Separation and Purication Technology 104 (2013) 1725

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Separation and Purication Technology

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Extraction characteristics and optimal parameters of anthocyanin from blueberry

powder under microwave-assisted extraction conditions
Xianzhe Zheng a,, Xiangwen Xu a, Chenghai Liu a, Yu Sun a, Zhen Lin a,b, Haijun Liu a,c
a

College of Engineering, Northeast Agricultural University, Harbin 150030, China

College of Building Materials and Industrial Technology, Heilongjiang College of Construction, Harbin 150025, China
c
College of Food, Heilongjiang Bayi Agricultural University, Daqing 163319, China
b

a r t i c l e

i n f o

Article history:
Received 16 September 2011
Received in revised form 6 November 2012
Accepted 7 November 2012
Available online 17 November 2012
Keywords:
Microwave
Extraction
Anthocyanin
Blueberry
Characteristics
Optimization

a b s t r a c t
This study investigates the extraction characteristics and optimal parameters of the microwave-assisted
extraction of anthocyanin from blueberry powder. Extraction time, extraction temperature, ethanol concentration, and solid-to-liquid ratio were selected as the extraction parameters. The effect of each extraction parameter on anthocyanin extraction rate showed an initially positive and subsequently negative
trend, which obeyed the Gaussian equation with convex trend curves. The kinetic constants of extraction
time, extraction temperature, ethanol concentration, and solid-to-liquid ratio were 2.44, 58.99, 45.39 and
24.47, respectively. The solid-to-liquid ratio has the most important effect on the anthocyanin extraction
kinetic from powdered blueberry, followed by the ethanol concentration and extraction temperature, and
the least is the extraction time. The optimum extracting parameters to achieve the highest anthocyanin
extraction rate of 73.73% from blueberry powder was obtained under an extraction time of 7 min, extraction temperature of 47 C, ethanol concentration (v/v) of 55.5%, and solid-to-liquid ratio of 1:34 (g/mL).
Three similarity criterions of microwave assisted extraction parameters were developed to extend the
criterion magnitudes to the similar conditions.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Blueberries are grown in many areas worldwide including
North America, Northeast China, and Russia. Blueberry cultivation
is highly favored due to their nutritional value, attractive blue pigment, and special aroma. Fresh blueberry fruit contain high levels
of phenolic compounds [1,2], especially in anthocyanins [3].
Anthocyanins, which are water-soluble polyphenols with strong
resistance to oxidation [4], potentially have great healthbenets
[5] such as anti-inammation [6], ghting cardiovascular disease,
and anti-aging [7]. The molecular structure of anthocyanin consists
of phenolic groups [8], formatted by phenolic hydroxyl and alkyl
groups, as shown in Fig. 1.
Anthocyanin is applied in many areas. For example, pharmaceuticals contain anthocyanosides as the most effective free radical
scavenging agents in the medicinal industry, and food additives
contain anthocyanin as a natural pigment. The extraction of
anthocyanin from blueberries with high yield, high efciency and
premium quality is a valuable topic for researchers and manufacturers. Microwave assisted extraction (MAE) is a feasible method

Corresponding author. Tel.: +86 451 55191606; fax: +86 451 55190677.
E-mail address: zhengxz@neau.edu.cn (X. Zheng).
1383-5866/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2012.11.011

for the extraction of anthocyanin from blueberries. Rapid extraction

and efcient separation are the typical characteristics of MAE technology. These characteristics are attributed to the disruption of cell
structure caused by the penetrating volume heating from microwave irradiation. The diffusion capacities of target components
may be improved by the increment of solvent temperature under
microwave heating [9]. A large number of studies have been performed on the MAE methods for the extraction of biological compounds including silymarin from milk thistle seeds [10], active
pharmaceutical ingredients from solid dosage [11], total phenols
from black tea powder [12], thymol from seeds of trachyspermum
ammi [13], and microalgal pigments [14]. Compared to conventional extraction methods, such as solidliquid, reux and supercritical uid extraction, MAE has advantages of high efciency,
low solvent consumption and superior product quality at lower cost
[10,15,16], hence also known as green extraction technology.
Therefore, MAE is a potential method for the extraction of anthocyanin from milled blueberries.
Determination of extraction characteristic contributes to reveal
the extraction mechanisms related to the change and transfer of
the target component. The optimization of extraction parameters
is helpful to systematically understand the interaction between
independent factors and obtain the optimal parameter combination for optimum extraction. Little information has been published

