Separation Science and Technology

ISSN: 0149-6395 (Print) 1520-5754 (Online) Journal homepage: http://www.tandfonline.com/loi/lsst20

Purification of anthocyanins from red cabbage

using semi interpenetrating network hydrogel
beads in a packed bed column

Balaji Shankaranarayanan & Ekambaram Nakkeeran

To cite this article: Balaji Shankaranarayanan & Ekambaram Nakkeeran (2018): Purification of
anthocyanins from red cabbage using semi interpenetrating network hydrogel beads in a packed
bed column, Separation Science and Technology

To link to this article: https://doi.org/10.1080/01496395.2018.1520728

Published online: 19 Sep 2018.

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SEPARATION SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/01496395.2018.1520728

Purification of anthocyanins from red cabbage using semi interpenetrating

network hydrogel beads in a packed bed column
Balaji Shankaranarayanan and Ekambaram Nakkeeran
Research Laboratory, Department of Biotechnology, Sri Venkateswara College of Engineering (Autonomous), Sriperumbudur Tk, Tamil Nadu,
India

ABSTRACT ARTICLE HISTORY

Anthocyanins are polyphenols, water-soluble pigments that have increased acceptability to food Received 21 November 2017
and pharmaceutical products. In this investigation, semi interpenetrating network hydrogel beads Accepted 4 September 2018
were developed to purify anthocyanins present in red cabbage. Effect of bead size and refluxing KEYWORDS
on anthocyanin purification and carbohydrates elimination were studied. Hydrogel bead with 1% Anthocyanins; red cabbage;
gelatin, 5% sodium alginate, and 1% calcium chloride resulted in maximum two-fold increase in purification; hydrogel bead;
anthocyanin purity with 35 mg/g carbohydrate elimination. Further, hydrogel bead size and packed bed column
refluxing exhibited inversely proportional anthocyanin purity and carbohydrate elimination.
Thereby, confirmed that anthocyanin purity is highly dependent on sugars. The results suggested
that the developed hydrogel beads could be suitable for purifying bioactive compounds.

Introduction methods result in low recovery of anthocyanins with

greater purity.[10] Hence a need exists to develop eco-
Anthocyanins are glycosylated polyhydroxy derivatives
nomic and non-toxic purification methods for obtain-
of 2-phenylbenzopyrilium (flavium) salts found in most
ing high purity anthocyanins from red cabbage.
fruits and leaves.[1] Their water-soluble nature and col-
Adsorption processes using commercial resins such as
oring ability simplifies its incorporation into various
Amberlite, Dowex, and Silica gel were used for the
food systems.[2] In addition, anthocyanins are also
purification of anthocyanins.[11] However, the produc-
widely used as nutraceuticals in pharmaceutical pre-
tion and regeneration of adsorbents are expensive and
parations because of their anti-oxidant property and a
energy intensive which resulted in an increasing inter-
range of biological activities including anti cancerous,
est to develop novel adsorbents.[12] Novel cellulose
anti-inflammatory, and vasoprotective effects.[3–5]
nanoparticles could be used as alternative adsorbents
Sources of anthocyanins include berries, colorized vege-
as they are much efficient than the non-conventional
tables such as radish, curly kale, black carrot, purple
adsorbents developed using rice husk, fly ash, hazelnut
sweet potato, eggplant, red bean, violet cauliflower, red
shell, banana peel, orange peel, kaolin etc.[13] Their
lettuce, red onion, and red cabbage.[6] Red cabbage
application in industrial scale is however limited due
(Brassica oleracea L.) is a promising source of antho-
to its difficulties in regeneration.[12] To avoid this lim-
cyanins for coloration of foods. Anthocyanins from red
itation, nano-materials could be replaced by polymeric
cabbage are unique colorants over a very broad pH
adsorbent materials such as hydrogels.[14,15]
range.[7,8] Currently, red cabbage anthocyanins are
Hydrogels are cross-linked polymeric networks that
used to color various beverages, candies, dry mixed
can adsorb and retain large amounts of water.[16] They
concentrates, chewing gums, yoghurts, and sauces.[9]
form the central component in numerous applications
Conventional purification methods of anthocyanins
such as separation membranes, biosensors, elimination
from red cabbage are non-selective and the resulting
materials, and drug delivery systems.[17] Despite of its
pigment solutions consist of large amounts of impuri-
unique characteristics, their applications are largely
ties/byproducts such as sugars, organic acids, amino
restricted due to poor mechanical properties which
acids, and proteins. These methods use solvents such
results from the microinhomogeneties of polymeric
as ethanol, methanol, and acetone for extraction and
topological structure created by cross-linking.[18] As
purification. However, besides being toxic, these

