Modeling of carbohydrate elimination process from anthocyanin rich red cabbage

extracts using semi interpenetrating network hydrogel beads loaded in a packed

bed column

Shankaranarayanan Balaji and Nakkeeran Ekambaram*

Research Laboratory, Department of Biotechnology, Sri Venkateswara College of

Engineering (Autonomous), Sriperumbudur Tk – 602 117, India

*Email Id: nakkeeran@svce.ac.in

Abstract

Anthocyanins are natural pigments that are commonly found in most fruits and
vegetables. In this investigation, attempts were made to separate carbohydrate
impurities present in the anthocyanin rich red cabbage extracts using semi
interpenetrating network hydrogel beads in a packed bed column. Two kinetic models,
Thomas, Yoon-Nelson, were applied to predict the effect of flow rate on the
breakthrough curves for elimination of carbohydrates on the hydrogel bead packed bed
column. The models revealed the relationship between flow rate and elimination
properties of the column. The elimination of carbohydrates was found to increase with
increase in flow rate. Further, the dissolution and hydration studies showed the stability
of the beads in neutral pH and room temperature conditions respectively. The results
suggested the suitability of the column in purifying anthocyanin rich red cabbage
extracts maintained at neutral pH conditions.

Keywords: Anthocyanins, Carbohydrate impurities, Separation, Hydrogel bead, Packed

Bed Column, Kinetic model, Breakthrough curve.
Introduction

Edible pigments are important additives that increase overall acceptability of

foods (Chethana et al., 2007). The pigments can be either synthetic or natural. In

recent times, the safety of synthetic pigments is scrutinized due to their potential side

effects caused upon consumption (McDougall et al., 2007; Patil et al., 2009). Thus,

there is greater focus on natural pigments due to their ecofriendly nature and lack of

toxicity. Natural pigments such as betalains from beetroot, carotenoids from carrot and

anthocyanins from grape and red cabbage are some examples that are currently

applied in various food systems (Mazza et al., 1990).

Red cabbage is one of the rich sources of anthocyanins. Compared to other

sources, red cabbage anthocyanins are unique as they exhibit various colours in

different pH ranges. The stability of anthocyanin pigment is influenced by various

factors including concentration of carbohydrates, presence of oxygen, pH, storage

temperature and light (Dorota et al., 2007). Removal of carbohydrates from red

cabbage extract plays a major role in determining the anthocyanin stability as its

presence leads to Maillard reaction (Marainne et al., 2001).

Several separation techniques are used to remove the carbohydrates present in

the red cabbage extract (Chandrasekher et al., 2012; Arapitsas et al., 2008). However,

in case of methods such as column chromatography and electrophoresis, the industrial

scale up is highly expensive (Chandrasekher et al., 2015). Hence a need exists to

develop an economical separation technique to remove the carbohydrates present in

the red cabbage extract.

 Hydrogels are defined as polymeric materials that exhibit the ability to swell in the

presence of water and retain a significant fraction of water within their structure without

dissolving (Druvy et al., 2003). The physical and chemical properties depend on the

type of monomer and cross linker from which they are synthesized (Rakmai et al.,

2015). Currently hydrogels are extensively used to separate toxic materials such as

industrial dyes, metals, toxins, hazardous substances etc. from waste water (Apel et al.,

1993; Papageorgiou et al., 2006). The major advantage behind using hydrogels is the

ease of applicability and high regeneration efficiency after uptake of contaminants.

In light of the above, attempts were made in this investigation to separate

carbohydrates from red cabbage juice using semi interpenetrating network hydrogel

beads prepared using gelatin and sodium alginate. Further, the separation process was

analyzed using kinetic models.

Materials and Methods

Materials

Analytical grade sodium alginate, gelatin, calcium chloride, sodium hydroxide and

hydrochloric acid were procured from Sisco Research laboratories, India. Red cabbage

and apple vinegar (food grade) were procured from a local market in Chennai.

Methods

Extraction of Anthocyanin Rich Red cabbage Extract

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 maintained 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 carbohydrate separation employing semi

interpenetrating network hydrogels in a packed bed column. 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

1% of gelatin (w/w) was added to a preheated solution of sodium alginate (5%

(w/w)) and was made into a homogenous mixture. 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 0.1% formaldehyde (w/w). The size of the beads was

maintained at 400 µm.