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X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

for the microwave extraction of anthocyanin from blueberry

powder. This limitation restricts the advantages of MAE of anthocyanin from blueberries in industrial application. Therefore, the
objectives of this study are as follows:
(1) To analyze the extraction characteristics of anthocyanin
from blueberries under microwave conditions in terms of
extraction time, extraction temperature, ethanol concentration, and solid-to-liquid ratio.
(2) To determine the optimum parameters for the highest
anthocyanin extraction rate and consider its scale-up
application.
2. Materials and methods
2.1. Plant material and reagents
Fresh wild blueberries were supplied by the Multi-berry Fruit
Group (Harbin, China) in August, 2010. Ripe blueberries free of foreign matter were selected as raw material. The raw blueberries
were stored at 18 C in a freezer (BC/BD-272SC, Haier Group,
Qingdao, China). The frozen blueberries were taken out to thaw
1012 h in room temperature (2528 C). The thawed blueberries
were pureed using a household electrical grinder (SJ260C, Lanpu
Electrical Equipment Factory, Guangzhou, China), and were frozen
in a freezer. The frozen blueberry puree was dried in a freeze vacuum dryer (Shanghai Instrument Co. Ltd., Shanghai, China) for 24 h
to get dehydrated blueberry slices with the moisture content below 0.5% (w.b.). The dehydrated blueberry slices were pulverized
with a cyclone mill passed through a 40-mesh sieve to produce
blueberry powder. The powder was kept in an airtight desiccators
for further extraction experiments.
Anthocyanin standards were provided by Tianjing Jianfeng Natural Products Research and Development Company (Tianjin, China).
Analytical grade methanol, ethanol, acetone, vanillic aldehyde, and
concentrated hydrochloric acid were purchased from Tianjin Tianxin Fine Chemical Factory (Tianjin, China). Distilled water was supplied by a local distilled water company (Harbin, China).

Miscible liquid of color reagent A and B was prepared at volume

ratio of 1:1 to nal constant volume of 100 mL for the further
experiment.
(2) Determination of standard curve
An anthocyanin standard solution of 1.2 mg/mL was prepared.
Five samples of 1, 2, 3, 4 and 5 mL were taken, respectively, in
methanol to get nal constant volume of 100 mL. 5 mL color reagent was added in each 1 mL anthocyanin solution (1 mL methanol as blank space), shaken, and stored in a dark location. Before
the colorimetric test, these standard samples were kept in a
30 1 C water bath for 30 min. The absorptions A of standard
samples were measured at 500 nm wavelength. The plotting curve
was developed by tting the absorption A and the concentration C
(mg/mL). Thus, a standard curvilinear equation was tted
(R2 = 0.9989) as:

A 1:934C  0:0582

The content of anthocyanin was expressed by:

C  V  n  1:2
 100%
W

where D is anthocyanin extraction rate, %; C is the same meaning as Eq. (1); V is the constant volume, mL; n is the multiple factor
of dilution; W is the total content of anthocyanin in 1 g raw material, mg. The anthocyanin content of fresh raw blueberry in this
study is 17.1558 mg/gdrysolid.

2.3.2. MAE procedures

In this MAE sequence, microwave irradiation conditions were
selected as microwave power, extraction time, extraction temperature, ethanol concentration, and solid-to-liquid ratio, as shown in
Tables 1 and 2. 1.0000 0.0005 g of dehydrated blueberry powder
was placed into the extraction vial to implement the microwave
extraction experiments. The cells loading extracted material were
heated to a preset temperature within 5 min followed by a static
extraction process under microwave extraction conditions.

