Optimization of chitin and chitosan extraction from by product from white leg shrimp (penaeus vannamei) industry in vietnam to improve its quality and efficiency

1

INTRODUCTION
1. The rationale
Vietnam is one of the leading shrimp production in the world with two main species of black tiger
(Penaeus monodon)and white leg shrimps (Penaeus vannamei). The production of cultured shrimp reached more
than 480,000 tons in 2012, among them 130,000 tons came from white shrimp and it will be more in future.
Annual amount of by-products from shrimp production was estimated up to 200,000 tones which consists of
head, shell and broken meat. Shrimp heads and shells is composed of protein, fat, chitin, protease and pigments,
astaxanthin. Therefore, the efforts to convert those wastes into useful products, especially bioactive molecules,
are rational and important because environment pollution will be prevented as well as more benefits was
achieved.
The by-products from shrimp industry was actually used for chitin and animal feed production in
Vietnam and commercial chitin is mainly isolated from crustacean shells through chemical treatments.
Consequently, added value and sensitive by-products such as protein hydrolyzates and pigments were not
recovered. Moreover, the chemical procedures caused the side effects on chitin quality and serious chemical
pollution. Therefore, a great interest still exists for the innovation and optimization of the recovery bioactive
compounds from shrimp wastes, especially from white shrimp - the new farmed species, that will facilitate
Vietnam's chitin industry following up the sustainable development.
Integration between chemical and biological methods in recovery of chitin and other bioactive
compounds from crustaceous wastes were explored increasingly, however, in oder to put it into practice more
information relevant to the kinetics and the extra assistance need to be cleared.
At the present, the trend of technology innovation is paying more attention on applying physic factors
on chemical and biological process. Of these factors, ultrasound was topics of universal interests. Ultrasonic
waves was proved that is one of green and efficient energy sources on several fields including textile, food and
chemicals industries. Studying of applying sonication on chitin and chitosan production is expected eagerly
opening a new way for innovation of recovery bioactive compounds.
The dissertation "Optimization of chitin and chitosan extraction from by-product from white leg
shrimp (Penaeus vannamei) industry in Vietnam to improve its quality and efficiency" was conducted with
the aim of finding out the way to integrate enzymatic and chemical methods with physic method which support
the innovation of chitin - chitosan production technology in Vietnam.

2. The scope and objectives
In the dissertation, three main steps in the chitin and chitosan production, including deminerilization,
proteinization and deacetylation, were optimized by using integrating technology in order to improve product
quality, reduce consumption of chemicals, recovery protein and prevent environment pollution.
The objectives include: (1) determine the components of white leg shrimp (mass, proximate, amino acid
and minerals components); (2) optimize chitin recovery process from shrimp heads and shells; (3) study the
kinetics of deproteinization under the catalysis of pepsin; (4) optimize and characterize the heterogeneous
deacetylation under the facilitation of sonication; and (5) recommend the efficient processes to recovery chitin
and protein simultaneously as well as chitosan through applying integrated technology.
2

3. The objects of the study
The main objects were shrimp heads and shells collected after the manufacturing of white shrimp
(Penaeus vannamei), in the average size of 81-120 bodies/kg.
4. The scientific and realistic significances and innovations
- The data of the composition of chemicals, amino acids, minerals and heavy metals of three main parts
of white leg shrimp (heads, shell and meat) as well as the effects of pH and temperature on the activity of the
endogenous proteases from heads of white shrimp leg cultivated in Khanh Hoa province were collected.
- Gaining the optimization condition for recovering chitin and protein hydrolysates from white leg
shrimp shells with pepsin. In addition, the information relevent to kinetics of the process and the linkage
between protein, minerals and chitin in shrimp shell were cleared.
- New data and information about the supporting capacities of ultrasound in chitin enzymatic extraction
and heterogeneous deacetylation were collected.
- The simple procedures to recovery chitin and protein efficiently through simultaneous combination of
autolysis with the endogenous protease and physical force were put forward.
- The benefits of the integration technology between physics (mechanical force and ultrasound), enzyme
(endogenous and commercial proteases) and chemicals (NaOH, HCl) in chitin and chitosan production were
demonstrated. The processes of chitin and chitosan production were controlled by the mathematic equations.
5. The structure
The main content of the dissertation was divided into three chapters (142 pages) accompanied with the

conclusions (3 pages), the references (19 pages) and the appendix (56 pages).
CHAPTER: LITERATURE REVIEW
1.1. The components and value of by-products from manufacturing shrimps
By-products from shrimp production consists of head, shell and a minor amount of broken meat.
Although the ratio between them was dependent on species, ages, seasons and methods of processing the total of
them was in range of 40-60% of the whole of raw materials.
The proximate components of different shrimp species is not the same, however, protein always is the
majority (33-49.8% dry basis), follwing by minerals and chitin (respectively 21,6-38% and 13,5-20% , db).
Therefore, shrimp by-products were the value source to recovery of both chitin and protein.
There are a significant amount of endogenous enzymes in shrimp head, especially proteases. These
proteases include both endoprotease and exoprotease and their activities were equal to commercial proteases
however they were easy to be lost due to denaturation and drift out. In case of white leg shrimp (Penaeus
vannamei), the favourate condition of proteases was temperature of approximately 60
o
C and pH of 7,5-8, which
was the same pH of fresh shrimp heads. Therefore, utilization of endogenous proteases in shrimp heads for
recovery of chitin and protein hydrolysate will be more economic than using commercial enzymes.
1.2. Pepsin and its application on recovery of protein and chitin
Pepsin was an endopeptidase and belong to aspartate protease. Pepsin is most efficient in
cleaving peptide bonds between hydrophobic and preferably aromatic amino acids such
as phenylalanine, tryptophan, and tyrosine. Pepsin is a monomeric, two domain, mainly β-protein, with a high
percentage of acidic residues (43 out of 327) leading to a very low pI. The catalytic site is formed by two
3

aspartate residues, Asp32 and Asp215, one of which has to be protonated, and the other deprotonated, for the
protein to be active. This occurs in the 1–5 pH interval, dependent on substrates. In the 5–7 pH interval the
conformation of pepsin is poorly characterised. Above pH 7, pepsin is in a denatured conformation that retains
some secondary structure. This denaturation is not fully reversible. The bioactive capacity of hydrolysates from
pepsin were more than that from other proteases such as Alcalase , α-chymotrypsin, or trypsin. Based on the
mentioned characteristics pepsin has a potential of application in chitin production to combine deprotenization

with deproteinization which will support for time - saving and recovery bioactive compounds.
Commercial pepsin is extracted from the glandular layer of hog stomachs through conventional method
therefore the price is rather high in comparison with other commercial proteases which were recovered from
mass of microorganisms. With the latest success in seeking new sources of pepsin (from fish viscera or
microorganisms such as Botrytis cinerea or Aspergillus niger) and innovation in purifying enzymes based on
Aqueous two-phase system it is expected that the price of pepsin will become reasonable in near future.
1.3. Ultrasound and its potential application
Ultrasound is an oscillating sound pressure wave with a frequency greater than the upper limit of the
human hearing range (>20kHz). In fields of food and biotechnology, ultrasound with low frequency - high
power (20-100kHz) were applied widely, especially for extraction and adjusting physical and chemical
characteristics of materials as well as the activity of enzymes. Generally, the mechanism of ultrasonic is based
on the high energy waves that create cavitations in the liquid solution. Dependent on the feature of the system
sonicated (characteristics of liquid, presence of air and solid debris) as well as sonication condition
(manipulation of wave duty cycle, time of exposure and acoustic power of ultrasonic system) the mechanism can
be changed.
Replying on multifunctional mechanism, sonication is able to create a change in spatial structure of
objectives (materials or enzymes) or/and increase the contact between them. This effect leads to facilitate
reaction rate and time-saving significantly.
Ultrasonication offers great potential in the processing of liquids - solid system, by improving the
mixing and chemical reactions in various applications and industries. Application of ultrasound is enable to cut
down the severity of reaction condition (temperature, time, chemicals), improve quality along with cost-saving.
1.4. Shortcomings in chitin-chitosan production in Vietnam
Heads and shells after manufacturing black tiger and white leg shrimps were the materials for chitin
production. In shrimp processing enterprises, due to the sensitive characteristics to deterioration shrimp heads
were always separated from the whole after receiving (exclusive HOSO product). Whereas, shrimp shells were
separated later which was dependent on types of products. At the end, they were mixed together and kept for
long time (interval of 4 to 8 hours) at ambient temperature in waste house. The by-products were often
deteriorated seriously before transferring to the place where fishmeal and chitin were produced. This way of
treatment caused the recovery of useful compounds to be lost the efficiency and to pollute environment
concurrently.

