OPTIMIZATION OF THE ENZYME ASSISTED EXTRACTION OF

BETALAIN FROM DRAGON FRUIT (HYLOCEREUS UNDATUS)

PEEL BY PECTINASE PREPARATION
Nguyen Thanh Sang1 , Hoang Kim Anh2 , Le Van Viet Man1
1

Department of Food Technology, Ho Chi Minh City University of Technology

2

Department of Food Technology, Saigon Technology University

*E-mail: lvvman@hcmut.edu.vn
Received: XX September 2014, Accepted: XX October 2014
ABSTRACT

Pectinex Ultra SP-L with polygalactorunase activity was used for betalain extraction from
dragon fruit (Hylocereus undatus) peel. Firstly, Plackett-Burman model was used to select the
technological factors for extraction optimization. Material : solvent ratio and extraction time
were the factors that hightly affected the betalain yield. Response surface methodology (RSM)
was then applied for maximizing the betalain yield. The optimal material : solvent ratio and
biocatalytic time were 1:28 and 66 min, respectively. Under these conditions, the maximal
betalain yield achieved 1,46 mg/g material dry weight and this value was 2,1 times higher than
that in the control without enzymatic treatment.
Keywords: betalains,
surfacemethodology.

dragon

fruit

peel,

pectinase,

Plackett-Burman,

response

1. INTRODUCTION
In recent years, natural colorants have attracted great interest mainly because of the
apparent lack of toxicity and eco-friendliness. In addition, natural colorants are found to be
nutritional antioxidants and their presence in the diet can reduce the risk of cardiovascular
diseases, cancer and diseases associated with ageing [1].
Betalains are water-soluble nitrogen-containing pigments, which consist of the red-violet
betacyanins and the yellow-orange betaxanthins. Dragon fruit (Hylocereus undatus) peel is rich
in betalains [2]. However, pectin in dragon fruit peel increased solvent viscosity during the
extraction and reduced the betalain yield [3].The application of pectinase preparation to betalain
extraction from dragon fruit peel is therefore essential. Our preliminary study showed that
material : solvent ratio, enzyme concentration, pH, temperature and biocatalytic time were
important factors affecting extraction efficiency. There has been no report on optimization of
enzyme-assisted extraction of betalains from dragon fruit peel.

The objective of this study was to optimize the extraction variables for maximizing the
betalain yield from dragon fruit peel.
2. MATERIALS AND METHODS
2.1. Materials
Peel of dragon fruit (Hylocereus undatus) was obtained from Rong Xanh company (Phan
thiet, Vietnam).
Pectinex Ultra SP-L produced by Aspergillus aculeatus was obtained from Novozymes
(Switzerland). The preparation contains polygalacturonase and other activities such as
pectinlyase, cellulase, polyphenol oxidase, -glucosidase. The catalytic activity of Pectinex Ultra
SP-L is 4188 polygalacturonase unit (PGU) per mL. Theoptimal temperature and pH of this
preparation are 50o C and 4,5; respectively.
Distilled water - an eco-friendly solvent - was used for betalain extraction.
2.2. Experimentation
Peel of dragon fruit was firstly washed and the blanched in hot water at 90-95o C for 3 min.
The peel was then ground in a blender (Panasonic, Malaysia). The peel mash was then sieved
through a 0,5 mm-mesh screen, kept at -18o C and used for the experimentation. Before use, the
peel mash of dragon fruit was mixed with distilled water at the appropriate ratio and adjusted to
pH 4,5 for further enzyme-assisted extraction of betalains. For each assay, samples of 30g peel
mash were taken and placed into 250 mL beakers, which were covered with aluminium-foil
paper to prevent the oxidative change from light.
2.2.1. Selection of technological factors of the enzyme-assisted extraction by PlackettBurman
design
Different technological factors of the enzyme-assisted extraction including material :
solvent ratio (Z1 ) (g material dry weight per mL solvent), pectinase concentration (Z2 ) (PGU/g
material dry weight), extraction temperature (Z3 ) (oC), pH of solvent (Z4 ) and biocatalytic time
(Z5 ) (min) were tested using PlackettBurman design [5]. Each technological factor was
examined at two levels: low (-) and high (+) values. The values of two levels were selected
according to our preliminary experimental results (Data not shown).The Plackett-Burman design
is shown in Table 1.
2.2.2. Optimization of the enzyme-assisted extraction by RSM
Based on the results of section 2.2.1, two technological factors which had the highest
impact on the betalain yield were selected for optimization of the enzyme-assisted extraction.
RSM with central composite circumscribed (CCC) design was used. In this experiment, a twofactor, ve-level (
, 1, 0, +1,
) CCC design with 13 runs were employed. The two
technological factors were denoted as X1 and X2 . The dependant variable was betalain yield
(mg/g material dry weight).
2.3.Analytical methods

