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Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization
Process Biochemistry 47 (2012) 2032–2039
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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 Laboratory of Industrial Chemistry, National School of Engineering, BP W 3038 Sfax, Tunisia c European Synchrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Cedex 9, France d Research Centre of Natural Macromolecules (CERMAV-CNRS) affiliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France b
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 11 May 2012 Accepted 14 July 2012 Available online 1 September 2012 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 Box–Behnken 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 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, 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 dissolved 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 cellulose, and its derivatives like chitosan are widely recognized to have immense applications in many fields [1]. They are widely used in the food industry, medicinal fields, 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 fibers 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: [email protected] (I. Younes). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.017
level for specific applications. Conventionally, preparation of chitin from such shellfish 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 final inconsistent physiological properties [5]. The chemical treatments also create waste disposal problems, because neutralization and detoxification 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 treatment by proteolytic enzymes which have been applied for the deproteinization of crustacean wastes [7,8]. The aim of this work is to investigate the influence of several operating parameters such as, enzyme/substrate ratio, temperature and incubation time on the deproteinization degree of shrimp shells by non-commercial Bacillus mojavensis A21 crude enzyme. Response surface methodology (RSM) is useful for designing experiments, building models and analysing the effects of several 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 determine a suitable polynomial equation that describes the response
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surface, RSM can be employed to optimize the process for gathering research results better than classical one-variable-at-a-time or full-factorial experimentation. In this work, a Box–Behnken 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 followed 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 20 min at 90 ◦ C. The cooked sample was drained and homogenized in a Moulinex® blender for about 2 min then used for moisture determination and kept at −20 ◦ C until further use. Commercial chitosan [39280-86-9] was provided by MP Biomedicals LLC France; 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, respectively, 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 Sfax city by Haddar et al. [13] and Agrebi et al. [14], respectively. Bacillus licheniformis NH1 was isolated by El Hadj Ali et al. [15] from an activated sludge reactor treating fishery 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 identified on the basis of the 16S rRNA gene sequencing and biochemical properties. The medium used for the isolation of A21, A26, NH1, MP1 and J1 strains was Luria–Bertani broth medium [19] composed of (g/l): peptone, 10; yeast extract, 5; NaCl, 5 (pH 7.0). The medium used 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 (pH 9.0). Production of proteases was carried out in optimized medium of each microbial strain. Media were autoclaved at 120 ◦ C for 20 min. Cultivations were performed on a rotatory shaker in optimal conditions for each microbial strain, in 250 ml Erlenmeyer flasks with a working volume of 25 ml. The cultures were centrifuged 5 min at 10,000 rpm, and the cell-free supernatants were recovered and concentrated by the addition of solid ammonium sulfate to 80% saturation. Protease activity was measured by the method described by Kembhavi et al. [20] using casein as a substrate. 2.4. Deproteinization of shrimp waste by proteases Microbial crude enzyme preparations were tested for their deproteinization efficiency. 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 (300 ml). Shrimp waste homogenate (15 g) were mixed with 45 ml distilled water. The pH and temperature of the mixture were adjusted to the optimum conditions for each enzyme: pH 10.0, 50 ◦ C for A21, NH1 and MP1 enzymes; pH 8.0, 40 ◦ C for A26; pH 11.0, 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 with crude enzymes. The reaction was then stopped by heating the solution at 90 ◦ C during 20 min 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]: [(PO × O) − (PR × R)] × 100 %DDP = PO × O
(1)
where PO 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.
