Food Chemistry 141 (2013) 30343041

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Optimisation of aqueous two-phase extraction of anthocyanins from

purple sweet potatoes by response surface methodology
Xingli Liu, Taihua Mu , Hongnan Sun, Miao Zhang, Jingwang Chen
Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-Products Processing, Ministry of Agriculture, No.
2 Yuan Ming Yuan West Road, Haidian District, P.O. Box 5109, Beijing 100193, China

a r t i c l e

i n f o

Article history:
Received 11 March 2013
Received in revised form 14 May 2013
Accepted 25 May 2013
Available online 4 June 2013
Keywords:
Anthocyanins
Purple sweet potato
Aqueous two-phase
Response surface methodology

a b s t r a c t
Aqueous two-phase extraction (ATPE) method was investigated for extraction of anthocyanins from purple sweet potatoes using response surface methodology (RSM). Results showed that the optimal conditions for anthocyanin extraction were that, 45:1 (mL/g) liquidsolid ratio, 25% (W/W) ethanol, 22%
(W/W) concentration of ammonium sulphate and pH3.3; the anthocyanin yield and partition coefcient
under the optimal conditions were 90.02% and 19.62, respectively. The result of HPLC-ESI-MS analysis
revealed eight kinds of compounds, and the major anthocyanins as cyanidi-caffeoy-fumaroy-sophoroside-3-O-glucoside, peonidin-caffeoyl-hydroxybenzoyl-3-O-glucoside, peonidin-caffeoyl-sophoroside-3O-glucoside, and peonidin-caffeoyl-fumaroyl-sophorosid-3-O-glucoside. Meanwhile, we found a
compound as a dimer of galloyl procyanin. These results suggest that ATPE is efcient in extracting
anthocyanins and has the potential to be used in natural anthocyanin extraction industry.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, interest in natural pigments has increased considerably, mainly because of their apparent non-teratogeneses,
non-carcinogenicity, non-mutation, and eco-friendliness characteristics. As a natural pigment, anthocyanins belong to the group
of avonoids and are responsible for the orange, red, and blue colours of owers, fruits and vegetables. These pigments are expected
to substitute synthetic pigments because of their attractive color
and physiological functionality (He & Giusti, 2010).
Purple sweet potato contains a high level of anthocyanins compared to white, yellow, and orange ones. Anthocyanins in purple
sweet potato are mono-acylated or di-acylated forms of cyanidin
and peonidin (Montilla et al., 2010). Purple sweet potato anthocyanins (PSPAs) have good heat stability compared to other sources
of anthocyanins, and recent studies showed that PSPAs have many
physiological functions, such as the antioxidant (Zhang et al.,
2009), hepato-protection (Hwang et al., 2011), and memory
enhancing (Lu et al., 2012).
It is well known that anthocyanins are soluble in polar solvents
and commonly extracted by aqueous mixtures of organic solvents
such as ethanol, methanol or acetone (Kano, Takayanagi, Harada,
Makino, & Ishikawa, 2005). The addition of a small amount of
hydrochloric acid or formic acid is recommended to prevent
the degradation of the non-acylated compounds. Besides the
Corresponding author. Tel./fax: +86 10 62815541.
E-mail address: mutaihuacaas@126.com (T. Mu).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.05.119

conventional solvent extraction, new methods based on more advanced extraction techniques were reported, such as microwave
(Yang & Zhai, 2010; Sun et al., 2007) (Yang, Fan, Gu, Han, and Chen
(2008)) and ultrasonic methods (Ghafoor, Choi, Jeon, & Jo, 2009;
Ghafoor, Hui, & Choi, 2011). However, these methods have
drawbacks due to the higher cost, special equipment and stringent
operating condition.
Aqueous two-phase extraction (ATPE) has been widely applied
to the separation of biomacro molecules, such as proteins (Klomklao, Benjakul, Visessanguan, Simpson, & Kishimura, 2005) and antibiotics (Paula, Rosa, & Azevedo, 2007), because of its mild
conditions and high capacity. Up until now, most ATPE was based
either on polyethylene glycol (PEG)/salt or polymer/polymer (e.g.,
PEG/dextran) systems. However, because of the high cost of the
polymers and difculty in isolating the extracted molecules from
the polymer phase by back extraction, these systems can be hardly
used for large-scale production (Ozlem, Emine, & UIku, 2011).
Recently, short-chain alcohol/inorganic salt systems have been
used as a novel ATPE system to purify natural compounds (Jiang,
Li, Dai, Zhang, & Xiu, 2009). This ATPE system has many advantages
such as low cost, low interfacial tension, good resolution, high
yield, high capacity, and simple scale-up (Rito-Palomares, 2004).
Moreover, because of its structure, these are suitable for hydrophilic compounds. Short-chain alcohols (ethanol, methanol, and
2-propanol) could form stable and adjustable ATPE system with
inorganic salts (e.g., phosphate and sulfate) (Gunduz, 2000). This
might be because of the salting-out effect and the low solubility
of inorganic salt in alcohols (Albertsson, 1986). When an ATPE

