Characteristics of deacetylation and depolymerization of β-chitin from jumbo squid (Dosidicus gigas) pens

Article abstract of DOI:10.1016/j.carres.2011.05.021

This study evaluated the deacetylation characteristics of β-chitin from jumbo squid (Dosidicus gigas) pens by using strongly alkaline solutions of NaOH or KOH. Taguchi design was employed to investigate the effect of reagent concentration, temperature, time, and treatment step on molecular mass (MM) and degree of deacetylation (DDA) of the chitosan obtained. The optimal treatment conditions for achieving high MM and DDA of chitosan were identified as: 40% NaOH at 90 °C for 6 h with three separate steps (2 h + 2 h + 2 h) or 50% NaOH at 90 °C for 6 h with one step, or 50% KOH at 90 °C for 4 h with three steps (1 h + 1 h + 2 h) or 6 h with one step. The most important factor affecting DDA and MM was temperature and time, respectively. The chitosan obtained was then further depolymerized by cellulase or lysozyme with cellulase giving a higher degradation ratio, lower relative viscosity, and a larger amount of reducingend formations than that of lysozyme due to its higher susceptibility. This study demonstrated that jumbo squid pens are a good source of materials to produce β-chitosan with high DDA and a wide range of MM for various potential applications.

Full text of DOI:10.1016/j.carres.2011.05.021

Carbohydrate Research 346 (2011) 1876–1884
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Characteristics of deacetylation and depolymerization of b-chitin
from jumbo squid (Dosidicus gigas) pens
⇑
Jooyeoun Jung, Yanyun Zhao
Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331-6602, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This study evaluated the deacetylation characteristics of b-chitin from jumbo squid (Dosidicus gigas) pens
by using strongly alkaline solutions of NaOH or KOH. Taguchi design was employed to investigate the
effect of reagent concentration, temperature, time, and treatment step on molecular mass (MM) and
degree of deacetylation (DDA) of the chitosan obtained. The optimal treatment conditions for achieving
high MM and DDA of chitosan were identified as: 40% NaOH at 90 °C for 6 h with three separate steps
Received 19 January 2011
Received in revised form 13 May 2011
Accepted 18 May 2011
Available online 26 May 2011
(
(
2 h + 2 h + 2 h) or 50% NaOH at 90 °C for 6 h with one step, or 50% KOH at 90 °C for 4 h with three steps
1 h + 1 h + 2 h) or 6 h with one step. The most important factor affecting DDA and MM was temperature
Keywords:
Jumbo squid pens
b-Chitosan
Deacetylation
Depolymerization
and time, respectively. The chitosan obtained was then further depolymerized by cellulase or lysozyme
with cellulase giving a higher degradation ratio, lower relative viscosity, and a larger amount of reducing-
end formations than that of lysozyme due to its higher susceptibility. This study demonstrated that
jumbo squid pens are a good source of materials to produce b-chitosan with high DDA and a wide range
of MM for various potential applications.
Ó 2011 Elsevier Ltd. All rights reserved.
8
9
1
. Introduction
use of an organic solvent, flash treatment, and enzymatic deacet-
1
0
ylation. Among them, the alkaline deacetylation using strong
alkaline reagents of NaOH or KOH has been mostly used, in which
the specific method is usually named by the name of the principal
researcher, such as the Broussignac method using KOH, and
Kurita method using NaOH. Both alkaline treatments hydrolyze
Chitin is a natural polysaccharide with a monomer of (1?4)-
linked 2-acetamido-2-deoxy-
ond-most abundant polymer after cellulose. Chitin exists in three
forms as , b, and , in which -chitin is mainly from shrimp, crab,
and krill shells, b-chitin is from squid pens, and -chitin is usually
from fungi and yeast with its morphology appearing as a combina-
tion of the and b structures. b-Chitin is more reactive and has an
D-glucose. It is known as the sec-
1
11
1
2
a
c
a
ꢁ
c
the acetyl groups from NAG, thus generating –CH
–NH
result in different deacetylating processes, obtaining chitosan with
3
COO and
2
groups on the polymer. However, using NaOH or KOH can
a
2
,11,12
affinity toward solvents due to its parallel structure and no inter-
different functional properties.
This may be due to the
hydrogen bonds, which gives rise to loose binding between the
solubility differences between NaOH and KOH in organic solvents
because of their different dielectric constants. For examples, the
dielectric constant of NaOH is 80.1 in water, which is much higher
than that of KOH, which is 25.3 in ethanol and 41.4 in ethylene gly-
col at 298 K when used for deacetylating chitosan. In the transition
state of the deacetylation reaction, the development of ionic
molecules, while
hydrogen bonds.
a-chitin has anti-parallel structure with inter-
2
,3
Chitin is insoluble in most organic solvents due to the highly ex-
tended hydrogen-bonded semicrystalline structure dominated by
the strong COꢀ ꢀ ꢀHN hydrogen bonds that show a distance of about
4
,5
ꢁ
0
.47 nm.
3
The acetyl groups (COCH ) in chitin are removed
charges is initiated by the nucleophilic attack of the HO on the
through a process called ‘deacetylation’ to obtain chitosan, that
is, soluble in dilute acids. When the majority of the N-acetyl- -glu-
cosamine (NAG) units are converted to -glucosamine (DG) units
with free –NH groups, the polymer becomes highly soluble in di-
lute acids. Several approaches may be employed to deacetylate chi-
carbonyl groups of the amide functions and is favored by the high
dielectric constant solvent.
The functionality of chitosan is affected by several factors,
including the source of the raw material, molecular mass (MM),
2
D
D
2
degree of deacetylation (DDA), and its physical state (conforma-
6
7
13–15
tin, including alkaline deacetylation, intermittent water washing,
tion, particle size, etc.).
Among them, MM and DDA may be
most critical. Therefore, it is sometimes necessary to depolymerize
native chitosan into polymers of lower molecular mass, a process
called ‘depolymerization’. Low MM chitosan has performed better
⇑
Corresponding author.
E-mail address: yanyun.zhao@oregonstate.edu (Y. Zhao).
1
6
for drug delivery because of its high solubility.