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Table 1 Design of experiment-levels of various process parameters of the Box–Behnken design. Parameter
X1 : enzyme/substrate ratio (U/mg) X2 : temperature (◦ C) X3 : incubation time (h)
Level −1.0
0.0
1.0
0 40 1
5 50 3.5
10 60 6
2.5. Experimental design and statistical analysis In order to describe the nature of the response surface in the experimental region, a Box–Behnken design was applied. As presented in Table 1, the experimental design involved three parameters (U1 : enzyme/substrate, U2 : temperature and U3 : incubation time), each at three levels for low, middle and high concentrations. Table 2 represents the design matrix of a 17 trials experiment. For predicting the optimal point, a second order polynomial function was fitted to correlate the relationship between independent variables and response. For the three factors this equation is yˆ = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where yˆ is the predicted response, b0 model constant; X1 , X2 and X3 are independent variables; b1 , b2 and b3 are linear coefficients; b12 , b13 and b23 are cross product coefficients and b11 , b22 and b33 are the quadratic coefficients. Xj : coded variables related to the natural variables Uj by the following equation: Xj = (Uj − U0j )/Step of variation where: U0j = (Uj,high – Uj,low )/2 Step of variation of j = (Uj,high + Uj,low )/2 Uj,high and Uj,low : two extreme levels (high and low) given for each natural variable Uj . The coded variables Xj are equal to −1 and +1 when the levels of natural variable Uj are Uj,low and Uj,high , respectively. The model coefficients were estimated by a least squares fitting of the model to the experimental results obtained in the design points (runs no. 1–12). The five 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 1.5 M HCl in 1:10 (w/v) ratio for 6 h at 50 ◦ C under constant stirring (150 rpm). The chitin product was filtered through four layers of gauze with the aid of a vacuum pump and washed to neutrality with deionized water and then dried for 1 h at 60 ◦ C. Demineralization was expressed as percentage and computed by the following equation [21]: %DDM =
[(MO × O) − (MR × R)] × 100 MO × O
(2)
where MO and MR are ash contents (%) before and after demineralization; while, O and R represent the mass (g) of deproteinized shell and demineralized residue in dry weight basis, respectively. 2.7. Deacetylation of chitin The purified chitin was treated with 12.5 M NaOH in 1:10 (w/v) ratio at 140 ◦ C for 4 h until it was deacetylated to chitosan. After filtration, 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 resonance) 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 (field of 7.04 T). CP/MAS sequence was used with the following parameters: the 13 C spin lattice relaxation time was 5 s; powdered samples were placed in an alumina rotor used for the double airbearing-type MAS system and spun as fast as 8 kHz; contact time was 8 ms.
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Table 2 The actual design of experiments and response of deproteinization. Design pointa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Enzyme/substrate ratio (X1 )
0 10 0 10 0 10 0 10 5 5 5 5 5 5 5 5 5
Temperature (X2 )
Incubation time (X3 )
40 40 60 60 50 50 50 50 40 60 40 60 50 50 50 50 50
3.5 3.5 3.5 3.5 1 1 6 6 1 1 6 6 3.5 3.5 3.5 3.5 3.5
Deproteinization (%) Experimentalb
Predicted
26 54 38 79 30 75 36 82 68 75 76 86 69 71 68 76 70
26 59 33 79 32 72 39 80 66 76 73 87 71 71 71 71 71
Bold values represent the five replicates at the center point used to estimate the pure error. a Experiments were conducted in a random order. 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[CH3 ] × 100 (I[C1 ] + I[C2 ] + I[C3 ] + I[C4 ] + I[C5 ] + I[C6 ])/6
(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), Staphylococcus aureus (ATCC 25923) Salmonella typhi and Enterococcus faecalis (ATCC 29212). Antibacterial activity assays were performed according to the method described by Berghe and Vlietinck [24]. Chitosan was dissolved at 50 mg/ml in 0.1% acetic acid (pH 4.68). The inoculum suspension (200 l) of the tested microorganisms, containing 106 colony forming units (CFU/ml) of bacteria cells were spread on Muller–Hinton agar. The inoculums were allowed to dry for 5 min. Then, bores (3-mm depth, 6-mm diameter) were made using a sterile borer and were loaded with 50 l of each sample. 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, firstly, for 1 h at 4 ◦ C, and then were incubated for 24 h at 37 ◦ C. Antibacterial activity was evaluated by measuring the diameter of the growth inhibition zones in millimeters (including well diameter of 6 mm) 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 deviation errors are reported. Mean separation and significance were analyzed using the SPSS software package (SPSS, Chicago, IL). Correlation and regression analysis was carried out using the EXCEL program.