Betalain content in the extract was determined by spectrophotometric method [3]. The
results were expressed as mg of betalain per 1 g of dry weight of dragon fruit peel.
2.4.Statistical analysis
Modde version 5.0 was used for experimentation design as well as for regression and
graphical analysis of the experimental data obtained.
3. RESULTS AND DISCUSSION
3.1.Selection of technological factors of the enzyme -assisted extraction by Plackett
Burman design
The experimental results of Plackett-Burman design are visualized in Table 1. Analysis of
the regression coefficients and P values of 5 factors is shown in Table 2. The factor with confidence
level above 95% is considered as significant factor. It was clear that variables Z1 , Z2 , Z4 , and Z5
were the significant factors, while factor Z2 was considered insignificant. According to the
affecting coefficient, Z1 , Z4 , Z5 had positive effects on betalain yield whereas Z2 had negative
effects. The absolute value of Z1 and Z5 were the highest. Therefore, the material : solvent ratio
(Z1 ) and biocatalytic time (Z5 ) were selected for further optimization. In a previous study,
material : solvent ratio and extraction time were also considered as important variables and used
to optimize oil extraction from sunflower seeds [4]. Although three factors Z2 , Z3 , Z4 were not
selected for optimization but their impacts on the extraction yield must be confirmed.
Table 1.Results of Plackett-Burman model for the effect of variables on the betalain
yield (mg/100g of material dry weight)
Run
1
2
3
4
5
6
7
8

Technological factors
Z1
Z2
Z3
Z4 Z5
1:30
5,2
25
6
30
1:30 15,6
25
4
90
1:30 15,6
35
4
30
1:20 15,6
35
6
30
1:30
5,2
35
6
90
1:20 15,6
25
6
90
1:20
5,2
35
4
90
1:20
5,2
25
4
30

Betalain
yield
1,19
1,11
1,06
1,02
1,28
1,08
1,12
0,99

Run
9
10
11
12
13
14
15
16

Technological factors
Z1
Z2
Z3
Z4 Z5
1:30
5,2
25
6 30
1:30 15,6
25
4 90
1:30 15,6
35
4 30
1:20 15,6
35
6 30
1:30
5,2
35
6 90
1:20 15,6
25
6 90
1:20
5,2
35
4 90
1:20
5,2
25
4 30

Table 2.Statistical analysis of Plackett-Burman model for the effectsof variables

onbetalain yield
Tech. factors
Z1
Z2
Z3
Z4
Z5

Affecting coefficient PValue at 5% significant level

0,529
0,0000159*
-0,288
0,0018*
0,072
0,3188
0,337
0,0006*
0,412
0,0001*

Betalain
yield
1,17
1,10
1,05
0,94
1,26
1,10
1,07
0,92

3.2.Optimization of the enzyme-assisted extraction by RSM

Table3.Experimental design and results
Run

X1

X2

1
2
3
4
5
6
7

1:20
1:30
1:20
1:30
1:17
1:32
1:25

30
30
90
90
60
60
18

Betalain yield
(mg/100g dry weight)
0,48
1,17
0,74
1,11
0,51
1,37
0,66

Run

X1

X2

8
9
10
11
12
13

1:25
1:25
1:25
1:25
1:25
1:25

102
60
60
60
60
60

Betalain yield
(mg/100g dry weight)
1,25
1,35
1,34
1,34
1,40
1,39

The experimental design and the results are presented in Table 3. Multiple regression
analysis was used to analyze the data and thus a second-order polynomial equation was derived
as follow:
Table 4 shows that the coecient of determination (R2 ) was calculated as 0,956 indicating
good agreement between the experimental and the predicted values [5]. Material : solvent ratio
(X1 ) and biocatalytic time (X2 ) had significantly positive effects on betalain yield, while their
quadratic effects were also observed, but negative. Material : solvent ratio (X1 ) had higher effect
on betalain yield than biocatalytic time (X2 ).
Table 4.Coefficient list for response surface
quadratic model
Betalain
content
Constant

