Synthesis and Swelling Kinetic Study of BSA-based Hydrogel Composite by Subcritical Water Technology

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 756-761

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Synthesis and Swelling Kinetic Study of BSA-based

Hydrogel Composite by Subcritical Water

Technology

Zahra M. Esfahan * , Shamsul Izhar * , Mohd Halim Shah Ismail , Hesam Kamyab , Yoshida

Hiruyuki , Razif Harun

Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, 43300 Serdang, Selangor, Malaysia

Department of Engineering, Razak Faculty of Technology and Informatics, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia

Received: 29/08/2019

Accepted: 15/04/2020

Published: 20/05/2020

Abstract

This study, for the first time, aimed to analyse ‘Bovine serum albumin’ hydrogel composite by simple and quick method (SCWT)

since not only there had been some indications that the low temperature subcritical water treatment may be as valuable products as its

high temperature treatment, but also, the positive outcome would put a stop to the waste of energy and money. For BSA-based hydrogel

at first, the optimum conditions were identified by assessing the effect of different influential parameters (SCWT temperature, time).

SCWT was done using a batch subcritical reactor. Additionally, the characterization tests were carried out on the BSA-based hydrogels

which was produced by this unique method. BSA-based Hydrogel preparation condition by SCWT accurately was investigated and

optimized SCWT condition according to maximum ESR (50%). Schott kinetic swelling model provided evidence to approve two-step

water diffusion mechanism in BSA-based hydrogel by SCWT.

Keywords: Bovine Serum Albumin, Subcritical Water, Batch Reactor, Hydrogel

Introduction ¹

Biomaterials have been proven to be quiet beneficial to

motivated researchers to investigate on replacing the time

consuming, toxic methods by clean and safe SCW technology.

According to the literature, it is assumed that BSA solution

under Sub-CW treatment, has aggregation-decomposition-

aggregation-decomposition pathway (15). In this case, this

study assumes that BSA coagulates to BSA solid network

(BSA-based hydrogel). So based on this, Sub-CW treatment

of BSA can be a great, novel method for producing BSA-based

hydrogel without using any chemicals and crosslinker in a very

short time.

Continuous efforts have been made to produce strong and

biocompatible bovine serum albumin hydrogels with retained

protein function; for example, the ability to bind, and release

types of well-defined molecules (16, 17, 18, 19). The BSA-

based hydrogels made by the thermal method have already been

well established and have been working on the induction gel

mechanism of proteins such as BSA (20, 21, 22). The main

drawback of heat-inducing method is the broadly protein

denaturation, with the risk of compromising protein

functionality and biocompatibility (23). Moreover, besides the

disadvantages of the denaturation problem, the time consumed

by the incubation process is another drawback (24, 25).

Electrostatically-triggered serum albumin hydrogels is another

preparation method of albumin hydrogels introduced very

recently (25). Despite the fact that this proposed method

reduced the time consumption and solved the denaturation

problem, still it is long and needs chemicals that are not

human beings as they have a diverse range of applications in

food, medicine, and many other areas (1, 2, 3). For instance, the

vast range of proteins has been used as a good source of

protein-based biomaterials (4). It is not surprising that the

demand for natural and organic products has increased

significantly worldwide as they are considered less harmful in

comparison with non-organic products. Nowadays, there are

numerous natural products in the market for cosmetics, food

additives, and medicinal purposes (5). Many of the above-

mentioned biomaterials are protein-based (6, 7). Protein-based

hydrogels are the most common biopolymers shaped from

proteins and have medical applications in the drug delivery

system, tissue engineering, ect (8, 9). However, the quality and

difficulty of each method varies significantly for different

protein sources (10, 11, 12). Therefore, given the afore-

mentioned importance of biomaterials for natural products,

finding highly efficient isolation methods for protein-based

biomaterials as well as understanding and identifying the

applications and properties for each protein-based biomaterial

is vitally important. Subcritical water technology is a novel

method that recently take an attention of researchers to

produced variable biomaterials such as bioplastic and biomass

without using additive, catalyst or any toxic chemicals (13, 14).

Additionally, promising properties of water in high temperature

and pressure range (SCW) and coagulation of the protein

structure under subcritical water treatment conditions

Corresponding author: Shamsul Izhar, Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, 43300

Serdang, Selangor, Malaysia. Email: z_m_esfahani@yahoo.com and shamizhar@upm.edu.my.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 756-761

environmental friendly. So, there is still a need for a faster and

environmental friendly method to replace it. Protein-based

biomaterials such as hydrogels produced with Subcritical

Water Treatment (SCWT) are a new promising method, which

is safe, fast, economical, and environmental friendly in

comparison with other new methods that are used to produce

protein-based biomaterials. Treatment of BSA by Sub-CW

does not use any additives or catalyst; it is cheap and

environmental friendly (15). Furthermore, Sub-CW treatment

has been shown to require significantly short process time (few

minutes) and utilises lower amount of raw material.