in Fig. 1, high deproteinization degrees were obtained with the crude enzymes of A21, A26, J1 and MP1 (at about 76 ± 4%), while the two others NH1 and ES1 were exerting significantly lower values (65 ± 3% and 59 ± 3% respectively). B. mojavensis A21 strain, rarely described in the literature, which produces at least six different proteases, was retained. Otherwise, commercial enzymes, bromelain and alcalase, were used as control group. These enzymes were chosen as control for deproteinization experiments. Deproteinization degrees obtained with bromelain and alcalase were lower than those obtained with crude enzymes; it reached 67 ± 3% and 54 ± 3% for bromelain and alcalase, respectively. Secondly, the enzymatic deproteinization of shrimp shells by B. mojavensis A21 crude enzyme was carried out under several experimental 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 38 ± 2%. Indeed, some proteins associated to chitin by electrostatic 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 treatments [25]. The deproteinization rate with an E/S ratio of 1 was high (71 ± 2%), which shows the effectiveness of the enzyme preparation of B. mojavensis A21, and further increase in enzyme concentration increased slightly (from 71 ± 2% to 77 ± 2%) the deproteinization rate. Beyond 20 U/mg the deproteinization rate remained constant. B. mojavensis A21 crude alkaline protease characterization [13] showed an optimal activity at pH 8.0–11.0 and at 60 ◦ C, using casein
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. mojavensis A21, B. subtilis A26, A. clavatus ES1, B. licheniformis MP1, B. licheniformis NH1 and V. metschnikovii J1 were tested for their deproteinization efficiency. Deproteinization tests were conducted for 3 h under conditions of optimal enzyme activity and stability with enzyme/substrate ratios equal to 20 U/mg of protein. As shown
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 fit of experimental data to response surface model. Source of variation
Sum of squares (SS)
Regression Residual Lack of fit Pur error
5706.44 153.80 115.00 38.80
Degrees of freedom (DF) 9 7 3 4
Total
5860.24
16
Mean square
Fexp
634.04 21.97 383.33 9.70
28.85 3.95
Significance 0.0102*** 10.9 NS
***: indicates significant at the level 99.9%. N.S.: indicates non-significant at the level 95.0%
as a substrate. It was extremely stable in the pH range of 7.0–11.0. In this work, the preliminary study showed that pH has a little impact on the deproteinization degree in the range of activity and stability of enzymes (pH 8.0–11.0) 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 (U1 ), temperature (U2 ) and incubation time (U3 ) 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 Box–Behnken design and carried out under several experimental conditions including various E/S ratio (in the range of 0–10 U/mg), temperature (in the range of 40–60 ◦ C) and incubation time (in the range of 1–6 h). 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 coefficients of the postulated model were calculated on the basis of the experimental responses (Table 2) by the least square regression using the NEMROD W software. The fitted model, expressed in coded variables, is represented by the equation: yˆ = 70.4 + 20 X1 + 6.75 X2 + 4 X3 + 3.25 X1 X2 + 0.25 X1 X3
deproteinization degree is shown in Fig. 2. The right parts and the left parts of both plots refer to the maximization and the minimization 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 codified variables, of the points of plot 2a. As it can be seen in Fig. 2a, the deproteinization degree significantly increases from the 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 (X3 ) and the temperature (X2 ) than that of E/S ratio (X1 ). 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 figures shows that, an important effect on the deproteinization efficiency was provided by the E/S ratio (X1 ). Indeed, Fig. 3a shows that when the E/S ratio is low, between 0 and 5 U/mg, the temperature has no significant effect on the deproteinization degree. However, temperature has a very significant positive effect on the response when the E/S ratio is beyond 5 U/mg. In this way, a deproteinization degree of above 85% is obtained with an E/S ratio of 7.5 U/mg and a temperature of 60 ◦ C. The same could be said for the reaction time effect, when compared to that of temperature. Fig. 3b represents the isocontour plots when the level of the temperature is fixed to its average level (50 ◦ C). It showed that yields higher than 85% could be reached by fixing the E/S ratio and incubation time at levels higher than 7.5 U/mg and 6 h, respectively. Finally, it could be seen from Fig. 3c that, by keeping X1 at its average level (5 U/mg), high deproteinization degrees are obtained with high levels of both temperature and incubation time.
+ 0.75 X2 X3 − 20.825 X12 − 0.325 X22 + 6.175 X32 It is necessary to make an analysis of the variance (ANOVA) using the F-test to attest the good quality of the fitting [26]. Results of the analysis of variance for the fitted model are summarized in Table 3. They clearly indicated that the regression sum of squares is statistically significant at the level 99.9%. Moreover, the coefficient of determination, R2 , for the conversion yield was 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 fitted the experimental data. On the other hand, the validity of the model has been established by comparing the variance related to the lack of fit to that of pure error which demonstrated the non-significance of the lack of fit (Table 3). The valid model was used to predict response values in the studied 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 fitted for the
3.2.3. Comparison between model and experimental results From this model analysis, the optimal experimental conditions were fixed at: E/S ratio 7.75 U/mg, temperature 60 ◦ C and incubation time 6 h which allow to obtain 94 ± 4% of yield. In order to confirm this prediction, an additional independent run was conducted using these selected variable levels. Results obtained showed that the observed deproteinization degree (88 ± 5%) was not significantly different from the predicted value. This result confirmed that the empirical model derived from RSM can 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 protease 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 habitually higher deproteinization rate. Busto and Healy [27] compared the effects of microbial and enzymatic deproteinization. A maximum value of 82% was achieved with Pseudomonas maltophilia after six days and 64% with purified 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 conditions by P. aeruginosa A2 were optimized by response surface methodology and maximal deproteinization of 89% was achieved after five 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 7.75 U/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, from the 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].
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 suitability of removal of calcium carbonate and other minerals from the raw material. There were no significant differences in the moisture content and ash among the two chitins (p > 0.05). An important difference concerns the protein content significantly higher in the chitin isolated after enzymatic deproteinization (p < 0.05); complete 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.
