Chitin and Chitosan Preparation From Shrimp Shells Using Optimized Enzymatic Deproteinization Younes I

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ARTICLE IN PRESSG ModelPRBI-9612; No. of Pages 8Process Biochemistry xxx (2012) xxxxxx

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com

Chitin hedeprot

Islem Yo f ChMoncef Na Laboratory of 3038 Sb Laboratory ofc European Synd Research Cen y, BP5

a r t i c l

Article history:Received 21 JaReceived in reAccepted 14 JuAvailable onlin

Keywords:Shrimp shellsChitinChitosanEnzymatic deproteinizationBacillus mojavensis A21Response surface methodology

ere ap leve

and i optimion tiions,

agreement with the prediction and larger than values generally given in literature. The deproteinizedshells were then demineralized to obtain chitin which was converted to chitosan by deacetylation andits 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 bacteriatested.

1. Introdu

Chitin, tlose, and ithave immeused in thetextiles, waof raw matecrustaceansshells consiproteins. Thof mineral quantitativehigh puritymethods caand mineradegree cann[4]. Therefothe extractisame time,

CorresponE-mail add

1359-5113/$ http://dx.doi.oe this article in press as: Younes I, et al. Chitin and chitosan preparation from shrimp shells using optimized enzymaticization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.2012.07.017

2012 Elsevier Ltd. All rights reserved.

ction

he second most abundant biopolymer next to cellu-s derivatives like chitosan are widely recognized tonse applications in many elds [1]. They are widely

food industry, medicinal elds, chemical industries,stewater treatment plants, etc. [2]. The main sourcesrial for the production of chitin are cuticles of various, principally crabs and shrimps. However, crustaceanst of compact matrices of chitin bers interlaced withese matrices are reinforced through the depositionsalts, mainly those of calcium [3]. They have to bely removed to achieve good accessibility and the

necessary for biological applications. Although manyn be found in the literature for the removal of proteinsls, effects on the molecular weight and acetylationot be avoided with any of these extraction processesre, a great interest still exists for the optimization ofon to minimize the degradation of chitin, while, at the

bringing the impurity levels down to a satisfactory

ding author. Tel.: +216 74 274 088; fax: +216 74 275 595.ress: [email protected] (I. Younes).

level for specic applications. Conventionally, preparation ofchitin from such shellsh wastes involves deproteinization anddemineralization with strong bases and acids. However, the useof these chemicals may cause a partial deacetylation of the chitinand hydrolysis of the polymer, resulting in nal inconsistent phys-iological properties [5]. The chemical treatments also create wastedisposal problems, because neutralization and detoxication of thedischarged wastewater are necessary. Furthermore, the interest ofthe protein hydrolysate is reduced due to the presence of sodiumhydroxide [6]. To overcome the defects of chemical treatments,some efforts have been directed toward its substitution by moreeco-friendly processes such as bacterial fermentation and treat-ment by proteolytic enzymes which have been applied for thedeproteinization of crustacean wastes [7,8].

The aim of this work is to investigate the inuence of severaloperating parameters such as, enzyme/substrate ratio, temperatureand incubation time on the deproteinization degree of shrimp shellsby non-commercial Bacillus mojavensis A21 crude enzyme.

Response surface methodology (RSM) is useful for designingexperiments, building models and analysing the effects of sev-eral independent variables [9,10]. The main advantage of RSM isthe reduced number of experimental trials needed to evaluatethe effect of multiple factors on the response. In order to deter-mine a suitable polynomial equation that describes the response

see front matter 2012 Elsevier Ltd. All rights reserved.rg/10.1016/j.procbio.2012.07.017and chitosan preparation from shrimp seinization

unesa,, Olfa Ghorbel-Bellaaja, Rim Nasria, Monceasri a

Enzyme Engineering and Microbiology, National School of Engineering, P.O. Box 1173- Industrial Chemistry, National School of Engineering, BP W 3038 Sfax, Tunisiachrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Cedex 9, Francetre of Natural Macromolecules (CERMAV-CNRS) afliated with Joseph Fourier Universit

e i n f o

nuary 2012vised form 11 May 2012ly 2012e xxx

a b s t r a c t

Different crude microbial proteases wdesign with three variables and threeenzyme/substrate ratio, temperaturemojavensis A21 crude protease. Thesea temperature of 60 C and an incubatimentally, in these optimized condit/ locate /procbio

lls using optimized enzymatic

aabounib, Marguerite Rinaudoc,d,

fax, Tunisia

3, 38041 Grenoble Cedex 9, France

plied for chitin extraction from shrimp shells. A BoxBehnkenls was applied in order to approach the prediction of optimalncubation time on the deproteinization degree with Bacillusal conditions were: an enzyme/substrate ratio of 7.75 U/mg,

me of 6 h allowing to predict 94 4% deproteinization. Exper-a deproteinization degree of 88 5% was obtained in good

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ARTICLE IN PRESSG ModelPRBI-9612; No. of Pages 82 I. Younes et al. / Process Biochemistry xxx (2012) xxxxxx

surface, RSM can be employed to optimize the process for gather-ing research results better than classical one-variable-at-a-time orfull-factorial experimentation.