Chitosan
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.021

J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
1877
depolymerized by different enzymes showed different antimicro-
bial functions, in which chitosan is depolymerized by chitinase
had stronger inhibition against Gram-negative bacteria, while
lysozyme-depolymerized chitosan was more effective against
the chitosan molecules. The MM range obtained in this study
was similar to that by Chandumpai et al. (9110–10,240 kDa) in
which 0.17 M acetic acid solution was used to measure MM,3 but
was highly different from the results by Tolaimate et al. (450–
595 kDa) in which 0.3 M acetic acid/0.2 M sodium acetate solution
Gram-positive bacteria.17 In a MM range of 5 ꢂ 10 –9.16 ꢂ 10 ,
3
4
2
8
the antimicrobial activity of chitosan increased along with
was used for measuring MM. As stated in the experimental sec-
tion, the molecular mass measured in this study was ‘viscosity-
average molecular mass’ and is highly related to the solubility of
chitosan in solvents, and in turn, the type of solvent used for dis-
solving the chitosan samples. Previous studies indicated that MM
values measured by dissolving chitosan in acetic acid/sodium chlo-
ride and acetic acid/sodium acetate were much lower than that by
4
6
increased MM, while in the MM range of 9.16 ꢂ 10 –1.08 ꢂ 10 ,
it decreased as MM increased.18 Chitosan products of different
MM (1.74, 2.36, and 3.07 g/mol) also showed different antifungal
activities against Rhizopus stolonifer, in which the lower MM was
more active against mycelia growth, while the higher MM, prod-
ucts inhibited mold germination.19 Chitosan with an MM of
2
,3,28
7
1 kDa was less inhibitory against Bacillus cereus and Escherichia
dissolving in diluted acid solution,
probably due to the electro-
20
coli than those with MM values of 4.74–10 kDa.
Therefore,
static repulsion of chitosan as the polycationic polymer in an acidic
solvent. The resolving process of chitosan is initiated by the bind-
controlling the MM of chitosan is necessary for achieving the most
effective antimicrobial activity. The degree of deacetylation (DDA)
of chitosan also impacts its properties. Chitosan with 99% DDA
showed the highest inhibition against the growth of both Gram-
negative and Gram-positive bacteria,21 and 90% DDA chitosan
had higher reactive oxygen scavenging activity than that of 50%
ing between the hydrogen ions and free amine group to form a cat-
þ
ion ion (NH3 ) when pH is below its pK
a
. Therefore, the amount of
cation ions is important in determining the solubility and viscosity
due to their electrostatic repulsions. The viscosity of a solution is
þ
increased along with the increased amount of NH3 , because it
2
2
DDA chitosan. In addition, increasing the DDA improved the
mechanical properties (tensile strength, elongation, and Young’s
modulus) of b-chitosan-based films.23
makes larger spaces between the polymers for a water trap, thus
2
9
forming longer linear polymers that are more stretched out.
ꢁ
ꢁ
3
However, anions, such as Cl or CH COO , as in sodium chloride
Depolymerization may be achieved by enzymatic, chemical, or
physical methods or combinations thereof. Chemical depolymeriza-
tion has limited control over the extent of depolymerization due to
its harsh conditions along with environmental concern of using high
concentrations of chemical reagents.24 The physical method, such as
ultrasonically assisted treatment, consistently results in irregular
or sodium acetate could block the electrostatic repulsion between
cations in chitosan, thus decreasing its intrinsic viscosity.30 The rel-
atively low MM observed in low-DDA samples might also be due to
the rigid crystal structure of the chitosan samples, resulting in low-
31,32
er solubilities.
Though there was no statistical difference on MM among nine
treatment conditions except the 1st and the 9th runs (Table 1),
the MM generally decreased in the severe treatment conditions
of using a higher NaOH concentration (50%) or longer reaction time
at 90 °C (Table 1). Similarly, Chandumpai et al. reported that the
MM of chitosan gradually decreased along with increased treat-
2
5
molecular masses. In contrast, the enzymatic method is more
26
applicable due to its controlled extent of reaction.
Previous studies on b-chitosan from squid pens were from squid
species of Loligo lessoniana, Loligo formosana, Loligo vulgaris,
Ommasterphes bartrami, and Illex argentines.2
,3,12,23,27
However,
3
the catch of jumbo squid (Dosidicus gigas) had increased signifi-
cantly during 1991–2002, and became the third largest amount
of squid processed worldwide in 2002 (406,356 tons, 12.8%) after
Illex argentines (511,087 tons, 16.1%) and Todarodes pacificus
ment time from 2 to 8 h in 50% NaOH at 100 °C. Tolaimate et al.
found that b-chitin deacetylated by 40% NaOH at 80 °C for 6 h
has a larger MM than that treated under the same conditions for
2
8
9 h. Hasegawa et al. also indicated that the MM decreases along
with increased concentrations of reagents and temperatures.33
Therefore, the higher concentration of deacetylation reagent and
longer reaction time served to further degrade the polymer. In con-
trast, Ottey et al. did not show further polymer degradation by ex-
(
504,438 tons, 15.9%). In spite of its increased production, studies
using jumbo squid pens as material for producing chitin and chito-
san have been scarce. As it is well known, the raw materials signif-
icantly impact the deacetylation process of chitin and the
functionality of the resulting chitosan. Therefore, this study aimed
to investigate the optimal deacetylation procedure of b-chitin from
jumbo squid pens and the enzymatic depolymerization of the b-
chitosan thus obtained to produce a series of low-MM chitosan
material. Different factors potentially contributing to the deacety-
lation process of chitosan, including the type and concentration of
alkaline reagents, reaction temperature, time, and treatment step
were statistically considered. Based on our best knowledge, no
study has reported the deacetylation and deploymerization charac-
teristics of b-chitin from jumbo squid pens, where all these major
contributing factors were statistically considered.
3
4
tended reaction time.
2.1.2. Optimal deacetylation conditions
Through the Taguchi design method the average values of
three measured parameters (DDA, intrinsic viscosity, and MM)
and the rank of each contribution factor on these parameters
were obtained (Table 1). Since the intrinsic viscosity directly re-
lated to MM, it is not separately discussed here. The R
for the NaOH concentration was the lowest among all tested con-
tributing factors on MM and DDA, R values of temperature and
i
value
i
time on DDA were, respectively, ranked first and second, and
were second and first on MM, respectively. ANOVA results indi-
cated that the NaOH concentration had no significant effect on
all measured parameters, but both temperature and time signifi-
cantly affected DDA, but not MM (P <0.05). Therefore, regardless
of the treatment factors and their levels applied in this study
while using the Kurita method, no polymer degradation (change
of MM) occurred in the deacetylation process. Based on this
study, it may be concluded that the optimal deacetylation condi-
tions to obtain chitosan with DDA values over 95% and without
significant polymer degradation is to use 40% NaOH at 90 °C for
2
2
2
1
. Results and discussions
.1. Deacetylation of chitin by the Kurita method12
.1.1. Characteristics of deacetylation
The DDA ranged from 45% to 99%, and the MM from 5362 to
1,684 kDa (Table 1). High DDA values (>90%) for chitosan were
all obtained at 90 °C treated with either 40% or 50% NaOH for at
least 4 h, in which relatively low MM was observed, indicating that
the severe treatment conditions removed more acetyl groups from
chitin, resulting in higher DDA values and further degradation of
6
h in three divided steps (2 h + 2 h + 2 h) or use 50% NaOH at
9
0 °C for a period of 6 h.