2.2. Equipment
Extraction experiments were performed in an Advanced Microwave Digestion System (Ethos1, Milestone Inc., Italy). A maximum
power of 1600 W was delivered to a chamber with 10 Teon vials
embodied in a rotating sample tray. The holding capacity of each
vial was 100 mL. One of the vials was used as a control for monitoring temperature and pressure. Anthocyanin content was measured
by using a UV/Vis spectrometer (Lambda 35, Perkinelmer, Singapore) with a variable wavelength Vis detector. The solvents were
removed from the extracting solution using a rotary evaporator
(RE-52AA, Shanghai Yarong Biochemistry Instrument Factory,
Shanghai, China). Residual solvent in the extracts was evaporated
using a vacuum freeze drier (Shanghai Instrument Co. Ltd., Shanghai, China).

Fig. 1. General structure of anthocyanins (aglycons).

2.3. Experimental methods

2.3.1. Determination of anthocyanin extraction rate using a visible
spectrophotometer
(1) Preparation of color reagent
 A: 1% vanillin solution (1.000 g vanillin dissolved in methanol to
get nal constant volume of 100 mL).
 B: 8% hydurochloric (8 mL concentrated hydrochloric acid in
methanol and nal constant volume of 100 mL).

Table 1
Factors and levels of single factor experiment.
Factors

Levels

Time (min)
Temperature (C)
Ethanol concentration (%(v/v))
Solid to liquid ratio (g mL1)

2
30
20
1:10

5
50
40
1:20

8
70
60
1:30

11
90
80
1:40

14
110
100
1:50

Note: values marked with asterisk () held constant level when other parameters
were tested.

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X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

Table 2
Factors and levels of quadratic orthogonal rotation regression combination design.
Levels

Factors

2
1
0
1
2

Time, X1 (min)

Temperature, X2 (C)

Ethanol concentration, X3 (%)

Solid to liquid ratio, X4 (g mL1)

1
3
5
7
9

30
40
50
60
70

40
50
60
70
80

1:10
1:20
1:30
1:40
1:50

2.3.3. Experimental design

2.3.3.1. Single factor experiment design. One gram (1.0000 0.0005 g)
material was used under different microwave conditions as shown
in Table 1. In a single experiment, each experimental operation
was performed three times, and an average of the three groups of
data was reported as nal data. The experimental data were veried
statistically using ANOVA analysis (Minitab Inc., Ver. 15.0, State
College, PA, US). The signicance of differences between treatments
was determined by the one-factor analysis of variance using the
Duncan multiple range test (p < 0.05).
Mathematical models of extraction process contribute to the
understanding of extraction characteristics and the application of
research results. Based on the determination coefcient (R2) and
the standard estimation error (SEE) under the signicance level
(p-value), all experimental data for each factor were non-linearly
tted for the equations from the Equation Category of Regression
Wizard embodied in SigmaPlot software (Release 11.0, SPSS, Inc.,
Chicago, US). Both high determination coefcient (R2 closed to 1)
and low standard error of estimate (SEE) are the selection basis
to estimate the accreditation of each parameters of equations in
special case. A Gaussian type equation was selected (Eq. (3)), as
the most reasonable equation in this study, to elucidate quantitatively the extraction characteristics of single factors on the anthocyanin extraction rate.


x  x 2 
0
y a exp 0:5
k

where y is the anthocyanin extraction rate (%); a is the maximum

anthocyanin extraction rate (y peak value); x is the level of factor
studied among single factors; x0 is the taken value of studied
factor when y achieve the peak value; k is the extraction kinetic
constant, which reects the extraction ability of factor studied.
The parameters estimations of Eq. (3) were obtained under different microwave extraction conditions based on nonlinear regression method.
2.3.3.2. Quadratic orthogonal rotation regression combination design.
A full second-order polynomial model of the design was used to
evaluate the anthocyanin extraction rate (response variable, Y) as
a function of independent variables (X), namely extraction time
(X1), extraction temperature (X2), ethanol concentration (X3) and
solid to liquid ratio(X4) and their signicant interacting terms. Response surface methodology (RSM) was adopted to generate
regression models for the independent variables for the response
variables. Value range for each factor was selected based on the results from the single factor experimental data. The factors and their
levels are shown in Table 2.
In all cases, data were analyzed using the procedures of DesignExpert 7.0 (Stat-Ease, Inc., Minneapolis, MN, US) and tted to a second-order polynomial equation to optimize the conditions of
extraction. The generalized second-order polynomial model used
in the response surface analysis was as follows:

Y b0

4
4
4
X
X
XX
bi X i
bii X 2i
bij X i X j
i1

i1

ji1

where b0, bi, bii, and bij are the regression coefcients for intercept,
linear, quadratic and interaction terms, respectively. Xi and Xj are
the independent variables. The Design-Expert software was used
to generate response surfaces and contour plots.
3. Results and discussion
3.1. Extraction properties of anthocyanin from blueberry powder
under various MAE conditions
In the MAE process, the content of target component n in raw
material with extraction time t may be represented by [17]:

dns
b
fcs 1 aDs /2 cs;0 expmt
dt

In Eq. (5), the residual amount of target component in unit time

(dns/dt) negatively depends on the variables involving diffusion
coefcient (Ds), particle diameters of raw material (/), concentration of solvent in real time (cs) and initial value (cs,0), enhancement
factor (a), as well as constant b, f, m. Gradient constant m in exponent equation represents the concentration gradient at the diffusion surface, and m < 0, m = 0, and m > 0 refers to the decrease,
unchanged and increase of concentration gradient with time,
respectively. The yield of target component in the extract has positive correlation with the variables in Eq. (5). However, the reduction rate of target component in raw material is not equal to its
yield in extract in microwave extraction process. It was attributed
to the degradation of target component caused by the microwave
irradiation [18].
3.1.1. Effect of extraction time on anthocyanin extraction rate
As shown in Fig. 2, the extraction rate increased, and then signicantly decreased until the completion of microwave extraction
process (p < 0.001).
According to Eq. (5), this result was explained that the extracted
amount of anthocyanin content in extract rise to achieve highest
level due to the sufciency extraction. And then the anthocyanin
was degraded by the heat accumulation of microwave energy

Fig. 2. Effect of extraction time on anthocyanin extraction rate in conditions of

extraction temperature of 50 C, ethanol concentration of 60% and solid to liquid
ratio of 1:30.

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X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

destroying and oxidizing phenol structure and the anthocyanin.

From Eq. (6), it was found the highest anthocyanin extracted rate
of 66.87% may be obtained at the extraction time of 5.3 min under
single factor condition with high reliability.
3.1.2. Effect of extraction temperature on anthocyanin extraction rate
As shown in Fig. 3, the extraction rate notably increased in the
range of 3050 C, and decreased with temperature up to 110 C.
The phenomenon may be attributed to the increment of diffusion
rate within material particles and solubility capacity in extract solvent with the rising temperature based on Eq. (5). It is the
improvement of the intrinsic diffusion coefcient Ds caused by
the extraction temperature that promotes the anthocyanin extraction rate in extract according to Arrhenius equation (7).



Ea
Ds D0 exp 
RT

However, the amount of anthocyanin in extract solvent declined

due to the degradation rate higher than that of extraction rate with
the increase of extraction temperature. As explained by Eq. (8),
when the extraction temperature is lower than the transition
temperature (Ttransition), the extraction process of anthocyanin act
on dominant action. Otherwise, the degradation process of
anthocyanin plays an important role. And the kinetic of extraction
and degradation depends on the difference of reciprocal extraction
temperature and reciprocal reference temperature based on Eq.
(9) [19].
t

dn
k1 k2 nt at
dt

ki k0 exp

k1 0 when T t > T transition

k2 0 when T t < T transition



Ea 1
1

R T t T ref

)
8

As a thermal sensitive material, degradation of anthocyanins is

to be a logarithmic relation with increasing temperature due to the
endothermic process [20]. In addition, it was found in this study
that the extract solvent was appeared burnt under the extraction
temperature of 110 C. Based on Eq. (10) embodied in Fig. 3, the
highest anthocyanin extracted rate of 60.87% was obtained at
extraction temperature of 58.43 C under this single factor condition with high reliability (R2 = 0.94). This result is inferred that
highest amount of anthocyanin extracted was achieved at the transition temperature, which is the lowest difference between positive acquisition and negative degradation of anthocyanin in
ethanol solution.

Fig. 3. Effect of extraction temperature on anthocyanin extraction rate in conditions of extaction time of 5 min, ethanol concentration of 60% and solid to liquid
ratio of 1:30.

Fig. 4. Effect of ethanol concentration on anthocyanin extraction rate in conditions

of extaction time of 5 min, extraction temperature of 50 C and solid to liquid ratio
of 1:30.