In this work, a BoxBehnken design [11] was employed toestablish thdeproteinizemployed thigher deprlowed by thwas then cantibacteriawas investi

2. Materials

2.1. Raw mate

The shrimfrom a shrimpthoroughly withen cooked foin a Moulinexkept at 20 C

Commerciits degree of a

2.2. Chemical

The moisttively, accordinitrogen conteCrude proteinof 6.25. Lipidssamples with

2.3. Microbial

B. mojavenSfax city by HaNH1 was isolaing shery waseawater fromwastewater owastewater. Aing and biochNH1, MP1 andpeptone, 10; yES1 strain was(v/v) and bactein optimized m20 min. Cultivaeach microbiaThe cultures wwere recoveresaturation.

Protease ausing casein a

2.4. Deprotein

Microbial cciency. Two cowere chosen awere carried ohomogenate (of the mixture50 C for A21, N8.5, 40 C for Eproteins were ing the solutiowashed and thwas expressed

%DDP = [(PO

where PO andwhile, O and Rdry weight ba

Table 1Design of experiment-levels of various process parameters of the BoxBehnkendesign.

Parameter Level

zyme/pera

ubati

erime

rder ta Boxesignincubble 2 mal pship b

is

b1 X1

23 X2 X

is thes; b1, nts anoded v

U0j)

0j = (U of va

h and

codedj,low anmodeperims at t.softwdratic

by soonse

mical

inera after

tio forthrougality wressed

[(MO

O andpreseht ba

cetyla

purintil it onizer at 5

CP/M

osan structural analysis was carried out by 13C NMR (nuclear magnetic res- with CP/MAS technique (cross-polarization, magic-angle-spinning) usingR-ASX300 instrument. NMR spectra were recorded at a 13C frequency ofz (eld of 7.04 T). CP/MAS sequence was used with the following parame-13C spin lattice relaxation time was 5 s; powdered samples were placed inina rotor used for the double airbearing-type MAS system and spun as fast; contact time was 8 ms.e this article in press as: Younes I, et al. Chitin and chitosan prization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procb

e relationship between the reaction variables and theation degree. Furthermore, the ridge analysis has beeno optimize the experimental conditions permitting theoteinization degree. The purity level of chitin was fol-e evaluation of the mineral and protein contents. Chitinonverted to chitosan by chemical deacetylation. Thel activity of the acid-soluble chitosan of shrimp wastegated.

and methods

rial

p (Metapenaeus monoceros) shells were obtained in fresh condition processing plant located in Sfax, Tunisia. Shell waste were washedth tap water, mixed with distilled water at a ratio of 1:2 (w/v) andr 20 min at 90 C. The cooked sample was drained and homogenized

blender for about 2 min then used for moisture determination and until further use.al chitosan [39280-86-9] was provided by MP Biomedicals LLC France;cetylation, determined by 13C NMR, was 0.22.

analysis of shrimp waste homogenate

ure and ash content were determined at 105 C and 550 C, respec-ng to the AOAC [12] standard methods 930.15 and 942.05. Totalnt of shrimp waste was determined by using the Kjeldahl method.

was estimated by multiplying total nitrogen content by the factor were determined gravimetrically after soxhlet extraction of driedhexane.

strains and enzymes preparation

sis A21 and Bacillus subtilis A26 were isolated from marine water inddar et al. [13] and Agrebi et al. [14], respectively. Bacillus licheniformisted by El Hadj Ali et al. [15] from an activated sludge reactor treat-stewater. Bacillus licheniformis MP1 [16] was isolated from polluted

Sfax port. Vibrio metschnikovii J1 [17] was isolated from an alkalinef the soap industry. Aspergillus clavatus ES1 [18] was isolated fromll strains were identied on the basis of the 16S rRNA gene sequenc-emical properties. The medium used for the isolation of A21, A26,

J1 strains was LuriaBertani broth medium [19] composed of (g/l):east extract, 5; NaCl, 5 (pH 7.0). The medium used for the isolation of

consisted of (g/l): peptone, 5.0; yeast extract, 3.0; skimmed milk 25%riological agar, 12.0 (pH 9.0). Production of proteases was carried outedium of each microbial strain. Media were autoclaved at 120 C fortions were performed on a rotatory shaker in optimal conditions for

l strain, in 250 ml Erlenmeyer asks with a working volume of 25 ml.ere centrifuged 5 min at 10,000 rpm, and the cell-free supernatantsd and concentrated by the addition of solid ammonium sulfate to 80%

ctivity was measured by the method described by Kembhavi et al. [20]s a substrate.