1
878
J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
Table 1
Intrinsic viscosity, molecular mass and degree of deacetylation of deacetylated chitin using the Kurita method and estimated parameters
from the Taguchi design method
Run
Measured parameters
Intrinsic
Viscosity-average
Degree of
viscosity (mL/g)
molecular mass (kDa)
deacetylation (%)
b+
5362b
52.7cd
1
2
3
4
5
6
7
8
3281
5708
ab
ab
ab
9730
8328
6583
7288
94.2
98.8
62.7
ab
ab
a
4923
ab
ab
c
3957
ab
ab
ab
4362
95.6
ab
ab
d
4963
8392
45.0
a
ab
81.0b
6109
5502
10464
ab
ab
ab
9357
90.0
a
a
ab
9
6766
11684
83.2
Factors
A
Levels
+
+A5357
4696
A
A
K
K
A1
9114
82.0
B
A
A
A2
7905
73.3
+
++
R
i
661
1209
8.7
A
A
A
B
C
K
K
R
B1
B2
i
4577
7700
60.3
A
A
B
5452
9277
92.4
875
1577
32.0
A
A
B
K
C1
K
C2
K
C3
R
i
4582
7703
62.5
A
A
AB
5477
9332
80.0
A
A
A
5131
8693
91.8
896
1629
29.3
A
A
A
D
K
D1
K
D2
K
D3
R
i
4803
8111
77.2
A
A
A
5593
9529
73.4
A
A
A
4794
8089
83.8
799
1440
10.4
Rank++++
C > B > D > A
C > B > D > A
B > C > D > A
+
Means followed by the lowercase letter in the same column within nine treatments were not significantly different (P >0.05).
Means preceded by the same capital letter in the same column within each factors were not significantly different (P >0.05).
+
+
+
++
R
i
was the difference between the highest and lowest values.
+
+++
Ranks were based on the order of R values.
i
2
2
.2. Deacetylation of chitosan using the Broussignac method11
In respect to DDA, 90 °C for 4 h was a minimal condition re-
quired to obtain chitosan with DDA over 90%, and the multiple-
step process helped increase the DDA as shown by the fact that
4 h with three divided steps (1 h + 1 h + 2 h) gave a 93% DDA versus
87% DDA obtained through a single 4-h treatment. The DDA was
over 90% when samples were treated at 120 °C, and there was no
significant difference in DDA in all 120 °C treated samples regard-
less of time or a multiple-step process (P >0.05). The lowest DDA
(57%) was observed on samples treated at 90 °C for 2 h with two
divided steps. Similar to our results, Tolaimate found that a 2-h
treatment at 120 °C results in a DDA over 96%, confirming the
important role of temperature, and a multi-step process resulted
in a higher DDA compared to a single step process for the same
.2.1. Characteristics of deacetylation
Our preliminary studies found that when using the Broussignac
method, samples treated at 60 °C were visibly insoluble in 0.1 M
acetic acid (data not shown); thus increasing the reaction temper-
ature to 90 and 120 °C was necessary. The DDA and MM ranged be-
tween 57% and 99% DDA and 34 and 11,934 kDa, respectively
(Table 2). The higher MM chitosan products were obtained at
9
0 °C for at least 4 h in 1 or 3 steps (run number of 5, 6 and 8 in
Table 2), while the MM from the treatment at 120 °C was signifi-
cantly (P <0.05) lower than those at 90 °C except for the one for
2
two divided steps was significantly lower than that for the other
samples treated at the same temperature (P <0.05), probably own-
ing to the lower solubility of chitosan as shown by the low DDA of
h (P >0.05). The MM (1797 kDa) obtained at 90 °C for 2 h in
2
treatment time. In this study, samples treated at 120 °C for 6 h
with three divided steps had a 99% DDA, which was close to the
value for a fully deacetylated chitosan.
5
7%. Similar to what was observed in the Kurita method, the rigid
structure with hydrogen bonds in low-DDA chitosan might induce
a lower solubility in the chitosan, thus affecting the measurement
of MM. The MM at 90 °C for 4 h with three divided steps
2.2.2. Optimal deacetylation conditions
i
The R value for temperature was the highest among all contrib-
uting factors (Table 2), and temperature significantly affected MM
and DDA (P <0.05). These results indicated that while the high tem-
perature treatment increases the DDA to a value over 95%, chitosan
depolymerization simultaneously occurred. Time was the second-
most contributory factor on DDA, but did not show a significant
effect on the MM. Hence, controlling treatment time in the Brous-
signac method may provide better control on the MM and DDA of
chitosan than temperature as it could satisfy our goal to obtain
chitosan with a high DDA, but with less polymer degradation. A
multiple-step process was the least contributing factor on DDA,
and the second-most contributory effect on MM. It did not affect
the DDA (P >0.05), but the MM, which was different from the
results of the Kurita method. This result was also inconsistent with
(
1 h + 1 h + 2 h) was significantly (P <0.05) higher than that from
the same conditions with one straight step. Similarly, the MM of
chitosan deacetylated for 6 h in three divided steps
(
2 h + 2 h + 2 h) was higher than that with two divided steps. This
might be explained as the multiple-step treatment helped prevent
polymer degradation. In the Kurita method, the highest MM was
also obtained at 90 °C for 4 h. Hence, the treatment conditions to
obtain the highest MM were at 90 °C for 4 h in both methods. Com-
pared with the Kurita method, the MM range obtained from the
Broussignac method was wider, in which the higher treatment
temperature may contribute to the significant degradation of
chitosan as evidenced by a decrease in MM.

J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
1879
Table 2
Intrinsic viscosity, molecular mass, and degree of deacetylation of deacetylated chitin using the Broussignac method
and estimated parameters from the Taguchi design method
Run
Measured parameters
Intrinsic
Viscosity-average
Degree of
viscosity (mL/g)
molecular mass (kDa)
deacetylation (%)
cd+
c
98.7a
1
2
3
4
5
6
7
8
859
111
122
1273
d
c
ab
143
157
96.1
99.1
57.2
93.2
d
c
a
c
c
c
1797
6903
2850
a
a
ab
11934
10645
ab
ab
ab
6207
94.3
94.0
d
c
ab
116
149
b
b
b
4789
8067
87.4
d
c
a
9
30
35
97.9
Factors
A
Levels
+
+B248
4924
B
A
K
K
A1
352
8375
97.2
A
A
B
A2
83.0
+
++
R
i
4676
8023
14.2
A
A
A
B
C
K
K
K
R
B1
B2
B3
i
924
1424
83.3
A
A
A
3934
6715
92.2
A
A
A
2120
3613
97.1
3010
5291
13.8
A
A
A
K
C1
K
C2
K
C3
R
i
3951
6662
93.5
B
B
A
646
1010
83.7
AB
AB
A
2380
4080
95.5
3305
5652
11.7
++++Rank
A > C > B
A > C > B
A > B > C
+
Means followed by the lowercase letter in the same column within nine treatments were not significantly
different (P >0.05).