The industry of chitin and chitosan production in Vietnam has not been developed and still employed
backward technology. The majority of chitin processing factories were in Mekong delta and the South. The
annual average output of a factory is approximately 2,000 tons. Chemical extraction were used prevalently while
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the procedures which were combined between chemical and biological methods have been applied in a quite
limitative level. HCl and NaOH were the main reagent used to liquidate minerals and protein, respectively. The
minerals was removed in the condition of HCl 4-6% at ambient temperature for one day. The solution of NaOH
4-5% was used to exclude protein at room temperature or at higher one. Infact, energy was only used in case of
having a demand of high quality chitin. The protein liquids were collected, conveyed to containing towers,
concentrated into thick liquid which were not in good quality and used for animal feeds, after that.
The primary product was chitin but its quality was still poor and not stable; the residuals of protein and
minerals remained high, over 1%, in addition it was easy to be changed into bad color and cost price was so high
therefore its ability of application and marketing was low. Fewer and fewer factories which produce chitin could
be alive. A large number of them must be closed due to violating the regulation of environment. The
predominant reasons of polluting came from off-odor, protein drain and chemical wastes. The urgent
requirements involve in finding out solutions which enable to solve thoroughly the pollution and improve the
quality.
In brief, by-products from shrimp processing industry was only utilized to recovery chitin. Up to now no
much attention was pay on recovery protein with its biological value. The products has not been competitive and
limitative in application. Besides, the studies conducted have only focused on establishment parameters of chitin
extracting procedures, the process kinetics as well as the interaction between process factors have not been
investigated.
CHAPTER II: MATERIALS AND METHODS
2.1. Materials
White leg shrimp (Penaeus vannamei), cultivated in Khanh Hoa province, were used in two forms: (1)
whole shrimp to determine the mass components (size of 60-160 bodies/kg), the proximate component, the
composition of amino acid amine and minerals (size of 81-120 bodies/kg); and (2) shrimp by-products (size of
81-120 bodies/kg, head and shell separately) to recovery chitin and protein. Materials were used in fresh
condition after collecting from NhaTrang Seafoods Company (F17), Nha Trang, Khanh Hoa.

2.2. Methodology
The figures and data were collected through experimental methods which were combined between one-
variable-at-a-time technique and response surface methodology; Data analysis conducted by using specialized
soft wares.
The research objects were characterized on the component of mass, the proximate component, and the
composition of acid amine and minerals as well as the changes during storing time when they were kept in the
conditions imitating the real parameters at the shrimp factory.
Finding out the procedures recovering chitin were conducted on heads and shell separately. The aim was
to be estimate the capacity of integrating enzymatic and chemical methods with physical methods.
Demineralization were carried out with HCl in the way how to reduce the side effects of the acidity on the
polysaccharide of chitin. Removing protein were implemented by biological methods: using endogenous
proteases for heads and commercial pepsin for shells. The outcomes were the optimization procedures for
applying autolysis and pepsin process to exclude protein in solid parts and recovery bioactive protein
hydrolysates. Chitin were converted into chitosan through heterogeneous deacetylation in the presence of
5

ultrasound. Sonication were used to facilitate deacetylation process at two point: previous and during the
process. In addition, the kinetic information relevant to protein hydrolyzing with pepsin and deacetylation were
collected.
Based on the data collected from my own experiments and from liturature review, procedures for chitin
and chitosan production were proposed. The products were characterized through deterniming the criteria
involving to purity, molecular weight, degree of acetyl/deacetyl, spectrum of IR, X-ray and NMR as well as
some important physicochemical fuctions; hydrolysates were analyzed its antioxidant capacity through DPPH
and total reducing power tests.
The quality of chitin and chitosan produced were evaluated and compared with the chitin and chitosan
standards which have been promulgated by two companies, AxioGen (India) and Ensymm (Germany). The
differences of the amount of consumption chemicals between that of the proposed procedures and that of the
reference procedure were used to estimated the efficiency, specially focussing on environment aspect.
2.3. Analytical methods
Data were collected through standardized and modern methods, including HPLC, X-ray, FT-IR,

H
1
NMR, and SEM.
2.4. Statistics analysis
Experiments were run in triplicate using three different lots of sample. The statistically differences
between means (p<0.05) were tested using analysis of variance (ANOVA) with the Pairwise Multiple
Comparison Procedures (Tukey Test). SigmaPlot, Origin Pro 8.0, Design Expert 8.0.7, and MINTAB 16.1 were
used to design experiments and analyze data.
2.5. Chemicals and equipments
Pepsin was 107185 0100 from Merck (Germany). Chemicals and reagents were purchaed from Merck or
LoBa company (India).
Ultrasound was creared by ultrasound bath (Model S15-S900H, Elma Co., Germay) and has the
frequency of 37kHz and RMS of 35W.
CHAPTER III: RESULTS AND DISCUSSION
3.1. Characteristics of the white leg shrimp by - product
The mass average ratio of head and shell of shrimp in range of 81-120 bodies/kg was 27.5±3.93 and
11.21± 2.63 (%), respectively, thus the estimative amount of by-products was 38.70±6.46 percentage of the total
number of raw materials processed.
The main constituents of shrimp head and shell were ash, protein, and chitin. Although there is no
significant difference in ash content between the head and shell of shrimp (size of 81-120 bodies/kg): 25.6 % to
32 % dry weight, respectively, the chitin and protein contents of head and shell are largely different. The chitin
content of shell and head of white shrimp were 27.37 and 11.40%, respectively. The chitin content in the shell
was three times higher that than in the head but the heads have up to 50% higher in protein content than the
shell.
The amount of amino acid in heads and shells was approximate 50 and 30 percentage of that in shrimp
meat, respectively. In general, there were slight differences in amino acid composition among three parts of
shrimp and most of essential amino acids were present. Glycine/Arginine, Glutamic/Glutamine,
6

Aspartic/Asparagine, and Alanine predominated of the amino acid profile. However, the amount of Tyr, Phe,

Leu and Val of head and shell part were higher than that of meat one. The contents of K and Cu in the shell and
head were nearly the same whereas the contents of Na, Ca and Fe were significantly different. A small amount
of heavy metal amount (As, Cd, and Pb) were detected in head and under the restricted levels to food. In the
shell, only Pb was found and the level was equal to that in the head. The contents of Se and Hg were under the
limit of detection.
Therefore, both protein and chitin should be recovered from by-product from the production of white leg
shrimps through reasonable procedures to keep their biological functions and to improve their quality as well as
the process efficiency.
3.2. Recovey of chitin and bioactive hydrolysates from white leg shrimp heads
3.2.1. Effects of storage time
The quality of shrimp heads declined seriously when the time of keeping them at room temperature (27-
30
o
C), was increased. The TVB-N value of shrimp head increased continously and nearly exceeded the level of
restriction to food after 4 hours (28.7 mg/100g in compared with the limited level of 30 mg/100). In
consequences, the loss of protein and total weight were rather significant (5.08±1.26% and 15.59±0.44% after 4
hours, respectively). Therefore, shrimp heads should be handled as soon as posible, not more than 4 hours after
removing out of the body so that the quality and pollution were controlled.
Time of delay (h)
0 2 4 6 8
Loss of weight (%)
0
5
10
15
20
25
Content of TVB-N (mg/100g)
0
10

20
30
40
50
Total weight
Protein
TVB-N
a
a
b

bcd
cd

d
bc

B
C
D
DE
E
F
A
E
a

Figure 3.1: Effects of storing time at room temperature (27-30
o
C) on the weight and protein losses of

heads of white leg shrimp and the changes of TVB-N value. Different letters indicate significant differences (p < 0.05).
3.2.2. Studying procedure to recovery proteinand chitin from heads of white leg shrimp
Data corresponding to the zero-hour samples in Figure 3.2 and Figure 3.3 shown that the combination of
using physical force for 2 mimutes to stir strongly shrimp heads and filtering the mixture through net having the
pore size of 1mm was the efficient manner which helped to divide shrimp heads into two parts: the solid was
carapaces and the liquid wad protein. The liquid part contained more than 70 percentage of the total protein
amount of heads wheeras the mass of the solid part was about 7,45± 1,89 percentage of the whole weight of
heads and its protein content was only 20% (db).
However, simultaneous combination of autolysis and physical force enabled not only to improve the
efficiency of nitrogen recovery in the liquid part but also to reduce the protein content of the solid one to
significantly lower level than that in case of using physical force individually. Increasing treatment time, the
7

efficiency of nitrogen recovery, the ratio of antioxidant products, and the degree of deproteinization from shrimp
heads became better and better at any level of supplied water. In spite of that, at the ratio of water to shrimp
heads was 1:1 (v/w) the efficiency of protein recovery, including both nitrogen recovery and antioxidant
products, was in the better tendency, its value was always the highest one corresponding with all of the water
ratios used as well as the protein residue on the carapaces was lowest. Protein hydrolysate collected after two
hour treatment at this ratio had the best capacity of scavenging DPPH radical (Figure 3.5). When the autolysising
time was more than two hours the efficiency of protein recovery and degree of deproteinization at the ratio of 1:1
did not increase significantly on the contrary the antioxidant capacity was in the decreasing trend.
Reaction Time (h)
0 1 2 3 4
Yield of nitrogen recovery (%)
30
40
50
60
70
80