ization of shrimp waste by proteases

rude enzyme preparations were tested for their deproteinization ef-mmercial enzymes, bromelain (Smart city) and alcalase (Novozyme)s control for deproteinization experiments. Deproteinization testsut in a thermostated stirred Pyrex reactor (300 ml). Shrimp waste

15 g) were mixed with 45 ml distilled water. The pH and temperature were adjusted to the optimum conditions for each enzyme: pH 10.0,H1 and MP1 enzymes; pH 8.0, 40 C for A26; pH 11.0, 40 C for J1, pH

S1, pH 8.0, 50 C for alcalase and bromelain. Then, the shrimp wastedigested with crude enzymes. The reaction was then stopped by heat-n at 90 C during 20 min to inactivate enzymes. The solid phase wasen pressed manually through four layers of gauze. Deproteinization

as percentage and computed by the following equation [21]:

O) (PR R)] 100PO O (1)

PR are the protein concentrations (%) before and after hydrolysis; represent the mass (g) of original sample and hydrolyzed residue insis, respectively.

X1: enX2: temX3: inc

2.5. Exp

In oregion, mental dand U3: tions. Tathe optirelationequation

y = b0 +

+ b

where yvariablecoefcie

Xj: c

Xj = (Uj

where: UStep

Uj,higable Uj .

The Uj are U

The to the exreplicatevariance

The and quaobtainedand resp

2.6. Che

Demobtained(w/v) raltered to neutrwas exp

%DDM =

where Mand R redry weig

2.7. Dea

The for 4 h uwith deiincubato

2.8. 13C

Chitonance)a BRUKE75.5 MHters: thean alumas 8 kHz1.0 0.0 1.0substrate ratio (U/mg) 0 5 10ture (C) 40 50 60on time (h) 1 3.5 6

ntal design and statistical analysis

o describe the nature of the response surface in the experimentalBehnken design was applied. As presented in Table 1, the experi-

involved three parameters (U1: enzyme/substrate, U2: temperatureation time), each at three levels for low, middle and high concentra-represents the design matrix of a 17 trials experiment. For predictingoint, a second order polynomial function was tted to correlate theetween independent variables and response. For the three factors this

+ b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3

3 + b11 X21 + b22 X22 + b33 X23 predicted response, b0 model constant; X1, X2 and X3 are independentb2 and b3 are linear coefcients; b12, b13 and b23 are cross productd b11, b22 and b33 are the quadratic coefcients.ariables related to the natural variables Uj by the following equation:

/Step of variation

j,high Uj,low)/2riation of j = (Uj,high + Uj,low)/2

Uj,low: two extreme levels (high and low) given for each natural vari-

variables Xj are equal to 1 and +1 when the levels of natural variabled Uj,high, respectively.l coefcients were estimated by a least squares tting of the modelental results obtained in the design points (runs no. 112). The ve

he center point were carried out in order to estimate the pure error

are NEMROD W [22] was used for experimental design data analysis model exploitation. The optimal conditions for deproteinization werelving the regression equation and also by analyzing the isoresponse

surface contour plots using the same software.

demineralization

lization was carried out in a dilute HCl solution. Solid fractions hydrolysis by A21 crude protease were treated with 1.5 M HCl in 1:10

6 h at 50 C under constant stirring (150 rpm). The chitin product wash four layers of gauze with the aid of a vacuum pump and washedith deionized water and then dried for 1 h at 60 C. Demineralization

as percentage and computed by the following equation [21]:

O) (MR R)] 100MO O (2)

MR are ash contents (%) before and after demineralization; while, Ont the mass (g) of deproteinized shell and demineralized residue insis, respectively.

tion of chitin

ed chitin was treated with 12.5 M NaOH in 1:10 (w/v) ratio at 140 Cwas deacetylated to chitosan. After ltration, the residue was washedd water, and the crude chitosan was obtained by drying in a dry heat0 C overnight.

AS-NMR spectroscopic analysis

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ARTICLE IN PRESSG ModelPRBI-9612; No. of Pages 8I. Younes et al. / Process Biochemistry xxx (2012) xxxxxx 3

Table 2The actual design of experiments and response of deproteinization.

