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Chitin and chitosan preparation from shrimp shells using optimized enzymatic
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Contents lists available at SciVerse ScienceDirect
Process Biochemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ pr ocbi o
Chitin and chitosan preparation from shrimp shells using optimized enzymatic
deproteinization
Islem Younes
a,
, Olfa Ghorbel-Bellaaj
a
, Rim Nasri
a
, Moncef Chaabouni
b
, Marguerite Rinaudo
c,d
,
Moncef Nasri
a
a
Laboratory of Enzyme Engineering and Microbiology, National School of Engineering, P.O. Box 1173-3038 Sfax, Tunisia
b
Laboratory of Industrial Chemistry, National School of Engineering, BP W3038 Sfax, Tunisia
c
European Synchrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Cedex 9, France
d
Research Centre of Natural Macromolecules (CERMAV-CNRS) afliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France
a r t i c l e i n f o
Article history:
Received 21 January 2012
Received in revised form11 May 2012
Accepted 14 July 2012
Available online xxx
Keywords:
Shrimp shells
Chitin
Chitosan
Enzymatic deproteinization
Bacillus mojavensis A21
Response surface methodology
a b s t r a c t
Different crude microbial proteases were applied for chitin extraction from shrimp shells. A BoxBehnken
design with three variables and three levels was applied in order to approach the prediction of optimal
enzyme/substrate ratio, temperature and incubation time on the deproteinization degree with Bacillus
mojavensis A21 crude protease. These optimal conditions were: an enzyme/substrate ratio of 7.75 U/mg,
a temperature of 60

C and an incubation time of 6h allowing to predict 94 4% deproteinization. Exper-

imentally, in these optimized conditions, a deproteinization degree of 88 5% was obtained in good
agreement with the prediction and larger than values generally given in literature. The deproteinized
shells were then demineralized to obtain chitin which was converted to chitosan by deacetylation and
its antibacterial activity against different bacteria was investigated. Results showed that chitosan dis-
solved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria
tested.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Chitin, the second most abundant biopolymer next to cellu-
lose, and its derivatives like chitosan are widely recognized to
have immense applications in many elds [1]. They are widely
used in the food industry, medicinal elds, chemical industries,
textiles, wastewater treatment plants, etc. [2]. The main sources
of raw material for the production of chitin are cuticles of various
crustaceans, principally crabs and shrimps. However, crustacean
shells consist of compact matrices of chitin bers interlaced with
proteins. These matrices are reinforced through the deposition
of mineral salts, mainly those of calcium [3]. They have to be
quantitatively removed to achieve good accessibility and the
high purity necessary for biological applications. Although many
methods can be found in the literature for the removal of proteins
and minerals, effects on the molecular weight and acetylation
degree cannot be avoided with any of these extraction processes
[4]. Therefore, a great interest still exists for the optimization of
the extraction to minimize the degradation of chitin, while, at the
same time, bringing the impurity levels down to a satisfactory

Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595.
E-mail address: issslem@hotmail.com (I. Younes).
level for specic applications. Conventionally, preparation of
chitin from such shellsh wastes involves deproteinization and
demineralization with strong bases and acids. However, the use
of these chemicals may cause a partial deacetylation of the chitin
and hydrolysis of the polymer, resulting in nal inconsistent phys-
iological properties [5]. The chemical treatments also create waste
disposal problems, because neutralization and detoxication of the
discharged wastewater are necessary. Furthermore, the interest of
the protein hydrolysate is reduced due to the presence of sodium
hydroxide [6]. To overcome the defects of chemical treatments,
some efforts have been directed toward its substitution by more
eco-friendly processes such as bacterial fermentation and treat-
ment by proteolytic enzymes which have been applied for the
deproteinization of crustacean wastes [7,8].
The aim of this work is to investigate the inuence of several
operatingparameters suchas, enzyme/substrateratio, temperature
andincubationtimeonthedeproteinizationdegreeof shrimpshells
by non-commercial Bacillus mojavensis A21 crude enzyme.
Response surface methodology (RSM) is useful for designing
experiments, building models and analysing the effects of sev-
eral independent variables [9,10]. The main advantage of RSM is
the reduced number of experimental trials needed to evaluate
the effect of multiple factors on the response. In order to deter-
mine a suitable polynomial equation that describes the response
1359-5113/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.07.017
Please cite this article in press as: Younes I, et al. Chitin and chitosan preparation from shrimp shells using optimized enzymatic
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surface, RSMcan be employed to optimize the process for gather-
ing research results better than classical one-variable-at-a-time or
full-factorial experimentation.
In this work, a BoxBehnken design [11] was employed to
establish the relationship between the reaction variables and the
deproteinization degree. Furthermore, the ridge analysis has been
employed to optimize the experimental conditions permitting the
higher deproteinization degree. The purity level of chitin was fol-
lowed by the evaluation of the mineral and protein contents. Chitin
was then converted to chitosan by chemical deacetylation. The
antibacterial activity of the acid-soluble chitosan of shrimp waste
was investigated.
2. Materials and methods
2.1. Raw material
The shrimp (Metapenaeus monoceros) shells were obtained in fresh condition
from a shrimp processing plant located in Sfax, Tunisia. Shell waste were washed
thoroughly with tap water, mixed with distilled water at a ratio of 1:2 (w/v) and
then cooked for 20min at 90