X. Liu et al. / Food Chemistry 141 (2013) 30343041

system is formed, the top phase is rich in alcohol and the bottom
phase is rich in inorganic salt, two phases are both with water content of 80% or more and show very low surface tension (Yuan, Raza,
Huang, & Shen, 2011). Ethanolammonium sulphate is a common
and an economic ATPE system, which has been applied to extract
of anthocyanins from Mulberry (Wu et al., 2011) and piceid, emodin, resveratrol (Wang, Dong, & Xiu, 2008).
Response Surface Methodology (RSM) is an effective statistical
tool for the optimisation of multiple variables for the prediction
of best performance conditions with a minimum number of experiments, which has been widely applied for optimizing conditions in
food industry.
In the present study, the applicability of ATPE formed by ethanolammonium sulphate to the extraction of PSPAs was investigated using the BoxBehnken design combined with the RSM
and the structures were measured by the HPLC-ESI-MS. To the best
of our knowledge, ATPE of PSPAs and its further optimisation using
RSM models have not been previously reported elsewhere.
2. Materials and methods

3035

where Ct and Cb were the equilibrium concentrations of PSPAs in the

top phase and bottom phase, respectively, Mtotal was the total
amount of PSPAs. The partition coefcient (Y2) of the PSPAs was calculated using the equation:

Y 2 C t =C b

2.4. Estimation of purple sweet potato anthocyanins

The total anthocyanin content was determined according to the
spectrophotometric pH-differential method (Hosseinian, Li, & Beta,
2008). Briey, an aliquot (1 mL) of the extract was mixed with
0.025 M potassium chloride buffer (pH 1.0, 4 mL) and 0.4 M sodium acetate buffer (pH 4.5, 4 mL), respectively. The absorbance
of the mixture was measured at 520 and 700 nm using an UV-Vis
spectrophotometer model UI-trospec 2000 (Amersham Pharmacia
Biotech, Dubendorf, Switzerland). Absorbance was calculated as
A = [(A520  A700) at pH 1.0]  [(A520  A700) at pH 4.5] with a molar
extinction coefcient of 26,900 for anthocyanins. The total anthocyanin content was calculated as cyanidin-3-glucoside equivalents
as Eq. (3):

2.1. Materials

C Af =eL  103  MW

Purple sweet potatoes (Ipomoea batatas Lam) (variety YAN No.

176) were offered by the academy of agricultural and forestry sciences in Hebei province. They were harvested in the early October
and then stored at 1014 C. Within 2 weeks after harvest, the entire batch of purple sweet potatoes was washed, cut; freeze dried
and then smashed for getting purple sweet potato powders; and
then stored in dark place at 4 C. Deionized water was used
throughout all experiments. Ammonium sulphate [(NH4)2SO4],
ethanol (CH3CH2OH), potassium chloride (KCl), sodium acetate
(CH3COONa) are analytical grade. Triuoroacetic acid (TFA) and
acetonitrile (C2H3N) are HPLC grade.

where A is absorbance, MW is the molecular weight of cyanidin3-glucoside (449.2 Da), f is the dilution factor, e is the cyanindin3-glucoside molar absorbance (26,900), L is the cell path length
(1 cm).

2.2. Chemical analysis of purple sweet potato powders

The purple sweet potato powders were analysed for ash, fat,
protein, starch, crude ber and anthocyanin content. The ash content was determined by placing the sample in a mufe furnace set
at 550 C for 8 h (AOAC method 923.03). Proteins were analysed
according to the Kjeldahl procedure, a factor of 6.25 was used for
conversion of the nitrogen content to crude protein values (AOAC
method 955.04). The starch content was determined using the
amyloglucosidase and a-amylase method (AOAC method 996.11).
Crude ber was determined according to the AOAC method
991.43. The anthocyanin content was determined according to
Lee, Durst, and Wrolstad (2005).