1,36401

Standard
Error
0.0432

X1

0,28153

0.0341

7,57E-005

X2

0,13055

0.0341

0,00655

2
X1
2
X2

-0,23266

0.0366

0,00039

-0,22266

0.0366

0,00051

X1X2

-0,07750

0.0483

0,15293

N = 13

Coefficient

DF = 7

Q = 0,702

Pvalue
8,31E-009

R = 0,956

Figure 1. Effect of material:solvent ratio and

extraction time onbetalainyield (A: twodimensional contour plots, B: three-dimensional
response surface plots)

Figure 1 shows the response surface plots and their corresponding contour plots. An
important increase in Y could be achieved when the value of material : solvent ratio decreased
from 1:17 to 1:28 g material dry weight/mL solvent, and the biocatalytic time increased 18 to 66
min.The predicted maximal betalain yield was 1,45 mg/g material dry weight at the material :
solvent ratio of 1:28 and biocatalytic time of 66 min. In order to verify the accuracy of the
model, three independent replicates were conducted under the optimal conditions. The betalain
yield was 1,460,004 mg/g material dry weight. The experimental values were therefore similar
to the predicted value from equation (1). The betalain yield obtained was 2,1 times higher than
that in the control without enzymatic treatment. Recently, Maran et al. (2013) optimized the
conditions of thebetalain extraction from prickly pear fruits and showed that the maximal
betalain yield was 0,42 mg/g material [1]. Our results showed higher extraction yield; that was

probably due to different distributions of the solutes in different plant materials and enzyme
application to betalain extraction.
4. CONCLUSION
In the enzyme-assisted extraction of betalains from dragon fruit peel, material : solvent
ratio and biocatalytic time were the most important technological factors. Maximal betalain yield
achieved 1,46 mg/g material dry weight when the material : solvent ratio and biocatalytic time
were 1:28 and 66 min, respectively. The betalain yield obtained from RSM was 2,1 times higher
than that in the control without enzymatic treatment.
REFERENCES
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from Opuntiacus-indica using BoxBehnken design with desirability function,Industrial
Crops and Products 49 (2013): 304-311.
2. Wybraniec S., Nowak-Wydra B., Mitka K., Kowalski P., MizrahiY. - Minor betalains in
fruits of Hylocereusspecies, Phytochemistry 68 (2007): 251-259.
3. SchweiggertR.M., Villalobos-Gutierrez M.G., Esquivel P., Carle R. - Development and
optimization of low temperature enzyme-assisted liquefaction for the production of colouring
foodstuff from purple pitaya (Hylocereus sp.[Weber] Britton & Rose), European Food
Research and Technology 230 (2009): 269-280.
4. Sineiro J., o gue H., Nez M.J., Lema J.M. - Optimization of the enzymatic treatment
during aqueous oil extraction from sunflower seeds, Food Chemistry 61 (1998): 467-474.
5. ZhouJ.,Yu X., Ding C., Wang Z., Zhou Q., Pao H., Cai W.- Optimization of phenol
degradation by Candida tropicalis Z-04 using Plackett-Burman design and response surface
methodology, Journal of Environmental Sciences 23 (2011): 22-30.
TM TT
TI U HA QU TRNH X L ENZYME TRCH LY BETALAIN T V QU
THANH LONG (HYLOCEREUS UNDATUS)VI CH PHM PECTINASE

Nguyn Thanh Sang1 , Hong Kim Anh2 , L Vn Vit Mn1

1

B mn Cng ngh thc phm, Khoa K thut ha hc, Trng i hc Bch khoa thnh ph
H Ch Minh
2

Khoa Cng ngh thc phm, Trng i hc Cng ngh Si Gn

*E-mail: lvvman@hcmut.edu.vn
n ta son: XX September 2014, Chp nhn: XX October 2014

Pectinex Ultra SP-L vi hot t h polygalactoru ase c s d g trch ly betalain t v

qu thanh long (Hylocereus undatus). u tin, m hnh Plackett-Bur a c s dng chn

cc yu t cng ngh cho th nghim ti u ha qu tr h trch ly. T l nguyn liu:dung mi v

thi gian trch ly l nhng yu t h hng nhiu n hiu sut trch ly betalain. Tiptheo,
ph g php p ng b mt c p d g hiu sut trch ly betalain t gi tr cc i. T l
nguyn liu:dung mi v thi gian xc tc ti u ca enzyme ln lt l 1:28 v 66 pht. Khi,
h lng betalain ti a thu c l 1,46 mg/g cht kh nguyn liu v gi tr y cao h gp
2,1 ln so vi mu i chng khng x l enzyme.
T kha: betalains, v thanh long, pectinase, Plackett-Bur a , ph g php p ng b
mt