90
100
Yield of antioxidant recovery (%)
3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
4.3
Nitrogen recovery at the ratio of 1:0
Nitrogen recovey at the ratio of 1:1
Nitrogen recovery at the raio of 1:2
Antioxidant recovery at the ratio of 1:0
Antioxidant recovery at the ratio of 1:1
Antioxidant recovery at the ratio of 1:2
a
a
a
a
bc
ab

cde cd
bcd
e e
cde
e

e

Figure 3.2: Effects of treatment time and water ratios
used on the efficiency of recovery of nitrogen and
antioxidant products when autolysising shrimp head
at temperature of 60
o
C and native pH
Time (h)
0 1 2 3 4
Protein content (%)
8
10
12
14
16
18
20
22
Degree of deproteinization (%)
72
74
76
78
80
82
84
86
88
90

92
Protein content at the ratio of 1:0
Protein content at the ratio of 1:1
Protein content at the ratio of 1:2
DP at the ratio of 1:0
DP at the ratio of 1:1
DP at the ratio of 1:2
a
a
a
b
bc
b
bcd

ef
cde
def
g
ef
ef
g

f

Figure 3.3: Effects of treatment time and water
ratios used on protein residues and degree of
deproteinization when autolysising shrimp head
at temperature of 60
o

C and native pH
Different letters indicate significant differences (p < 0.05).


Reaction time (h)
0 1 2 3 4
DPPH (M/g materials)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
a
b b bcd
bcde bcde
bcdef
cdef
def ef
bcdefbcdef
f
bc
bcd
A

Reaction time (h)
0 1 2 3 4
OD at 700nm
0.04

0.06
0.08
0.10
0.12
0.14
0.16
0.18
a
cde
cde
a ab
abc
abc
abcd
abcde
abcde
de
bcde
abc
e
cde
The ratio at 1:0 The ratio of 1:1 The ratio of 1:2
B

Figure 3.5: Effects of treatment time and water ratios used on the capacity of scavenging DPPH radials
(A) and total reducing power (B) of the hydrolysate. Different letters indicate significant differences (p < 0.05).
The protein and minerals content of the carapaces which were collected after autolysising at the optimal
condition (Temperature of 60
o
C, the ratio of water to shrimp heads was 1:1, native pH, 2 hours) and separated by

physical force were 13,78± 0,75%, and 34,23±0,2% % (db), respectively. These carapaces were handled more
deeply to recover chitin. According to the literature, the carapaces were proposed to handle under the condition
combining deminerilization of HCl 0,25M at room temperature during 12h with deproteinization of NaOH 1% at
70
o
C for 8h. The content of protein and minerals in chitin extracted by the proposed procedure were under 1%
(0,59 ± 0,17% and 0,45±0,12%, respectively).
8

In a word, the combination of autolysising at proper condition (Temperature of 60
o
C, the ratio of water
to shrimp heads was 1:1, native pH, 2 hours) and using physical force to stir and filter allowed to recover
protein hydrolysate having antioxidant activity along with chitin efficiently from fresh shrimp heads: the
recovery efficiency of nitrogen and antioxidant products were approximately 86,19±1,67% and 4,09±0,12%,
respectively, moreover, nearly 90 percentage of total shrimp heads could be prevented to treat with chemicals.
However, it is need to carry out more deep studies on bioactive capacity of shrimp protein hydrolysate collected
to seek solutions that enable to commercialize them.
3.3. Recovery of chitin and bioactive hydrolysates from wwhite leg shrimp shells
3.3.1. Treatment with HCl
The curve displaying relationship between the contents of mineral and protein with time during the
process of HCl 0,25M (Figure 3.6) shown that deminerilazation mostly happened in the period of first two hours,
there were 96 percentage of minerals eliminated, after that only a small amount of them were excluded and the
rate of deminerilazation left off at the tenth hour; the remaining contents of protein and minerals were 32,26 and
2,61 percentages (db), respectively. When 96 percentage of minerals were removed out of the shells pH of the
mixture also reached the stable value (around pH value of 1,77±0,06). Therefore, shells were demineralized in
the condition of 0.25M HCl (1:4, v/w), at room temperature, for 2h.
Reaction time (h)
0 2 4 6 8 10 12 14 16 18 20 22 24
Content of minerals (%)

0
5
10
15
20
25
30
Degree of demineralization (%)
40
50
60
70
80
90
100
Content of Minerals
Degree of demineralization

Figure 3.6: The curve displaying relationship between the contents of mineral and protein with time
during the process of HCl 0,25M at room temperature (27-30
o
C)
3.3.2. Estimating the posibility of pepsin
Results in Figure 3.7 shown that the catalysis activity of pepsin facilitated remarkably demineralization
and deproteinization, the increasing level were dependent on the concentration of pepsin used. At the pepsin
concentration of 5U/g protein, extra 40% of protein and 20% of minerals were emilinated in compared to the
controll sample and when the pepsin concentration was 25U/g protein degree of deproteinization and
demineralization reached the maximum with the value of 85,93±0,25% and 90,34±0,9%, respectively. If the
action of HCl were included, the total degrees were 91,16±0,65%; and 99,79± 0,02%, respectively. Although the
difference of the total degree of demineraiazation in two cases of with and without pepsin were not considerable

the disproportion had an significant meaning due to the minerals were removed strictly which made the minaeral
residue were under 1% and the product met the quality criteria of high-value chitin.
9

Pepsin concentration (U/g protein)
0 5 10 15 20 25 30 35
Degree of Deproteinization/Demineralization (%)
0
20
40
60
80
100
120
DP of Pepsin
DM of Pepsin
Total DP
Total DM

Figure 3.7: Effects of pepsin concentration on degree of deproteinization and demineralization
The SEM images of shrimp shell in Figure 3.8 shown that the shell morphology were changed after
treated with HCl, alot of pores appeared in the shell which might support for pepsin penetrating into deeper
layers.

A

B

Figure 3.8: SEM (20kV) images of shrimp shell before (A) and after (B) treated with HCl 0,25M for 2h
3.3.3. Optimization of pepsin process

After analysising experimental data (Table 3.8) through the function of respone surface methodology
(RSM) in software Design Expert 8.0.7 an quadratic model was expoited. Equation (3-2) expressed the
relationship between degree of deproteinization with independent variables including temperature (X
1
, in range
of 30-40
o
C), E/S ratio (X
2
, in the range of 5-25U/g protein) and incubation time (X
3
, in range of 6 - 18h). The
fitted model, expressed in coded variables, is represented by the equation: = 65.33 + 21X
1
+9.875X
2
+
11.375X
3
+ 5.75X
1
X
2
+ 3,75X
1
X
3
– 9.417X
1
2

– 8.167X
2
2
– 11.667X
3
2
(Equation 3-2).
Because the second degree coefficients in Equation (3-2) were all negative, the surface response is
elliptic parabolic with a maxium point. The regression sum squares (R-square) and the adjusted coefficient (R
square-adjusted) were at the level more than 99.9% and the value of lack of fit was 0.47 as well as the results in
Table 3.9 clearly indicated that the predicted model well fitted the experimental data. The Pred-R
Squared
, of 0.958
meant that the data estimated by Equation (3-2) had the accuracy of 95.8% in compared to experimental data.
Results in Table 3.9 were in agreement. This once again confirmed the reliability of Equation (3-2) and it was
able to be used for controlling the process of handling shrimp shell by pepsin in reality.
The optimal conditions were temperature at 40
o
C, reaction time of 16h, E/S ratio of 20U/g protein at
pH=2. After treatment, approximately 92% of protein in shrimp shells were removed and the residues of protein
and minerals were 8,2±1,6% and 0,56±0,04%, respectively
10

Table 3.8: The Box-Behnken design of the experiments and response of deproteinization
N
o

X
1
,

o
C
X
2
, U/g.protein
X
3
, h
Y, (%)
*

N
o

X
1
,
o
C
X
2
, U/g.protein
X
3
, h
Y, (%)
*

1
-1

-1
0
41,89
9
0
-1
-1
41,08
2
1
-1
0
65,30
10
0
1
-1
58,57
3
-1
1
0
45,64
11
0
-1
1
57,11
4
1