Design pointa Enzyme/substrate ratio (X1) Temperature (X2) Incubation time (X3) Deproteinization (%)

Experimentalb Predicted

1 3.5 26 262 3.5 54 593 3.5 38 334 3.5 79 795 1 30 326 1 75 727 6 36 398 6 82 809 1 68 6610 1 75 7611 6 76 7312 6 86 8713 3.5 69 7114 3.5 71 7115 3.5 68 7116 3.5 76 7117 3.5 70 71

Bold values rea Experimenb The values

The degreintensity of ththe resonancefollowing rela

%DA =(I[C1] +

I is the intensi

2.9. Antimicro

The micro(ATCC 4698), EKlebsiella pneucus aureus (ATAntibacterial aBerghe and Vli4.68). The inoc106 colony foragar. The inocudiameter) werple. Well with 3.25). Gentamfor 1 h at 4 C, evaluated by mters (includingcontrols. The mreplications, a

2.10. Statistic

All experimdeviation errothe SPSS softwwas carried ou

3. Results

3.1. Enzymproteases

Firstly, wrole on enzythe most efsis A21, B. licheniformideproteinizfor 3 h undwith enzym

1, hnzymhers % aned ises, wd alc

as cgreebtaimelaondlnsis l cont te0 Ct 38ic forent. H

thei [25].%), wojaveed s0 40 10 40 0 60

10 600 50

10 50 0 50

10 50 5 40 5 605 405 60 5 50 5 50 5 50 5 50 5 50

present the ve replicates at the center point used to estimate the pure error.ts were conducted in a random order.

given in the table are the average of three independent experiments.

e of acetylation (DA) of the samples was determined by dividing thee resonance of the methyl group carbon by the average intensity ofs of the glycosyl ring carbon atoms. The DA was calculated using thetionship [23]:

I[CH3] I[C2] + I[C3] + I[C4] + I[C5] + I[C6])/6

100 (3)

ty of the particular resonance peak.

bial activity of chitosan

organisms used for antimicrobial activity were Micrococcus luteusscherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),moniae (ATCC 13883), Bacillus cereus (ATCC 11778), Staphylococ-CC 25923) Salmonella typhi and Enterococcus faecalis (ATCC 29212).ctivity assays were performed according to the method described byetinck [24]. Chitosan was dissolved at 50 mg/ml in 0.1% acetic acid (pHulum suspension (200 l) of the tested microorganisms, containingming units (CFU/ml) of bacteria cells were spread on MullerHintonlums were allowed to dry for 5 min. Then, bores (3-mm depth, 6-mme made using a sterile borer and were loaded with 50 l of each sam-only acetic acid (without chitosan) was used as a negative control (pHycin was used as positive reference. The Petri dishes were kept, rstly,and then were incubated for 24 h at 37 C. Antibacterial activity waseasuring the diameter of the growth inhibition zones in millime-

well diameter of 6 mm) for the test organisms and comparing to theeasurements of inhibition zones were carried out for three sample

nd values are the average of three replicates.

in Fig.crude etwo ot(65 3describprotealain anchosention dethose ofor bro

Secmojaveimentadiffereout at 5of aboutrostattreatmbonds,ments(71 2of B. mincrease this article in press as: Younes I, et al. Chitin and chitosan preparaization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.20

al analysis

ents were carried out in triplicate, and average values with standardrs are reported. Mean separation and signicance were analyzed usingare package (SPSS, Chicago, IL). Correlation and regression analysist using the EXCEL program.

and discussion

atic deproteinization of shrimp waste by microbial

e tried to point out the main parameters playing amatic deproteinization. In this view, we tried to select

fective enzymes. Microbial proteases from B. mojaven-subtilis A26, A. clavatus ES1, B. licheniformis MP1, B.s NH1 and V. metschnikovii J1 were tested for theiration efciency. Deproteinization tests were conducteder conditions of optimal enzyme activity and stabilitye/substrate ratios equal to 20 U/mg of protein. As shown

rate. BeyonB. mojav

showed an

Fig. 1. DeprotBromelain; A2MP1; A26: B. sigh deproteinization degrees were obtained with thees of A21, A26, J1 and MP1 (at about 76 4%), while the

NH1 and ES1 were exerting signicantly lower valuesd 59 3% respectively). B. mojavensis A21 strain, rarelyn the literature, which produces at least six differentas retained. Otherwise, commercial enzymes, brome-

alase, were used as control group. These enzymes wereontrol for deproteinization experiments. Deproteiniza-s obtained with bromelain and alcalase were lower thanned with crude enzymes; it reached 67 3% and 54 3%in and alcalase, respectively.y, the enzymatic deproteinization of shrimp shells by B.A21 crude enzyme was carried out under several exper-nditions, including various enzyme/substrate ratios,mperatures, pH and incubation times. Reaction carried

without enzymes resulted in a deproteinization degree 2%. Indeed, some proteins associated to chitin by elec-ces or hydrogen bonds, could be dissociated by thermalowever, other proteins are linked to chitin by covalent