+
+
Means preceded by the same capital letter in the same column within each factors were not significantly
different (P >0.05).
+
++
R
i
was the difference between the highest and lowest values.
+
+++
Ranks were based on the order of R value.
i
the findings by Tolaimate et al., in which the multi-step treatment
prevented the depolymerization of MM when the temperature was
at 80 °C. The difference might be due to the low solubility of some
of the chitosan samples, which affects the accuracy in MM mea-
surement as previously stated. In future work, the solubility of
deacetylated chitosan should be considered as a contributing fac-
tor in the Taguchi design to obtain a more accurate result.
2.2.3. Depolymerization of chitosan by commercial enzymes
Enzyme type (ET), initial MM (IM), and depolymerization time
(DT) were all significant (P <0.05) factors affecting depolymeriza-
tion of chitosan, and there were significant (P <0.05) interactions
between ET and DT, and IM and DT (P <0.05) (Table 3). High molec-
ular mass (HMM) (2100 kDa) and low molecular mass (LMM)
(594 kDa) chitosan had about 89% and 56% degradations, respec-
tively, by cellulase (EC3.2.1.4) in the first hour, another 15–20%
degradation in the following hour, but no further degradation after
2 h (Fig. 1). Lysozyme (EC 3.2.1.17) resulted in about 56% and 68%
degradation in HMM chitosan during the first and second hour
treatment, but did not induce further degradation after that. While
the reaction on LMM chitosan was significantly slower, no degra-
dation occurred until after 5 h (15% and 47% degradation at 5
and 6 h, respectively). Therefore, chitosan depolymerization by en-
zymes was significantly induced during the first 1–2 h of the reac-
tion except for lysozyme treatment on LMM chitosan. The initial
2
Based on the results from this study, it may be concluded that
the Broussignac method was more applicable when lower MM
and higher DDA chitosans were targeted, while the Kurita method
was more suitable for obtaining chitosan with higher MM and
higher DDA. This conclusion was similar to that reported by Tola-
imate who compared the Kurita and Broussignac methods in the
2
deacetylation of b-chitin from Loligo vulgaris squid pens. However,
it should be noted that the KOH used in the Broussignac method is
more expensive than the NaOH used in the Kurita method; thus it
might be of less interest in commercial applications.
Table 3
Analysis of variance (ANOVA) results (P = 0.05) for analyzing independence or interactions among treatment factors during the deploymerization of chitosan
Source of variation
Molecular mass (MM, kDa)
Degree of deacetylation (DDA, %)
df
F value
P value
df
F value
P value
Linear terms
Enzyme types (ET)
Initial MM (IM)
Depolymerized time (DT)
1
1
7
111.57
54.73
56.75
<.0001
<.0001
<.0001
1
1
7
0.06
28.42
0.69
0.8158
<.0001
0.6759
Interaction terms
ET X IM
ET X DT
IM X DT
Model
1
7
7
24
39
63
3.17
31.09
2.42
0.0829
<.0001
0.0368
<.0001
1
7
7
24
39
63
5.72
0.31
0.92
1.99
0.0217
0.9428
0.5020
0.0274
33.39
Error
Corrected total

1
880
J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
1
1
20
00
2.2.4. Physicochemical properties of depolymerized chitosan
LMM-Cellulase
LMM-Lysozyme
HMM-Cellulase
HMM-Lysozyme
Changes of relative viscosity and reducing-end formation dur-
ing the depolymerization of chitosan are illustrated in Figures 2
and 3, respectively. Initial viscosity of HMM (2137 kDa) and LMM
(594 kDa) chitosan were 2048 cP and 48 cP, respectively. While
the MM in HMM chitosan was about four times higher than that
in LMM, the viscosity of HMM chitosan was about 50 times higher
than that of LMM chitosan. Similar to the changes in MM, the rel-
ative viscosity of HMM chitosan decreased much faster than that of
LMM chitosan, showing an approximate 95% reduction in the first
80
60
40
20
0
1
0 min and no change after that. This was observed on HMM chito-
san treated by either cellulose or lysozyme. On the other hand, the
relative viscosity of the LMM chitosan depolymerized by cellulase
or lysozyme gradually decreased during the first hour, reaching
about 73% and 37% reduction at 1 h, respectively, and was stable
after that. These observations were similar to those from a previ-
ous study that showed that the viscosity change was mostly in-
duced during the initial 10–20 min as a result of enzymatic
0
1
2
3
4
5
6
7
8
Time (hours)
2
5
Figure 1. The ratio of the depolymerization in chitosan treated with 1% cellulose or
lysozyme for 7 h (n = 2). Data were mean of two replications. HMM = higher
molecular mass; LMM = lower molecular mass.
action. The reduction of relative viscosity was faster and higher
in LMM chitosan treated by cellulase than that by lysozyme since
the high DDA in LMM showed lower susceptibility on lysozyme
treatment.
fast degradation was probably due to the predominant endo-action
of cellulase and lysozyme that broke internal bonds (the (1?4)-
glycosidic linkage of the polysaccharide) of chitosan.17 Previous
studies reported that chitosan depolymerized by cellulase shows
Theoretically, the relative viscosity is proportional to MM, con-
centration, and the chain entanglement of chitosan, and can be
highly increased when MM is higher than a certain critical molec-
3
6
ular mass (M ) of a polymer. The strong dependence of viscosity in
C
8
3.5% degradation in only 5 min, and 95.3% degradation in 4 h with
the high MM samples can be explained as the effect of chain entan-
3
5
37
a final MM of 24 kDa.
glement in the polymer. For example, the viscosity is propor-
3
.4
1<a<2.5
The types of enzyme had significantly different impact on the
depolymerization of HMM and LMM chitosan (P <0.05). MM values
of lysozyme-treated samples were significantly (P <0.05) higher
than those from cellulose-treated ones regardless of the difference
in the initial MM (Table 4). According to Lin, lysozyme had less
susceptibility than cellulase in terms of chitosan degradation since
it only recognizes the site existed NAG(3–5) site of N-acetyl-
tional to MM if MM > MC and to MM
if MM < M , and no
Therefore, the rel-
C
3
7,38
distinct M could be up to MM of 1000 kDa.
C
ative viscosity of HMM chitosan was significantly higher than that
3
7
of LLM chitosan due to its stronger dependence on MM. The way
the viscosity is computed may also attribute to the high viscosity
value in HMM chitosan as the relative viscosity was expressed as
the viscosity at sampling time relative to the initial viscosity of
chitosan prior to enzyme treatment. The initial viscosity of HMM
chitosan was considerably higher than that of LMM chitosan,
resulting in a higher decreasing rate even though the same number
of viscosity reduction occurred in both HMM and LMM chitosan.