Packed Bed Column Experiments

A 30 cm long glass tube of 2 cm diameter was used as the column to carry out

the experiments. 35 g of the synthesized hydrogel beads were taken and loaded into

the column. Glass wool was placed at both ends to prevent the expansion of the

hydrogel beads under vacumn. Silicone tubing was connected to both the ends to

facilitate the transport of the red cabbage juice into and out of the column. Prior to

carrying out the experiment, water was passed through the column for 20 min to

evacuate air bubbles and impurities present on the hydrogel bead surface. 500 ml of

red cabbage juice was passed from the bottom using a peristalitic pump at a desired

flow rate. Effluent samples were collected at regular intervals from the top of the

column. The carbohydrate content in the feed and effluent was measured using phenol
sulfuric acid method (Dubois et al., 1956). All the tests were carried out at room

temperature. The performance of the hydrogel bead packed bed column at various flow

rates was analyzed using break through curves obtained from two kinetic models,

Thomas and Yoon Nelson.

Modeling of Column Data

Thomas Model

Thomas model is used to identify the effect of feed flow rate on carbohydrate

elimination capacity of the hydrogel bead column. The assumptions of Thomas model

are: (i) negligible axial and radial dispersion in the packed bed column; (ii) the

elimination is described by a pseudo second-order reaction rate principle; (iii) constant

column void fraction; (iv) constant physical properties of the composition of the solution

passed through; (v) isothermal and isobaric process conditions; (vi) negligible intra

particle diffusion and external resistance during the mass transfer processes. The

linearized form of the Thomas model is expressed as (Dolphen et al., 2007):

C0 k qw
ln(  1)  Th 0  k Th C0 t
Ct ν

where, kTh (ml/(min.mg)) is the Thomas rate constant, q0 (mg/g) is the equilibrium

carbohydrate uptake per g of the hydrogel bead, C0 (mg/L) is the influent carbohydrate

concentration, Ct (mg/L) is the effluent concentration at time t, w (g) is the mass of

hydrogel beads used and ν (ml/min) represents the flow rate. The values of q0 and kTh

are determined using the intercept and slope of the linear plot between ln[(C0/Ct)−1]

against time (t).

Yoon Nelson Model

The Yoon Nelson model is based on the assumption that rate of decrease in the

probability of elimination of carbohydrates is proportional to product of probability of

carbohydrate elimination and the probability of carbohydrate elimination breakthrough

on the adsorbent. Compared to other models, the Yoon Nelson model is less

complicated as it requires no detailed data and it does not consider characteristics of

adsorbate, the type of adsorbent, and the physical properties of the packed bed column.

The linearized form of the model is expressed as (Yoon et al., 1987):

Ct
ln  k YN t  k YN
C0  C t

where, kYN (L/min) is the rate velocity constant,  (min) is the time in required for 50%

carbohydrate elimination breakthrough. The values of  and kYN are identified from the

slope and intercept of the linear plot of ln [Ct / (C0−Ct)] against sampling time (t).

Maximum Carbohydrate Holding Capacity

For a given feed carbohydrate concentration and feed flow rate, the maximum

column holding capacity qtotal (mg/l) for retention of carbohydrates was calculated using

the area under the curve of the plot between concentration of carbohydrates adsorbed

with respect to time. The area under the curve was calculated using the following

equation (Han et al., 2009):

Q ttotal
qtotal   Cad dt
1000 0
where, Ce, ttotal and Q are the eliminated carbohydrate concentration (mg/l), total flow

time through the column and volumetric flow rate (ml/min) respectively. The amount of

carbohydrate eliminated was found using the following equation:

Ce  Co  C t

where, Co and Ct are the concentration of carbohydrates in the feed and effluent

respectively.

Dissolution and Hydration Rate Tests

The hydrogel beads were tested for the dissolution property and hydration rate.

In the dissolution tests, the 10 g of the beads were placed in 10 ml of HCl and NaOH

solutions prepared at various pH ranges. The weight of the beads was determined at

regular intervals. The rate of dissolution of the beads was identified using the following

equation:

w
De swelling rate (%)  f x100
w
i

where, W i and W f denote the initial and final weights of the hydrogel bead respectively.

To determine the hydration rate of the beads, 10 g of hydrogel beads were taken and

subjected to drying at room temperature and at 121°C. The rate of hydration of the

beads was calculated using the following equation (Li et al., 2005):

Wh - Wd
Hydration Rate (%)  x100
Wh

where, W h is the weight of the beads before the drying and W d is the weight of

dehydrated beads. Hydration rate indicates the mass and water contents in the
hydrogel beads. Smaller hydration rate, greater is the mechanical strength of the

hydrogel beads.