C. The cooked sample was drained and homogenized

in a Moulinex

blender for about 2min then used for moisture determination and
kept at 20

C until further use.

Commercial chitosan[39280-86-9] was providedbyMP Biomedicals LLCFrance;
its degree of acetylation, determined by
13
C NMR, was 0.22.
2.2. Chemical analysis of shrimp waste homogenate
The moisture and ash content were determined at 105

C and 550

C, respec-
tively, according to the AOAC [12] standard methods 930.15 and 942.05. Total
nitrogen content of shrimp waste was determined by using the Kjeldahl method.
Crude protein was estimated by multiplying total nitrogen content by the factor
of 6.25. Lipids were determined gravimetrically after soxhlet extraction of dried
samples with hexane.
2.3. Microbial strains and enzymes preparation
B. mojavensis A21 and Bacillus subtilis A26 were isolated from marine water in
SfaxcitybyHaddar et al. [13] andAgrebi et al. [14], respectively. Bacillus licheniformis
NH1 was isolated by El Hadj Ali et al. [15] from an activated sludge reactor treat-
ing shery wastewater. Bacillus licheniformis MP1 [16] was isolated from polluted
seawater from Sfax port. Vibrio metschnikovii J1 [17] was isolated from an alkaline
wastewater of the soap industry. Aspergillus clavatus ES1 [18] was isolated from
wastewater. All strains were identied on the basis of the 16S rRNA gene sequenc-
ing and biochemical properties. The medium used for the isolation of A21, A26,
NH1, MP1 and J1 strains was LuriaBertani broth medium [19] composed of (g/l):
peptone, 10; yeast extract, 5; NaCl, 5 (pH 7.0). The mediumused for the isolation of
ES1 strain was consisted of (g/l): peptone, 5.0; yeast extract, 3.0; skimmed milk 25%
(v/v) and bacteriological agar, 12.0 (pH9.0). Production of proteases was carried out
in optimized mediumof each microbial strain. Media were autoclaved at 120

C for
20min. Cultivations were performed on a rotatory shaker in optimal conditions for
each microbial strain, in 250ml Erlenmeyer asks with a working volume of 25ml.
The cultures were centrifuged 5min at 10,000rpm, and the cell-free supernatants
were recovered and concentrated by the addition of solid ammoniumsulfate to 80%
saturation.
Protease activity was measuredby the methoddescribedby Kembhavi et al. [20]
using casein as a substrate.
2.4. Deproteinization of shrimp waste by proteases
Microbial crudeenzymepreparations weretestedfor their deproteinizationef-
ciency. Two commercial enzymes, bromelain (Smart city) and alcalase (Novozyme)
were chosen as control for deproteinization experiments. Deproteinization tests
were carried out in a thermostated stirred Pyrex reactor (300ml). Shrimp waste
homogenate (15g) were mixed with 45ml distilled water. The pH and temperature
of the mixture were adjusted to the optimumconditions for each enzyme: pH 10.0,
50

C for A21, NH1 and MP1 enzymes; pH8.0, 40

C for A26; pH11.0, 40

C for J1, pH
8.5, 40

C for ES1, pH 8.0, 50

C for alcalase and bromelain. Then, the shrimp waste

proteins were digested withcrude enzymes. The reactionwas thenstopped by heat-
ing the solution at 90