1
0
86,95
12
0
1
1
76,09
5
-1
0
-1
34,99
13
0
0
0
73,15
6
1
0
-1
61,91
14
0
0
0
73,17
7
-1

0
1
46,77
15
0
0
0
73,76
8
1
0
1
85,55





*
Mean ± SD (n=3)
Table 3.9: Observed and predicted values of the confirmation experiments

Trials

Condition
DP (%)
*

Observed
Predicted

1
X
1
=40
o
C ; X
2
= 10U/g.pro; X
3
=15h
82,41 ± 0,97
81,25
2
X
1
=40
o
C ; X
2
= 12,5U/g.pro; X
3
=14h
86,58 ± 0,51
85,39
3
X
1
=40
o
C ; X

2
= 15U/g.pro; X
3
=15h
89,87 ± 0,19
89,52
4
X
1
=40
o
C ; X
2
= 15U/g.pro; X
3
=16h
90,22 ± 0,14
89,74
5
X
1
=40
o
C ; X
2
= 20U/g.pro; X
3
=16h
93,29 ± 0,16
92,48

*
Mean ± SD (n=3)
3.3.4. The posibility of using sonication to facilitate pepsin activity in chitin extraction
Figure 3.12 shown that sonicating time (at 37kHz, RMS=35W) had a significant impact on the activity
of pepsin, in the interval of first 25 minutes, the catalysis of pepsin were directly proportion to time, the acivity
was increased by 8% after 20-25 minutes of treatment, however, extending time caused opposite effect, pepsin
activity trend to go down (p<0,05), after 40 minutes of unbroken sonication pepsin activity was no more
different with that in case of no sonication and if time prolonged more catalysis of pepsin were lower than that of
the control (p<0,05).
Time of treatment (min)
0 20 40 60 80 100
Enzyme activity (U/mg)
36
38
40
42
44
46
With sonication
Without sonication

Figure 3.12: Effects of sonication time on pepsin
activity (37kHz, 35W)
Time of treatment (min)
0 5 10 15 20 25 30
Enzyme activity U/mg)
36
38
40
42

44
46
With sonication
Without sonication
ab a
abc
abcde cde bcde

ef
def
abc
abc
abc
abcd abcd

Figure 3.17: Effects of sonicating pepsin on
deproteinization from shrimp shell (20U/g protein,
40
o
C, pH=2).
Different letters indicate significant differences (p < 0.05).
Figure 3.17 shown that degree of deproteinization that were achieved after 14h of treatment with 25min-
sonicated pepsin and that gained after 16h of treatment with non-sonication pepsin was no significant and
prolonging processing time with sonicated pepsin more than 25 min did not bring any better results (Figure
11

3.17, the small). Therefore, sonicating pepsin for 25 min before deproteinization from shrimp shell enabled to
reduce processing time to 2 hours
3.3.5. Improving the proposed procedure to recover chitin and protein from white leg shrimp shells
The second step to deproteinize was ininitated by NaOH 1% (with the ratio of materials to solution =

2:1, v/w), for 8 hours at 70
o
C. Chitin produced was refined upon a high level of purity (both ash and protein
contents were under <1%, respectively, 0,56±0,04% and 0,79±0,02%); structure of polysacchride chains was
almost not attacted (DA= 97,01±0,85% and viscosity average molecular weight of chitin M
v
= 1652Da); the
product was suitable for processing further chitin derivaties which had bioactivity and very useful, such as N-
acetyl glucosamine. Protein hydrolysate was valuable to produce bioactive substrates having antioxidant
capacity with the yield of 3,52±1,54% (Table 3.10).
Table 3.10: Data relevant to protein recovery from shrimp shell by pepsin


Chỉ tiêu

Nitrogen
recovery
a

(%)
Ratio of antioxidant
recovery
b
(%)
Antioxidant capacity
For solution having
concentration of 1mg/ml
In comparison to
BHA (1mg/ml) (%)
64,2±2,7

3,52±1,54
DPPH
(mM)
TNLK
(OD
700nm
)
DPPH

TNLK

0,13±0,01
0,1640±0,015
46,79±4,19
46,02±1,67
a
In comparison to total of nitrogen in raw materials;
b
In comparison to the weight of raw materials.
3.4. Kinetics of deproteinization by pepsin
The logarithmic variation of the percentage of protein remaining in chitin which was plotted as a
function of the deproteinization time at 40
o
C by pepsin in Figure 3.22 revealed that the deproteinization from
shrimp shells appears to obey first-order reaction kinetics. The deproteinization mechanism could be described
by a first-order equation dP/dt = -kP in each of three domains, where P represents the protein contain remaining
in the shrimp shell, t the treatment time, and k the reaction rate constant.
Time (h)
0 2 4 6 8 10 12 14 16 18 20 22 24
Ln (P/Po)

-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
k
2
k
3
k
1

Figure 3.22: Logarithmic variation of the protein content in chitin as a function of the deproteinization
time performed at the optimal condition (Pepsin concentration of 0.42g/L, pH2, 40
o
C). The initial protein
concentration is 15g/L equally 250g of demineralized shrimp shells L
-1
.
The results in Table 3.11 shown that the rate constants kept the same value within every phase and
decreased abruptly when change to next phase with k
1
= 0.72x10
-2
, k
2
=3.05x10
-3

, k
3
=6.5x10
-4
and the rate
constant of the second phase was only a half of that one of the first phase, whereas at the third phase the rate
constant reduced nearly ten times. The abrupt change of rate constant shown that the binding between protein
and chitin could be structured with different layers.
12

The behavior of the kinetic model in our study shown the same trend as the models which were
established for reaction with NaOH. However, in our work the curve was broken into three fragments with the
difference in rate constants. The study of Percot (2003) for NaOH deproteinization shown that in the first stage
the values of rate constants were higher than those of pepsin; In the second stage, the latter was a half of the
former at 50
o
C or both were nearly the same at 70
o
C but in the third the rate constant of pepsin was sinificantly
higher (Table 3.11).
Table 3.11: Comparison of rate constants between deproteinization by pepsin and NaOH treatment
Rate constants
Pepsin treatment
([E] =0.42 g/L, E/S = 1.68/1000 g/g
demineralized shrimp shells)
*

NaOH treatment
(1M, 15mL/g demineralized shrimp shells)
**

40
o
C
50
o
C
70
o
C
k
1
(10
-2
min
-1
)
0.72 ± 0.092 (r = 0.98)
2.10 ± 0.04
2.68 ± 0.05
k
2
(10
-3
min
-1
)
3.05 ± 0.57 (r = 0.98)
3.12 ± 0.16
1.52 ± 0.08

k
3
(10
-4
min
-1
)
6.5 ± 0.01 (r = 0.83)
0.48 ± 0.1
1.53 ± 0.27
*
Mean ± SD (n=3);
**
According to Percot, 2003.
Deproteinization by pepsin mostly took place in the two first hours of the process; The results relation to
kinetics and regression analysis allowed to draw that degree of deproteinization (DP) and its rate (r) in this
period obeyed Equation (3-9) and Equation (3-10), respectively and the value of the rate constants were k
2
=
40,983 (min
-1
) and k
d
(=k
3
*K
m
) = 1,535 (min
-1
).

 


     




    (Equation 3-9)
   




  

 



 (Equation 3-10)
3.5. Enhancement of heterogenous deacetylation
3.5.1. Effects of chitin pretreatment
Figure 3.27 shown that pretreatment of chitin before handling with NaOH considerably facilitated
deacetylation. Degree of deacetylation of the samples which were pretreated by soaking with hot water or by
sonication were 20% higher than that of the control when all of them were deacetylated by conventional
procedure (NaOH 60% (w/w), 3h). There were not significantly differenct between the efficiency of two
manners of pretreatment (p>0,05).
Samples
DD (%)

50
60
70
80
90
100
With sonication
With hot water
Control
a
a
b

Figure 3.27: Effects of chitin pretreatment on deacetylation.
Different letters indicate significant differences (p < 0.05).
SEM images in Figure 3.28 shown that the surface of chitin sonicated rougher and had more wrinkles
than that of chitin treated with hot water as well as the results were drawn from XRD spetra by Origin Pro 8.0
13

software (Table 3.13) indicated that in compared with the control Crystalline Index (χ
cr
) of chitin which were
pretreated with ultrasound and hot water reduced by 1,38 and 2,54%, respectively. It was supposed that
pretreatment of chitin enable to decrease crystal area therefore the efficience of deacetylation were improved.
Thus, proposed pretreatment was that soaking chitin in hot water of 60
o
C for 60 min.
A