CONTACT Ekambaram Nakkeeran nakkeeran@svce.ac.in Research Laboratory, Department of Biotechnology, Sri Venkateswara College of
Engineering (Autonomous), Sriperumbudur Tk, Tamil Nadu 602 117, India
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsst.
© 2018 Taylor & Francis
2 B. SHANKARANARAYANAN AND E. NAKKEERAN
the networks cannot behave cooperatively, they begin
to break from the weakest link and thereby reducing
the whole mechanical strength. Thus, to improve the
mechanical strength, doping of the polymer with
another copolymer is carried out to form a semi inter-
penetrating network hydrogel which is stable toward
physical stimuli.[19] Also, hydrogel networks are useful
for purification processes which require materials that
have good compatibility with aqueous solvents. In light
of the above, attempts were made in this investigation
to optimize and model the purification of anthocyanins
from red cabbage using semi interpenetrating network
hydrogel beads in a packed bed column.
Materials and methods
Materials
The reagents including gelatin, sodium alginate, for-
maldehyde and calcium chloride required for synthesis
of hydrogel beads were procured from Sisco Research
Laboratories, India. Apple vinegar (Food Grade) and
red cabbage was procured from a local market in
Chennai.
Extraction of anthocyanin from red cabbage
1 kg of red cabbage was taken and shred into small
pieces. The cut leaves were subjected to blanching at
100°C in a 0.1% (w/w) solution of apple vinegar for a
period of 15 min. The solid to liquid ratio was main-
tained at 1:2 (w/v). The leaves were minced together
and juice was extracted from minced leaves. The
extracted juice was subjected to filtration through
Whatmann No.1 filter paper to obtain a clear juice.
The juice was used as the crude for further purification
employing semi interpenetrating network hydrogels in
a packed bed column with an internal diameter of
2.5 cm, height of 30 cm and a bed volume of 24 cm.
In order to prevent degradation during storage, 0.1%
(w/w) ascorbic acid was added to the juice.
Synthesis of semi interpenetrating network
hydrogel beads
Various concentrations of gelatin were taken and added
drop wise on a pre-heated solution of sodium alginate
maintained at 60°C and made into a homogenous solu-
tion. The mixture was added drop wise over a solution
of 5% (w/w) calcium chloride (CaCl2) maintained at
37°C. The beads of calcium alginate were formed under
constant mixing at 100 rpm. The formed beads were
subjected to curing with a solution of formaldehyde of
various concentrations for 24 h at room temperature.
Diameter of the beads was varied to optimize the bead
size. After 24 h, the beads were stored in the formalde-
hyde solution for further use.
Purification of anthocyanins using the semi
interpenetrating network hydrogel beads
35 g of synthesized hydrogel beads of various composi-
tion and diameters were taken and loaded into a glass
column. Both the ends of the column were plugged
using glass wool to retain the beads under pressure.
Anthocyanin was passed from the bottom of the col-
umn and collected through the top using a peristaltic
pump at both ends. The peristaltic pump was curated
according to the flow rate. The purity of anthocyanin
was determined using the following equation:[20]
CV
Purity ¼ (1)
W
where, C is the concentration of anthocyanins in the
solution, V is the volume of the sample obtained, and W
is the total weight of solution obtained. Comparative study
between the purities of the feed and purified sample was
carried out and the fold increase in purity was calculated.
Estimation of anthocyanin
Anthocyanin concentration was determined using pH
differential method by using the following equation:[21]
Anthocyanin Concentration ðmg=LÞ
A x DF x MW x L
¼ X 1000 (2)
ε
where, Absorbance (A) = (A530− A700)pH 1.0− (A530−
A700)pH 4.5, MW is molecular weight of anthocyanin
(449.2 g/mol), DF is dilution factor, ε is the extinction
coefficient (26,900 L/cm mol), and L is the path length
(1 cm). It was considered that red cabbage anthocya-
nins are all derived from cyanidin glycoside and quan-
titative data were expressed as cyanidin-3-glycoside.[22]
The absorbance was measured using UV- vis
spectrophotometer.
Estimation of carbohydrate elimination capacity
The total carbohydrate content was estimated using
phenol–sulphuric acid method. The carbohydrate elim-
ination capacity of the hydrogel beads was calculated
using the following equation:[23]
Co  Ce
qe ðmg=gÞ ¼ x Vi (3)
W
 SEPARATION SCIENCE AND TECHNOLOGY 3