The absorbance value at 420 nm indicates the amount of reduc-
ing ends formed by the depolymerization of chitosan (Fig. 3). The
absorbance value in chitosan depolymerized by cellulase was sig-
nificantly higher than that by lysozyme in both HMM and LMM
chitosan. There was no significant difference in the absorbance be-
tween HMM and LMM chitosan (P >0.05). The absorbance in-
creased fast in the initial 30 min and then increased slowly after
that. The difference in the reducing end formation of chitosan
depolymerized by cellulase or lysozyme was probably due to dif-
ferent specificities of enzymes for cleaving at different active
D-glucosamine (NAG), while cellulase can randomly cleave the
(
1?4)-glycosidic linkages of chitosan regardless of the type of
1
7
monomers. Kurita found that lysozyme had higher susceptibility
27
in 57% DDA chitosan than in higher DDA chitosan. The initial
DDA values of HMM and LMM chitosan were about 85% and 95%,
respectively, and may explain the diminished degradation
observed in LMM chitosan when depolymerized by lysozyme.
DDA values were measured during the polymerization process to
investigate the potential impact of the treatment factors
(Table 5). The initial MM was the only factor affecting the DDA (P
<0.05), clearly due to the initial difference in DDA for the HMM
and LMM chitosans. After one hour of deploymerization, the differ-
ence in DDA no longer existed among products of the different treat-
ment conditions. A significant (P <0.05) interaction between enzyme
type and initial MM on DDA was observed (Table 3). This interaction
is probably attributable to the different susceptibility of the enzyme
to chitosan that had different MM and DDA values.
1
7,27
sites.
As discussed in the changes of MM and relative viscosity,
the DDA values of chitosan impacted the specificities of enzymes
on the formation of the reducing ends that were formed by the
Table 4
Effect of initial molecular mass of chitosan, type of enzyme, and depolymerization time on the molecular mass of chitosan during depolymerization
Initial MM+++ (IM)
Enzyme types (ET)
Depolymerization time (h)
P value
0
1
2
3
4
5
6
7
+
A2100a
B
298b
B
165b
B
137b
B
145b
B
139b
B
133b
B
124b
HMM
LMM
Cellulase
Lysozyme
Cellulase
Lysozyme
<.0001
0.0003
<.0001
0.0012
A
B
a++
A
B
b
A
B
b
A
B
b
A
B
b
A
B
b
A
B
b
A
B
b
2100
670
633
533
645
625
718
611
a
b
a
b
a
b
a
b
a
167b
136b
317b
128b
279b
594
594
264
686
148
604
175
553
152
584
B
a
A
A
A
A
A
ab
B
B
505
P value
0.0180
0.0036
0.0001
0.001
0.0003
0.0080
0.0012
0.0023
+
+
Means preceded by the same capital letter in the same column within each treatment were not significantly different among different treatments (P >0.05).
Means followed by the lowercase letter in the same row within each treatment were not significantly different among different depolymerization times (P >0.05).
HMM = higher molecular mass, LMM = lower molecular mass.
+
+
++

J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
1881
Table 5
Effect of initial molecular mass of chitosan, type of enzyme, and depolymerization time on the degree of deacetylation (DDA) of chitosan during depolymerization
Initial MM+++ (IM)
Enzyme types (ET)
Depolymerization time (h)
P value
0
1
2
3
4
5
6
7
+
B
a++
A
a
a
a
a
A
a
a
a
a
A
a
a
a
a
A
a
a
a
a
A
a
a
a
a
A
a
a
a
a
A
a
a
a
a
HMM
LMM
Cellulase
Lysozyme
Cellulase
Lysozyme
85.5
87.2
83.1
88.8
87.6
85.0
87.0
82.9
0.2107
0.1885
0.3451
0.8213
B
a
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
85.5
95.1
95.1
90.6
92.2
90.0
89.9
95.4
85.2
89.7
95.3
90.9
89.7
91.5
92.1
87.0
90.1
92.2
89.0
94.2
90.4
85.3
93.1
91.1
A
A
a
a
P value
0.0001
0.8167
0.0521
0.1274
0.7391
0.2036
0.2550
0.1969
+
+
Means preceded by the same capital letter in the same column within each treatment were not significantly different among different treatments (P >0.05).
Means followed by the lowercase letter in the same row within each treatment were not significantly different among different depolymerization times (P >0.05).
HMM = higher molecular mass, LMM = lower molecular mass.
+
+
++
was about 85% in HMM and 96% in LMM. Because of the relatively
1
1
20
00
higher DDA in both HMM and LMM chitosan with fewer NAG
monomers, chitosan depolymerized by lysozyme produced a smal-
ler amount of reducing ends due to lower susceptibility, compared
with chitosan depolymerized by cellulase. Cellulase is one of
enzymes with the highest chitosan depolymerizing activity.26
Studies of chitosan depolymerized by pepsin, cellulase, lipase A,
and chitosanase also showed the fast generation of the reducing
LMM-Cellulase
LMM-Lysozyme
HMM-Cellulase
HMM-Lysozyme
80
60
40
20
0
2
6
ends during the first hour. The number of reducing ends was
more dependent on the type of enzyme than the initial properties
of the chitosan, probably due to the relatively higher DDA in both
LMM and HMM chitosan.
3
. Experimental
-20
0
50
100
150
200
250
3.1. Materials
Time (minutes)
The b-chitin from jumbo squid (Dosidicus gigas) pens was pro-
vided by Dosidicus LLC, USA. Samples were ground into about 18
mesh (ASTM) size by a grinder (Glenmills Inc., USA) and stored
in a desiccator until deacetylation treatment was carried out.
NaOH and KOH were purchased from the Mallinckrodt Chemicals
Figure 2. The decreasing ratio of viscosity in 1% (w/v) chitosan solution treated
with 1% cellulose or lysozyme for 210 min. The decreasing ratio was expressed as
the viscosity at the sampling time relative to the initial viscosity of the higher or
lower molecular mass chitosan without enzyme treatment (n = 2). HHM = higher
molecular mass; LMM = lower molecular mass.
Co., (USA). N-Acetyl-D-glucosamine and monoethylene glycol were
from Sigma–Aldrich Chemical Co., (USA). Toluidine Blue indicator
and 1/400 potassium polyvinyl sulfate (PVS) were from Wako
Chemicals (USA). Hen egg-white lysozyme and cellulase from
Aspergillus niger were obtained from Fordras S.A. (SWISS) and TCI
America (USA), respectively. All chemicals were of reagent grade.
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
LMM-Cellulase
LMM-Lysozyme
HMM-Cellulase
HMM-Lysozyme
3
.2. Deacetylation of chitin
In this study, two different alkaline deacetylation processes
were carried out: the method of Kurita et al. using NaOH as re-
1
2
11
agent and the method of Broussignac using KOH Depending
on the type of chitin and alkaline reagents employed, the deacety-
lation process may respond differently. Using NaOH might result in
b-chitosan with higher DDA and MM under moderate treatment
conditions (40% NaOH at 80 °C) as compared with the use of
2
,11
KOH.