B

Figure 3.28: SEM (10kV) images of chitin treated for 60 min at 60
o
C with hotwater (A) and
ultrasound(B).
Table 3.13: Crystalline Index of chitin after treatment
Sample
CrI
020
(%)
CrI
110
(%)
χ
cr
(%)
Control
90,09
95,85
74,12
Hot water
88,68
95,05
71,58
Ultrasound
90,13
95,97
72,74
3.5.2. Screening the supporting posibility for deacetylation of sonication
Figure 3.30 shown that when chitin was deactylaed with NaOH solution having concentration in range

of 35-65%, w/w, ultrasound had impact on both solubility and degree of deacetylation but they were in different
extent. The influence of sonication on degree of deacetylation was significant when NaOH concentration was
NaOH ≤45%, DD of the sonicated samples were considerably higher than those of the controls (p<0,05), the
value of DD were in direct proportion to NaOH concentrations. However, when NaOH concentration were
NaOH ≥50% the effects of sonication and NaOH concentration were not significant, DD of the sonicated
samplea and controls were not significant different (p>0,05). In the same deacetylation condition of NaOH
concentration and reaction time,the solubility of the soncated samples were always higher than those of the
controls. However, the influence decreased when the NaOH concentration were increased and when the
concentration reached to 65% there was no remarkable differnce (p>0,05). In comparison with DD, sonication
had more impact on solubility, the impact was continued until NaOH concentration was up to 60%.
NaOH concentration (%, w/w)
35 40 45 50 55 60 65
Degree of deacetylation (%)
20
30
40
50
60
70
80
90
NaOH concentration (%, w/w)
35 40 45 50 55 60 65
Solubility (%)
20
40
60
80
100
With sonication

Without sonication
A B
a
b
a
c
c
e
ef d
fg g
efg efg efg efg
a b

c
e c
f hi ij ijhg

Figure 3.30: The relationship between DD and solubility with NaOH concetration and deacetylating
means as function of time during 6h at 80
o
C.
Different letters indicate significant differences (p < 0.05).
14

The properties of chitosan were produced in the present of sonication and in condition without
sonication (80
o
C, 4h, NaOH=60%, w/w) were characterised by XDR and FT-IR spectra. XRD spectra in Figure
3.31 indicated that there was a small change in case of the sonicated sample at the position of 020, from
2θ=9,54

o
to 2θ=10,08
o
which was accompanied with a slight reduction of Crystalline Index, after 4 hours of
deacetylation degree of crystallinity (χ
cr
) of the sonicated and control samples were 71,38% and 72,81%,
respectively.

Figure 3.31: XRD spectra of deacetylated products in conditions of [NaOH]=60% at 80
o
C for 4h with (A)
and without sonication (B)


Figure 3.32: FT-IR spectra of deacetylated products in condition of [NaOH]=60% at 80
o
C for 4h with (red
line) and without sonication (black lie)
FT-IR spetra of chitosan samples treated in condition with and without sonication in Figure 3.32 shown
that these of two chitosans had similar peaks as those which were characterised in the spectum of chitosan by
Rinaudo (2006). It means that ultrasound at the frequency of 37kHz (35W) did not have any signifficant impact
on chemical bonds in chitosan moleculars. However, in the FT-IR of chitosan deacetylated with sonocation the
absorption bands at position of 1560 and 1312 cm
-1
(ascribed to amide II and amide III) were lower and the peak
at 1415cm
-1
was sharper, all of those proved that sonication facilitated deacetylation and there was an increasing
DD in the sample sonicated.

3.5.3. Kinetics of deacetylation process with sonication
The changes of DD values of chitosan deacetylated with and without sonication (37kHz, RMS 35W) in
the condition of 80
o
C, 4h, NaOH=35-60%, w/w, were in the same pattern: DD rapid increased as a function of
time until it reached the maximum and leveled off; the happening moment was indirect proportion to NaOH
concentration: the higher NaOH concentration the faster, except NaOH concentration of 35%, w/w. In the same
0 10 20 30 40 50
-20
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50
-20
0
20
40
60
80
100
120
140
160
10.08273

20.1758
38.59359
9.54498
20.21717
Lin (counts)
2THETA
Lin (counts)
2THETA
A
B
3448.70
2922.26
1657.23
1421.75
1380.14
1321.87
1262.14
1154.35
1082.15
1030.95
896.01
798.13
773.65
669.51
630.70
612.42
583.04
543.54
531.02
500600700800900100012001400160018002000230026002900320035003800

Wavenumber cm-1
40 45 50 55 60 65
Transmittance [%]
15

condition of NaOH concentration and time, the samples treated in the presence of sonication always leveled off
sooner and reached the higher maximum value of DD in compared with those of the control samples (Figure
3.33).
Reaction time (min)
0 60 120 180 240 300 360
DD (%)
0
10
20
30
40
50
Reaction time (min)
0 60 120 180 240 300 360
DD (%)
0
20
40
60
80
Reaction time (min)
0 60 120 180 240 300 360
DD (%)
0
20

40
60
80
100
Reaction time (min)
0 60 120 180 240 300 360
DD (%)
0
20
40
60
80
100
Reaction time (min)
0 60 120 180 240 300 360
DD (%)
0
20
40
60
80
100
A
B E
Without sonication
With sonication
D C

Figure 3.33: The DD of the resulting chitosans during heterogenous alkaline deacetylation reactions in
condition with and without of sonication at 80°C and different NaOH concentrations
(A) 35%, (B) 40%, (C) 45%, (D) 50% và (E) 60% (w/w).
*
Mean ± SD (n=3)
The rate values of deacetylation reaction in different conditions (Table 3.16) shown that deacetylation
process reduced the rate as function of time and it nearly happened in the first hour, especially in the first fifteen
minutes. In the first period (0-15 min), the rate of deacetylation always was in bigger values when NaOH
concentration was increased as well as when sonication was applied whereas in the latter periods (2, 3 and 4) the
rates were in reverse tendency it went down when deacetylation were carried out in the condition of higher
NaOH concentration and sonication, except the case of NaOH 35%. After 4 hour of deacetylation, the rates of
the reactions were too small, reaching nearly zero even if sonication was used.
Table 3.16: The rate of deacetylation reaction (%/min) (x10
2
)
a
Period
b

Deacetylation condition
NaOH con centration (%) (w/w)
Without soncation
With sonication
35
40
45
50
60

35
40
45
50
60
1
18,58
44,53
173,58
271,19
311,32
78,58
196,35
252,32
355,94
416,37
2
54,41
63,81
51,87
49,23
59,66
55,58
50,62
42,19
37,62
37,51
3
2,25
11,30

10,76
4,96
3,14
1,61
8,43
8,70
2,56
1,41
4
0,24
8,08
3,90
3,24
1,87
0,16
3,77
6,55
3,53
1,88
a
Mean ± SD (n=3) ;
b
Period 1: 1 (0-15 min); 2 (15-60 min); 3 (60-240 min); 4 (240 -360 min)
Deacetylation processes in condition with and without sonication followed Pseudo-first order kinetics
and were expressed by the equation dX/dt = k* X, where X was DD value of the sample at the moment t of
deacetylation and k was the rate constant; however, the value of k always changed in NaOH concentration, time
and the manner of deacetylation (with or without of sonication) and it had the same trend as that described above
for the rate of deacetylation (Table 3.17)

16

Table 3.17: The rate constant of deacetylation reaction (min
-1
) (x10
3
)
a
Period
b

Deacetylation condition
NaOH con centration (%) (w/w)
Without soncation
With soncation
35
40
45
50
60
35
40
45
50
60
1
43,81
78,11
151,36
178,58
187,19

106,36
158,77
174,11
195,62
205,55
2
36,78
30,63
13,11
9,12
9,60
22,01
11,82
8,48
5,83
5,10
3
0,70
2,36
1,75
0,71
0,40
0,39
1,35
1,29
0,34
0,17
4
0,07
1,27

0,53
0,42
0,22
0,04
0,52
0,82
0,44
0,22
a
Mean ± SD (n=3) ;
b
Period 1: 1 (0-15 min); 2 (15-60 min); 3 (60-240 min); 4 (240 -360 min)
Therefore, ultrasound (37kHz, 35W) had the capacity for supporting deacetylation, it increased the rate
of the process and the uniform of products, facilitated the solubility but not changed the nature of deacetylation
as well as chemical bonds in chitosan molecular in comparison with conventional deacetylation.
3.5.4. Effects of NaOH concentration, temperature and reaction time on DD and solubility of chitosan
produced by heterogenous deacetylation with the facility of sonication
The influcence of temperature (70-80
o
C), reaction time (2-6h), and NaOH concentration (40-60%) on
deacetylation process in the presence of sonication was investigated with two-level factors model and the results
shown in Table 3.19 and Table 3.20 revealed that all of NaOH concentration, time and temperature as well as
interaction of temperature and time, interaction of concentration and time and interaction of temperature and
concentration played dominant role on DD and solubility of chitosan within the experiment range (p<0.05);
Among of them, NaOH concentration was the most important term with its level of 26,73% and 52,65% on DD
and solubility, respectively. The second important term on solubility was temperature but in case of DD that was
the term of time. The effect levels of temperature on DD and solubility were 11,10% and 3,73%; whereas those
of time on DD and solubility were 5,85% and 10,13%, respectively.
Table 3.19: Analysis of variance from chitosan’s
data for response degree of deacetylation (