(SR) = W

(1)

The swelling behavior of hydrogel in different temperature

range of distilled water was explored. The swelling tests were

performed in ionic medium solutions, prepared as explained by

Wang et al. (28). In a typical experiment about 0.2 g of sample

was weighed (W

for 6 hours, after which they were removed and blotted dry, and

their weights were recorded (W ). The equilibrium swelling

) and immersed in different swelling media

ratio (ESR) of the hydrogel in distillate water was computed

with the equation below:

ESR (gr⁄gr) = (W

-W

) ⁄ W

(2)

Materials and method

.1 Materials

.5 BSA-based hydrogel swelling kinetic model

In this study, two kinetic models were used to study the

In this study, couples of chemical materials were used for

different purposes. Bovine serum albumin (BSA, fraction V)

was used as a model protein in SCWT experiments provide by

Wako, Japan. Chloric acid and sodium hydroxide (Sigma

Aldrich) were used to study the effect of pH on BSA-based

hydrogel swelling behavior in acidic and base media. Ionic

solutions (eg. different concentration of NaCl) was used to

discuss the impact of ionic strength on BSA-based hydrogels.

kinetic models and the mechanism of swelling for BSA-based

hydrogel by SCWT process. (Table 1).

Table1. Applied swelling kinetic models for BSA-based

hydrogels

Plotting

Kinetic

parameters constant(s)

Model

Equation

Fickian diffusion

f = M

∞

= K

lnf vs. t

.2 BSA-based hydrogel preparation

For producing BSA-based hydrogel, 10gr BSA was

weighted by AND balance series GR-200 from Japan and was

gently dissolved in Milli-Q water in a volumetric flask with

shaking. For solution of BSA, subcritical water reaction was

Schotts second-

order

2 ∞

dS/dt = k (S - S) t/s vs. t

done in the range of 80-200 C for 30 seconds to 30 minutes and

Result and Discussion

gel-like bovine serum albumin biopolymer (GBSA) was made

in a batch reactor. Hydrogel solid was formed by composition

of BSA in sub-critical water process. In high concentration of

initial BSA solution, at some temperature range only gel phase

was produced depending on the reaction time as well. A batch

laboratory-scale sub critical fluid system was used for this

research. The reactor was a stainless steel batch reactor（

SUS316, i.d 7.5 mm×150.4 mm) length, capped with Swagelok

fittings. 3ml BSA solution charged into the reactor tube of

which one was side capped with Swagelok fitting. After

purging argon gas, Sealing of the reactor with Swagelok fitting

was done followed by immersion into a preheated oil bath

thermometer inspecting Celsius M type (Thomas Kagaku Co.

Ltd., Japan) that contained silicone oil for reaction temperature

BSA-based hydrogels were synthesized by self-coagulation

of ‘Bovine serum albumin’ solutions under SCWT temperature

from 80 C and above. Coagulation of BSA started at 90 C and

gelation process was continued by rising temperature to 120 C

Figure1). Although by increasing SCWT temperature, more

gel network was formed, since at 130 C that the accumulated

gel decomposes to the smaller molecules and by increasing

temperature. It totally decomposed to smaller molecules that

will discuss them in our next paper.

(

lower than 200 C. The synthesis reactions were performed in

the range of 80-130°C. After the desired reaction time, the

reactor was immediately removed from salt bath or oil bath and

cooled by immersion in a chilled water (20L) bath.

.3 Freeze-drying

For removing water from the product the method of Robert

et al. was followed (26). After removing water, put samples in

Figure 1: BSA-based hydrogel, prepared at a) 90 C, b) 100 C, c)

the refrigerator (-1 C) over a night and after that kept at -40 ºC

1100C, d) 120 C by SCWT, Freezer dried BSA-based hydrogel

for further precipitation. After 24 hrs, the precipitate was

collected and dried using freeze drying system (FTS) for 72 hr

27).

prepared at e) 90 C, f) 100 C, g) 110 C and h) 120 C

(

3.1 Effect of coagulation temperature on ESR of hydrogel

The lower range of temperature was the lowest temperature

that BSA molecules start to coagulate (i.e. 80 C) that has the

ability to bring the water to the boiling point (near subcritical

point) (29). The gel preparation temperature increased from

100 C to 120 C, the equilibrium swelling ratio (ESRs)

decreased from 49 to 40, demonstrating the swelling behavior

was reliant on SCWT temperature (Figure 2). In addition,

incomplete gelation at the minimum temperature may be

accounted for by the slower BSA coagulation during process

which is essential for forming of hydrogel bonds. Similar result

.4 BSA-based hydrogel behavior

After reaction, remove whole solid from the reactor

carefully and prepare predetermined amounts of solid (present

weight of dry sample, W ). The swollen hydrogels were

removed from water and weighed at regular interval times (W ).

