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Article history:
Received 6 April 2010
Received in revised form 1 September 2010
Accepted 5 September 2010
Keywords:
Quaternary ammonium
Choline
Deep eutectic solvent
Hyaluronic acid
Adsorption
Hydrophobic interaction
a b s t r a c t
Quaternary ammonium containing compounds (QACs) such as cetylpyridinium chloride (CPC) is
commonly employed in hyaluronic acid (HA) production process as an HA precipitating agent. 3(Trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, a Si containing QAC (Si-QAC) generally
used to modify the surface of cotton bers for the preparation of nonleaching antibacterial textiles, has
a chemical structure very similar to CPC. Choline, a natural QAC, can form a deep eutectic solvent with
urea and be grafted onto cotton surface when incubating cotton in the deep eutectic solvent. Both of the
QAC-modied cottons demonstrated antibacterial activity against Gram () Escherichia coli and Gram
(+) Bacillus subtilis along with HA adsorption capacity. The HA adsorption isotherm could be well-tted
with Langmuir model with maximum adsorption capacities of 184 mg/g and 351 mg/g for Si-QAC and
choline surface-functionalized cotton bers, respectively. In the presence of high concentration of contaminants, HA could be directly recovered from a B. subtilis culture with a capacity of 15 mg/g by using
Si-QAC modied antimicrobial cotton ber.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Hyaluronic acid (HA) is a linear polysaccharide consists of alternating d-glucuronic acid and N-acetyl-d-glucosamine subunits. The
chain lengths of HA can reach up to 30,000 repeating disaccharide
units, corresponding to a molecular weight of 106 Da. HA is highly
hydrophilic, and in aqueous solutions shows viscoelastic behavior and water binding capacity due to the high molecular weight
and the high number of charged groups. HA has many signicant
structural, rheological, physiological, and biological functions in the
body, such as providing a cushion effect in human joints, stimulating the immune system and maintaining a smooth, elastic skin [1].
These important functions have led to a wide range of applications,
such as in cosmetics, ophthalmic surgery, arthritis treatment, postsurgical adhesions, tissue engineering scaffolds for wound healing,
and drug delivery devices [2].
Commercially, HA is produced through extraction from rooster
combs or via bacterial fermentation but the latter is gradually
replacing extraction as the preferred source of HA [3], because the
bacterial process presents the opportunity to optimize the product yield and quality through metabolic engineering and control of
culture conditions. The separation and purication of HA from bacterial fermentation broth generally involves the precipitation of HA
with quaternary ammonium compound (QAC) which is a cationic
surface-active agent with antibacterial activity [46]. Cetylpyri-
45
Fig. 1. Reaction schemes for grafting cotton surface with (a) 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium; (b) choline in deep eutectic solvent.
46
Table 1
Elemental analysis and grafting ratio of modied cotton bers.
Samples
C (%)
H (%)
N (%)
Unmodied
Si-QAC modied (SMC)
Choline modied (CMC)
41.02 0.16
46.14 1.53
47.97 0.63
6.62 0.02
7.94 0.48
8.29 0.28
0.08 0.01
0.59 0.08
1.16 0.28
0
25.05 2.57
14.61 1.34
CMC). Each experiment was carried out in duplicate and the results
are shown with error bars.
The antibacterial activity of modied cotton bers was evaluated against E. coli and B. subtilis by the viable cell counting method
[16]. Prior to the antibacterial tests, all cotton bers samples were
sterilized with 70% ethanol solution and kept under UV-light for
overnight. One-loop full of the bacteria from a colony on agar
plate was inoculated into 5 mL liquid culture of Luria Bertani (LB)
medium and incubated for 15 h at 37 C. The bacteria-containing
broth was centrifuged (5000 rpm, 2 min) and the obtained cells
pellets were washed twice with Ringer saline solution (8.6 g/L
NaCl, 0.3 g/L KCl, 0.33 g/L CaCl2 ). The bacteria stock solution was
diluted with saline solution to give cell number of approximately
107 108 CFU/mL for antimicrobial activity test. Five milliliters of
bacteria suspension was then added to the each test tube containing 20 mg of unmodied cellulose bers (UMC), SMC, and CMC,
respectively and incubated at 37 C for 3 h with 200 rpm shaking. A
control culture without cotton bers was also treated in the similar
way. At the predetermined time, 1 mL of bacteria culture was taken
and serially diluted down to 107 . Aliquots (100 L) of the diluted
samples were then spread, in duplicate, onto nutrient agar plate.