3.3. Chemical demineralization
3.5. Preparation of chitosan
In the recovery of chitin from shrimp waste, associated minerals should be removed as a second stage. As a consequence, shrimp waste deproteinized by enzymatic treatment was subjected to mild acid treatment in order to remove minerals. The demineralization was completely achieved within 6 h at 50 ◦ C after treatment with 1.5 M HCl solution at a ratio of 1:10 (w/v). One of the factors determining the good quality of chitin is the low mineral content [29]. Chitin obtained in this work present content 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 18.5 ± 2.3% of its initial dry mass as a water insoluble white fibrous 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].
The major procedure for obtaining chitosan is based on the alkaline deacetylation of chitin with strong alkaline solution. In this study, chitosan was prepared from chitin obtained by a treatment with 12.5 M NaOH in 1:10 (w/v) ratio at 140 ◦ C for 4 h. The efficiency 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 chitosan sample by the chemical shifts and the intensities of the 13 C absorption peaks [33].
3.4. Chitin characterization The same raw material was treated by alkali (NaOH 1.25 M after 4 h 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 (33.5 ± 2.3%) and ash (33.2 ± 0.2%). These results are comparable with those reported by previous studies [29,31]. The
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 conformation 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). %
Raw material
Chitin (1)
Chitin (2)
Moisture Chitin Ash Protein Lipid Appearance
67.0 ± 1.1 33.6 ± 2.3 33.2 ± 0.2 27.1 ± 1.3 6.0 ± 0.3 –
4.6 ± 1.1 18.5 ± 2.3 1.9 ± 0.1 12.0 ± 1.6 – White flakes
3.9 ± 0.5 20.0 ± 2.0 1.4 ± 0.1 6.2 ± 1.3 – Yellowish flakes
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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 3.5 h (a) E/S ratio and incubation time at constant temperature 50 ◦ C. (b) Temperature and incubation time at constant E/S ratio 5.00 U/mg (c).
reactions and other structural and physical properties under different 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 carbon atoms of chitosan. The C1-C6 carbons of N-acetylglucosamine monomeric unit are observed between 50 and 110 ppm, indicating high structural homogenity. The carbonyl group is around 173 ppm, while the methyl group of the acetyl group produced a peak at around 23 ppm. The less the peaks of carbonyl and acetyl groups are, the more efficient the deacetylation reaction is. Fig. 4 shows the
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Fig. 4. 13 C CP/MAS-NMR solid-state spectra of chitosans. (1) Chitosan prepared from chitin 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 173 ppm, that correspond to the CH3 and C O groups, respectively. The other peaks correspond to C1 (ı104.63), C2 (ı55.71), C3 (ı73.93), C4 (ı83.63), C5 (ı76.14) and C6 (ı61.46). Note in the spectrum that the removal of proteins were efficient 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 possible 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 70–90% and low protein content is considered as a good final 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 inhibited the growth of all Gram-negative and Gram-positive bacteria tested excepted P. aeruginosa and E. faecalis. Chitosan obtained by deproteinization with B. mojavensis proteases categorized as 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 negatively 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 inhibition zones (mm) Chitosana
Chitosanb
Gram −
Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae Salmonella typhi
12.4 ± 1.5 R 12 ± 1.2 10 ± 0.6
7 ± 1.2 R 7 ± 0.8 9 ± 0.4
Gram +
Staphylococcus aureus Bacillus cereus Enterococcus faecalis Micrococcus luteus
8 ± 0.5 9.5 ± 0.5 R 9 ± 1.2
7 ± 0.6 9 ± 0.5 R 8.4 ± 0.8
Diameter well: 6 mm; R = resistant. a Chitosan prepared from chitin obtained by enzymatic deproteinization with B. mojavensis proteases and chemical demineralization b Commercial chitosan obtained by chemical deproteinization and chemical demineralization.