3.1.3. Effect of extraction ethanol concentration on anthocyanin

extraction rate
As shown in Fig. 4, the anthocyanin extraction rate was lower in
distilled water than the extract liquid. This result attributed to
higher solubility of anthocyanin in ethanol than that in water, particularly evident in the microwave eld. With the increase of ethanol concentration, the amounts of anthocyanin extracted
presented a rising trend (p < 0.001), as explained by Eq. (5). When
the ethanol concentration was higher than 50%, the amount of
anthocyanin extracted declined with the concentration. Therefore,
the extraction abilities of ethanol are variable in a MAE system.
Both the dielectric constant and dielectric loss factor of water
are higher than that of ethanol. Under the same irradiated intensity
of microwave output power, water may absorb more thermal than
ethanol to drive target component (anthocyanin) to diffuse within
blueberry particles toward. However, the driving forces decrease
with the increase of ethanol concentration. The anthocyanins extracted from blueberry particles are polyphenols and contain a
large number of polar hydroxyl. The increases of ethanol concentrations result in the addition of the polarity molecular to close
that in anthocyanin. Thus, the anthocyanin solubility may be improved with the ethanol concentration based on the principle that
similar substances are more likely dissolved by each other. It is explained that ethanol plays a major role to rupture the hydrogen
bonds and hydrophobic bonds existing between anthocyanins
proteins and anthocyaninscellulose in the waterethanol system
[21]. For ethanol concentrations between 60% and 100%, the continuous reduction of anthocyanin solubility is due to the increasing
polar difference between anthocyanin and ethanol. At higher
concentrations of 60100%, the diffusion coefcient decreased
due to the lower solubility of anthocyanin in alcohol than that in
water [22]. Therefore, the more appropriate ethanol concentration
is around 60%. According Eq. (11) embodied in Fig. 4, the highest
anthocyanin extraction rate was obtained at 73.22% in the conditions of ethanol concentration of 54.69% with relatively high reliability (R2 = 0.90).
3.1.4. Effect of extraction solidliquid on anthocyanin extraction rate
As shown in Fig. 5, the anthocyanin extraction rate signicantly
increased up to 74.31% at the solid-to-liquid ratio of 1:30. The
anthocyanin content decreased with the further increase of the solid-to-liquid ratio. The solubility of material depends on the intermolecular or interionic forces of solute and solvent. The molecules
of solvent have sufcient attraction to break up the hydrogen
bonds between molecules or the dispersion forces between molecules in the solvent for the solute particles [23]. As concentration
gradients constant m in Eq. (5), increasing solid to liquid ratio enhances the concentration gradients, which promotes the diffusion

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X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

Table 3
Experimental design and results.
Run

Fig. 5. Effect of solid to liquid ratio on anthocyanin extraction rate in conditions of

extaction time of 5 min, extraction temperature of 50 C and ethanol concentration
of 60%.

of anthocyanin within the material to elevate the anthocyanin

extraction rate. With the further increase of solid-to-liquid ratio,
the activity of certain polar phenolics (other than anthocyanin)
within blueberry particles were improved to diffuse, which weakened the dissolution of the anthocyanin in ethanol liquid. Similar
result was obtained in Yangs research [24]. In conventional extraction, such as pressurized liquid extraction or heat reux extraction,
the positive impact of solvent-to-solid ratio was found on the
enhancement of target component yields [25]. From Eq. (12), the
highest anthocyanin extraction rate of 74.31% was obtained with
high reliability under the solid-to-liquid ratio of 1:30.
According to the kinetic constant k determined by Eqs. (6) and
(10)(12), the inuence on the extraction kinetic from high to
low is as follows: extraction temperature, ethanol concentration,
solid-to-liquid ratio, and extraction time.
3.2. Optimization of MAE of anthocyanin from blueberry

Dependent factor Y

X1

X2

X3

X4

1
2
0
1
0
1
1
1
1
1
1
2
0
1
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0

1
0
2
1
2
1
1
1
1
1
1
0
0
1
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0

1
0
0
1
0
1
1
1
1
1
1
0
2
1
1
1
1
2
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0