r removal requires severe chemical or enzymatic treat- The deproteinization rate with an E/S ratio of 1 was highhich shows the effectiveness of the enzyme preparationnsis A21, and further increase in enzyme concentrationlightly (from 71 2% to 77 2%) the deproteinizationtion from shrimp shells using optimized enzymatic12.07.017

d 20 U/mg the deproteinization rate remained constant.ensis A21 crude alkaline protease characterization [13]optimal activity at pH 8.011.0 and at 60 C, using casein

einization of shrimp waste (%) by protease preparations. Alcalase;1: B. mojavensis A21; J1: V. metschnikovii J1; MP1: B. licheniformisubtilis A26; NH1: B. licheniformis NH1 and ES1: A. clavatus ES1.

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Table 3Analysis of Variance (ANOVA) for the t of experimental data to response surface model.

Source of variation Sum of squares (SS) Degrees of freedom (DF) Mean square Fexp Signicance

Regression 5706.44 9 634.04 28.85 0.0102***ResidualLack of tPur error

Total

***: indicates s

as a substrathis work, ton the deprof enzymestime coulddegree.

With remojavensis (U2) and incvariables ona response conditions

3.2. Optimiproteases

The optwaste deprand carriedvarious E/Srange of 40pH was maconditions establish a and the deg

3.2.1. ModeThe coe

the basis osquare regrexpressed i

y = 70.4 + + 0.75

It is necethe F-test tthe analysiTable 3. Thestatisticallyof determinthat 97.4% effects. Thuthe experim

On the oby comparierror which(Table 3).

The validied domainsurfaces.

3.2.2. GrapThe rep

the ridge a

einizrts oon o

of thm res the

of pl signary (rlose ve tore (X

mus isorg the

cons show

was the E

signraturhe E

of aerat

samto thhen. It sng thg an

ally, e levigh le

Compm thxed

timermcted d thanic

thattely

shevens

depas beiniz153.80 7 115.00 3 38.80 4

5860.24 16

ignicant at the level 99.9%. N.S.: indicates non-signicant at the level 95.0%

te. It was extremely stable in the pH range of 7.011.0. Inhe preliminary study showed that pH has a little impactoteinization degree in the range of activity and stability

(pH 8.011.0) and only temperature and incubation have a considerable impact on the deproteinization

gards to these results, for the selected enzyme B.A21 crude alkaline protease, E/S ratio (U1), temperatureubation time (U3) were selected as effective operating

the deproteinization degree. In this view, we appliedsurface methodology allowing to predict the optimumfor enzymatic deproteinization of shrimp shells.

zation of the shrimp waste deproteinization using A21

imization of the experimental conditions of shrimpoteinization was achieved using a BoxBehnken design

out under several experimental conditions including ratio (in the range of 010 U/mg), temperature (in the60 C) and incubation time (in the range of 16 h). Theintained at 9.0. Table 2 shows the real experimentaland the measured responses. These data are used tomathematical relation between the set of parametersree of deproteinization.

l equation and validationfcients of the postulated model were calculated onf the experimental responses (Table 2) by the leastession using the NEMROD W software. The tted model,n coded variables, is represented by the equation:

20 X1 + 6.75 X2 + 4 X3 + 3.25 X1X2 + 0.25 X1X3 X2X3 20.825 X21 0.325 X22 + 6.175 X23

ssary to make an analysis of the variance (ANOVA) usingo attest the good quality of the tting [26]. Results ofs of variance for the tted model are summarized iny clearly indicated that the regression sum of squares is

signicant at the level 99.9%. Moreover, the coefcientation, R2, for the conversion yield was 0.974. This meansof the observed variation is attributed to the variables, it is concluded that the predicted model well ttedental data.

ther hand, the validity of the model has been establishedng the variance related to the lack of t to that of pure

demonstrated the non-signicance of the lack of t

model was used to predict response values in the stud-

deprotleft pamizaticenteroptimudisplaypointsdegreeboundthat, csensitiperatufactors

Theplottinis heldguresciencywhen has notempewhen tdegreea temp

Thepared plots w(50 C)by xi7.5 U/m

Finaveragwith h

3.2.3. Fro

were bationto concondushowenot sigrmedadequashrimpB. moja

Thetease wDeprote this article in press as: Younes I, et al. Chitin and chitosan preparaization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.20

and to draw isoresponse contour plots and response

hical interpretation of the response surface modelresentation of the optimum path computed fromnalysis [26] of the response surface tted for the

better thanually highethe effects imum valuafter six dasame condi21.97383.33 3.95 10.9 NS