[36]. However, No et al. [37] showed stronger bactericidal effects of chitosan toward Gram-positive than Gram-negative bacteria. 4. Conclusion A Box–Behnken design with three variables and three levels was applied for the determination of deproteinization efficiency of shrimp shells with enzymatic treatment by B. mojavensis A21 proteases. A21 proteases were found to remove up to 88 ± 5% of the shell proteins in agreement with optimization using response surface methodology. The optimal conditions for deproteinization were: an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 ◦ C and an incubation time of 6 h. Chitin obtained by enzymatic deproteinization was then converted to chitosan, with a low degree of acetylation, which was found to exhibit remarkably antibacterial activities. Acknowledgment This work was supported by grants from Ministry of Higher Education and Scientific Research, Tunisia.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
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 Laboratory of Industrial Chemistry, National School of Engineering, BP W 3038 Sfax, Tunisia c European Synchrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Cedex 9, France d Research Centre of Natural Macromolecules (CERMAV-CNRS) affiliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France b
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 11 May 2012 Accepted 14 July 2012 Available online 1 September 2012 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 Box–Behnken 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 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, 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 dissolved 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 cellulose, and its derivatives like chitosan are widely recognized to have immense applications in many fields [1]. They are widely used in the food industry, medicinal fields, 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 fibers 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: [email protected] (I. Younes). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.017
level for specific applications. Conventionally, preparation of chitin from such shellfish 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 final inconsistent physiological properties [5]. The chemical treatments also create waste disposal problems, because neutralization and detoxification 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 treatment by proteolytic enzymes which have been applied for the deproteinization of crustacean wastes [7,8]. The aim of this work is to investigate the influence of several operating parameters such as, enzyme/substrate ratio, temperature and incubation time on the deproteinization degree of shrimp shells by non-commercial Bacillus mojavensis A21 crude enzyme. Response surface methodology (RSM) is useful for designing experiments, building models and analysing the effects of several 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 determine a suitable polynomial equation that describes the response
I. Younes et al. / Process Biochemistry 47 (2012) 2032–2039
surface, RSM can be employed to optimize the process for gathering research results better than classical one-variable-at-a-time or full-factorial experimentation. In this work, a Box–Behnken 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 followed 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 20 min at 90 ◦ C. The cooked sample was drained and homogenized in a Moulinex® blender for about 2 min then used for moisture determination and kept at −20 ◦ C until further use. Commercial chitosan [39280-86-9] was provided by MP Biomedicals LLC France; 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, respectively, 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 Sfax city by Haddar et al. [13] and Agrebi et al. [14], respectively. Bacillus licheniformis NH1 was isolated by El Hadj Ali et al. [15] from an activated sludge reactor treating fishery 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 identified on the basis of the 16S rRNA gene sequencing and biochemical properties. The medium used for the isolation of A21, A26, NH1, MP1 and J1 strains was Luria–Bertani broth medium [19] composed of (g/l): peptone, 10; yeast extract, 5; NaCl, 5 (pH 7.0). The medium used 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 (pH 9.0). Production of proteases was carried out in optimized medium of each microbial strain. Media were autoclaved at 120 ◦ C for 20 min. Cultivations were performed on a rotatory shaker in optimal conditions for each microbial strain, in 250 ml Erlenmeyer flasks with a working volume of 25 ml. The cultures were centrifuged 5 min at 10,000 rpm, and the cell-free supernatants were recovered and concentrated by the addition of solid ammonium sulfate to 80% saturation. Protease activity was measured by the method described by Kembhavi et al. [20] using casein as a substrate. 2.4. Deproteinization of shrimp waste by proteases Microbial crude enzyme preparations were tested for their deproteinization efficiency. 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 (300 ml). Shrimp waste homogenate (15 g) were mixed with 45 ml distilled water. The pH and temperature of the mixture were adjusted to the optimum conditions for each enzyme: pH 10.0, 50 ◦ C for A21, NH1 and MP1 enzymes; pH 8.0, 40 ◦ C for A26; pH 11.0, 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 with crude enzymes. The reaction was then stopped by heating the solution at 90 ◦ C during 20 min 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]: [(PO × O) − (PR × R)] × 100 %DDP = PO × O
(1)
where PO 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.