After incubation at 37 C for 12 h, the number of colony formed
was counted. In order to study the effect of antibacterial activity
on the cell growth, the bacteria were grown in 50 mL of LB at 37 C
and shaken at 200 rpm along with 0.2 g of cotton bers samples
for 15 h. The cell density was measured by spectrophotometer at
wavelength 600 nm.
Wg Wo
100%
Wo
(1)
2.5. HA adsorption
3. Results and discussion
Adsorption of HA by the antimicrobial cotton bers was carried
out by mixing 1 mL of HA solution of various concentrations with
5 mg of dry cotton bers. Prior to the addition of cotton bers, the
HA solutions were placed in a water bath for 30 min to achieve the
specic adsorption temperature. HA concentration in the supernatant was analysed by carbazole method [17]. The effect of pH on
HA adsorption onto cotton bers was studied at pH in the range of
38 at 37 C and ionic strength 0.1 M. The effect of temperature was
investigated at 4, 18, and 37 C at pH 4 (for SMC) and at pH 7 (for
3.1. Characterization
Table 1 lists the results of elementary analysis of cotton bers
before and after modication. The higher nitrogen and carbon contents detected after modication suggested that both Si-QAC and
choline have been grafted onto the bers. The grafting either SiQAC or choline to the hydroxyl groups of cotton causes an increase
in weight as represented by grafting ratio in Table 1. Although a
(a)
8
7
6
5
4
3
2
0
20
40
60
80
100
120
140
Time (min)
10
(b)
9
3.3. HA adsorption
7
6
5
5
(a)
3
3
2
0
20
40
60
80
100
120
140
Time (min)
OD600
47
2
Fig. 2. Viable cell number of (a) E. coli and (b) B. subtilis vs. time incubated with modied cotton bers as estimated by spread plate method. The bacteria were incubated
in 5 mL of salt solution and shaken at 200 rpm at 37 C with () no bers addition,
0.02 g of () unmodied bers, () choline modied bers, () Si-QAC modied
bers.
0
0
10
12
14
16
10
12
14
16
Time (h)
5
(b)
4
OD600
0
0
Time (h)
Fig. 3. Growth curves of (a) E. coli and (b) B. subtilis in 50 mL of LB media () without
bers, () 0.2 g unmodied bers, () 0.2 g choline modied bers, () 0.2 g Si-QAC
modied bers.
48
HA concentration (mg/mL)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
30
60
90
120
150
180
210
240
270
Time (min)
Fig. 4. Time courses of hyaluronic acid adsorption onto () unmodied bers,
() choline modied bers, () Si-QAC modied bers. [HA]initial = 1 mg/mL;
ionic strength = 0.1 M; T = 37 C; pHinitial = 4; shaker speed = 200 rpm; cotton bers
dose = 5 mg/mL.
100
HA adsorbed (%)
80
60
40
20
0
2
pH
Fig. 5. Effect of pH on the equilibrium adsorption of hyaluronic acid onto
() unmodied bers, () choline modied bers, () Si-QAC modied bers.
[HA]initial = 1 mg/mL; ionic strength = 0.1 M; T = 37 C; t = 3 h; pHinitial = 4; shaker
speed = 200 rpm; cotton bers dose = 5 mg/mL.
Fig. 6. Schematic representations of (a) hydrophobic attraction (dashed line) on SiQAC modied cotton bers, and (b) electrostatic interaction (dotted line) on choline
modied bers with hyaluronic acid. Shadowed hexagonal, circles, and squares represent hydrophobic patches, acetamido, and carboxylate groups, respectively, in
hyaluronic acid structure.
Table 2
Temperature effect on Langmuir model for hyaluronic acid adsorption by modied
cotton.