The swelling ratios of solid matrices were established from the

weight change before and after swelling: swelling ratio

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 756-761

was also reported by other studies that used low temperature

range of Sub-CW treatment of BSA. For example, Aida et al.

point. The equilibrium swelling ratio of BSA-based hydrogel

was measured for hydrogels that prepared at 1-20 minutes but

ESR% did not change considerably after 9 minutes of Sub-CW

treatment. Thus, coagulation and gelation of BSA was

considered to be completed after 9 mins reaction time, when

the ESR curve reaches the steady state.

(2017) demonstrated that the lower temperature of BSA Sub-

CW treatment at 5 minutes as a result of slow-heating and mass

transfer that caused protein aggregation in batch reactor. The

decrease of ESR in 120 C accounted for by starting

decomposition of the sample as the result of hydrolysis of

formed solid.

3.3 Optimization of SCWT operational condition

The results provided evidence that for reaching the highest

percentage of swelling ratio in BSA-based hydrogel, SCWT

temperature should be high enough to provide sufficient

temperature for coagulating the BSA while not resulting in any

undesirable reactions such as decomposition and hydrolysis

(they can be undesirable for producing hydrogel). Additionally,

the result confirmed the suggested mechanism of Sub-CW

treatment of protein that the accumulation of protein is

commerce when the SCWT temperature is sufficient for water

to be in subcritical region and protect the sample completely

from the direct heat and at the same time prevent

denaturation(29)( drawback of direct heating gelation of BSA.

The result showed that for obtaining highest possible

equilibrium swelling ratio, the SCWT time should only be long

enough to let all BSA molecules to coagulated (32). Obviously,

longer treatment time is not necessary and it is only the wastes

of energy since no significant changes in swelling ratio of BSA-

based hydrogel.

Figure 2: Relation between Equilibrium swelling ratio (ESR) of BSA-

based hydrogel and BSA-based hydrogel preparation temperature in

different time course

The decomposition, and hydrolysis are the common

possible reactions that may happen during SCWT method that

perform at high temperatures or long decomposition times (30,

.4 Swelling kinetic model

1). So, it is essential to identify the best practical SCWT time

For a better understanding of the swelling model of BSA-

and temperature for coagulation the best possible ESR of BSA-

based hydrogel occurrence of undesirable reactions. In short,

the coagulation process was more efficient at medium

based hydrogel prepared by SCWT at different operational

conditions, the mechanism of the swelling was assessed using

two different models. The swelling behavior of BSA-based

hydrogels was analyzed with these models to find the best

model for describing the swelling behavior in neutral pH levels.

temperatures (i.e. 100 or 110 CUse the "Insert Citation" button

to add citations to this document). As the result, the prepared

BSA-based hydrogel was better coagulated from the Bovine

serum albumin at SCWT temperature of 100-120 C.

.2 Effect of Sub-CW coagulation time course on ESR of

hydrogel

Duration of Sub-CW treatment is another important

parameter in BSA coagulation process. It should be long

enough to all protein in the initial solution coagulated and form

the gel network. In order to find the optimum time for the

complete gelation of BSA, the equilibrium swelling ratio (ESR)

of BSA-based hydrogel at coagulation temperature of 80-

20 C were measured during the coagulation time until the

Figure 4: Swelling test of GBSA100, GBSA110, GBSA120 in

distillated water

maximum percentage of swelling ratio was observed (Figure

Swelling kinetic of GBSA100, GBSA110, GBSA120 were

investigated in distillated water (pH= 7) and result observed in

figure 4. The parameters of the two kinetic models and their R²

(coefficient of determination) were calculated using plotting

method. It is obvious that Fickian diffusion model showed the

lowest fit to the experiment data compared to Schott model

(Table 2). So, it was concluded that this model cannot properly

explain the swelling of BSA-based hydrogel by Sub-CW

treatment.