9.70

ation degree is shown in Fig. 2. The right parts and thef both plots refer to the maximization and the mini-f the response, respectively. The distance (r) from thee design is indicated in abscissas. Fig. 2a shows that thesponse reached as a function of the distance r. Fig. 2b

coordinates for each factor, in codied variables, of theot 2a. As it can be seen in Fig. 2a, the deproteinizationicantly increases from the center of the domain to the

= 1) where it reaches 88.87%. In addition, Fig. 2b showsto the maximum, the deproteinization degree is more

the variations of the incubation time (X3) and the tem-2) than that of E/S ratio (X1). To reach the maximum, allt tend toward relatively high values.esponse curves and the response surface were drawn by

response variation against two factors, while the thirdtant at its mean level (Fig. 3). The examination of theses that, an important effect on the deproteinization ef-

provided by the E/S ratio (X1). Indeed, Fig. 3a shows that/S ratio is low, between 0 and 5 U/mg, the temperatureicant effect on the deproteinization degree. However,e has a very signicant positive effect on the response/S ratio is beyond 5 U/mg. In this way, a deproteinizationbove 85% is obtained with an E/S ratio of 7.5 U/mg andure of 60 C.e could be said for the reaction time effect, when com-at of temperature. Fig. 3b represents the isocontour

the level of the temperature is xed to its average levelhowed that yields higher than 85% could be reachede E/S ratio and incubation time at levels higher thand 6 h, respectively.it could be seen from Fig. 3c that, by keeping X1 at itsel (5 U/mg), high deproteinization degrees are obtainedvels of both temperature and incubation time.

arison between model and experimental resultsis model analysis, the optimal experimental conditions

at: E/S ratio 7.75 U/mg, temperature 60 C and incu- 6 h which allow to obtain 94 4% of yield. In order

this prediction, an additional independent run wasusing these selected variable levels. Results obtainedt the observed deproteinization degree (88 5%) was

antly different from the predicted value. This result con- the empirical model derived from RSM can be used to

describe the relationship between the factors and thells deproteinization response using the crude enzyme ofis A21.roteinization activity of B. mojavensis A21 crude pro-etter than many proteases reported in previous studies.ation rate obtained using A21 proteases was evention from shrimp shells using optimized enzymatic12.07.017

values reached using fermentation which gives habit-r deproteinization rate. Busto and Healy [27] comparedof microbial and enzymatic deproteinization. A max-e of 82% was achieved with Pseudomonas maltophiliays and 64% with puried microbial protease under thetion. Fermentation using the culture supernatant from

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optim

Pseudomona78% after a ditions by Pmethodologafter ve dalso appliedshrimp wasobtained byof 7.75 U/mis explainedprotected blayer of shrfrom the attoccur [25]. be linked cochitins [25]

3.3. Chemic

In the reshould be rwaste deproacid treatmwas comple1.5 M HCl so

One of tlow mineratent of minaround of 1

The treawaste allowas a water igood yield fwere presenthe extracti

3.4. Chitin c

The samafter 4 h in(Table 4). Inprepared byalkaline trewaste befoof chitin (3comparable

eralizt to pduce32]. emoaterintennce cisolaemovieveatureoug

ed byd maown

epar

majacetchito2.5 M

of thn deC RM

chito abso

C CP/Fig. 2. Ridge analysis: optimal response plot (a) and

s aeruginosa K-187 allows a deproteinization rate ofseven-day incubation at 37 C [28]. Fermentation con-. aeruginosa A2 were optimized by response surfacey and maximal deproteinization of 89% was achieved

ays incubation [7]. Bacillus cereus SV1 proteases were for enzymatic deproteinization. Protein removal fromte reached the same value of deproteinization degree

A21 proteases but using higher E/S ratio (20 insteadg) [8]. The fact that deproteinization cannot reach 100%

by the non-accessibility of enzymes to some proteinsy chitin and minerals. Indeed, the proteins in the innerimp shell waste are protected by the outer layer chitin,ack by proteases and thus, no further proteolysis couldIn addition, some portions of peptides are suggested tovalently to a small number of the C-2 amino groups of.

al demineralization

covery of chitin from shrimp waste, associated mineralsemoved as a second stage. As a consequence, shrimpteinized by enzymatic treatment was subjected to mildent in order to remove minerals. The demineralizationtely achieved within 6 h at 50 C after treatment withlution at a ratio of 1:10 (w/v).

he factors determining the good quality of chitin is thel content [29]. Chitin obtained in this work present con-erals as low as those reported in other works [30] at.9%.tments employed to extract chitin from the shrimped the recovery of 18.5 2.3% of its initial dry mass

demincontenwas reet al. [ity of rraw mture codifferechitin plete rnot achin liter