Hence, it is necessary to investigate which alkaline solvent
performs more appropriately on b-chitin from jumbo squid pens.
0
50
100
150
200
250
1
2
While using the Kurita method, NaOH was first diluted to tar-
geted concentrations by dissolving it in distilled water. Chitin was
then added to the NaOH solution at a ratio of 1:20. Four treatment
factors, including NaOH concentration (40%, 50%), temperature (60,
Time (minutes)
Figure 3. The reducing end formation from 1% chitosan solution treated with 1%
cellulose or lysozyme for 210 min (n = 2). Ferricyanide absorbance corresponded to
the amount of the reducing ends as a function of time. HHM = higher molecular
mass; LMM = lower molecular mass.
9
0 °C), time (2, 4, 6 h), and treatment step (1, 2, 3 times) were
investigated. The typical deacetylation conditions for -chitin from
shrimp or squilla shells were using 40–50% NaOH at 80–100 °C for
a
2
6
–12 h. Since the squid pens from the species of Dosidicus gigas
enzymatic depolymerization of polysaccharide. It is known that
the active site of cellulase is in (1?4)-glycosidic linkages regard-
less of the type of monomers, while lysozyme cleaves the active
site occupied by the NAG(3–5) bindings. The initial DDA of chitosan
are much smaller and thinner than other ones frequently used
(Loligo or Illex pens), the mild treatment conditions could be ap-
plied. The multiple treatment steps at same treatment time were
evaluated for the possible prevention of chitosan degradation

1
882
J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
during deacetylation.2 After NaOH treatment under given condi-
tions, samples were washed with distilled water to reach neutral
pH, and the residue that remained was washed with MeOH and
acetone. Samples were then dried at 50 °C in a drying oven (Preci-
sion Scientific Inc., USA) for 24 h.
3.4. Measurement of degree of deacetylation (DDA)
The DDA was measured by using the colloidal titration meth-
6
od. A 50 mg sample of deacetylated chitosan (0.5%, w/w) was dis-
solved in 10 mL of 5% (v/v) HOAc solution, and then 1 g of solution
was transferred into a flask and diluted up to 30 mL with distilled
water. After adding 100 lL of Toluidine Blue indicator, the solution
In the Broussignac method,11 KOH (50% w/w) was dissolved in a
mixture of 96% EtOH (25% w/w) and monoethylene glycol (25%
w/w) solution. Chitin was then added to the solution at a ratio of
was titrated with 1/400 potassium polyvinyl sulfate (PPVS) till the
1
:20. According to the previous study, the Broussignac process re-
solution color changed from blue to violet. The DDA was calculated
as:
2
quired minimal treatment conditions of 90 °C for 2 h in 1% HOAc
to obtain soluble chitosan. Therefore, three treatment factors
including temperature (90, 120 °C), time (2, 4, 6 h), and treatment
step (1, 2, 3 times) were considered. After the treatment, the same
washing and drying procedures as those used in the Kurita method
were applied.
The Taguchi experimental design with orthogonal arrays was
applied for each method (Table 6). By using orthogonal arrays, it
was expected that the optimal treatment conditions for obtaining
chitosan with high DDA and desirable MM values can be identified
with minimal treatment combinations (9 in this study) in each
method. The factors that contributed most to achieve desirable re-
sults and their levels could then be identified through the Taguchi
design.
DDAð%Þ ¼ ðX=161Þ=ðX=161Þ þ ðY=203Þ
ð1Þ
ð2Þ
1
1
X ¼ 4
ꢂ
ꢂ F ꢂ 161 ꢂ V
00
1000
1
Y ¼ 0:5 ꢂ 1 ꢁ X
ð3Þ
00
where X was the weight of the
D-glucosamine residue, g; F was the
factor of 1/400 PPVS; V was the volume of consumed PPVS, mL; Y
was the weight of N-acetyl- -glucosamine residue, g; and 161 and
203 in (Eq. 1) were the molecular weight of -glucosamine and N-
acetyl- -glucosamine (2-acetamido-2-deoxy- -glucose), respectively.
D
D
D
D
3.5. Determination of intrinsic viscosity and viscosity-average
molecular mass
3
.3. Depolymerization of chitosan
The intrinsic viscosity of chitosan was determined by using a
Ubbelohde Dilution Viscometer (Cannon instrument Co., USA) that
has a capillary size of 0.58 mm in a water bath at 25 °C. Viscosity-
average molecular mass (MM) was then calculated from the mea-
sured intrinsic viscosity. Solutions used for measuring the MM of
the chitosan samples from deacetylation and depolymerization
treatments were different. This is mainly because the chitosan
samples obtained from the deacetylation and depolymerization
experiments had different DDA values, thus requiring different
strengths of acid to completely dissolve and ensure accurate MM
measurements. Our preliminary studies (data not shown) evalu-
ated three types of acid solutions that are commonly used for
measuring the MM in chitosan: 0.1 M HOAc, 0.1 M HOAc/0.2 M
The HMM and LMM chitosan prepared from the deacetylation
study were depolymerized by cellulase or lysozyme, known to
have different susceptibilities based on the properties of chitosan.
The chitosan samples were dissolved in 5% HOAc solution at a ratio
of 2:100 (chitosan:solvent). Cellulase or lysozyme was added into
the solution at 1% (w/w) and allowed to react for up to 7 h. A 20-
mL aliquot was taken out hourly and boiled for 10 min to stop
the enzymatic reaction. NaOH was then added into the solution
to reach a final pH 11 for precipitation. The precipitated samples
were washed with distilled water to remove other residues and
dried in an oven at 42 °C for 24 h. The MM and DDA of the depoly-
merized chitosan were measured at each sampling time.
Table 6
L9 standard orthogonal array of different contributing factors with different levels applied in the Kurita [A] and the Broussignac [B] methods1
1,12
Experimental run
Contributing factors (i)
Temperature (°C)
Concentration (%)
A
Time (h)
C
Multiple-steps
D
B
[
A]—Kurita method using NaOH as reagent
1
2
3
4
5
6
7
8
9
1 (40)
1 (40)
1 (40)
2 (50)
2 (50)
2 (50)
1 (40)
2 (50)
1 (40)
1 (60)
2 (90)
2 (90)
1 (60)
2 (90)
1 (60)
1 (60)
2 (90)
2 (90)
1 (2)
2 (4)
3 (6)
2 (4)
3 (6)
1 (2)
3 (6)
1 (2)
2 (4)
1
2
3
3
1
2
2
3
1
Experimental run
Contributing factors (i)
Temperature (°C)
B
Time (h)
C
Multiple-steps
D
[
B]—Broussignac method using KOH as reagent
1
2
3
4
5
6
7
8
9
1 (120)
1 (120)
1 (120)
2 (90)
2 (90)
2 (90)
1 (120)
2 (90)
1 (120)
1 (2)
2 (4)
3 (6)
1 (2)
2 (4)
3 (6)
1 (2)
2 (4)
3 (6)
1
2
3
2
3
1
3
1
2

J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
1883
NaCl, and 0.3 M HOAc/0.2 M NaOAc, and found that 0.1 M HOAc is
3.7. Experimental design and data analysis
able to dissolve all the chitosan samples. The 0.1 M HOAc was thus
chosen for measuring the MM of all chitosan samples in the
deacetylation study. The same test was carried out for identifying
the solution in measuring the MM of depolymerized chitosan by
focusing on finding a more precise measurement of MM using a
commercial chitosan with known MM as an indicator. The 0.1 M
HOAc/0.2 M NaCl solution provided the closest MM value to that
reported on the commercial chitosan.