2.5. Experimental design for selection of extraction parameters

The purple sweet potato powder was treated by various temperatures (2060 C), concentrations of ethanol (2030% w/w)
and concentrations of ammonium sulfate [(NH4)2SO4, 1722%
w/w], with a ratio of liquidsolid (mL/g) (ranging from 10:1 to
100:1 v/w), for a given time (from 20 to 120 min), while the pH
value of deionized water ranged from 1 to 5. The anthocyanin yield
(Y1) and the partition coefcient (Y2) were indexes for evaluating
the effects of different parameters.
2.6. Experimental design for optimisation of extraction parameters
Response surface methodology was applied to determine the
working conditions of anthocyanin extraction from purple sweet
potato based on the single-factor experiment results. BoxBehnken
design with four independent factors (X1, liquidsolid ratio; X2,
ethanol concentration; X3, ammonium sulphate concentration;
X4, pH) set at three variation levels was carried out (Table 1). The
correspondence between the coded and uncoded values was obtained using the following equation:

2.3. Aqueous two-phase extraction (ATPE)

X i X i  X 0 =DX i

An aqueous two-phase system was prepared according to Li,

Teng, and Xiu (2010). A predetermined quantity of ammonium sulphate was dissolved in water, and then certain volumes of ethanol
and purple sweet potato powders were added to the ammonium
sulphate solution, and mixed well to form two phases. The mixture
was mingled thoroughly and held until the two phases were completely separated. The PSPAs concentrations in both the top and
bottom phases were analyzed and residues accumulated at the
interface of two phases were discarded.
The yield (Y1) was the ratio of the PSPAs partitioned in the top
phase to the total amount of PSPAs. It was calculated using the following equations:

where xi is the coded value of the variable; Xi the actual value of

variable; X0 the actual value of Xi at the centre point; and DXi the
step change value.
The complete design consisted of 27 experiments including 24
factorial experiments and three replicates at the centre point.
Experiments at the centre of the design were undertaken to estimate the possibility of pure error. All the experiments were carried
out in random to minimize the effect of unexplained variability in
the observed responses owing to systematic errors. The experimental results were tted to the second-order regression (Eq. (5)):

Y 1 C t  V t =Mtotal

Y b0 b1 X 1 b2 X 2 b3 X 3 b4 X 4 b11 X 211 b22 X 222

b33 X 233 b44 X 244 b12 X 1 X 2 b13 X 1 X 3 b14 X 1 X 4
b23 X 2 X 3 b24 X 2 X 4 b34 X 3 X 4

3036

X. Liu et al. / Food Chemistry 141 (2013) 30343041

Table 1
Design approach and experimental result of response surface methodology.
Run

Parameters
*

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

Factor 1 (X1)

Factor 2 (X2)

Factor 3 (X3)

Factor 4 (X4)

50
60
50
50
50
60
40
60
50
60
40
40
50
50
40
50
50
50
50
40
50
50
60
60
40
50
50

23
27
23
27
25
23
25
25
25
25
23
25
27
25
25
25
25
23
27
25
23
25
25
25
27
27
25

21
21
20
21
21
21
20
21
21
20
21
21
20
22
22
20
20
22
21
21
21
22
22
21
21
22
21

3 (1)
3.5 (0)
3.5 (0)
3 (1)
3.5 (0)
3.5 (0)
3.5 (0)
4 (1)
3.5 (0)
3.5 (0)
3.5 (0)
4 (1)
3.5 (0)
3 (1)
3.5 (0)
4 (1)
4 (1)
3.5 (0)
4 (1)
3 (1)
4 (1)
4 (1)
3.5 (0)
3 (1)
3.5 (0)
3.5 (0)
3.5 (0)

(0)
(1)
(0)
(0)
(0)
(1)
(1)
(1)
(0)
(1)
(1)
(1)
(0)
(0)
(1)
(0)
(0)
(0)
(0)
(1)
(0)
(0)
(1)
(1)
(1)
(0)
(0)

(1)
(1)
(1)
(1)
(0)
(1)
(0)
(0)
(0)
(0)
(1)
(0)
(1)
(0)
(0)
(0)
(0)
(1)
(1)
(0)
(1)
(0)
(0)
(0)
(1)
(1)
(0)

(0)
(0)
(1)
(0)
(0)
(0)
(1)
(0)
(0)
(1)
(0)
(0)
(1)
(1)
(1)
(1)
(1)
(1)
(0)
(0)
(0)
(1)
(1)
(0)
(0)
(1)
(0)

Y1*(%)

Y2*

89.56 0.28
81.46 1.01
83.80 0.17
88.13 0.58
85.44 0.44
85.99 0.68
71.40 1.23
64.26 2.11
86.46 0.78
82.78 0.38
88.49 0.22
68.14 1.14
80.26 0.75
83.78 1.11
89.65 0.17
62.19 1.36
68.62 0.41
88.16 0.27
68.62 1.28
84.98 0.76
73.99 1.58
68.12 0.94
71.81 0.88
82.42 0.53
85.97 0.54
83.86 0.29
85.97 0.47