C during 20min to inactivate enzymes. The solid phase was

washed and then pressed manually through four layers of gauze. Deproteinization
was expressed as percentage and computed by the following equation [21]:
%DDP =
[(P
O
O) (PR R)] 100
P
O
O
(1)
where P
O
and PR are the protein concentrations (%) before and after hydrolysis;
while, O and R represent the mass (g) of original sample and hydrolyzed residue in
dry weight basis, respectively.
Table 1
Design of experiment-levels of various process parameters of the BoxBehnken
design.
Parameter Level
1.0 0.0 1.0
X
1
: enzyme/substrate ratio (U/mg) 0 5 10
X
2
: temperature (

C) 40 50 60
X
3
: incubation time (h) 1 3.5 6
2.5. Experimental design and statistical analysis
In order to describe the nature of the response surface in the experimental
region, a BoxBehnken design was applied. As presented in Table 1, the experi-
mental design involved three parameters (U
1
: enzyme/substrate, U
2
: temperature
and U
3
: incubation time), each at three levels for low, middle and high concentra-
tions. Table 2 represents the design matrix of a 17 trials experiment. For predicting
the optimal point, a second order polynomial function was tted to correlate the
relationship between independent variables and response. For the three factors this
equation is
y = b
0
+b
1
X
1
+b
2
X
2
+b
3
X
3
+b
12
X
1
X
2
+b
13
X
1
X
3
+b
23
X
2
X
3
+b
11
X
2
1
+b
22
X
2
2
+b
33
X
2
3
where y is the predicted response, b
0
model constant; X
1
, X
2
and X
3
are independent
variables; b
1
, b
2
and b
3
are linear coefcients; b
12
, b
13
and b
23
are cross product
coefcients and b
11
, b
22
and b
33
are the quadratic coefcients.
X
j
: coded variables related to the natural variables U
j
by the following equation:
X
j
= (U
j
U
0j
)/Stepof variation
where: U
0j
=(U
j,high
U
j,low
)/2
Step of variation of j =(U
j,high
+U
j,low
)/2
U
j,high
and U
j,low
: two extreme levels (high and low) given for each natural vari-
able U
j
.
The coded variables X
j
are equal to 1 and +1 when the levels of natural variable
U
j
are U
j,low
and U
j,high
, respectively.
The model coefcients were estimated by a least squares tting of the model
to the experimental results obtained in the design points (runs no. 112). The ve
replicates at the center point were carried out in order to estimate the pure error
variance.
The software NEMROD W[22] was used for experimental design data analysis
and quadratic model exploitation. The optimal conditions for deproteinization were
obtained by solving the regression equation and also by analyzing the isoresponse
and response surface contour plots using the same software.
2.6. Chemical demineralization
Demineralization was carried out in a dilute HCl solution. Solid fractions
obtained after hydrolysis by A21 crude protease were treated with 1.5MHCl in 1:10
(w/v) ratio for 6h at 50

C under constant stirring (150rpm). The chitin product was

ltered through four layers of gauze with the aid of a vacuum pump and washed
to neutrality with deionized water and then dried for 1h at 60

C. Demineralization
was expressed as percentage and computed by the following equation [21]:
%DDM =
[(M
O
O) (MR R)] 100
M
O
O
(2)
where M
O
and MR are ash contents (%) before and after demineralization; while, O
and R represent the mass (g) of deproteinizedshell and demineralized residue in
dry weight basis, respectively.
2.7. Deacetylation of chitin
The puried chitin was treated with 12.5MNaOH in 1:10 (w/v) ratio at 140

C
for 4h until it was deacetylated to chitosan. After ltration, the residue was washed
with deionized water, and the crude chitosan was obtained by drying in a dry heat
incubator at 50