(p=0,05)
Parameters
Effect (%)
Coefficient
Prob>F
X
0


70,080
0,001
X
1

3,751
1,875
0,001
X
2

10,138
5,069
0,001
X
3

26,731
13,365
0,001
X
1
X
3

-1,967
-0,984
0,004
X
2
X
3

-9,017
-4,509
0,001

Table 3.20: Analysis of variance from chitosan’s
data for response solubility (



(p=0,05)
Parameters
Effect (%)
Coefficient
Prob>F

X
0


68,225
0,001
X
1

11,100
5,550
0,001
X
2

5,850
2,925
0,007
X
3

52,650
26,325
0,001
X
1
X
3

-6,300

-3,150
0,005
X
2
X
3

-3,650
-1,825
0,031

X
1
: Temperature (
o
C); X
2
:Time (h); X
3
: Concentration (%);
The best-fit regression equations for the relationship of these terms with DD (


) (%) and solubility (


)
(%) obtained from the statistical analysis were Equation (3-11) and Equation (3-12), respectively.




70,081

+ 1,875 X
1
+5,069 X
2
+13,365 X
3
- 0,984 X
1
X
3
- 4,509 X
2
X
3
(Equation 3-11)



= 68,225

+ 5,55 X
1
+2,925 X
2
+26,325 X
3
- 3,154 X

1
X
3
- 1,825 X
2
X
3
(Equation 3-12)
Respond surface for the changes in DD and solubility as a function of NaOH concentration, time and
temperature indicated that in order to produce chitosan having DD > 70% and solubility > 85% the deacetylation
must be conducted in condition of NaOH≥ 50% and reaction time ≥ 4h at 80
o
C.
3.5.5. Optimal conditions for deacetylation with sonication
17

In order to investigate the optimal condition of deacetylation, DD and solubility of samples obtained
from various conditions (Table 3.21) were statistically analyzed using respone surface methodology with
MINITAB 16.1 software. The best fit regression Equation (3-13) and Equation (3-14) for the optimum DD (


)
(%) and solubility (


) (%), respectively, within the experiment range were obtained.



= 85,8035+ 3,0240 X

1
+2,069 X
2
- 1,2260X
1
2
- 1,2865X
2
2
-0,5759X
1
X
2
(Equation 3-13)



= 96,5179+ 3,0600 X
1
+1,3300 X
2
+0,6400X
1
2
- 0,5100X
2
2
-1,2175X
1
X

2
(Equation 3-14)
Table 3.21: Results of the experimental matrix with Central Composite design

X
1
: Concentration (%); X
2
:Time (h)
Results of statistical analysis summaried in Table 3.23 allowed to confirm the accuracy of Equation (3-
13) and Equation (3-14). These two Equation could explain 97,85% and 95,76% for the experimental figures
corresponding to the respones of DD and solubility, respectively.
Table 3.23: Results of statistical analysis for Equation (3-13) and (3-14), (p=0,05)
Terms
Respone
Degree of deacetylation



, %)
Degree of Solubility



, %)
PRESS
5,62
5,43
R-Sq (%)
98,84

97,72
R-Sq(pred) (%)
94,18
93,03
R-Sq(adj) (%)
97,85
95,76
Lack of fit
0,48
0,91
Table 3.25: Effects of deacetylation condition on the viscosity average molecular weight of
chitosan (Mv, kDa)
Deacetylation
condition
Viscosity average molecular weight of
chitosan (M
v
), (kDa)
With sonication
Without sonication
Temperature (
o
C)
80
70
80
NaOH Concentration (%)
50
60
50

50
60
Time (h)

0
1652, 00
1652,00
1652,00
1652,00
1652,00
2
592,61
539,12
-
-
-
4
468,53
404,21
-
-
-
6
421,82
361,84
473,26
439,90
378,53
8
377,37

323,55
-
-
-
*
Mean of duplicate
Due to deacetylation and depolymerization reactions always happened simultaneously and affected on
the properites of chitosan, the average molecular weight were studied in the resulting chitosan. Results in Table
3.25 shown that viscosity average molecular weight of chitosan (M
v
) was dependent on the condition of deacetylation
(temperature, time, concentration and the presence of sonication).
N
o

X
1
,
%
X
2
, h
DD
(%)
Solubility
(%)
N
o

X

1
, %
X
2
, h
DD
(%)
Solubility

(%)
1
-1
-1
78,31
91,56
8
0
0
80,89
93,65
2
+1
-1
85,41
100,00
9
+1
0
87,13
100,00

3
-1
+1
83,32
96,43
10
0
-1
81,60
94,12
4
+1
+1
88,12
100,00
11
0
+1
86,30
97,23
5
0
0
86,34
96,14
12
0
-1
85,95
95,78

6
0
0
85,98
97,13
13
0
0
84,94
95,78
7
0
0
86,12
97,45
14
0
0
85,76
96,76
18

In the same other condition of deacetylation (for 6h, NaOH concentration of 60%, (w/w)) viscosity
average molecular weight of chitosan produced at 70
o
C were significant higher than that of chitosan collected at 80
o
C
(473,26 so với 421,82kDa); chitosan deacetylated in conventional condition, without sonication, had the average
molecular higher than that in case of sonication but the differences were not remarkable (439,90 and 378,53 kDa

in compared with 421,82 and 361,84 kDa regarding to NaOH concentration of 50% and 60%, respectively at the
temperature of 80
o
C for 6h).
Average molecular weight of chitosan reduced when reaction time incerased in some extent and the rate
of reduction was different and dependent on duration of deacetylation. The rate of depolymerization was rather
high during the first two hour, the average molecular weight reduces about 64-67% only after two hours of
deacetylation (from 1652kDa to 592,61 and 539,12 kDa regarding to NaOH concentration of 50% and 60%,
respectively). After that, the rate of depolymer had a slower trend, reduction of approximate 20 and 25% were
observed after 6h and 8h, respectively, when deacetylation were conducted at NaOH concentration of both 50%
and 60%.
The results of regression analysis of logarithmic variation of of avarage molecular weight as a function
of the deacetylation time performed during the period of 2 to 8 hours revealed that the trentency of deacetylation
reaction in case of sonication was the same as that in case of conventional deacetylation and they followed
Pseudo-first-order reaction (Figure 3.38).
Time (h)
2 4 6 8
Lg(Mv)
2.4
2.5
2.6
2.7
2.8
2.9
y = 2,8198-0,0317*t (R=0,9573)
y = 2,7800-0,0352*t (R=0,9363)
NaOH 50%
NaOH 60%

Figure 3.38: Effects of NaoH concentration and time on viscosity average molecular weight of

chitosan deacetylated in the presence of sonication
Equation (3-15) and (3-16) indicated the rule of the change in average molecular weight as a function of
time when deacetylation was carried out in the condition of 80
o
C, NaOH 50% and 60%, respectively. These
equations allowed to determine the exact deacetylation condition (time and NaOH concentration) to collect
chitosan having required molecular weight.



= 2,8501 - 0,0404*t (Equation 3-15)



= 2,7767 - 0,0388*t (Equation 3-16)
Therefore, four equations marked from Equation (3-13) to (3-16)) enablde to be controlled deactylation
at 80
o
C in the presence of sonication (37kHz) to achieve chitosan having desirable DD, Mv and solubility.
3.6. Proposing the procedures applied new technology to recover chitin, chitosan and protein and
estimating their benefits
3.6.1. The proposed procedures to recover chitin, chitosan and protein
19

Based on the results achieved in my own study it declare that the proposed technology which were
integrated by physical, enzymatic and chemical methods allowed to innovate the production of chitin and
chitosan from by-products of the manufacturing white leg shrimp. The procedues were put forward in Figure
3.39, Figure 3.40 and Figure 3.41.
Fresh shrimp heads after removing out of shrimp body were conveyed as soon as possible to reaction
vessels having propellers. They were mixed with an equal amount of clean water, and heated up to 60

o
C and
then kept for 2 hours to conduct autolysisat at native pH. When finishing the autolysis, the mixture was stirred
strongly at the rate of approximate 1000 r/min and filtered through sieve plates having the pore size of 1mm to
retain the head carapace (solid phase). The liquid phase was centrifugate at 10,000xg at 4
o
C for 10 min. The
suspernatant was a solution containing the protein hydrolysate with antioxidant activity. It depended on the
purpose of use the supernatant was dried and purified futher. The solid phase (chitinous residue) was used for
chitin extraction and chitosan production. They were treated with HCl 0,25M (at the ratio of 1:4, w/v) for 12h at
room temperature and then washed to neutral pH and drained. Next, they were mixed with NaOH 1% (at the
ratio of 1:2, w/v) to deproteinization for 8h at temperature of 70
o
C. Chitin were collected, washed to neutral pH
and dried at the moisture of lower 10% before storage in dry place.