For Fickian swelling kinetic model, when gel swells in

media, three steps occur: to start with water diffusion into the

BSA-based hydrogel structure; then, the swelled chains rest;

and finally, the hydrogel grows. ln(f) versus ln (t) of selected

hydrogels were driven. In distillate water, diffusion exponent n

and characteristic constant K were computed from slopes and

intercepts of the lines are recorded in Table 2. The n values

were all in the range from 0.45– 0.47 territory, showing the

swelling conduct at the underlying swelling stage was

Figure 3: Relation between Equilibrium swelling ratio (ESR) of BSA-

based hydrogel and preparation time in different SCWT temperature

The maximum swelling achieved in 100 C after 9 minutes,

9%. BSA-based hydrogels by SCWT have the high swelling

at 100, 110 and 120 C after samples reached the equilibrium

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 756-761

overwhelmed by Fickian dispersion. Linear relations of t/S

with t are plotted; the kinetic parameters computed from the

slopes and intercepts of the lines are listed in Table 2. It can be

seen that the related coefﬁcients (R) are all beyond 0.998,

indicating the Schotts second-order kinetic equation was

appropriate in describing the whole swelling process. The

results provided evidence that both models of Fickian diffusion

and second-order schotte model fitted with experiment results.

As it can be seen in Table 2, The Fickian diffusion model’s

kinetic constants had increased by the increase of SCWT

temperature. Despite the fact that Fickian kinetic model was

better fitted to the data, model of second order diffusion had

significantly higher R² (0.998 and above).

results, the coagulation of BSA-based hydrogel from BSA

solution follows second order Schott model through the whole

swelling process. For Fickian dispersion, three stages

happening in progression are proposed when dry gel swells in

water: to start with, water particles diffuse into the polymer

structure; second, the hydrated polymer chains rest; and third,

the polymer arrange grows to the encompassing. In the first

swelling step, (Mt/M∞≤60%), the main equation that describe

permeation in the second step of swelling: f = Mt/M∞ = Ktn,

where Mt was the mass of water absorbed at time t and M∞ was

the gel mass at equilibrium, K was a characteristic constant

showed network structure of gels and n was the diffusion

exponent. . For n < 0.5, Fickian diffusion would be the best

model to explain swelling producer; for 0.5 < n < 1, the

transport would be anomalous (non-Fickian diffusion); and for

n = 1, the relaxation of polymer chains would control the

system. Charts of ln (f) versus ln (t) of selected hydrogels were

driven in Fig. 5.

The Fickian diffusion kinetic model describing a swelling

mechanism that occurs in fast water diffusion processes using

one rate constant (k; Table 2;(33)) whereas model of second

order schott describes the gelation using a kinetic constant for

slow and fast stage of water diffusion process (k and S∞; Table

;(34, 35)). Second-order schott and Fickian diffusion kinetic

models were one of the best kinetic models for explaining the

protein-based swelling mechanism (35). On the basis of the

proposed model, the water mechanism in BSA-based dry

hydrogel consists of two stages. First is the fast penetration of

the water on or near the particle surface and second is the slow

diffusion through the particles.

Table 2: Values of the kinetic parameters

Fickian diffusion model Schotts model

Sample

-1

(min ) R

∞

GBSA100 0.47 0.179 0.998

GBSA110 0.45 0.177 0.998

GBSA120 0.45 0.159 0.991

33.33

9.09

12.82

0.999 50

0.999 47.62

0.998 43.48

These two stages can be easily identified in the figure 4.

The fast swelling can be identified during the beginning of

swelling process (first 3th hours), in which the swelling ratio

increases rapidly due to high-capacity purses and mass transfer.

As it is clear, swelling in acidic media grows more sharply in

gel compare to the pure water. In short, the swelling kinetic

model provided evidence that coagulation temperature (i.e.

SCWT temperature) affects the gelation process, equilibrium

swelling ratio of hydrogel, and the kinetic of swelling

hydrogels. The result of swelling kinetic modelling showed that

the gelation of BSA-based hydrogel from BSA solution under

Sub-CW treatment follows Fickian diffusion model in early

hours of swelling. Based on the swelling kinetic modelling

Conclusion

In the current study BSA-based hydrogels was successfully

synthesized with Subcritical water technology. The result in

general indicated that subcritical water treatment (SCWT)

method is a better alternative as a current method of BSA-based

hydrogel preparation since the BSA-based biomaterials using

SCWT were obtained in significantly shorter time without

adding any reagents or crosslinkers (toxic materials). It was

found that temperature was the most influential parameter of

preparing BSA-based hydrogel as well as initial BSA solution

concentration.

(a)

(b)

Figure 5: (a) kinetic theory of Fickian, (b) Schotts second-order kinetic model

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 756-761

[

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