C overnight.
2.8.
13
C CP/MAS-NMR spectroscopic analysis
Chitosan structural analysis was carried out by
13
C NMR (nuclear magnetic res-
onance) with CP/MAS technique (cross-polarization, magic-angle-spinning) using
a BRUKER-ASX300 instrument. NMR spectra were recorded at a
13
C frequency of
75.5 MHz (eld of 7.04T). CP/MAS sequence was used with the following parame-
ters: the
13
C spin lattice relaxation time was 5s; powdered samples were placed in
an alumina rotor used for the double airbearing-type MAS systemand spun as fast
as 8kHz; contact time was 8ms.
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Table 2
The actual design of experiments and response of deproteinization.
Design point
a
Enzyme/substrate ratio (X
1
) Temperature (X
2
) Incubation time (X
3
) Deproteinization (%)
Experimental
b
Predicted
1 0 40 3.5 26 26
2 10 40 3.5 54 59
3 0 60 3.5 38 33
4 10 60 3.5 79 79
5 0 50 1 30 32
6 10 50 1 75 72
7 0 50 6 36 39
8 10 50 6 82 80
9 5 40 1 68 66
10 5 60 1 75 76
11 5 40 6 76 73
12 5 60 6 86 87
13 5 50 3.5 69 71
14 5 50 3.5 71 71
15 5 50 3.5 68 71
16 5 50 3.5 76 71
17 5 50 3.5 70 71
Bold values represent the ve replicates at the center point used to estimate the pure error.
a
Experiments were conducted in a randomorder.
b
The values given in the table are the average of three independent experiments.
The degree of acetylation (DA) of the samples was determined by dividing the
intensity of the resonance of the methyl group carbon by the average intensity of
the resonances of the glycosyl ring carbon atoms. The DA was calculated using the
following relationship [23]:
%DA =
I[CH
3
]
(I[C
1
] +I[C
2
] +I[C
3
] +I[C
4
] +I[C5] +I[C
6
])/6
100 (3)
I is the intensity of the particular resonance peak.
2.9. Antimicrobial activity of chitosan
The microorganisms used for antimicrobial activity were Micrococcus luteus
(ATCC 4698), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),
Klebsiella pneumoniae (ATCC 13883), Bacillus cereus (ATCC 11778), Staphylococ-
cus aureus (ATCC 25923) Salmonella typhi and Enterococcus faecalis (ATCC 29212).
Antibacterial activity assays were performed according to the method described by
Berghe andVlietinck [24]. Chitosanwas dissolvedat 50mg/ml in0.1%acetic acid(pH
4.68). The inoculum suspension (200l) of the tested microorganisms, containing
10
6
colony forming units (CFU/ml) of bacteria cells were spread on MullerHinton
agar. The inoculums were allowed to dry for 5min. Then, bores (3-mmdepth, 6-mm
diameter) were made using a sterile borer and were loaded with 50l of each sam-
ple. Well with only acetic acid (without chitosan) was used as a negative control (pH
3.25). Gentamycin was used as positive reference. The Petri dishes were kept, rstly,
for 1h at 4

C, and then were incubated for 24h at 37

C. Antibacterial activity was

evaluated by measuring the diameter of the growth inhibition zones in millime-
ters (including well diameter of 6mm) for the test organisms and comparing to the
controls. The measurements of inhibition zones were carried out for three sample
replications, and values are the average of three replicates.
2.10. Statistical analysis
All experiments were carried out in triplicate, and average values with standard
deviationerrors are reported. Meanseparationandsignicance were analyzedusing
the SPSS software package (SPSS, Chicago, IL). Correlation and regression analysis
was carried out using the EXCEL program.
3. Results and discussion
3.1. Enzymatic deproteinization of shrimp waste by microbial
proteases
Firstly, we tried to point out the main parameters playing a
role on enzymatic deproteinization. In this view, we tried to select
the most effective enzymes. Microbial proteases from B. mojaven-
sis A21, B. subtilis A26, A. clavatus ES1, B. licheniformis MP1, B.
licheniformis NH1 and V. metschnikovii J1 were tested for their
deproteinization efciency. Deproteinization tests were conducted
for 3h under conditions of optimal enzyme activity and stability
withenzyme/substrateratios equal to20U/mgof protein. As shown
in Fig. 1, high deproteinization degrees were obtained with the
crude enzymes of A21, A26, J1andMP1 (at about 764%), while the
two others NH1 and ES1 were exerting signicantly lower values
(653% and 593% respectively). B. mojavensis A21 strain, rarely
described in the literature, which produces at least six different
proteases, was retained. Otherwise, commercial enzymes, brome-
lain and alcalase, were used as control group. These enzymes were
chosen as control for deproteinization experiments. Deproteiniza-
tiondegrees obtainedwithbromelainandalcalase were lower than
those obtained with crude enzymes; it reached 673%and 543%
for bromelain and alcalase, respectively.
Secondly, the enzymatic deproteinization of shrimp shells by B.
mojavensis A21 crude enzyme was carried out under several exper-
imental conditions, including various enzyme/substrate ratios,
different temperatures, pH and incubation times. Reaction carried
out at 50