Results and Discussion

Thomas Model

The column data was fitted to the Thomas model to determine the Thomas rate

constant and the maximum solid phase concentration (qe). Linearization was carried

out to determine the level of significance (R2). Figure 2 shows the Thomas rate curve

for the process and Table 1 summarizes the Thomas rate constant and the maximum

solid phase concentrations obtained for different flow rate conditions. It was identified

that as the flow rate was increased from 1 to 5 ml/min, the values of qe and kTh

increased. Maximum solid phase concentration of 7.19 mg/g of carbohydrates was

obtained when flow rate was maintained at 5 ml/min. This inferred that the driving force

for retention of carbohydrates is the time of contact between the feed and the hydrogel

bead. Further, from the R2 values, it was identified that stability of the model increased

with increase in flow rate. Thus, it was concluded that the hydrogel bead packed bed

column should be operated at higher flow rates to improve the column performance and

hence the separation of carbohydrate impurities from the feed.

 Figure 1 Linearized Thomas model at various flow rate conditions

Table 1 Parameters of Thomas model using linear regression analysis and the

equilibrium carbohydrate uptake

Flow Rate Kth (x 10-3) qe R2

(ml/min) (ml/(min.mg)) (mg/g)

1 1.09 0.56 0.9097

5 1.4 7.19 0.9865

Yoon Nelson Model

The effect of interaction between carbohydrates and the hydrogel beads was

predicted using the values of Yoon Nelson rate constant and time required for 50%

breakthrough (  ) (Figure 2). The values of kyn and  are listed in table 2. It was

identified that Yoon Nelson rate constant increased with increase in flow rate. In
contrast,  decreased with increase in flow rate. This showed the poor carbohydrate

elimination efficiency of the beads at low flow rate conditions. Thus it was concluded

from the model that flow rate showed direct proportionality with carbohydrate elimination

rate.

Figure 2 Linearized Yoon Nelson model curve at various flow rate conditions

Table 2 Parameters of Yoon-Nelson model using linear regression analysis and

the equilibrium carbohydrate uptake

Flow Rate Kyn (x 10-3)  R2

(ml/min) (ml/(min.mg)) (min)

1 0.17 1805 0.9097

5 1.24 175 0.9865

Maximum Holding Capacity of Packed Bed Column

The maximum carbohydrate holding capacity of the hydrogel bead packed bed

column for feed flow rate of 5 ml/min was calculated using the area under the curve of

the plot between amount of carbohydrate eliminated with respect to time (Figure 3).

From the plot, a maximum holding capacity of 762 mg/l of carbohydrates was obtained.

The R2 value of 0.98 showed the significance of the experiment.

Figure 3 Plot to identify column holding capacity

Dissolution and Hydration Rate Tests

The rate of dissolution of hydrogel beads with respect to change in pH was

identified. It was found that, a change in pH effected dissolution of the beads and 100%

dissolution of the beads was seen at extreme pH conditions of 1 and 12 respectively. In

contrast, it was identified that at neutral pH, the beads remained highly stable indicating

the suitability of the beads to separate impurities from neutral foods. Considering the

hydration rate, a linear relationship was seen between drying temperature and hydration

rate. With increase in drying temperature, the weight of the beads decreased indicating
the loss of water molecules from the hydrogel beads. After a period of 60 min, a

maximum of 94% hydration rate was observed at 121°C. However, at room

temperature, no hydration was seen. Hence, it was concluded that the synthesized

beads showed high degree of stability at room temperature conditions.

Conclusions

A method for separation of carbohydrate impurities from anthocyanin rich red

cabbage extract was developed using semi interpenetrating network hydrogel beads

loaded in a packed bed column. Breakthrough analysis using kinetic models such as

Thomas model and Yoon Nelson model were applied to model the data. The results

revealed that flow rate was directly proportional to the carbohydrate retention. Further,

the time for 50% breakthrough reduced by 40% when the flow rate was increased from

1 ml/min to 5 ml/min. Overall, the hydrogel bead packed bed column showed a

maximum carbohydrate holding capacity of 762 mg/l. Dissolution and hydration studies

revealed the stability of the beads under neutral pH and room temperature conditions

respectively.

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