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Table 1 Design of experiment-levels of various process parameters of the Box–Behnken design. Parameter
X1 : enzyme/substrate ratio (U/mg) X2 : temperature (◦ C) X3 : incubation time (h)
Level −1.0
0.0
1.0
0 40 1
5 50 3.5
10 60 6
2.5. Experimental design and statistical analysis In order to describe the nature of the response surface in the experimental region, a Box–Behnken design was applied. As presented in Table 1, the experimental design involved three parameters (U1 : enzyme/substrate, U2 : temperature and U3 : incubation time), each at three levels for low, middle and high concentrations. Table 2 represents the design matrix of a 17 trials experiment. For predicting the optimal point, a second order polynomial function was fitted to correlate the relationship between independent variables and response. For the three factors this equation is yˆ = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where yˆ is the predicted response, b0 model constant; X1 , X2 and X3 are independent variables; b1 , b2 and b3 are linear coefficients; b12 , b13 and b23 are cross product coefficients and b11 , b22 and b33 are the quadratic coefficients. Xj : coded variables related to the natural variables Uj by the following equation: Xj = (Uj − U0j )/Step of variation where: U0j = (Uj,high – Uj,low )/2 Step of variation of j = (Uj,high + Uj,low )/2 Uj,high and Uj,low : two extreme levels (high and low) given for each natural variable Uj . The coded variables Xj are equal to −1 and +1 when the levels of natural variable Uj are Uj,low and Uj,high , respectively. The model coefficients were estimated by a least squares fitting of the model to the experimental results obtained in the design points (runs no. 1–12). The five 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 1.5 M HCl in 1:10 (w/v) ratio for 6 h at 50 ◦ C under constant stirring (150 rpm). The chitin product was filtered through four layers of gauze with the aid of a vacuum pump and washed to neutrality with deionized water and then dried for 1 h at 60 ◦ C. Demineralization was expressed as percentage and computed by the following equation [21]: %DDM =
[(MO × O) − (MR × R)] × 100 MO × O
(2)
where MO and MR are ash contents (%) before and after demineralization; while, O and R represent the mass (g) of deproteinized shell and demineralized residue in dry weight basis, respectively. 2.7. Deacetylation of chitin The purified chitin was treated with 12.5 M NaOH in 1:10 (w/v) ratio at 140 ◦ C for 4 h until it was deacetylated to chitosan. After filtration, 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 resonance) 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 (field of 7.04 T). CP/MAS sequence was used with the following parameters: the 13 C spin lattice relaxation time was 5 s; powdered samples were placed in an alumina rotor used for the double airbearing-type MAS system and spun as fast as 8 kHz; contact time was 8 ms.
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I. Younes et al. / Process Biochemistry 47 (2012) 2032–2039
Table 2 The actual design of experiments and response of deproteinization. Design pointa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Enzyme/substrate ratio (X1 )
0 10 0 10 0 10 0 10 5 5 5 5 5 5 5 5 5
Temperature (X2 )
Incubation time (X3 )
40 40 60 60 50 50 50 50 40 60 40 60 50 50 50 50 50
3.5 3.5 3.5 3.5 1 1 6 6 1 1 6 6 3.5 3.5 3.5 3.5 3.5
Deproteinization (%) Experimentalb
Predicted
26 54 38 79 30 75 36 82 68 75 76 86 69 71 68 76 70
26 59 33 79 32 72 39 80 66 76 73 87 71 71 71 71 71
Bold values represent the five replicates at the center point used to estimate the pure error. a Experiments were conducted in a random order. 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[CH3 ] × 100 (I[C1 ] + I[C2 ] + I[C3 ] + I[C4 ] + I[C5 ] + I[C6 ])/6
(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), Staphylococcus aureus (ATCC 25923) Salmonella typhi and Enterococcus faecalis (ATCC 29212). Antibacterial activity assays were performed according to the method described by Berghe and Vlietinck [24]. Chitosan was dissolved at 50 mg/ml in 0.1% acetic acid (pH 4.68). The inoculum suspension (200 l) of the tested microorganisms, containing 106 colony forming units (CFU/ml) of bacteria cells were spread on Muller–Hinton agar. The inoculums were allowed to dry for 5 min. Then, bores (3-mm depth, 6-mm diameter) were made using a sterile borer and were loaded with 50 l of each sample. 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, firstly, for 1 h at 4 ◦ C, and then were incubated for 24 h at 37 ◦ C. Antibacterial activity was evaluated by measuring the diameter of the growth inhibition zones in millimeters (including well diameter of 6 mm) 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 deviation errors are reported. Mean separation and significance were analyzed using the SPSS software package (SPSS, Chicago, IL). Correlation and regression analysis was carried out using the EXCEL program.