1
0
0
1
0
1
1
1
1
1
1
0
0
1
1
1
1
0
1
1
1
2
1
2
0
0
0
0
0
0
0
0
0
0
0
0

62.89
58.64
57.88
59.92
59.34
62.60
60.85
66.51
45.70
49.72
38.41
66.63
56.72
57.30
60.63
42.44
60.45
58.41
57.77
67.32
66.10
65.23
62.89
44.24
74.89
71.64
69.54
74.67
74.20
74.79
75.19
74.06
69.40
73.31
74.94
79.37

X1 Extraction time (min), X2 extraction temperature (C), X3 ethanol concentration (%), X4 solid to liquid ratio (g mL1), Y anthocyanin extraction rate (%).

The design matrix of 36 experiments and the anthocyanin

extraction rates are shown in Table 3. The effects of the parameters
were analyzed by using multiple regression techniques. Variance
analyses of experimental results are shown in Table 4.
A linear regression equation (Eq. (13)) was obtained from the
regression results of factorial experiment with the parameters selected at signicant level p < 0.05.

Y 73:83396 1:67339X 2  2:40200X 3 5:10275X 4

 2:93228X 21  3:93777X 22  4:20007X 23  4:90683X 24
 3:05655X 1 X 2  2:00005X 1 X 3 0:36066X 2 X 3
2:0878X 3 X 4

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36

Independent factors

13

The individual effects of temperature and solid-to-liquid ratio,

as well as the interaction effects of temperatureethanol concentration and ethanol concentrationsolid-to-liquid ratio were positive. Negative effects were observed in ethanol concentration and
the interaction of time and temperature, temperature and ethanol
concentration, as well as the quadratic items of four factors on the
anthocyanin extraction rate. The model could be explained about
87.2% of the variation in the anthocyanin extraction rate under
microwave extraction conditions studied. Thus about 12.6% of
the variation was due to other factors not included in the model.
3.2.1. Analysis of interaction between factors
The response surface plot (Fig. 6) indicated that the extraction
temperature and extraction time had signicant effects on the
anthocyanin extraction rate. The anthocyanin extraction rate
tended to the sharp rise with the increase of extraction tempera-

Table 4
Variance analysis of experimental results.
Parameter

Sum of squares

df

Mean square

F-Value

p-value

Model
X1
X2
X3
X4
X 21

3331.54
51.82
67.21
138.47
624.91
275.14

14
1
1
1
1
1

237.96
51.82
67.21
138.47
624.91
275.14

18.18
3.96
5.14
10.58
47.73
21.02

<0.0001
0.0598
0.0342
0.0038
<0.0001
0.0002

X 22

496.19

496.19

37.90

<0.0001

X 23

564.50

564.5

43.12

<0.0001

X 24
X1X2
X1X3
X1X4
X2X3
X2X4
X3X4
Residual
Lack of t
Pure error
Cor. total

770.46

770.46

58.85

<0.0001

149.48
64.00
13.17
2.08
44.35
69.72
274.94
195.10
79.64
3606.45

1
1
1
1
1
1
21
10
11
35

149.48
64.00
13.17
2.08
44.35
69.72
13.09
19.51
7.26

11.42
4.89
1.01
0.16
3.39
5.33

0.0028
0.0383
0.3274
0.6941
0.0799
0.0313

2.69

0.1600

X1 Extraction time (min), X2 extraction temperature (C), X3 ethanol concentration (%), X4 solid to liquid ratio (g mL1), Y anthocyanin extraction rate (%).

ture at extraction time of 5 min (at corresponding 0 level) to

achieve the peak value. And these were followed by the declines
in growth rate on the completion of extraction time, which depended on extraction temperature. During microwave extraction,

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X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

Fig. 6. Interaction of extraction time and temperature on anthocyanin extraction rate in the condition of ethanol concentration of 60% and solid to liquid ratio of 1:30.