Fresh
shrimpheads
Autolysis
(2h, 60
o
C, native pH, ratio of H
2
O to materials =1:1(v/w)
Stiring for 2 min, rate of 1000 r/min
Carapaces Protein hydrolysate
Treated with HCl 0,25M for 12 h at
room temperature, the ratio of HCl to
materials=4:1(v/w)
Treated with NaOH 1%, 8h,

70
o
C, the ratio of NaOH to
materials = 2:1(v/w)
Washing to neutral pH
Washing to neutral
pH
Drying Chitin
Drying
Filtering
(Pore size of 1mm)
The powder

Figure 3.39: Proposed procedue for recovering chitin
and protein from heads of white leg shrimp
Fresh shell
Demineralization
(HCl 0,25M for 2h, at room temperature, the
ratio of HCl to materials =4:1
Washing and pressing
Deproteinization by pepsin
at pH =2, for14h, 40
o
C
Sonicating pepsin solution
for 25min
SeparatingHydrolysates
Drying
Deproteinization by NaOH
1%, for 8h at 70

o
C, the ratio of
NaOH to materials = 2:1
Washing to
neutral pH
Drying
Chitin
Solid
Pressing
The powder
Preparation of pepsin
solution (20U/g protein)

Figure 3.40: Proposed procedue for recovering
chitin and protein from shells of white leg shrimp

Shrimp shell after pressing to remove dipping water were demineralization with HCl 0,25M (at the ratio
of 1:4, w/v) at room temperature for 2h. Then, the mixture was decanted and the solid phase was collected and
washed roughly before treatment with pepsin. The demineralized shells was soaked in the solution of pH2 (at the
ratio of 1:3, w/v), all were heated up to 40
o
C and then mixed with pepsin at the concentration of 20U/g protein.
The deproteinization process with pepsin was carried out at 40
o
C for 14 or 16h which depended on pepsin
previously submitted to ultrasound for 25 min or not. When the time was over, the mixture was handled to
seperate into the liquid and solid parts. The former part was treated in the same manner which was mentioned
above for that collected from shrimp heads. The latter part was mixed with NaOH 1% (at the ratio of 1:2, w/v) to
20

deproteinization for 8h at temperature of 70
o
C. Chitin were collected, washed to neutral pH and dried at the
moisture of lower 10% before storage in dry place.
According to the purpose of using chitiosan the required Mv, DD and solubity were determined and
from that the parameters of deacetylation at 80
o
C (reaction time and NaOH concentration) were calculated based
on Equation (3-13), Equation (3-14) and Equation (3-15) or Equation (3-16).
Chitin (flake form) was previously submitted to hot water treatment for 60 min at 60
o
C and decanted
before deacetylation reaction was conducted in the specified condition of NaOH concentration and time with the
ratio of chitin to NaOH solution 1:15, w/v. Sonication was applied during the deacetylation and the temperature
was constant at 80
o
C. When the deacetylation process was finished chitosan was soaked and washed to neutral
pH before drying to the moisture of less than 10%. The product was kept in dry and hermetic packaging

Soaking in hot water of 60
o
C for 60 min
Wasing to neutral pH
Drying
Chitosan
Chitin
Sonication
(37kHz, 35RMS)
Pressing
Determine the required values of DD

Mv and solubility of chitosan
Deacetyl at 80
o
C
Based on Equation (3-13), (3-14) ,
(3-15) or (3-16)
Calculating the reasonable condition
for deacetylation (Time, NaOH
concentration)
Clarify the purpose of use

Figure 3.41: Proposed procedue for determining parameters of deacetylation
3.6.2. Characteristics of chitin and chitosan produced according to the proposed procedures
Chitin and chitosan produced from shrimp heads and shells according to the proposed procedures in
Figure 3.39, Figure 3.40 and Figure 3.41 were characterized and their properties were presented in Table 3.26
and Table 3.27. The results in Table 3.26 shown that the quality of chitin produced from by-product of white leg
shrimps met the specifications of commercial chitin that were declared by AxioGen Co (India), all of the test
iterms were in range of required values.
Table 3.26: Quality of chitin produced according to the proposed procetures
Test Items
Specifications of commercial chitin
b

Test resuts

From shells
From heads
Colour
Off yellow
Pinky white

Pinky white
Ash (%, db)
<1
0,57±0,07
0,68±0,05
Protein (%, db)
-
0,66±0,1
0,79±0,04
Degree of acetyl (%)
>90
97,01±0,85
94,32±0,29
Moisture (%)
<10
8,70±0,79
8,07±0,56
Viscosity average molecular weight of
chitosan (M
v
, kDa)
-
1652
1232
Hevy metals (ppm)
≤ 15
0,29
Insoluble materials (%)
<1
<1

a


a
Disolved by NaOH 40% in addition of ure, for 72h at 6-10
o
C;
b
: According to AxioGen Co, Ấn Độ.
21

By the way, due to restriction of contact with HCl solution having high concentration the polysaccharide
chains of chitin produced according to the proposed procedures were affected insignificantly, the viscosity
average molecular weight of chitosan were rather high (1232 and 1652 kDa regarding to chitin from heads and
shells). The reduction in both of NaOH concentration used and its treatment times also made the benefit of
lessen the side effects of NaOH on the structure of chitin produced, their degree of acetylation were high ((94%
and 97% regarding to chitin from heads and shells). The chitin produced by the proposed procedures had the
properties suitable for N-acetyl glucosamine production, one of products has had high demand and high price.
The test results relevant to the properties of chitosan deacetylated in the condition of sonication (80
o
C,
4h, NaOH 60%) in Table 3.27 indicated that chitosan produced according to the proposed procedure was almost
met the required quality of commercial chitosan applied for food, medical and pharmacy aspects which were
announced by AxioGenCo. (India) and Ensymm (Germany) except only two specifications (Turbility and
Transition temperature). However, in order to sastify all of the specifications the chitosan needed to be improved
in bulk density.
Table 3.27: Quality of chitosan deacetylated in the presence of sonication at 80
o
C, [NaOH]=60% for 4h
(chitin collected from shells applying the proposed procedure)

Test Items
Specifications of commercial chitosan
a, b

Test results
Food
Medical
Pharmaceutical
Colour
Off white
White or yellow
White
White
Ash (%)
-
<0,5
-
0,66±0,05
Protein (%)
-
<0,3
<1
0,55±0,05
Degree of deacetyl (%)
-
66-99,8
90-95
84,84±1,22
Moisture (%)
-

<10
<8
8,7±0,7
Molecular weight (kDa)
-
< 4000
-
404,21
Bulk density (g/cm
3
)
0,50 - 0,6
1,35 - 1,40
0,50 - 0,6
0,62±0,04
Solubility (%)
-
>99
a
; >99,8
b

-
99,03±0,1
Turbility (NTU)
-
≤ 30
-
NA
Transition temperature (

o
C)
-
203
-
NA
Fat Binding Capacity (g/g)
≥5,0
-
≥5,0
6,89±0,5
Apparent viscosity (Dung dịch 1% trong axit 1%) (cP)
-
20 - 1600
-
1209±103
Asenic (ppm)
ND
**

<2
<2
ND
Cadmium (ppm)
ND
<0,2
<0,2
ND
Pb (ppm)
≤10,15

<1
<1
0,22
Hg (ppm)
ND
<0,05
<0,05
ND
Total plate count (cfu/g)
<50,000
<1000
<1000
750
E.Coli (cfu/g)
ND
ND
ND
ND
Listeria (cfu/25g)
ND
-
ND
ND
Pseudomonas (cfu/25g)
ND
-
ND
ND
Staphylococcus aureus (cfu/g)
ND