in Fig. 1, high deproteinization degrees were obtained with the crude enzymes of A21, A26, J1 and MP1 (at about 76 ± 4%), while the two others NH1 and ES1 were exerting significantly lower values (65 ± 3% and 59 ± 3% respectively). B. mojavensis A21 strain, rarely described in the literature, which produces at least six different proteases, was retained. Otherwise, commercial enzymes, bromelain and alcalase, were used as control group. These enzymes were chosen as control for deproteinization experiments. Deproteinization degrees obtained with bromelain and alcalase were lower than those obtained with crude enzymes; it reached 67 ± 3% and 54 ± 3% for bromelain and alcalase, respectively. Secondly, the enzymatic deproteinization of shrimp shells by B. mojavensis A21 crude enzyme was carried out under several experimental 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 38 ± 2%. Indeed, some proteins associated to chitin by electrostatic 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 treatments [25]. The deproteinization rate with an E/S ratio of 1 was high (71 ± 2%), which shows the effectiveness of the enzyme preparation of B. mojavensis A21, and further increase in enzyme concentration increased slightly (from 71 ± 2% to 77 ± 2%) the deproteinization rate. Beyond 20 U/mg the deproteinization rate remained constant. B. mojavensis A21 crude alkaline protease characterization [13] showed an optimal activity at pH 8.0–11.0 and at 60 ◦ C, using casein
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. mojavensis A21, B. subtilis A26, A. clavatus ES1, B. licheniformis MP1, B. licheniformis NH1 and V. metschnikovii J1 were tested for their deproteinization efficiency. Deproteinization tests were conducted for 3 h under conditions of optimal enzyme activity and stability with enzyme/substrate ratios equal to 20 U/mg of protein. As shown
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 fit of experimental data to response surface model. Source of variation
Sum of squares (SS)
Regression Residual Lack of fit Pur error
5706.44 153.80 115.00 38.80
Degrees of freedom (DF) 9 7 3 4
Total
5860.24
16
Mean square
Fexp
634.04 21.97 383.33 9.70
28.85 3.95
Significance 0.0102*** 10.9 NS
***: indicates significant at the level 99.9%. N.S.: indicates non-significant at the level 95.0%
as a substrate. It was extremely stable in the pH range of 7.0–11.0. In this work, the preliminary study showed that pH has a little impact on the deproteinization degree in the range of activity and stability of enzymes (pH 8.0–11.0) 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 (U1 ), temperature (U2 ) and incubation time (U3 ) 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 Box–Behnken design and carried out under several experimental conditions including various E/S ratio (in the range of 0–10 U/mg), temperature (in the range of 40–60 ◦ C) and incubation time (in the range of 1–6 h). 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 coefficients of the postulated model were calculated on the basis of the experimental responses (Table 2) by the least square regression using the NEMROD W software. The fitted model, expressed in coded variables, is represented by the equation: yˆ = 70.4 + 20 X1 + 6.75 X2 + 4 X3 + 3.25 X1 X2 + 0.25 X1 X3
deproteinization degree is shown in Fig. 2. The right parts and the left parts of both plots refer to the maximization and the minimization 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 codified variables, of the points of plot 2a. As it can be seen in Fig. 2a, the deproteinization degree significantly increases from the 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 (X3 ) and the temperature (X2 ) than that of E/S ratio (X1 ). 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 figures shows that, an important effect on the deproteinization efficiency was provided by the E/S ratio (X1 ). Indeed, Fig. 3a shows that when the E/S ratio is low, between 0 and 5 U/mg, the temperature has no significant effect on the deproteinization degree. However, temperature has a very significant positive effect on the response when the E/S ratio is beyond 5 U/mg. In this way, a deproteinization degree of above 85% is obtained with an E/S ratio of 7.5 U/mg and a temperature of 60 ◦ C. The same could be said for the reaction time effect, when compared to that of temperature. Fig. 3b represents the isocontour plots when the level of the temperature is fixed to its average level (50 ◦ C). It showed that yields higher than 85% could be reached by fixing the E/S ratio and incubation time at levels higher than 7.5 U/mg and 6 h, respectively. Finally, it could be seen from Fig. 3c that, by keeping X1 at its average level (5 U/mg), high deproteinization degrees are obtained with high levels of both temperature and incubation time.
+ 0.75 X2 X3 − 20.825 X12 − 0.325 X22 + 6.175 X32 It is necessary to make an analysis of the variance (ANOVA) using the F-test to attest the good quality of the fitting [26]. Results of the analysis of variance for the fitted model are summarized in Table 3. They clearly indicated that the regression sum of squares is statistically significant at the level 99.9%. Moreover, the coefficient of determination, R2 , for the conversion yield was 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 fitted the experimental data. On the other hand, the validity of the model has been established by comparing the variance related to the lack of fit to that of pure error which demonstrated the non-significance of the lack of fit (Table 3). The valid model was used to predict response values in the studied 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 fitted for the
3.2.3. Comparison between model and experimental results From this model analysis, the optimal experimental conditions were fixed at: E/S ratio 7.75 U/mg, temperature 60 ◦ C and incubation time 6 h which allow to obtain 94 ± 4% of yield. In order to confirm this prediction, an additional independent run was conducted using these selected variable levels. Results obtained showed that the observed deproteinization degree (88 ± 5%) was not significantly different from the predicted value. This result confirmed that the empirical model derived from RSM can 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 protease 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 habitually higher deproteinization rate. Busto and Healy [27] compared the effects of microbial and enzymatic deproteinization. A maximum value of 82% was achieved with Pseudomonas maltophilia after six days and 64% with purified 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 conditions by P. aeruginosa A2 were optimized by response surface methodology and maximal deproteinization of 89% was achieved after five 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 7.75 U/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, from the 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].
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 suitability of removal of calcium carbonate and other minerals from the raw material. There were no significant differences in the moisture content and ash among the two chitins (p > 0.05). An important difference concerns the protein content significantly higher in the chitin isolated after enzymatic deproteinization (p < 0.05); complete 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.