9.78 0.66
10.82 0.90
15.66 1.02
11.41 1.27
15.82 0.71
16.00 0.13
11.67 1.14
13.49 0.56
18.36 0.64
11.89 0.35
12.02 0.26
15.25 0.35
10.03 0.05
17.08 0.24
17.18 0.37
16.35 0.27
13.49 0.21
17.38 0.39
13.49 0.17
9.13 0.07
17.25 0.47
17.98 0.51
17.45 0.64
10.9 0.28
14.82 0.25
19.55 0.03
17.75 0.33

X1: liquidsolid ratio (mL/g); X2: ethanol concentration (w/w, %); X3: ammonium sulphate concentration (w/w, %); X4: pH value; Y1: anthocyanin yield (%); Y2: anthocyanin partition coefcient.

where Y is the predicted response (the yield and the partition coefcient), b0 is the intercept; b1, b2, b3, b4 are linear coefcients; b11,
b22, b33, b44 are squared coefcients; b12, b13, b14, b23, b24, b34 are
interaction coefcients. The goodness-of-t of the regression model
and the signicance of parameter estimates were determined by the
analysis of variance (ANOVA).

p < 0.05 was considered statistically signicant and p < 0.01was

very signicant.

2.7. Identication of anthocyanins from purple sweet potato by HPLCESI-MS

The starch, crude ber, ash, protein and fat contents of the purple sweet potato powder were 63.35, 4.87, 3.98, 8.21 and 0.7
g/100 g dry matter, respectively, which indicated that starch was
the predominant component of the powder followed by crude ber, ash, protein and fat. The anthocyanin content of purple sweet
potato powder was 311 mg/100 g, which was slightly lower than
grape skins anothocyanin content (435.8 mg/100 g) reported by
Margarita, Avelina, Peter, and Bernhard (2009). However, this value was obviously higher than that of the bokbunja (37.2 mg/
100 g) and purple corn (185.1 mg/100 g) (Ku & Mun, 2008; Yang
& Zhai, 2010). Thus make it a suitable raw material for the extraction of anthocyanins.

An Agilent 1200 series high-performance liquid chromatography (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent 6410 triple quadrupole mass spectrometer and an Agilent
Zorbax SB-C18 column (250 mm  4.6 mm I.D, 5 lm) was used
to identify anthocyanins in purple sweet potato extract basing on
that of Sariburun, Sahin, Demir, Trkben, and Uylaser (2010) method with some modication. Chromatographic separation was performed using a mixture of two eluents: solvent A, 0.1%
triuoroacetic acid (TFA) in water (V/V); and solvent B, acetonitrile.
The gradient elution program was used as follows: 05 min, 530%
B; 545 min, 3045% B; 4550 min, 4590% B. Column temperature were 25 C. Flow rate was 0.5 mL/min, and injection volume
was 20 lL. For the identication of anthocyanins, electrospray ionization (ESI) was operated in the positive ion mode in the mass
range 102000 (m/z) and under the following conditions: capillary
voltage, 4500 V; nebulizer pressure, 40 psi; drying gas temperature
at 350 C was used at a ow rate of 8.0 L/min.
2.8. Statistical analysis
All the experiments were carried out in triplicate, and the results were expressed as means SD (standard deviation). Statistical analyses were performed using the Statistical Analysis System
version 8.1 software (SAS Institute Inc., Cary, NC, USA). A value of

3. Results and discussion

3.1. Chemical composition of purple sweet potato powder

3.2. The results of selection of extraction parameters

The extraction process of chemical constituents from crude resources such as plants was affected by many factors. Given the
ATPE system, factors such as extraction temperature, ethanol concentrations, ammonium sulphate concentration, liquidsolid ratio,
extraction time and pH were usually considered to have signicant
effect on the extraction process. The effects of these parameters on
the anthocyanins yield (Y1) and the partition coefcient (Y2) were
studied during this research (data not shown). The results indicated that each selected parameter, except extraction temperature
and extraction time, had a signicant inuence on the extraction
process (P < 0.05). Considering the cost of production and economic benet, extraction temperature is room temperature and

3037

X. Liu et al. / Food Chemistry 141 (2013) 30343041

Y 2 18:31 0:041X 1  0:66X 2 2:81X 3 2:35X 4

Table 2
ANOVA of anthocyanin yield test.