C without enzymes resulted in a deproteinization degree

of about 382%. Indeed, some proteins associatedto chitinby elec-
trostatic forces or hydrogen bonds, could be dissociated by thermal
treatment. However, other proteins are linked to chitin by covalent
bonds, their removal requires severe chemical or enzymatic treat-
ments [25]. The deproteinizationrate withanE/S ratioof 1was high
(712%), whichshows theeffectiveness of theenzymepreparation
of B. mojavensis A21, and further increase in enzyme concentration
increased slightly (from 712% to 772%) the deproteinization
rate. Beyond 20U/mg the deproteinization rate remained constant.
B. mojavensis A21 crude alkaline protease characterization [13]
showedanoptimal activity at pH8.011.0andat 60

C, using casein
Fig. 1. Deproteinization of shrimp waste (%) by protease preparations. Alcalase;
Bromelain; A21: B. mojavensis A21; J1: V. metschnikovii J1; MP1: B. licheniformis
MP1; A26: B. subtilis A26; NH1: B. licheniformis NH1 and ES1: A. clavatus ES1.
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Table 3
Analysis of Variance (ANOVA) for the t of experimental data to response surface model.
Source of variation Sumof squares (SS) Degrees of freedom(DF) Mean square Fexp Signicance
Regression 5706.44 9 634.04 28.85 0.0102***
Residual 153.80 7 21.97
Lack of t 115.00 3 383.33 3.95 10.9 NS
Pur error 38.80 4 9.70
Total 5860.24 16
***: indicates signicant at the level 99.9%. N.S.: indicates non-signicant at the level 95.0%
as a substrate. It was extremelystable inthe pHrange of 7.011.0. In
this work, the preliminary study showed that pHhas a little impact
on the deproteinization degree in the range of activity and stability
of enzymes (pH 8.011.0) and only temperature and incubation
time could have a considerable impact on the deproteinization
degree.
With regards to these results, for the selected enzyme B.
mojavensis A21 crude alkaline protease, E/S ratio (U
1
), temperature
(U
2
) and incubation time (U
3
) were selected as effective operating
variables on the deproteinization degree. In this view, we applied
a response surface methodology allowing to predict the optimum
conditions for enzymatic deproteinization of shrimp shells.
3.2. Optimization of the shrimp waste deproteinization using A21
proteases
The optimization of the experimental conditions of shrimp
waste deproteinization was achieved using a BoxBehnken design
and carried out under several experimental conditions including
various E/S ratio (in the range of 010 U/mg), temperature (in the
range of 4060

C) and incubation time (in the range of 16h). The

pH was maintained at 9.0. Table 2 shows the real experimental
conditions and the measured responses. These data are used to
establish a mathematical relation between the set of parameters
and the degree of deproteinization.
3.2.1. Model equation and validation
The coefcients of the postulated model were calculated on
the basis of the experimental responses (Table 2) by the least
square regressionusing the NEMRODWsoftware. The ttedmodel,
expressed in coded variables, is represented by the equation:
y = 70.4 +20X
1
+6.75X
2
+4X
3
+3.25X
1
X
2
+0.25X
1
X
3
+0.75X
2
X
3
20.825X
2
1
0.325X
2
2
+6.175X
2
3
It is necessarytomakeananalysis of thevariance(ANOVA) using
the F-test to attest the good quality of the tting [26]. Results of
the analysis of variance for the tted model are summarized in
Table 3. They clearly indicated that the regression sumof squares is
statistically signicant at the level 99.9%. Moreover, the coefcient
of determination, R
2
, for the conversionyieldwas 0.974. This means
that 97.4% of the observed variation is attributed to the variable
effects. Thus, it is concluded that the predicted model well tted
the experimental data.
Ontheother hand, thevalidityof themodel has beenestablished
by comparing the variance related to the lack of t to that of pure
error which demonstrated the non-signicance of the lack of t
(Table 3).
The validmodel was usedto predict response values inthe stud-
ied domain and to draw isoresponse contour plots and response
surfaces.
3.2.2. Graphical interpretation of the response surface model
The representation of the optimum path computed from
the ridge analysis [26] of the response surface tted for the
deproteinization degree is shown in Fig. 2. The right parts and the
left parts of both plots refer to the maximization and the mini-
mization of the response, respectively. The distance (r) from the
center of the design is indicated in abscissas. Fig. 2a shows that the
optimum response reached as a function of the distance r. Fig. 2b
displays the coordinates for each factor, in codied variables, of the
points of plot 2a. As it can be seen in Fig. 2a, the deproteinization
degree signicantly increases fromthe center of the domain to the
boundary (r =1) where it reaches 88.87%. In addition, Fig. 2b shows
that, close to the maximum, the deproteinization degree is more
sensitive to the variations of the incubation time (X
3
) and the tem-
perature (X
2
) than that of E/S ratio (X
1
). To reach the maximum, all
factors must tend toward relatively high values.
The isoresponse curves and the response surface were drawn by
plotting the response variation against two factors, while the third
is held constant at its mean level (Fig. 3). The examination of these
gures shows that, animportant effect onthe deproteinizationef-
ciency was provided by the E/S ratio (X
1
). Indeed, Fig. 3a shows that
when the E/S ratio is low, between 0 and 5U/mg, the temperature
has no signicant effect on the deproteinization degree. However,
temperature has a very signicant positive effect on the response
whenthe E/S ratio is beyond 5U/mg. Inthis way, a deproteinization
degree of above 85% is obtained with an E/S ratio of 7.5U/mg and
a temperature of 60

