics Sonochemistry 18 (2011) 243–249 247

Fig. 8. Effect of electrical acoustic intensity on the extraction yield of all-trans-b-

carotene under UAE. Different letters on bars indicate significant differences
(p < 0.05).

Fig. 7. Effect of time on the extraction yield of all-trans-b-carotene under UAE and
CE. Different letters on bars indicate significant difference (p < 0.05).

extraction in CE easily arrives at extraction equilibrium and UAE

had a better extraction rate than CE. This may be explained in that
the effects of cavitation, including macro-turbulence generated by
the implosion of cavitation bubbles, and micro-jets caused by cav-
itation on the product surface facilitated the diffusing of all-trans-
b-carotene extracts of UAE to make the osmotic pressure between
the inside and the outside of the cell different. However, the diffus-
ing of all-trans-b-carotene extracts of CE was slow, so that the os-
motic pressure between the inside and the outside of the cell easily
arrived at equilibrium.

3.6. Effect of electrical acoustic intensity

The extraction of all-trans-b-carotene increased significantly

with electrical acoustic intensity ranging from 0 to 544.59 W/cm2
in a logarithmic function (R2 = 0.991, p < 0.001), then decreased
significantly with electrical acoustic intensity from 544.59 to
786.62 W/cm2 (p < 0.05). However, there was a slight increase with
electrical acoustic intensity ranging from 786.62 to 1028.86 W/cm2
(p < 0.05) (Fig. 8). The results may be attributed to the combination
of cavitation and thermal effects. However, when electrical acous-
tic intensity ranged from 0 to 786.62 W/cm2, the thermal effect can
be ignored because the heat produced by ultrasound may be al-
most completely diffused by the low-temperature thermostatic
ethanol bath in a short time. The variation of extraction yield with
electrical acoustic intensity can be explained that it was both eas-
ier to form cavitation bubbles and the bubbles collapsed more vio-
lently with increasing electrical acoustic intensity in the range of
0–544.59 W/cm2 [41]. The cavitation bubbles may grow too big
to collapse or collapse weakly which can cause the reduction of
the cavitation effect. Also, too many bubbles hamper the propaga-
tion of ultrasound waves as the electrical acoustic intensity ranged
from 544.59 to 786.62 W/cm2 [40]. The slight increase of extrac-
tion yield may be due to the combination of the absorption of heat
and reduction of the cavitation effect as mentioned above when
electrical acoustic intensity ranged from 786.62 to 1028.86 W/cm2.
3.7. Effect of liquid height
Fig. 9 shows that extraction yield decreased significantly with
an increase in the liquid height (p < 0.001). For example, the
Fig. 9. Effect of liquid height from the horn microtip to tube bottom on the
extraction yield of all-trans-b-carotene under UAE.
extraction yield at 2 cm and 12 cm height under UAE were sepa-
rately 84.5% and 12.3% higher than the extraction yield of CE
(2.51 lg/g) (p < 0.05). This may be due to the fact that the cavita-
tion intensity decreases with increasing height because of the
attenuation of the waves caused by absorption and scattering.
Other authors have also found that the maximum ultrasound
power was observed in the vicinity of the radiating surface of the
ultrasonic horn, and that ultrasonic intensity decreases rather
abruptly as the distance from the radiating surface increases
[42,26].
3.8. Effect of duty cycle of ultrasound exposure
Fig. 10 shows that the extraction yield increased significantly
with increasing of duty cycle until equilibrium at a duty cycle of
66.7% (p < 0.05). There was not a significant difference in extraction
yield as the duty cycle ranged from 66.7% to 100% (p > 0.05). The
results indicate that the proper use of pulse mode of ultrasound
can replace continuous irradiation by ultrasound in order to obtain
better extraction yields and reduce electrical energy consumption.
248 Y. Sun et al. / Ultrasonics Sonochemistry 18 (2011) 243–249
Fig. 10. Effect of duty cycle on the extraction yield of all-trans-b-carotene under
UAE. Different letters on bars indicate significant differences (p < 0.05).
However, the effect of duty cycle on extraction yield in the litera-
ture was inconsistent. Sivakumar [43] found that a duty cycle of
50% increased the extraction efficiency of the natural dye from
beetroot in comparison to continuous ultrasound. Herrera [44]
found that the duty cycle was a significant factor in the ultrasound
extraction of phenolic compounds from strawberries. Luque-García
[45], on the other hand, found that the duty cycle was not a signif-
icant factor for the extraction of total fat from oleaginous seeds.
The effect of duty cycle on extraction efficiency needs further
study.
4. Conclusions
The results of the present study indicate that each factor in the
ultrasound treatment had a significant influence on the extraction
yield of all-trans-b-carotene from Bendizao mandarin peel under
ultrasound-assisted extraction. Ultrasound significantly increased
the extraction yield when the particles sizes were greater than
0.28 mm compared to CE extraction. The solvent type influenced
the stability and extraction yield of all-trans-b-carotene under
ultrasound treatment. Dichloromethane caused degradation of
all-trans-b-carotene extracted under ultrasound treatment. Etha-
nol, generally recognized as a safe (GRAS) solvent, can be consid-
ered as an alternative solvent for use in ultrasound extraction,
although it showed a poor extraction yield under CE extraction.
The relatively low temperature of UAE could limit the degradation
of all-trans-b-carotene during extraction compared to the CE
extraction. Proper ultrasonic intensity and duty cycle were also
necessary to optimize extraction yield, given the optimum condi-
tions of particle size and solvent. Low extraction efficiency and
degradation during ultrasound-assisted extraction may occur if
conditions are not optimized. Therefore, much care should be ex-
erted in applying the technique for the extraction of carotenoids.
The current study should be very useful for optimizing ultrasound
extraction conditions for carotenoids according to the different
parameters studied.
Acknowledgments
This project was supported by the Chinese National Key Tech-
nologies R&D Program of 11th Five-year Plan (2006BAD27B06)
and the Key Cultural Project of Innovation of Ministry Education
(707034).
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