 1:99X 1 X 2 0:011X 1 X 3  0:88X 1 X 4 1:95X 2 X 3

Source

Sum of
squares

Degree
of
freedom

Mean
square

F-value

P-value

Model
X1
X2
X3
X4
X1X2
X1X3
X1X4
X2X3
X2X4
X3X4
X 21

1805.5
33.02
39.23
45.58
920.71
1.02
213.47
0.43
0.14
3.88
3.44
43.84

14
1
1
1
1
1
1
1
1
1
1
1

128.93
33.02
39.23
45.58
920.71
1.02
213.47
0.43
0.14
3.88
3.44
43.84

1472.97
77.19
448.08
520.71
10518.49
11.64
2438.076
4.93
1.6
44.38
39.32
500.88

<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0052
<0.0001
0.0465
0.2304
<0.0001
<0.0001
<0.0001

X 22

27.03

27.03

308.84

<0.0001

X 23

90.27

90.27

1031.33

<0.0001

X 24
Total error
Lack of t
Pure error
Total SS
Predicted R2 = 0.9977

347.20

347.20

3966.56

<0.0001

1.05
0.53
0.52
1806.10

12
10
2
26

1.088
0.053
0.26

0.20

0.9673

extraction time is 20 min. Therefore, liquidsolid ratio, ethanol

concentration, ammonium sulphate concentration, and pH were
selected as the four extraction parameters for the following RSM
design.
3.3. Optimisation of the procedure
3.3.1. Statistical analysis and model tting
Response surface methodology was used to optimize the
extraction conditions of anthocyanins. Experimental values obtained for anthocyanin yield (Y1) and the partition coefcients
(Y2) at the designed points are shown in Table 1. After application
of the response surface regression (RSREG) procedure, the predicted model can be described by the following equation:

Y 1 85:96  1:66X 1  1:81X 2 1:95X 3  8:76X 4  0:5X 1 X 2

 7:31X 1 X 3  0:33X 1 X 4  0:19X 2 X 3  0:99X 2 X 4
 0:93X 3 X 4  2:87X 21 2:25X 22  4:11X 23  8:07X 24

 1:35X 2 X 4  2:04X 3 X 4  2:99X 21  2:04X 22  0:63X 23

 3:14X 24

where Y1 is anthocyanin yield (%), Y2 is the partition coefcient. X1 is

the liquidsolid ratio, X2 is ethanol concentration (%), X3 is ammonium sulphate concentration (%), and X4 is pH value. Analysis of variance for quadratic model is required to test the signicance and
adequacy of the model. The results of ANOVA of model (6) and
(7) were shown in Tables 2 and 3, respectively.
3.3.1.1. Analysis of variance (ANOVA) for the extraction yield of purple
sweet potato anthocyanins. Analysis of variance (ANOVA) for the
model Y1 was shown in Table 2. The regression model was highly
signicant (P < 0.01), while the lack of t was not signicant
(P = 0.9673 > 0.05). The determination coefcient (R2) of the predicted model was 0.9977, indicating that only 0.23% of the total
variation was not explained by the model. Meanwhile, a very low
value of 0.37 for the coefcient of variation (CV) clearly indicated
that the experimental values were associated with a very high degree of precision and a good deal of reliability. Thus, the model explained the response adequately. The result of regression
coefcient analysis showed that the linear effects of all the variables were very signicant (P < 0.01). According to the signicance
of the regression coefcients of the quadratic polynomial model,
pH value was found to be the most signicant factor to affect the
anthocyanin yield, followed by ammonium sulphate concentration,
ethanol concentration and liquidsolid ratio. The quadratic term
(X 21 , X 22 , X 23 , X 24 ) was very signicant (P < 0.01). Among the interaction terms, the interactions between X1X3, X2X4 and X3X4
(P < 0.01) were very signicant with regard to the anthocyanin
yield, X1X2 and X1X4 (P < 0.05) were also signicant. The other
terms were not signicant (P > 0.05). In addition, the standard errors (SE) of all the regression coefcients were ranged from 0.085
to 0.17.
3.3.1.2. Analysis of variance (ANOVA) for the partition coefcient of
purple sweet potato anthocyanins. Table 3 showed the analysis of
partition coefcient variance (ANOVA) for the regression equation.
It revealed that the most relevant variable (p < 0.0001) concerning
the partition coefcient was pH value, followed by ammonium sulphate concentration and ethanol concentration, the liquidsolid

Table 3
ANOVA of anthocyanin partition coefcient test.
Source

Sum of squares

Degree of freedom

Mean square

F-value

P-value

Model
X1
X2
X3
X4
X1X2
X1X3
X1X4
X2X3
X2X4
X3X4
X 21

308.52
0.020
5.3
94.88
66.45
15.89
4.731E004
3.11
15.20
7.26
16.72
47.62

14
1
1
1
1
1
1
1
1
1
1
1

22.04
0.020
5.3
94.88
66.45
15.89
4.731E004
3.11
15.20
7.26
16.72
47.62

187.28
0.17
45.02
806.34
564.68
135.07
4.020E003
26.47
129.21
61.73
142.07
404.70

<0.0001
0.6890
<0.0001
<0.0001
<0.0001
<0.0001
0.9505
0.0002
<0.0001
<0.0001
<0.0001
<0.0001