Table 1. Experimental ranges and levels of factors used in the Table 2. ANOVA for effect of hydrogel composition and flow
factorial design. rate on anthocyanin purity.
Independent Variables Coded Symbol Range and Level Degree of Co- Sum of Contribution p-
−1 +1 Source Freedom Efficient Squares (%) Value
Gelatin A 1 5 Constant - 1.1271 - - 0.000
Sodium Alginate B 5 7 Main Effects
Calcium Chloride C 1 5 A 1 −0.1210 0.46883 26 0.022
Formaldehyde D 0.1 1 B 1 −0.0645 0.13331 7 0.195
Flow Rate E 1 5 C 1 −0.0076 0.00183 0 0.876
E 1 0.0353 0.03995 2 0.47
2–Way Interactions
AB 1 0.1061 0.36024 20 0.041
AE 1 0.1680 0.01146 1 0.003
where, qe is the carbohydrate elimination capacity, Co BC 1 −0.0189 0.10603 6 0.697
and Ce are the initial and final carbohydrate concentra- BE 1 0.0576 0.17149 9 0.697
3-Way Interactions
tions, Vi is the volume of solution used, and W is ABC 1 −0.0732 0.01528 1 0.654
amount of hydrogel beads used. ACE 1 −0.0219 0.14649 8 0.011
BCE 1 0.1368 0.18964 10 0.175
4-Way Interactions
ABCE 1 −0.0770 0.18964 10 0.126
Residual 17 - 0.18964 - -
Modelling by 2k full factorial method Error
Total 29 - 1.83419 - -
In order to optimize and model the purification pro- R-Sq 72% - - - -
cess, a number of factors influencing the process such
as composition of hydrogel beads as well as flow rate of
red cabbage juice through the column were identified.
anthocyanin extractability due the softening of pericarp
Studying the effect of each factor individually is quite
and enzyme inactivation. Further, Yue and Xu[27]
tedious and time consuming.[24] Hence, to minimize
reported that anthocyanin extraction at 100°C
the difficulties, 2k full factorial method of optimization
increased higher free radical scavenging capability,
was used. Factorial design helps to estimate the overall
thereby, increased the antioxidant activities.
main factor effects and interactions of different factors.
Literatures reported an increased extraction of antho-
Two levels for each factor were set in the experi-
cyanin at acidified water.[11] Generally, 1% (v/v) HCl
mental matrix is based on the preliminary studies
acidified water is used for extraction process but not
(Table 1). Purity fold of anthocyanin was taken as the
preferable for food applications. Hence, in this study,
response parameter. The full factorial method of opti-
blanching at 100°C in a 0.1% (w/w) solution of apple
mization was carried out with the help of MINITAB 16
vinegar for a period of 15 min is used to extract
software. The coded mathematical model for 25 factor-
anthocyanin.
ial designs is given below:[25]
Purity Fold ¼ X0 þ X1 A þ X2 B þ X3 C þ X4 D
þ X5 E þ ::: þ X32 ABCDE (4) Effect of hydrogel composition and feed flow rate
on anthocyanin purity
where, X0 and Y0 are the global variables and A, B, C, Analysis of variance
D, E, stand for concentration of gelatin, sodium algi- Generally, separation of bioactive compounds separated
nate, calcium chloride and formaldehyde and flow rate by chromatography technique is influenced by the bead
at which the red cabbage juice is passed through the (compositions and size) and flow rate. The contribution
column, respectively. The co-efficient Xi was found of the significant factors was predicted using the fol-
using the ANOVA Table 2. lowing equation:[28]
Individual Sum of Squares
% Contribution ¼ X 100
Total Sum of Squares
Results and discussion
(5)
Extraction of anthocyanin
From the ANOVA for purity of anthocyanin (Table 2),