Althobtainto avoibreakd

3.5. Pr

Theline destudy, with 1ciencychitosastate 13

of the the 13C

3.6. 13e this article in press as: Younes I, et al. Chitin and chitosan preparaization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.20

nsoluble white brous material, which indicates that aor the chitin extraction was attained and no pigmentst in the chitin. Other studies reported similar yields foron of chitin [29,30].

haracterization

e raw material was treated by alkali (NaOH 1.25 Mcubation at 80 C) [8]; this treatment gives chitin 2

Table 4, the characteristics of the raw material, chitin enzymatic treatment (chitin 1) and that obtained byatment (chitin 2) are compared. The ground shrimpre pre-treatment contained a relatively high contents3.5 2.3%) and ash (33.2 0.2%). These results are

with those reported by previous studies [29,31]. The

NMR ispolysacchanon-destruformation

Table 4Properties of t(1) and by alka

%

Moisture Chitin Ash ProteinLipid Appearanceal coordinate plot (b).

ation conditions used in this study reduce the mineralermissible limits in the chitin. Indeed, the ash contentd to about 1.9%. This was lower than that found by SiniThis low ash content for chitin indicated the suitabil-val of calcium carbonate and other minerals from theal. There were no signicant differences in the mois-t and ash among the two chitins (p > 0.05). An importantoncerns the protein content signicantly higher in theted after enzymatic deproteinization (p < 0.05); com-al of the residual protein associated with the chitin wasd even if the residual yield is lower than usually found.

h such deproteinization percentage is lower than that chemical treatment, enzymatic deproteinization helpsny drawbacks of chemical treatment, over-hydrolysis,

of chitin, etc.

ation of chitosan

or procedure for obtaining chitosan is based on the alka-ylation of chitin with strong alkaline solution. In thissan was prepared from chitin obtained by a treatment

NaOH in 1:10 (w/v) ratio at 140 C for 4 h. The ef-is treatment is evaluated by the acetylation degree oftermined by Nuclear Magnetic Resonance (NMR). SolidN spectroscopy was used in order to verify the purity

san sample by the chemical shifts and the intensities ofrption peaks [33].

MAS-NMR spectroscopic analysistion from shrimp shells using optimized enzymatic12.07.017

one of the most powerful tools in the study ofride composition and sequential structure. NMR is active method resulting in retained structure and con-of the polysaccharide, making it possible to monitor

he chitins obtained by deproteinization with B. mojavensis proteasesli deproteinization (2).

Raw material Chitin (1) Chitin (2)

67.0 1.1 4.6 1.1 3.9 0.533.6 2.3 18.5 2.3 20.0 2.033.2 0.2 1.9 0.1 1.4 0.127.1 1.3 12.0 1.6 6.2 1.36.0 0.3

White akes Yellowish akes

Please citdeprotein


Fig. 3. Three-dand incubation

reactions aferent solve

Solid stachanges in chitosan prchitosan pranalysis of e this article in press as: Younes I, et al. Chitin and chitosan preparaization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.20

imensional response surface and contour plots for the effect on the deproteinization de time at constant temperature 50 C. (b) Temperature and incubation time at constant E

nd other structural and physical properties under dif-nt conditions.te 13C CP/MAS-NMR is known to be very sensitive tothe local structure. 13C CP/MAS-NMR spectrum of theepared by enzymatic deproteinization and commercialepared by alkaline treatment, are shown in Fig. 4. NMRthe shrimp waste chitosan gave similar peak pattern

to that of cbon atoms monomerichigh structuwhile the maround 23 pare, the motion from shrimp shells using optimized enzymatic12.07.017

gree of: E/S ratio and temperature at constant time 3.5 h (a) E/S ratio/S ratio 5.00 U/mg (c).

ommercial chitosan. There are 8 signals for the 8 car-of chitosan. The C1-C6 carbons of N-acetylglucosamine

unit are observed between 50 and 110 ppm, indicatingral homogenity. The carbonyl group is around 173 ppm,ethyl group of the acetyl group produced a peak atpm. The less the peaks of carbonyl and acetyl groups

re efcient the deacetylation reaction is. Fig. 4 shows the

Please citdeprotein


Fig. 4. 13C CP/ d by edemineralizat emin

chitosan spsince therethe CH3 andto C1 (104and C6 (61teins were peaks, suggof some bacble by-prodchitosan (Fi

The degusing Eq. (commerciadeacetylateis considerethe higher d

3.7. Antimi

Antimicrspecies hasimportant applicationacetic acid four Gram-inhibited thria tested eby deproteidegree of acommerciaare in line antibacteriadegree. Thithan Gram-lipopolysacseemed to Gram-posittively charg

meters of inhibition zones against Gram-positive and Gram-negative.