C.
The same could be said for the reaction time effect, when com-
pared to that of temperature. Fig. 3b represents the isocontour
plots when the level of the temperature is xed to its average level
(50

C). It showed that yields higher than 85% could be reached

by xing the E/S ratio and incubation time at levels higher than
7.5U/mg and 6h, respectively.
Finally, it could be seen from Fig. 3c that, by keeping X
1
at its
average level (5U/mg), high deproteinization degrees are obtained
with high levels of both temperature and incubation time.
3.2.3. Comparison between model and experimental results
Fromthis model analysis, the optimal experimental conditions
were xed at: E/S ratio 7.75U/mg, temperature 60

C and incu-
bation time 6h which allow to obtain 944% of yield. In order
to conrm this prediction, an additional independent run was
conducted using these selected variable levels. Results obtained
showed that the observed deproteinization degree (885%) was
not signicantly different fromthe predicted value. This result con-
rmed that the empirical model derived fromRSMcan be used to
adequately describe the relationship between the factors and the
shrimp shells deproteinization response using the crude enzyme of
B. mojavensis A21.
The deproteinization activity of B. mojavensis A21 crude pro-
tease was better than many proteases reported in previous studies.
Deproteinization rate obtained using A21 proteases was even
better than values reached using fermentation which gives habit-
ually higher deproteinization rate. Busto and Healy [27] compared
the effects of microbial and enzymatic deproteinization. A max-
imum value of 82% was achieved with Pseudomonas maltophilia
after six days and 64% with puried microbial protease under the
same condition. Fermentation using the culture supernatant from
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Fig. 2. Ridge analysis: optimal response plot (a) and optimal coordinate plot (b).
Pseudomonas aeruginosa K-187 allows a deproteinization rate of
78% after a seven-day incubation at 37

C [28]. Fermentation con-

ditions byP. aeruginosa A2 were optimized by response surface
methodology and maximal deproteinization of 89% was achieved
after ve days incubation [7]. Bacillus cereus SV1 proteases were
also applied for enzymatic deproteinization. Protein removal from
shrimp waste reached the same value of deproteinization degree
obtained by A21 proteases but using higher E/S ratio (20 instead
of 7.75U/mg) [8]. The fact that deproteinization cannot reach 100%
is explained by the non-accessibility of enzymes to some proteins
protected by chitin and minerals. Indeed, the proteins in the inner
layer of shrimp shell waste are protected by the outer layer chitin,
fromthe attack by proteases and thus, no further proteolysis could
occur [25]. In addition, some portions of peptides are suggested to
be linked covalently to a small number of the C-2 amino groups of
chitins [25].
3.3. Chemical demineralization
Inthe recovery of chitinfromshrimp waste, associated minerals
should be removed as a second stage. As a consequence, shrimp
waste deproteinizedby enzymatic treatment was subjectedtomild
acid treatment in order to remove minerals. The demineralization
was completely achieved within 6h at 50