Fig. 7. Interaction of extraction time and ethanol concentration on anthocyanin extraction rate in the condition of extraction temperature of 50 C and solid to liquid ratio of
1:30.

increasing extraction temperature had a positive effect on the

anthocyanin extraction rate due to the improvement of diffusion
within blueberry particles and solubility in extract liquid of anthocyanin [26]. High extraction temperature resulted in the degradation of anthocyanin due to the thermal accumulation subjected to
the synergistic effect of extraction temperature and duration [27].
From the observed trend in Fig. 6, it could be deduced that the
50 C extraction temperature and 5 min time under ethanol concentration of 60% and solid to liquid ratio of 1:30 were the critical
level. This result depends on the predomination for acquirement
(below the point) or degradation (beyond the point) of anthocyanin from blueberry extractant.
The response surface plots (Fig. 7) indicated the obvious interactions of ethanol concentration and extraction on extraction yield.
With the increase of ethanol concentration on completion of
extraction time, anthocyanin extraction ratio rst increased up to
peak level at 60% ethanol concentration. This result may be attrib-

uted to the gradually close polar between the ethanol and anthocyanin molecular based on the principle of the similar substance to
be more likely dissolved. And then the anthocyanin extraction rate
consistently decline due to the decline of solubility caused by the
further increment of polar difference between ethanol and anthocyanin. It is found that the peak level of anthocyanin extraction ratio in each extraction time was shifted upward to the highest value
in range of extraction time of 57 min.
The response surface plots (Fig. 8) presented the interactions of
extraction temperature and solidliquid rate on the extraction
yield of anthocyanin. When extraction time and ethanol concentration were kept at center zero level, and extraction temperature was
at lower level, anthocyanin extraction rate was signicantly increased with the solid to liquid rate. It is explained that the increase of anthocyanin extraction rates attributed to the increase
of the concentration gradient within the material as driving force
to improve the mass transfer. Higher solid to liquid ratio resulted

X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

23

Fig. 8. Interaction of extraction temperature and solidliquid rate on anthocyanin extraction rate in the condition of extraction time of 5 min and ethanol concentration of
60%.

Fig. 9. Interaction of ethanol concentration and solidliquid rate on anthocyanin extraction rate in the condition of extraction time of 5 min and extraction temperature of
50 C.

in an increase of the diffusion rate of anthocyanin without thermal

degradation [28]. However, with extraction temperature further
increases to a higher level, anthocyanin extraction rate rst increased due to the dominant state of drive force from solid to liquid
ratio and diffusivity from temperature. And then extraction rate
decreased with the solidliquid rate in high temperature due to
the degradation caused by the microwave heating impact. The
anthocyanin content in extract is in equilibrium level under solid
to liquid ratio of 1:50 and temperature around 50 C.
The response surface plots described the interaction of ethanol
concentration and solidliquid rate on extraction yield, as shown
in Fig. 9. When extraction time and extraction temperature was
kept at center zero level, anthocyanin extraction rate was initially
increased and then decreased with the solidliquid ratio. It was
clearly demonstrated the positive interaction from solid to liquid

ratio resulting in the increment of drive potential of anthocyanin

within material and ethanol concentration. This case resulted in
the improvement of solubility of anthocyanin in extractant.
There was no need to analyze the interactions of extraction time
and solid to liquid ratio, temperature and ethanol concentration
due to the statistically no-signicance of each interaction, as
shown in Table 4.
3.2.2. The importance of inuence factors considering the interaction
The importance of experimental factor was determined according to the F-value. Higher F value of inuence factor indicated the
greater impact on the objective index. From variance analysis of
experimental results (in Table 4), the solid to liquid ratio had the
most important inuence on the anthocyanin extraction rate, followed by the ethanol concentration and extraction temperature,

24

X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

Table 5
The optimization results and validation values.
Factors

Extraction time (min)

Extraction temperature (C)

Ethanol concentration (%)

Solid to liquid ratio

Predict value(%)

Validation value

Optimization value
Validation value

7
7

47.1
47

55.5
55

1:33.9
1:34

74.01

73.73%

and the least is the extraction time. Compared to the kinetic constant k from single factor, the inuence importance of factor had
obvious difference due to the interaction effects and wide range
of value taken.