-
ND
ND
Coliform (cfu/g)
ND
ND
ND
ND
Salmonella (cfu/25g)
ND
ND
ND
ND
Mold and Yeast (cfu/g)
<1000
<100
<100
ND
*
NA: not determined;
**
ND: not detectd;
a
: According to AxioGen Co., India,
b
: According to Ensymm co., Germany.
3.6.3. Estimating the efficiency of the proposed procedures
The important data regarding to demineralization, deproteinization and deacetylation which were applied
to recover chitin, protein and chitosan according to the proposed procedures were presented in Table 3.28. These
figures shown that the quality of chitin produced by the proposed procedures was enhanced in compared with

that of chitin produced by the reference procedure, including: (1) higher purity (the residues of protein and
22

minerals of the former chitin were below 1% whereas the protein content of the latter chitin from heads was
above 1%); (2) higher degree of acetyl and viscosity average molecular weight.
The proposed procedures extracting chitin enabled to obtain more benefit of recovering protein in the
form having antioxidant activity as well as retricting a remarkable amount of chemicals consumed and wastes
(due to reducing a large amount of protein and chemicals in the effluent from demineralization and
deproteinzation), in details: the yield of antioxidant product was 3,52±1,54 và 4,09±0,12%; the amount of
concentrated HCl and the amount of anhydrous NaOH consumed when extracting chitin from heads and shells
were reduced 75,67 and 96,26%; 86,67 and 97,95%, respectively. The proposed procedues also allowed to
improve the yield of chitin, 32,87% and 2,8%, regarding to heads and shells. In addition, protein was recovered
thoroughly helped decreasing amount of protein in the effluent in chitin production which solved the
shortcomings in environment issues.
Table 3.28: Comparison between the products produced by proposed and reference procedures
(Estimated to 1000kg raw materials/bath)
Test Items
Proposed procedure
a

Reference procedure
b
[9]
Shells
Heads
Shells
Heads
CHITIN
Protein content (%, db)
0,66±0,1

0,79±0,04
0,81±0,10
1,57±0,10
Ash content (%,db)
0,57±0,07
0,68±0,05
0,66±0,10
1,33±0,10
Molecular weight (kDa)
1652
1232
959,58
735,6
Degree of acetyl (%)
97,01±0,85
94,32±0,29
88,17±1,54
83,44±0,57
Yield (%)
d

6,85 ± 0,09
3,84 ± 0,18
6,66±0,13
2,89 ± 0,11
Processing time (h)
c

24
22

36
36
Amount of concentrated HCl (L)
77,33
8,59
317,8
229,52
Amount of anhydrous NaOH (kg)
18
2
135
97,5
Amount of Pepsin (kg)
1,4
0
0
0
PROTEIN




Ratio of nitrogen recovery (%)
64,20±2,70
86,20±1,67
-
-
Recovery ratio of antioxidant products (%)
d

3,52±1,54
4,09±0,12
-
-
CHITOSAN
Protein content (%, db)
0,55±0,05
0,65±0,08
Ash content (%, db)
0,66±0,05
0,79±0,12
Viscosity average molecular weight of
chitosan (kDa)
404,21
253,32
Degree of deacetyl (%)
84,84±1,22
82,12±1,23
Solubility (%)
99,03±0,1
96,84±1,15
Yield of purified chitosan (%)
95,47±0,91
88,76±1,43
Amount of anhydrous NaOH (kg)
41,10
39,96
Processing time (h)
4
10

a
Chitosan deacetylated in the presence of sonication at 80
o
C, [NaOH]=60%, 4h;
b
Sample was produced following to the reference
procedure;
c
In case of sonication applied to pepsin,
d
In comparison with the weight of raw materials.
The benefits from the proposed procedures came from the innovation of combining physical force with
emzymatic technology (using endogenous enzymes and pepsin). In the reference procedure, pressing only
allowed to reduce nearly 35% of the initial volume of materials whereas the innovation in the proposed
procedure helped to decrease more than 90% of them. Therefore, amount of materials need to be treated with
chemicals to extract chitin went to the minimum and most of protein in shrimp heads did not contacted with
chemicals and its bioactivitive were kept. The higher yield of chitin recovey in the proposed procedure might be
due to deacetylation and the loss were controled.
23

Thanking to applying sonication during deacetylation solubility of chitosan was enhanced about 1,5%
although decetylation was conducted in less time and lower NaOH concentration in comparison with the
reference procedure. The improvement in solubility helped to increase the yield of purified chitosan by 10,63%.
Based on the figures in Table 3.28 the rough benefits of applying the innovated technology for 1000 kg
of shrimp by-products were estimated and summarized in Table 3.29. The proposed innovation in chitin
extraction brought the profit in both aspects of economy and environment. It helped to solve the shortcomings in
environment issues and create foundation for developing sustainably chitin and chitosan industry in Vietnam.
Table 3.29: Comparison of the benefits between applying integrated technology and conventional
procedure to recover chitin, chitosan and protein (Estimated to 1000 kg raw materials/bath)
Test Items

*


Materials

Heads

Shells
Extra amount of chitin (kg)

9,5

1,9
Extra amount of purified chitosan (kg)

-

5,2
Amount of protein recovered (kg)

83,4

46,7
Amount of antioxidant products (kg)

40,9

35,2
Amount of anhydrous NaOH reduced (kg)

77, 5

117,0
Amount of concentrated HCl reduced (L)

220,9

240,5
Time - saving (h)

20, 0

18,0
Amount of water reduced (m
3
)

3,6

0
Amount of protein prevented to wastewater (kg)

83,4

46,7

*Means of replicate and figures only collected from deproteinization, demineralization ans deacetylation.
Although the fesibility of using pepsin in chitin extraction was proved on technology and environmnet
areas its application still is not practical due to high price of pepsin. However, it was hoped that the progressive
technology on pepsin production from new resources and enzymatic refinement might help to bring the price of

pepsin to reasonable level to application in chitin industry in near future.
CONLUSIONS AND RECOMMENDATION
CONCLUSIONS
Based on the results of the dissertation, some conclusions can be made as follow:
1. The simple procedure to recovery chitin and protein efficiently through simultaneous combination of
autolysis with the endogenous protease and physical force were put forward: 60
o
C, native pH for 2h, the
ratio of water to materials = 1:1 (v/w).
2. The deproteinzation by pepsin was optimized and its kinetics also were clarified:
 The opitimum was the ratio of pepsin to substrate: 20U/g protein, for 16h, at 40
o
C, pH=2;
 Deproteinization conducted in shells of white leg shrimp by pepsin obeyed Pseudo-first-order and
protein existed in shrimp shell in a structure of layers. Degree of deproteinization (DP) and its rate (r) in
the period of first hours had the equation as following:
-  


 

    




    


    





  

 







2. Ultrasound (37kHz, RMS 35W) had capacity to facilitate the activity of pepsin (increase 8% after sonication
of 25 min) as well as intensify the rate and the uniform of heterogenous deacetylation
24

3. The heterogenous deacetylation in the present of sonication (37kHz, 35 RMS) at 80
o
C was optimized as well
as four equations which indicated the relationships between degree of deacetylation, solubility and molecular
weight with NaOH concentration and time were found.
4. Three processes to recovery chitin (02) and chitosan (01) accompanying with specific parameters and
mathematic equations were brought out that allowed to control efficiently the chitin and chitosan production.
Chitin and chitosan produced according to the proposed procedures had high quality that met the
specifications from the leading companies. In addition, the yield of chitin recovery was incereased by 2,8
and 32,87% respectively to shell and heads; Solubility of chitosan was incerased by 1,5% and và yield of
purified chitosan also raised about 10,63%; The amount of concentrated HCl used to extract chitin from shell
and heads decreased 75,67 and 96,26% ; the amount of anhydrous NaOH reduces 86,67 and 97,95% ,

respectively, in compared with those used in the reference procedures.
5. Concurrently, the data in relation to the composition of chemicals, amino acids, minerals and heavy metals
of three main parts of white leg shrimp (heads, shell and meat) (Penaeus vannamei) cultivated in Khanh Hoa
province were collecte:
- In case of shrimps in size of 60÷160 bodies/kg: the mass ratio of head and shell were average of
38,70±6,46 (%) in comparison with the mass of whole shrimps, whereas, the ratio of each part was
27,50±3,93 and 11,21±2,63 (%) respectively;
- In case of shrimps in size of 81÷120 bodies/kg, the content of protein, minerals and chitin of head and
shell parts were 44,39±1,84 and 26,11±1,15%; 26,66±1,63 and 25,61±1,79%; 11,40±1,69 and
27,37±1,16%, respectively; The content of amino acid was approximate of 50% and 30%, respectively
in compared with those of the meat part; the component of minerals were value with Ca, Na, K, Fe, Cu,
and Zn; all heavy metals found such as Pb, As, Cd, Hg, Se were under the limitation level for food.
RECOMMENDATION
To get more information, the following activities should be continued:
1. Studying procedures to produce and commercialize the antioxidant products from the protein hydrolysates.
2. Studying the impact of sonication on deacetylation of chitin from different sources
3. Scale-up the proposed technology to find out the reasonable values for mass production of chitin.