Diameter of inhibition zones (mm)

Chitosana Chitosanb

Escherichia coli 12.4 1.5 7 1.2Pseudomonas aeruginosa R RKlebsiella pneumoniae 12 1.2 7 0.8Salmonella typhi 10 0.6 9 0.4

+ Staphylococcus aureus 8 0.5 7 0.6Bacillus cereus 9.5 0.5 9 0.5Enterococcus faecalis R RMicrococcus luteus 9 1.2 8.4 0.8

r well: 6 mm; R = resistant.osan prepared from chitin obtained by enzymatic deproteinization with B.sis proteases and chemical demineralizationmercial chitosan obtained by chemical deproteinization and chemical dem-tion.MAS-NMR solid-state spectra of chitosans. (1) Chitosan prepared from chitin obtaineion. (2) Commercial chitosan obtained by chemical deproteinization and chemical d

ectrum, in which the deacetylation of chitin is evident, are tight peaks at 23 and 173 ppm, that correspond to

C O groups, respectively. The other peaks correspond.63), C2 (55.71), C3 (73.93), C4 (83.63), C5 (76.14).46). Note in the spectrum that the removal of pro-

efcient during the extraction, once there are no otheresting a great purity of the product [34]. The presencekground noise could be due to the presence of a possi-uct or impurity in the sample, especially in commercialg. 4).ree of acetylation in both chitosans was determined3). The degree of acetylation was 22% and 4% in thel sample and the prepared sample, respectively. Ad chitin with a rate of 7090% and low protein contentd as a good nal product, the best one being that withegree of deacetylation.

Table 5The diabacteria

Gram

Gram

Diametea Chit

mojavenb Com

ineralizae this article in press as: Younes I, et al. Chitin and chitosan preparaization. Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.20

crobial activity

obial activity of chitosans against several bacterial been recognized and is considered as one of the mostproperties linked directly to their possible biologicals. The antimicrobial activity of chitosan dissolved in0.1% was investigated against four Gram-positive andnegative bacteria. As shown in Table 5, both chitosanse growth of all Gram-negative and Gram-positive bacte-xcepted P. aeruginosa and E. faecalis. Chitosan obtainednization with B. mojavensis proteases categorized as lowcetylation showed higher inhibition activity than thel one with higher degree of acetylation. These resultswith the works of Chen et al. [35] who reported thatl activity increase in the order of chitin deacetylations inhibition is more important against Gram-negativepositive bacteria tested. Gram-negative bacteria, withcharide at the outer surface providing negative charges,be very sensitive to chitosan while the sensitivity ofive bacteria that can have variable amounts of nega-ed teichoic acids at their outer surface varied greatly

[36]. Howeof chitosan

4. Conclus

A BoxBwas applieof shrimp sproteases. Athe shell prsurface metwere: an e60 C and adeproteinizof acetylatioactivities.

Acknowled

This woEducation anzymatic deproteinization with B. mojavensis proteases and chemicaleralization.tion from shrimp shells using optimized enzymatic12.07.017

ver, No et al. [37] showed stronger bactericidal effects toward Gram-positive than Gram-negative bacteria.

ion

ehnken design with three variables and three levelsd for the determination of deproteinization efciencyhells with enzymatic treatment by B. mojavensis A2121 proteases were found to remove up to 88 5% of

oteins in agreement with optimization using responsehodology. The optimal conditions for deproteinizationnzyme/substrate ratio of 7.75 U/mg, a temperature ofn incubation time of 6 h. Chitin obtained by enzymatication was then converted to chitosan, with a low degreen, which was found to exhibit remarkably antibacterial

gment

rk was supported by grants from Ministry of Highernd Scientic Research, Tunisia.

Please cit eparadeprotein io.20


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Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization1 Introduction2 Materials and methods2.1 Raw material2.2 Chemical analysis of shrimp waste homogenate2.3 Microbial strains and enzymes preparation2.4 Deproteinization of shrimp waste by proteases2.5 Experimental design and statistical analysis2.6 Chemical demineralization2.7 Deacetylation of chitin2.8 13C CP/MAS-NMR spectroscopic analysis2.9 Antimicrobial activity of chitosan2.10 Statistical analysis

3 Results and discussion3.1 Enzymatic deproteinization of shrimp waste by microbial proteases3.2 Optimization of the shrimp waste deproteinization using A21 proteases3.2.1 Model equation and validation3.2.2 Graphical interpretation of the response surface model3.2.3 Comparison between model and experimental results

3.3 Chemical demineralization3.4 Chitin characterization3.5 Preparation of chitosan3.6 13C CP/MAS-NMR spectroscopic analysis3.7 Antimicrobial activity

4 ConclusionAcknowledgmentReferences

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Chitin and Chitosan Preparation From Shrimp Shells Using Optimized Enzymatic Deproteinization Younes I