C after treatment with

1.5MHCl solution at a ratio of 1:10 (w/v).
One of the factors determining the good quality of chitin is the
lowmineral content [29]. Chitin obtained in this work present con-
tent of minerals as low as those reported in other works [30] at
around of 1.9%.
The treatments employed to extract chitin from the shrimp
waste allowed the recovery of 18.52.3% of its initial dry mass
as a water insoluble white brous material, which indicates that a
good yield for the chitin extraction was attained and no pigments
were present in the chitin. Other studies reported similar yields for
the extraction of chitin [29,30].
3.4. Chitin characterization
The same raw material was treated by alkali (NaOH 1.25M
after 4h incubation at 80

C) [8]; this treatment gives chitin 2

(Table 4). In Table 4, the characteristics of the raw material, chitin
prepared by enzymatic treatment (chitin 1) and that obtained by
alkaline treatment (chitin 2) are compared. The ground shrimp
waste before pre-treatment contained a relatively high contents
of chitin (33.52.3%) and ash (33.20.2%). These results are
comparable with those reported by previous studies [29,31]. The
demineralization conditions used in this study reduce the mineral
content to permissible limits in the chitin. Indeed, the ash content
was reduced to about 1.9%. This was lower than that found by Sini
et al. [32]. This low ash content for chitin indicated the suitabil-
ity of removal of calcium carbonate and other minerals from the
raw material. There were no signicant differences in the mois-
ture content andashamong the two chitins (p>0.05). Animportant
difference concerns the protein content signicantly higher in the
chitin isolated after enzymatic deproteinization (p<0.05); com-
plete removal of the residual protein associated with the chitin was
not achieved even if the residual yield is lower than usually found
in literature.
Although such deproteinization percentage is lower than that
obtained by chemical treatment, enzymatic deproteinization helps
to avoid many drawbacks of chemical treatment, over-hydrolysis,
breakdown of chitin, etc.
3.5. Preparation of chitosan
The major procedure for obtaining chitosanis based onthe alka-
line deacetylation of chitin with strong alkaline solution. In this
study, chitosan was prepared fromchitin obtained by a treatment
with 12.5M NaOH in 1:10 (w/v) ratio at 140

C for 4h. The ef-

ciency of this treatment is evaluated by the acetylation degree of
chitosan determined by Nuclear Magnetic Resonance (NMR). Solid
state
13
C RMN spectroscopy was used in order to verify the purity
of the chitosansample by the chemical shifts and the intensities of
the
13
C absorption peaks [33].
3.6.
13
C CP/MAS-NMR spectroscopic analysis
NMR is one of the most powerful tools in the study of
polysaccharide composition and sequential structure. NMR is a
non-destructive method resulting in retained structure and con-
formation of the polysaccharide, making it possible to monitor
Table 4
Properties of the chitins obtained by deproteinization with B. mojavensis proteases
(1) and by alkali deproteinization (2).
% Rawmaterial Chitin (1) Chitin (2)
Moisture 67.01.1 4.61.1 3.90.5
Chitin 33.62.3 18.52.3 20.02.0
Ash 33.20.2 1.90.1 1.40.1
Protein 27.11.3 12.01.6 6.21.3
Lipid 6.00.3
Appearance White akes Yellowish akes
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Fig. 3. Three-dimensional response surface and contour plots for the effect on the deproteinization degree of: E/S ratio and temperature at constant time 3.5h (a) E/S ratio
and incubation time at constant temperature 50