X 22

22.22

22.22

188.82

<0.0001

X 23

2.12

2.12

18.04

0.0011

X 24
Total error
Lack of t
Pure error
Total SS
Predicted R2 = 0.9803

52.73

52.73

448.13

<0.0001

1.41
0.83
0.58
309.93

12
10
2
26

0.12
0.083
0.29

0.29

0.9803

3038

X. Liu et al. / Food Chemistry 141 (2013) 30343041

Fig. 1. Response surface and contour plots showing effects of the extraction parameters on anthocyanin yield. (a) at varyingnd Liquidsolid ratio and ammonium sulphate
concentration, (b) at varying Liquidsolid ratio and pH value, (c) at varying ethanol concentration and pH value, (d) at varying ammonium sulphate concentration and pH
value.

ratio was not signicant (P > 0.05); The quadratic terms

(X 21 , X 22 , X 23 , X 24 ) were very signicant (P < 0.01); In addition to the
interaction terms of X1X3 (P > 0.05), the others were highly relevant
to affect the partition coefcient (P < 0.01). The determination
coefcient (R2) of the model was 0.9803, which indicated that
the model had adequately represented the real relationship between the parameters chosen. The regression model was highly
signicant (P < 0.01), and the lack of t was not signicant
(P = 0.9298 > 0.05), CV was 2.38, and the standard errors (SE) of
all the regression coefcients were ranged from 0.099 to
0.20,which all proved the model can be used accurately.
3.3.2. Analysis of response surface
Response surface plots were plotted between two independent
variables while keeping the other independent variables at the
zero coded level. Effecting on the response functions (anthocyanin
yield and partition coefcient) were shown in Figs. 1 and 2,
respectively.
3.3.2.1. The interaction between the variables on anthocyanin yield
(Y1). The effect of different liquidsolid ratio (X1) and ammonium
sulphate concentration (X3) on the anthocyanin yield when the
ethanol concentrations (X2) and pH (X4) were xed at zero levels
was illustrated in Fig. 1(a). With the liquidsolid ratio and

ammonium sulphate concentration rising, the anthocyanin yield

increased, which was probably due to the fact that the anthocyanin
mostly combined with protein and polysaccharide (Florian &
Reinhold, 2004). With the liquidsolid ratio increasing, more solvent could enter cells and more anthocyanins could permeate into
the solvent (Prasad et al., 2009). When ammonium sulphate concentration increased from 20% to 21.5%, the anthocyanin yield
increasing from 70% to 85% was observed. This was probably due
to the enhanced salting-out effect. Our results were in good agreement with Li et al. (2010), where the 2,3-butanediol content
increased when ammonium sulphate concentration increased.
Fig. 1(b) showed the response surface plot of the effects of pH
(X4) and liquidsolid ratio (X1). The effects of pH (X4) and liquid
solid ratio (X1) were very signicant (P < 0.01) to anthocyanin yield
(Table 2), but the interaction between them was mainly the effect
of pH value, which probably because that pH can usually change
the electrical property of assigned materials, and then inuence
on the electric potential of ATPE system. With pH adding, a decline
in anthocyanin yield was observed, this may be because that
anthocyanins were stable in acidic condition and higher pH can
lead to the degradation of anthocyanins, which results in destroying the structure (Fan, Han, Gu, & Chen, 2008). While lower pH value would result in the hydrolysis of anthocyanins (Yang et al.,
2008), so appropriate pH value is needed in the extraction process.

X. Liu et al. / Food Chemistry 141 (2013) 30343041

3039

Fig. 2. Response surface and contour plots showing effects of the extraction parameters on anthocyanin partition coefcient. (a) at varyingnd Liquidsolid ratio and ethanol
concentration, (b) at varying ethanol concentration and ammonium sulphate concentration, (c) at varying ethanol concentration and pH value, (d) at varying ammonium
sulphate concentration and pH value.