Blanching is an intermediate thermal processing step it was found that except for main effects of gelatin
used for enhancing the food product quality. In this concentration, two-way interactions between gelatin
present study, blanching is used to extract anthocyanin and calcium chloride and three way interactions
pigment from red cabbage. Deylami et al.[26] reported between concentration of gelatin and sodium alginate
that blanching at 100°C exhibited positive effect on the and flow rate, all other factors and their interactions
4 B. SHANKARANARAYANAN AND E. NAKKEERAN
were insignificant in affecting the purity of anthocya-
nin. Concentration of gelatin showed maximum con-
tribution of 26% in influencing the purity of
anthocyanin. The main effect of formaldehyde and its
interaction effects with other parameters showed high
degree of insignificance. Hence, they were eliminated.
The result confirmed the inert nature of formaldehyde.
The type of effect induced by the significant parameters
was identified using the normal probability plot.
Considering the coefficients of significant parameters,
purity was modeled using the following equation:
Purity Fold ¼ 1:1271  0:1210 A þ 0:1061 AB
(6)
þ0:1680 AE  0:0219 ACE
The R-Square value of 72% showed a 28% deviation
from the linear model applied to the purification
process.
Normal probability plot
The type of effect induced by the significant parameters
on anthocyanin purity was found using the normal prob-
ability plot.[29] The points close to the fitted line does not
exhibit any significant effect on the model. Points away
from the line represent the significant effects on the
model.[30] The position of points representing the signifi-
cant effects determines the type of effect induced on
purity. On analyzing the normal probability plot for pur-
ity fold (Fig. 1), it was found that two way interactions
between gelatin and calcium chloride and three way
Figure 1. Normal probability plot for purity fold of anthocyanin (R-Sq: 72%).
interactions between concentration of gelatin and sodium
alginate and flow rate showed positive effect of above 70%
in affecting the anthocyanin purity. However, the con-
centration of gelatin showed negative effect on the purity.
Hence it was concluded that the concentration of gelatin
was inversely proportional to the purity of anthocyanin.
Optimization of response parameters for the
purification of anthocyanin
Optimization of the factors on purity of anthocyanin
was carried out using desirability function (D).[30]
Desirability function is used to find the optimal con-
dition that provides the most desirable response
value. The main objective of this work is to enhance
the purity of anthocyanin. Based on the results of
ANOVA table and normal probability plot, the fac-
tors were set at different ranges using response opti-
mizer to achieve maximum desirability. The
combination that gave maximum desirability is repre-
sented in the response optimization plot (Fig. 2). It
was found that two fold increase in purity of antho-
cyanins is obtainable with a desirability value of 1
when red cabbage juice was passed with a flow rate
of 5 ml/min through hydrogel column prepared with
1% gelatin combined with 5% sodium alginate and
1% calcium chloride. Concentration of formaldehyde
was eliminated since it was insignificant in affecting
the purity of anthocyanins. The result was confirmed
experimentally.
 SEPARATION SCIENCE AND TECHNOLOGY 5