C. (b) Temperature and incubation time at constant E/S ratio 5.00U/mg (c).
reactions and other structural and physical properties under dif-
ferent solvent conditions.
Solid state
13
C CP/MAS-NMR is known to be very sensitive to
changes in the local structure.
13
C CP/MAS-NMR spectrum of the
chitosan prepared by enzymatic deproteinization and commercial
chitosan prepared by alkaline treatment, are shown in Fig. 4. NMR
analysis of the shrimp waste chitosan gave similar peak pattern
to that of commercial chitosan. There are 8 signals for the 8 car-
bon atoms of chitosan. The C1-C6 carbons of N-acetylglucosamine
monomeric unit are observed between 50 and 110ppm, indicating
highstructural homogenity. The carbonyl groupis around173ppm,
while the methyl group of the acetyl group produced a peak at
around 23ppm. The less the peaks of carbonyl and acetyl groups
are, the more efcient the deacetylationreactionis. Fig. 4shows the
Please cite this article in press as: Younes I, et al. Chitin and chitosan preparation from shrimp shells using optimized enzymatic
deproteinization. Process Biochem(2012), http://dx.doi.org/10.1016/j.procbio.2012.07.017
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Fig. 4.
13
C CP/MAS-NMRsolid-state spectra of chitosans. (1) Chitosan prepared fromchitin obtained by enzymatic deproteinization with B. mojavensis proteases and chemical
demineralization. (2) Commercial chitosan obtained by chemical deproteinization and chemical demineralization.
chitosan spectrum, in which the deacetylation of chitin is evident,
since there are tight peaks at 23 and 173ppm, that correspond to
the CH
3
and C O groups, respectively. The other peaks correspond
to C
1
(104.63), C
2
(55.71), C
3
(73.93), C
4
(83.63), C
5
(76.14)
and C
6
(61.46). Note in the spectrum that the removal of pro-
teins were efcient during the extraction, once there are no other
peaks, suggesting a great purity of the product [34]. The presence
of some background noise could be due to the presence of a possi-
ble by-product or impurity in the sample, especially in commercial
chitosan (Fig. 4).
The degree of acetylation in both chitosans was determined
using Eq. (3). The degree of acetylation was 22% and 4% in the
commercial sample and the prepared sample, respectively. A
deacetylated chitin with a rate of 7090% and lowprotein content
is considered as a good nal product, the best one being that with
the higher degree of deacetylation.
3.7. Antimicrobial activity
Antimicrobial activity of chitosans against several bacterial
species has been recognized and is considered as one of the most
important properties linked directly to their possible biological
applications. The antimicrobial activity of chitosan dissolved in
acetic acid 0.1% was investigated against four Gram-positive and
four Gram-negative bacteria. As shown in Table 5, both chitosans
inhibitedthegrowthof all Gram-negativeandGram-positivebacte-
ria tested excepted P. aeruginosa and E. faecalis. Chitosan obtained
bydeproteinizationwithB. mojavensis proteases categorizedas low
degree of acetylation showed higher inhibition activity than the
commercial one with higher degree of acetylation. These results
are in line with the works of Chen et al. [35] who reported that
antibacterial activity increase in the order of chitin deacetylation
degree. This inhibition is more important against Gram-negative
than Gram-positive bacteria tested. Gram-negative bacteria, with
lipopolysaccharide at the outer surface providing negative charges,
seemed to be very sensitive to chitosan while the sensitivity of
Gram-positive bacteria that can have variable amounts of nega-
tively charged teichoic acids at their outer surface varied greatly
Table 5
The diameters of inhibition zones against Gram-positive and Gram-negative
bacteria.
Diameter of inhibitionzones (mm)
Chitosan
a
Chitosan
b
Gram Escherichia coli 12.41.5 71.2
Pseudomonas aeruginosa R R
Klebsiella pneumoniae 121.2 70.8
Salmonella typhi 100.6 90.4
Gram+ Staphylococcus aureus 80.5 70.6
Bacillus cereus 9.50.5 90.5
Enterococcus faecalis R R
Micrococcus luteus 91.2 8.40.8
Diameter well: 6mm; R=resistant.
a
Chitosan prepared fromchitin obtained by enzymatic deproteinization with B.
mojavensis proteases and chemical demineralization
b
Commercial chitosanobtainedby chemical deproteinizationandchemical dem-
ineralization.
[36]. However, No et al. [37] showed stronger bactericidal effects
of chitosan toward Gram-positive than Gram-negative bacteria.
4. Conclusion
A BoxBehnken design with three variables and three levels
was applied for the determination of deproteinization efciency
of shrimp shells with enzymatic treatment by B. mojavensis A21
proteases. A21 proteases were found to remove up to 885% of
the shell proteins in agreement with optimization using response
surface methodology. The optimal conditions for deproteinization
were: an enzyme/substrate ratio of 7.75U/mg, a temperature of
60

C and an incubation time of 6h. Chitin obtained by enzymatic

deproteinizationwas thenconvertedto chitosan, witha lowdegree
of acetylation, which was found to exhibit remarkably antibacterial
activities.
Acknowledgment
This work was supported by grants from Ministry of Higher
Education and Scientic Research, Tunisia.
Please cite this article in press as: Younes I, et al. Chitin and chitosan preparation from shrimp shells using optimized enzymatic
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