Fig. 1(c) showed the response surface plot of various ethanol

concentrations (X2) and pH (X4) values at ammonium sulphate concentration (X3) of 20% and liquidsolid ratio of 50:1 (mL/g) (X1).
The interaction effect was similar to Fig. 1(b). The inuence of ethanol concentration was not signicant, which was probably due to
the fact that ethanol concentration increased from 23% to 27%,
ATPE system was not changed signicantly. Acidied ethanol is a
traditional method of extraction anthocyanins. However, our results were obviously different with Jayaprakasha, Girennavar, and
Patil (2008), where the total polyphenol extraction yield of grape
increased when ethanol concentration increased. The reason may
be that the ethanol concentration over the range of 2327%, ethanol is redistributed in the top and bottom phase, the ethanol concentration in top phase can reach to 60%. Ghafoor et al. (2009) have
reported that the ethanol concentration of 60% was sufcient for
extraction high quantities of anthocyanins from grape seeds.
The inuence of the interaction between ammonium sulphate
concentration (X2) and pH (X4) value was illustrated in Fig. 1(d).
The interaction effect was similar to Fig. 1(b) and (c), which was
that the inuence of pH was signicant compared to ammonium
sulphate concentration. Therefore, we should control the pH value
strictly in the extraction process.

3.3.2.2. The interaction between the variables on anthocyanin partition coefcient (Y2). The effects of ethanol concentration (X2) and liquidsolid ratio (X1) shown in Fig. 2(a) demonstrated that the
anthocyanin partition coefcient increased rapidly with the increase of ethanol concentration and liquidsolid ratio. The reason
may be that the increasing of ethanol concentration can lead to
increase its concentration in top phase, which was in favor of the
distribution of anthocyanins to top phase. Guo, Han, Zhang, Wang,
and Zhou (2013) have reported that ethanol concentration increased from 15% to 19%, the partition coefcient of lignans increased from 17.05 to 74.93. Wang et al. (2008) obtained higher
partition coefcient piceid, resveratrol and emodin from Polygonumcuspidatum, when ethanol concentration of 25% was used.
Our results were in good agreement with these reports. The inuence of the liquidsolid ratio on the anthocyanin partition coefcient was not signicant (P > 0.05) (Table 3), but the interaction
with ethanol concentration was signicant, which was probably
due to that the extraction process was more complex and affected
by many factors. Although the liquidsolid ratio did not change the
aqueous two-phase system, when the liquidsolid ratio increased,
this can improve the rate of penetrating and spreading to purple
sweet potato cell. Thus accelerate the rate of damaging the

3040

X. Liu et al. / Food Chemistry 141 (2013) 30343041

Table 4
Molecular structure of purple sweet potato anthocyanin identied by HPLC-ESI-MS.
Peak number

tR (min)

[M+] (m/z)

Compounds

References

1
2
3
4
5
6
7
8

16.02
19.07
21.43
34.19
36.07
39.83
43.66
46.26

471
730
907
516
1111
949
1069
1125

Cyanidin-3-glucoside
Galloyl procyanin dimer
Peonidin-3-p-hydroxybenzoyl-sophoroside-5-O-glucoside
Peonidin-3-glucoside
Cyanidin-caffeoyl-fumaroyl-sophoroside-3-O-glucoside
Peonidin-caffeoyl-sophoroside-3-O-glucoside
Peonidin-caffeoyl-hydroxybenzoyl-3-O-glucoside
Peonidin-caffeoyl-fumaroyl-sophoroside-3-O-glucoside

Grant & Helleur (2008)

Sandhu & Gu (2010)
Kim et al. (2012)
Grant & Helleur (2008)
Harada et al. (2004)
Harada et al. (2004)
Harada et al. (2004)
Harada et al. (2004)

hydrophobic bond and diffusing to the top phase, thereby affected

the anthocyanin partition coefcient.
Fig. 2(b) showed the effect of ammonium sulphate concentration (X3) and ethanol concentration (X2) on the anthocyanin partition coefcient. The anthocyanin partition coefcient increased
from 13.29 to 19.05 with the increase of ammonium sulphate concentration from 20% to 22%. This is probably due to that the ion
dipole between salt ions and water molecules, which can reduce
the quantity of free water in bottom phase, thus crowd out ethanol
and target objects to assign to top phase (Wang, Han, Xu, Hu, &
Yan, 2010). Our results were consentience with Li et al. (2010),
with the ammonium sulphate concentration from 20% to 28%,
the partition coefcient of 2,3-butanediol increased from 4 to
8.29. In a certain range, with the increase of ethanol concentration
(2326%), anthocyanin partition coefcient increased, which was
identical to Fig. 2(a). However, with the further increase in ethanol
concentration, a decline in the partition coefcient was discovered
(Fig. 2(b)). This may be because that the content of anthocyanins in
top phase was closed to saturation, and the distribution in bottom
phase was more than top phase, which reduced the anthocyanin
partition coefcient.
With the increase pH (34) (X4) and ethanol concentration
(2327%) (X2), the partition coefcient increased from 9 to 17.
The impact of ethanol concentration was similar to Fig. 2(a). When
pH changed from 3 to 4, the partition coefcient improved from
9.52 to 17.46, which was possibly due to that with the decrease
of pH, anthocyanins gradually combined with H+, and weakened
the binding force of the top phase; which lessened the partition
coefcient. However, the increase in pH can increase the electric
potential (Rito-Palomares, 2004), while the electric potential was
proportional to partition coefcient (Cisneros, Benavides, Brenes,
& Rito-Palomares, 2004), but the impact of this effect was not dominant. Our results were supported by the results of Wu et al. (2011)
and Guo, Han, Zhang, Wang, and Zhou (2012).
Fig. 2(d) showed the effect of pH and ammonium sulphate concentration to anthocyanin partition coefcient, and with the increase in ammonium sulphate concentration, the partition
coefcient added from 7 to 18, which was resembled to Fig. 2(b)
and (c).