Figure 2. Response optimization plot for purity of anthocyanin.

Table 3. Hydrogel bead combinations.

Combination Number Gelatin (%) Alginate (%) Calcium Chloride (%) Formaldehyde (%) Flow Rate (ml/min)
1 1 5 1 0.1 1
2 1 5 1 1 1
3 1 5 5 0.1 1
4 1 5 5 1 1
5 5 5 1 1 1
6 1 7 1 0.1 1
7 5 7 1 0.1 1
8 1 7 1 1 1
9 5 7 1 1 1
10 5 5 1 0.1 1
11 5 5 5 0.1 1
12 1 7 5 0.1 1
13 5 7 5 0.1 1
14 5 5 5 1 1
15 1 7 5 1 1
16 5 7 5 1 1
17 1 5 1 0.1 5
18 1 5 1 1 5
19 1 5 5 0.1 5
20 1 5 5 1 5
21 5 5 1 1 5
22 1 7 1 0.1 5
23 5 7 1 0.1 5
24 1 7 1 1 5
25 5 7 1 1 5
26 5 5 1 0.1 5
27 5 5 5 0.1 5
28 1 7 5 0.1 5
29 5 7 5 0.1 5
30 5 5 5 1 5
31 1 7 5 1 5
32 5 7 5 1 5

Carbohydrate elimination capacity and elimination capacities are represented in Fig. 3.

Maximum carbohydrate elimination capacity of
The elimination capacity correlates with the properties
35 mg/g was obtained when feed flow rate was main-
of the hydrogel beads toward retaining carbohydrates.
tained at 5 ml/min and the hydrogel bead was prepared
Physical properties of the hydrogel played an important
using 1% gelatin, 5% sodium alginate, and 1% calcium
role in process of elimination of carbohydrates. Various
chloride.
hydrogel combinations used are represented in Table 3
6 B. SHANKARANARAYANAN AND E. NAKKEERAN

40

Carbohydrate Elimination Capacity (mg/g)

30

20

10

0
1 6 11 16 21 26 31
Combination Number

Figure 3. Carbohydrate elimination capacities of various hydrogel bead combinations.

Table 4. Experimental ranges and levels of factors used to at minimal levels to achieve greater purity and carbo-
optimize the effect of bead size and refluxing. hydrate elimination.
Independent Variables Coded Symbol Range and Level
−1 +1
Bead Size F 400 µm 3 mm
Refluxing G 1 5
Reusability of the hydrogel beads
Reusability of the beads was identified by continuously
Table 5. Effect of bead size and reflux rate on anthocyanin using the column for five times without washing the bed
purity and carbohydrate elimination.
column. It was found that the column could be used only
Bead Reflux Purity Carbohydrate Elimination Capacity
Size Rate (Fold) (mg/g) once without washing. Hence, it was concluded that in
3 mm 5 1 3.3 order to increase the reusability levels, the strength of the
0.4 µm 5 2 11.5 beads has to be increased to protect its integrity and prevent
0.4 µm 1 2 15.3
3 mm 1 1 11 its break down.

Effect of hydrogel bead size and refluxing on

Conclusions
anthocyanin purity and carbohydrate elimination
A method for purification of anthocyanin pigment from
To optimize the effects of bead size and refluxing on
red cabbage using semi interpenetrating network hydrogel
the purity of anthocyanin and carbohydrate elimination
beads loaded in a packed bed column was developed. A two
capacity, hydrogel beads of various sizes (Table 4) were
fold increase in anthocyanin purity could be obtained with
synthesized using the optimized composition. Refluxing
carbohydrate elimination capacity of 35 mg/g using the
was carried out by passing the processed juice continu-
developed method. This showed the efficiency of the devel-
ously through the column. From the experimental
oped method for obtaining pure anthocyanin extracts from
results (Table 5), it was found that, when hydrogel
red cabbage juice. Further, it was identified that the hydro-
beads were synthesized with bead size of 400 µm and
gel bead size, concentration of the dopant (gelatin) and the
when no refluxing was carried out, a two fold increase
refluxing were inversely proportional to the response fac-
in purity was obtained with a maximum carbohydrate
tors. The reusability studies on the hydrogel bead packed
elimination capacity of 15.3 mg/g. Thus, it was identi-
bed column showed that it could be used only once without
fied that bead size and refluxing should be maintained
any washing step.

Acknowledgments
Authors thank Prof. M. Sivanandham, Secretary, SVEHT and
SVCE for providing necessary facilities.
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