200
(a)
HA adsorbed (mg/g)
180
R2
160
Samples
T (K)
140
Si-QAC modied
(SMC)
pH 4
277
78.7
5.8
0.980
291
310
277
153.6
183.7
351.3
6.4
7.7
11.3
0.993
0.997
0.997
291
310
225.3
100.2
9.3
7.4
0.980
0.975
120
100
Choline modied
(CMC)
pH 7
80
60
4 oC
40
18 oC
37 oC
Langmuir model
20
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
(b)
HA adsorbed (mg/g)
49
300
200
100
4 oC
o
18 C
37 oC
Langmuir model
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
(2)
Qe (mg/g solid)
b (L/mg)
(3)
G0
(4)
H 0
S 0
+
RT
R
(5)
Table 3
Thermodynamic constants for the equilibrium adsorption of HA onto modied cottons.
Samples
G0 (kJ/mol)
277 K
291 K
310 K
4.0
5.6
4.5
5.4
5.2
5.2
H0 (kJ/mol)
S0 (kJ/mol K)
6.1
9.0
0.011
0.012
200
100
150
60
100
40
50
20
HA adsorbed (mg/g)
80
(a)
100
160
HA adsorbed
Desorption efficiency
Adsorption capacity
amount HA adsorbed
140
80
120
100
60
80
40
60
40
20
50
20
0
0
0
Number of cycle
400
30
200
20
100
10
HA adsorbed (mg/g)
HA adsorbed
Desorption efficiency
300
0
0
30
in culture broth
60
40
(b)
0
5 mg/mL
in buffer system
Number of cycle
Fig. 8. Repeated use of (a) Si-QAC modied cotton bers at pH 4, and (b) choline
modied bers at pH 7 incubation time = 3 h; shaker speed = 200 rpm; bers
dose = 5 mg/mL. One cycle of operation is dened as the bers incubated with HA
solution, washing, incubated with 1 N NaCl, pH 7 phosphate buffer for SMC and 1 N
NaCl, pH 3 glycineHCl buffer for CMC to desorb HA, and washed again with the
buffer used for adsorption.
Fig. 9. Effect of culture broth to the adsorption capacity and adsorption efciency
of hyaluronic acid adsorbed onto silane modied cellulose bers.
Table 4
Recovery of hyaluronic acid from Bacillus subtilis culture by using Si-QAC modied cotton ber.
Samples
HA conc. (mg/mL)
Yield (%)
Culture broth
Supernatant (after adsorption)
Eluted (after desorption)
1.054 0.023
0.158 0.032
0.402 0.013
0.194 0.002
0.162 0.015
0
100.00
14.99
38.14
SMC. Table 4 shows the HA purication table using SMC for adsorption. The initial HA and protein concentrations in the culture broth
were 1.054 and 0.194 mg/mL, respectively. After adsorption, only
0.158 mg/mL HA left in the supernatant. By incubating with 1 N
NaCl, pH 7 phosphate buffer for desorption, HA of 0.4 mg/mL free
of contaminant proteins was obtained. As much as 38% of HA in the
B. subtilis culture was recovered by using SMC for adsorption.
4. Conclusion
Modication of cotton bers by the antibacterial quaternary
ammonium compound (QAC) was addressed in this work for isolating HA from B. subtilis culture. Both Si-QAC and choline modied
cotton bers showed antibacterial activity against E. coli and B.
subtilis, with the former had better activity due to the presence
of long alkyl chain on the quaternary ammonium groups of SMC.
The adsorption of HA onto antibacterial cotton bers was a function of pH and temperature. Hydrophobic attractions are relatively
more important for HA adsorption by SMC at pH 4, while electrostatic interactions are more important for the adsorption of HA by
CMC at pH 7. The amount of adsorbed HA on SMC increases with
temperature, conversely, the adsorption capacity of CMC decreases
with temperature. The equilibrium adsorption isotherm can be well
described by Langmuir model. The maximum adsorption capacities for HA were 183.7 mg/g for SMC and 351.3 mg/g for CMC. The
adsorbed HA on SMC could be effectively desorbed as much as
90.11% by 1 N NaCl, pH 7 while changing the pH to 3 was effective
to desorbed 36.50% HA from CMC. SMC could be used to recover HA
directly from the B. subtilis culture about 15 mg of HA was recovered
from B. subtilis culture by 1 g of SMC.
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