3.3. Chemical demineralization
3.5. Preparation of chitosan
In the recovery of chitin from shrimp waste, associated minerals should be removed as a second stage. As a consequence, shrimp waste deproteinized by enzymatic treatment was subjected to mild acid treatment in order to remove minerals. The demineralization was completely achieved within 6 h at 50 ◦ C after treatment with 1.5 M HCl solution at a ratio of 1:10 (w/v). One of the factors determining the good quality of chitin is the low mineral content [29]. Chitin obtained in this work present content 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 18.5 ± 2.3% of its initial dry mass as a water insoluble white fibrous 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].
The major procedure for obtaining chitosan is based on the alkaline deacetylation of chitin with strong alkaline solution. In this study, chitosan was prepared from chitin obtained by a treatment with 12.5 M NaOH in 1:10 (w/v) ratio at 140 ◦ C for 4 h. The efficiency 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 chitosan sample by the chemical shifts and the intensities of the 13 C absorption peaks [33].
3.4. Chitin characterization The same raw material was treated by alkali (NaOH 1.25 M after 4 h 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 (33.5 ± 2.3%) and ash (33.2 ± 0.2%). These results are comparable with those reported by previous studies [29,31]. The
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 conformation 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). %
Raw material
Chitin (1)
Chitin (2)
Moisture Chitin Ash Protein Lipid Appearance
67.0 ± 1.1 33.6 ± 2.3 33.2 ± 0.2 27.1 ± 1.3 6.0 ± 0.3 –
4.6 ± 1.1 18.5 ± 2.3 1.9 ± 0.1 12.0 ± 1.6 – White flakes
3.9 ± 0.5 20.0 ± 2.0 1.4 ± 0.1 6.2 ± 1.3 – Yellowish flakes
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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 3.5 h (a) E/S ratio and incubation time at constant temperature 50 ◦ C. (b) Temperature and incubation time at constant E/S ratio 5.00 U/mg (c).
reactions and other structural and physical properties under different 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 carbon atoms of chitosan. The C1-C6 carbons of N-acetylglucosamine monomeric unit are observed between 50 and 110 ppm, indicating high structural homogenity. The carbonyl group is around 173 ppm, while the methyl group of the acetyl group produced a peak at around 23 ppm. The less the peaks of carbonyl and acetyl groups are, the more efficient the deacetylation reaction is. Fig. 4 shows the
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Fig. 4. 13 C CP/MAS-NMR solid-state spectra of chitosans. (1) Chitosan prepared from chitin 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 173 ppm, that correspond to the CH3 and C O groups, respectively. The other peaks correspond to C1 (ı104.63), C2 (ı55.71), C3 (ı73.93), C4 (ı83.63), C5 (ı76.14) and C6 (ı61.46). Note in the spectrum that the removal of proteins were efficient 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 possible 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 70–90% and low protein content is considered as a good final 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 inhibited the growth of all Gram-negative and Gram-positive bacteria tested excepted P. aeruginosa and E. faecalis. Chitosan obtained by deproteinization with B. mojavensis proteases categorized as 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 negatively 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 inhibition zones (mm) Chitosana
Chitosanb
Gram −
Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae Salmonella typhi
12.4 ± 1.5 R 12 ± 1.2 10 ± 0.6
7 ± 1.2 R 7 ± 0.8 9 ± 0.4
Gram +
Staphylococcus aureus Bacillus cereus Enterococcus faecalis Micrococcus luteus
8 ± 0.5 9.5 ± 0.5 R 9 ± 1.2
7 ± 0.6 9 ± 0.5 R 8.4 ± 0.8
Diameter well: 6 mm; R = resistant. a Chitosan prepared from chitin obtained by enzymatic deproteinization with B. mojavensis proteases and chemical demineralization b Commercial chitosan obtained by chemical deproteinization and chemical demineralization.
[36]. However, No et al. [37] showed stronger bactericidal effects of chitosan toward Gram-positive than Gram-negative bacteria. 4. Conclusion A Box–Behnken design with three variables and three levels was applied for the determination of deproteinization efficiency of shrimp shells with enzymatic treatment by B. mojavensis A21 proteases. A21 proteases were found to remove up to 88 ± 5% of the shell proteins in agreement with optimization using response surface methodology. The optimal conditions for deproteinization were: an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 ◦ C and an incubation time of 6 h. Chitin obtained by enzymatic deproteinization was then converted to chitosan, with a low degree of acetylation, which was found to exhibit remarkably antibacterial activities. Acknowledgment This work was supported by grants from Ministry of Higher Education and Scientific Research, Tunisia.
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