The Taguchi design method was used in the deacetylation study
to identify optimal deacetylation conditions. The orthogonal arrays
used in the Taguchi design had nine treatment trials for both the
Kurtia and Broussignac methods as shown in Table 6. Each exper-
imental run represented one trial. This array was designed for
determining (1) the contribution of individual treatment factor,
and (2) the level of each factor. The Taguchi design offered a simple
and systematic approach to optimize the experiments and signifi-
cantly reduced the number of treatment combinations when mul-
Four different concentrations of chitosan in a range of 0.05–0.1%
were used for measuring the viscosity of the samples. The intrinsic
viscosity was measured by the intercept between the Huggins (re-
4
0
tiple factors were considered.
n
c
sp
ln½
g
c
ꢄ
rel
duced viscosity,
ꢃ C) and Kraemer (relative viscosity,
ꢃ C)
In the Taguchi design, two parameters were applied to optimize
the treatment conditions. The first parameter (Kij) was the average
value of each measured functional parameter in level j (j = 1, 2, 3)
of each factor i (i = A, B, C, D) and expressed as
16
plots when the concentration was 0. Relative viscosity, reduced
viscosity, and intrinsic viscosity were determined as:
t
N
i
grel ¼ t
ð4Þ
ð5Þ
ð6Þ
X
1
0
K
ij ¼ N
yi;j
n
sp
i
u¼1
gsp
¼
grel ꢁ 1; gred
¼
c
ꢂ
ꢃ
ꢀn
ꢁ
where i represented the factor A, B, C, and D; j represented the level
1, 2, and 3; N is the number of trials for each factor, and y is the
measured values of factor i at level j. This parameter could explain
how targeted parameters were changed in different levels of each
treatment factor.
The second parameter, R
est and lowest values of Kij, and determined the most contributed
factor among all factors. R was calculated as
sp
ln½
g
ꢄrel =C
½
gꢄ ¼
¼
c¼0
i
i,j
c
c
c¼0
where t was the flow time measured for the sample solution at a gi-
ven time t (s); t (s) was the flow time of the solution (0.1 M HOAc
and 0.1 M HOAc/0.2 M NaCl) without chitosan sample; C was the
concentration of chitosan samples in diluted solution (g/mL),; and
] was intrinsic viscosity, mL/g.
0
i
, was the difference between the high-
i
[
g
ꢄ
ꢅ
ꢄ
ꢅ
The viscosity-average molecular mass (MM) of chitosan was
calculated by the Mark–Houwink–Sakurada (MHS) (Eq. 7):
R
i
¼ Kij
ꢁ Kij
max
min
where (Kij
)
max and (Kij
)
min indicated the highest and lowest values of
a
½
g
ꢄ ¼ KðMMÞ
ð7Þ
the measured parameter in each factor, respectively. As R values in-
creased among different factors, the factor which showed the high-
est value was the most contributed factor in determining the
characteristics of the samples. Hence, by using the Taguchi design,
the levels in each factor and the most contributed factor could be
identified by using only nine treatment combinations.
i
where K and a were the constants, K = 1.81 ꢂ 10ꢁ3, a = 0.93,11 and
[g] was the intrinsic viscosity obtained from the Huggins and Kra-
emer plots.
3
.6. Chitosan hydrolyzing activity assay
The deacetylation study was conducted in triplicate, and each
study was considered as a block. A one-way ANOVA was carried
out to determine the significant differences among different factors
and their levels, and the Tukey test was done for multiple compar-
isons in the Taguchi design method (SAS 9.2, SAS Institute, Inc.,
USA).
A completely randomized 2 ꢂ 2 ꢂ 8 factorial design was applied
in the depolymerization study with two replications. Three inde-
pendent factors were the type of enzymes (cellulase and lyso-
zyme), the initial MM of chitosan (HMM and LMM), and
depolymerized time (0–7 h). Interactions among the three factors
were determined. PROC GLM was applied to determine the signif-
icant difference (P <0.05) among treatment factors using the SAS
program (SAS 9.2, SAS Institute, Inc., USA).
The extent of hydrolyzing activity in chitosan was measured by
investigating the relative viscosity and the reducing ends. One per-
cent (w/w) of chitosan solution was prepared in 100 mM NaOAc
buffer at pH 4.5. Cellulase or lysozyme was added into the solution
in a ratio of 1:100 (w/w), respectively, and the reactions were al-
lowed to continue for 210 min in a 40 °C water bath to compare
the hydrolyzing activity between the two enzymes.
The viscosity of the chitosan solutions at different reaction
times with the enzymes were measured using a Brookfield DV-
III + viscometer (Brookfield Inc., USA). Changes in the viscosity
were expressed as the relative viscosity with respect to the control,
enzyme-free chitosan solution.26
Relative viscosityð%Þ ¼ ½ðInitial viscosityðcpÞꢁReduced viscosityðcpÞÞ
4
. Conclusions
=
Initial viscosityðcpÞꢄꢅ100
The absorbance of the reducing ends of chitosan was mea-
Under tested treatment temperature and time, the NaOH
39
sured using the method of Shales and Shales with some modi-
deacetylation of b-chitin obtained from the jumbo squid pens re-
sulted in b-chitosan with a wide range of degree of deacetylation,
but little change in molecular mass, while the KOH treatment pro-
duced b-chitosan with a high degree of deacetylation (>93%) and a
huge amount of polymer degradation at 120 °C. When using NaOH,
the deacetylation time and temperature were ranked the first- and
second-most contributing factors affecting molecular mass and de-
gree of deacetylation, respectively. In the KOH deacetylation, the
temperature was the most contributing factor that impacted both
the molecular mass and the degree of deacetylation of chitosan.
Chitosan could be depolymerized by cellulase and lysozyme, in
2
6
fications. Samples (3 mL) were extracted from the solution at
different reaction times, boiled for 10 min to inactivate the
enzymes, and reacted with 4 mL of a solution of 0.5 g/L potassium
2 3
ferricyanide dissolved in 0.5 M Na CO . Mixtures were then
boiled for 15 min for inducing color change by the amount of
reducing ends. The samples were cooled and then centrifuged
to remove the precipitated chitosan. Distilled water (1 mL) was
added to 2 mL of supernatant and the absorbance of the solution
was measured at 420 nm in a UV spectrophotometer (Shimadzu
UV 160U).