3.3.3. Optimisation of extraction condition and results of method

validation
The optimum extraction condition was determined by the
canonical analysis of response surface, which predicted that the
conditions of 47.34:1 liquidsolid ratio, 23.20% ethanol concentration, 21.56% ammonium sulphate concentration and 3.33 pH
would lead to the maximum anthocyanin yield (Eq. (6)), and
43.29:1 liquidsolid ratio, 27.00% ethanol concentration, 22.00%
ammonium sulphate concentration and 3.34 pH would result in
the maximum anthocyanin partition coefcient (Eq. (7)). According to Eqs. (6) and (7), and considering the cost and process
requirements comprehensively, we chose the best conditions for

PSPAs were 45:1 liquidsolid ratio, 25% ethanol, 22% concentration

of ammonium sulphate and pH 3.3. The verication experiment
was performed using the selected optimal conditions. The experimental anthocyanin yield was 90.02 0.01%, and anthocyanin partition coefcient was 19.62 0.02, which was in agreement with
the predicted value (90.12%, 19.73) using the equation; which indicating that the model was adequate for describing the extraction
process.
3.4. Anthocyanins compound identication
According to the HPLC chromatogram of purple sweet potato
anthocyanins extracted by ATPE (gure not shown), a total of eight
compounds was identied by their rentention times (tR), UV-Vis
spectra and mass spectra as compared with reported data in the
literature (Table 4).cyanidin-caffeoyl-fumaroyl-sophoroside-3-Oglucoside (Peak 5) was the major compound followed by
peonidin-caffeoyl-hydroxybenzoyl-3-O-glucoside (Peak 7), then
peonidin-caffeoyl-sophoroside-3-O-glucoside (Peak 6), peonidincaffeoyl-fumaroyl-sophoroside-3-O-glucoside (Peak 8), which
coincided with that found in previous studies (Harada, Kano,
Takayanagi, Yamakawa, & Ishikawa, 2004). Peak 1 and Peak 4 corresponded to cyanidin-3-glucoside, peonidin-3-glucoside (Grant &
Helleur, 2008), respectively. Peak 3 was associated with peonidin
acylated with p-hydroxybenzoic acid (Kim et al., 2012). One of
the original contributions of our work to the purple sweet potato
extraction was the identication of Peak 2 as galloyl procyanin dimer which was isolated from Vitisrotundifolia (Muscadine Grapes)
(Sandhu & Gu, 2010) but not reported in the purple sweet potato
anthocyanins.
4. Conclusions
Response surface methodology is a useful tool for determining
the optimal extraction conditions of anthocyanins from purple
sweet potato. The optimal anthocyanin yield and partition coefcient of 90.02%, 19.62 were obtained when the optimum extraction
conditions of anthocyanins were as follows, 45:1 liquidsolid ratio,
25% ethanol concentration, 22% ammonium sulphate concentration and pH3.3. Under these conditions, the experimental yield
agreed closely with the predicted yield of 90.12% and 19.73.
HPLC-ESI-MS/MS analysis results showed that the PSPAs were
mainly composed of peonidins and cyanidins acidylated by caffeic
acid, fumaric acid and hydroxy benzoic acid. The composition of
PSPAs was not altered obviously, which were consistent with the
previous research results of PSPAs structure. Aqueous two-phase
extraction was a mild method compared to conventional solvent
extraction. Meanwhile, this method can keep the original composition and structure. These results demonstrated the successful
extraction of anthocyanins with aqueous two-phase extraction,
providing potential benets for industrial extraction of anthocyanins from purple sweet potato.

X. Liu et al. / Food Chemistry 141 (2013) 30343041

Acknowledgements
The authors gratefully acknowledge the earmarked fund for the
China Agriculture Research System (CARS-11-B-19). We also thank
the Support Plan of National Science and Technology, the Suitability Evaluation and Special Varieties Screening of Sweet Potato
(2012BAD29B03-03).
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