0.45 mm), ethanol concentration C (%, dimensionless) and mass

of blueberry powder m (g, M).
According to the law of homogeneity on dimension and second
theorem of dimension [29], three similarity criterions in connection with these quantities were derived as p1 = y,

3.2.3. Optimization of extraction parameters

The optimum extraction parameter combination was obtained
by using the optimum module embodied in Design Expert software
(Table 5).
The optimum conditions predicts the highest extract yield at
extraction time 7 min, extraction temperature 47.1 C, ethanol
concentration (v/v) 55.5%, and solid to liquid ratio 1:33.9 (g/mL).
Three validation experiments were performed to verify the optimal
parameters and the average value was reported. Experimental value of anthocyanin extraction rate is 74.01%. Predictive value of
anthocyanin extraction rate was 73.73%. The relative error of the
actual value with the theoretical value was 0.28%. Therefore, the
experimental data showed a good t with the mathematical
regression model.
3.3. Construction of similarity criterions
Based on Eq. (5) and above-mentioned analysis, it was determined the physical quantities (unit, dimension) describing the process of microwave extracting anthocyanin from powdered
blueberry were as follows: extraction yield y (%, dimensionless),
extraction temperature T (C, ML2 T2), time t (min, T), ratio of solid
to liquid R (g/ML, ML3), diameter of particle / (mm, L) (equivalent
diameter of powder particle of blue berry with mean value of

p2 /T2 mt and

p3 R/
, which was expressed in general form equation (14).
mc
lg p1 0:0269 1:0470 lg p2 0:0006 lg p3
 0:1505 lgp2 2  0:0248 lgp3 2
R 0:5722; SEE 0:060; p < 0:05

14

p1 is named as anthocyanin extraction rate number, p2 and p3 is

named as temperature number and ratio number, respectively.
Thus, clustering these physical quantities into number groups
may reduce cumbersome modeling procedure. And the number
groups with special physical meaning related to the microwave assisted extracting anthocyanin from powdered blue berry contribute to the understanding of factors comprehensive effect and
extend of optimal parameters combination. As shown in Fig. 10,
an arch shape presented that the high anthcoyanin yield was obtained at intermediate level of p2.
The p1 as the function p2 and p3 were used to extend the
parameters of microwave extracting anthocyanin from milled blue
berry in trail research scale up to industrial practice. Three similarity criterion terms were deduced to characterize quantitative
dependency between inuencing factor groups, which may be applied to extend the criterion magnitudes in the similar case with
respect to the geometrical similarity, kinematics similarity, and dynamic similarity.

1.90

1.85

lg Pi1

1.80
1.75
1.70
1.65

lgP

1.60

4.4
4.2
4.0
3.8

1.55

3.6
1.0

.5

3.4
0.0

lgPi
3

-.5

3.2
-1.0

-1.5

3.0

Fig. 10. Curve surface of logarithm value of anthocyanin extraction rate Y (lg p1) as function of parameters criterions lg p2 and lg p3

p1 y; p2 /T2 mt and p3 R/
.
mc

X. Zheng et al. / Separation and Purication Technology 104 (2013) 1725

4. Conclusions
From a single-factor aspect, the inuence of extraction time,
extraction temperature, ethanol concentration and solid-to-liquid
ratio on the anthocyanin extraction rate showed similar trend with
an initial increase up to certain level and followed by decreasing
change. These processes may be presented by using the Gaussian
equations. Considering the extraction kinetic of the single factor,
the extraction temperature has the most signicant kinetic on
the anthocyanin extraction rate, followed by the ethanol concentration and solid to liquid ratio, and the least is the extraction time,
according to kinetic constant value of each factor of 58.9930,
45.3869 and 24.4661, 2.4388, respectively. However, considering
the interaction of these factors, the sequence that inuences the
extraction rate within the experimental model was as follows: solid-to-liquid ratio > ethanol concentration > extraction temperature > extraction time. The optimum conditions predicts the
highest extract yield in the conditions of extraction time 7 min,
extraction temperature 47.1 C, ethanol concentration 55.5% (v/
v), and solid to liquid ratio 1: 33.9 (g/mL). Under the optimal conditions, an average validation value of anthocyanin extraction rate
is 73.73%. Three similarity criterion terms of microwave assisted
extraction parameters were constructed to extend the criterion
magnitudes in the similar conditions.
Acknowledgements
The authors thank the nancial support provided by the National Natural Science Foundation of China (Nos.: 31071579 and
31271911) and the Key Program of the Natural Science Foundation
of Heilongjiang Province of China (No: ZD201013) for this research
project.
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