1
884
J. Jung, Y. Zhao / Carbohydrate Research 346 (2011) 1876–1884
which cellulase had higher susceptibility on chitosan, resulted in a
higher and faster chitosan degradation than that of lysozyme. Fur-
ther studies to investigate the antimicrobial and antioxidant func-
tions of b-chitosan from jumbo squid pens are underway.
16. Mao, S.; Shuai, X.; Unger, F.; Simon, M.; Bi, D.; Kissel, T. Int. J. Pharm. 2004, 281,
5–54.
4
1
1
7. Lin, S. B.; Lin, Y. C.; Chen, H. H. Food Chem. 2009, 116, 47–53.
8. Fei Liu, X.; Lin Guan, Y.; Zhi Yang, D.; Li, Z.; De Yao, K. J. Appl. Polym. Sci. 2001,
79, 1324–1335.
1
9. Hernández-Lauzardo, A. N.; Bautista-Baños, S.; Veláquez-del Valle, M. G.;
Méndez-Montealvo, M. G.; Sánchez-Rivera, M. M.; Bello-Pérez, L. A. Carbohydr.
Polym. 2008, 73, 541–547.
Acknowledgment
20. Vishu Kumar, B. A.; Varadaraj, M. C.; Tharanathan, R. N. Biomacromolecules
2
007, 8, 566–572.
The authors thank Mr. Mark Ludlow at Dosidicus LLC, USA for
the donation of b-chitin from jumbo squid pens.
2
1. Jung, E. J.; Youn, D. K.; Lee, S. H.; No, H. K.; Ha, J. G.; Prinyawiwatkul, W. Int. J.
Food Sci. Technol. 2010, 45, 676–682.
2
2
2. Park, P. J.; Je, J. Y.; Kim, S. K. Carbohydr. Polym. 2004, 55, 17–22.
3. Santos, C.; Seabra, P.; Veleirinho, B.; Delgadillo, I.; Lopes da Silva, J. A. Eur.
Polym. J. 2006, 42, 3277–3285.
References
2
2
4. Kim, S. K.; Rajapakse, N. Carbohydr. Polym. 2005, 62, 357–368.
5. Tsao, C. T.; Chang, C. H.; Lin, Y. Y.; Wu, M. F.; Han, J. L.; Hsieh, K. H. Carbohydr.
Res. 2011, 346, 94–102.
1
.
Knaul, J. Z.; Kassai, M. R.; Bui, T.; Creber, K. A. M. Can. J. Chem. 1998, 76, 1699–
706.
Tolaimate, A.; Desbrieres, J.; Rhazi, M.; Alagui, A. Polymer 2003, 44, 7939–7952.
Chandumpai, A.; Singhpibulporn, N.; Faroongsarng, D.; Sornprasit, P. Carbohydr.
Polym. 2004, 58, 467–474.
Minke, R.; Blackwell, J. J. Mol. Biol. 1978, 120, 167–181.
Rinaudo, M. Prog. Polym. Sci. 2006, 31, 603–632.
Chang, K. L. B.; Tsai, G.; Lee, J.; Fu, W. R. Carbohydr. Res. 1997, 303, 327–332.
Mima, S.; Miya, M.; Iwamoto, R.; Yoshikawa, S. J. Appl. Polym. Sci. 1983, 28,
1
2
3
.
.
2
6. Roncal, T.; Oviedo, A.; de Armentia, I. L.; Fernández, L.; Villarán, M. C.
Carbohydr. Res. 2007, 342, 2750–2756.
2
2
7. Kurita, K.; Kaji, Y.; Mori, T.; Nishiyama, Y. Carbohydr. Polym. 2000, 42, 19–21.
8. Tolaimate, A.; Desbrieres, J.; Rhazi, M.; Alagui, A.; Vincendon, M.; Vottero, P.
Polymer 2000, 41, 2463–2469.
9. Lu, S.; Song, X.; Cao, D.; Chen, Y.; Yao, K. J. Appl. Polym. Sci. 2004, 91, 3497–3503.
0. Li, L.; Hsieh, Y.-L. Carbohydr. Res. 2006, 341, 374–381.
4.
5.
6.
7.
2
3
3
3
3
3
3
3
1
909–1917.
1. Ilium, L. Pharm. Res. 1998, 15, 1326–1331.
8
9
.
.
Batista, I.; Roberts, G. A. F. Makromol. Chem. 1990, 191, 429–434.
Focher, B.; Beltrame, P. L.; Naggi, A.; Torri, G. Carbohydr. Polym. 1990, 12, 405–
4
2. Sannan, T.; Kurita, K.; Iwakura, Y. Makromol. Chem. 1976, 177, 3589–3600.
3. Hasegawa, M.; Isogai, A.; Onabe, F. Carbohydr. Res. 1994, 262, 161–166.
4. Ottey, M. H.; Vårum, K. M.; Smidsrød, O. Carbohydr. Polym. 1996, 29, 17–24.
5. Xie, Y.; Wei, Y.; Hu, J. Appl. Biochem. Biotechnol. 2010, 160, 1074–1083.
6. Ferry, J. D. Viscoelastic Properties of Polymers, 3rd ed.; John Wiley & Sons: New
York, 1980. pp. 177–315.
18.
0. Martinou, A.; Kafetzopoulos, D.; Bouriotis, V. Carbohydr. Res. 1995, 273, 235–
42.
1
2
1
1. Broussignac, P. Chim. Ind. Génie Chim. 1968, 99, 1241–1247.
1
2. Kurita, K.; Tada, T.; Ishii, S.; Nishinura, S. I.; Shimoda, K. J. Polym. Sci. 1993, 31,
37. Sato, N.; Ito, S.; Yamamoto, M. Macromolecules 1998, 31, 2673–2675.
38. Izuka, A.; Winter, H. H.; Hashimoto, T. Macromolecules 1992, 25, 2422–2428.
39. Schales, O.; Schales, S. S. Arch. Biochem. 1945, 8, 285–292.
40. Park, S. H. Robust Designed and Analysis of Quality Engineering, 1st ed.; Chapman
and Hall: London, 1996.
4
86–491.
3. Bough, W. A.; Satter, W. L.; Wu, A. C. M.; Perkin, B. E. Biotechnol. Bioeng. 1978,
0, 1931–1943.
1
2
1
1
4. Brine, C. J.; Austin, P. R. Comp. Biochem. Physiol. B 1981, 69, 283–286.
5. Shepherd, R.; Reader, S.; Falshaw, A. Glycoconjugate J. 1997, 14, 535–542.