ARTICLE IN PRESS
G Model BIOMAC 4125 1–9
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural differences between chitin and chitosan extracted from three different marine sources
1
2
3 4 5 6 7
Q1
Sawssen Hajji a,∗ , Islem Younes a , Olfa Ghorbel-Bellaaj a , Rachid Hajji b , Marguerite Rinaudo c , Moncef Nasri a , Kemel Jellouli a a
Laboratory of Enzyme Engineering and Microbiology, National School of Engineering of Sfax, University of Sfax, B.P. 1173, 3038 Sfax, Tunisia Laboratory of Solid State, Faculty of Science, University of Sfax, B.P. 802, 3018 Sfax, Tunisia c Biomaterials Applications, 6, rue Lesdiguières, 38000 Grenoble, France b
8
9
a r t i c l e
i n f o
a b s t r a c t
10 11 12 13 14
Article history: Received 18 November 2013 Received in revised form 11 January 2014 Accepted 17 January 2014 Available online xxx
15
20
Keywords: Chitin Penaeus kerathurus Carcinus mediterraneus Sepia officinalis
21
1. Introduction
16 17 18 19
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Three marine sources of chitin from Tunisia were investigated. Structural differences between ␣-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and -chitin from cuttlefish (Sepia officinalis) bones were indicated by the 13 C NMR, FTIR, and XRD diffractograms. The 13 C NMR analysis showed a splitting of the C3 and C5 carbon signals for ␣-chitin, while that of -chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of ␣-chitin in the region of O H, N H and CO stretching regions. IR and 13 C NMR were used to determine the chitin degree of acetylation. XRD analysis indicated that ␣-chitins were more crystalline polymorph than -chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molar masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source. © 2014 Published by Elsevier B.V.
Chitin is the second most abundant polysaccharide in biomass after cellulose. Chitin production was about 25,000 tons in 2006 [1]. This polymer is a linear chain consisting of poly (1–4) N-acetyld-glucosamine. Chitin is usually isolated from the exoskeletons of crustaceans and more particularly from shrimps and crabs where ␣-chitin isomorph is produced [2]. ␣-Chitin has a tightly compact structure due to its crystalline structure in which antiparallel chain favor strong hydrogen bonding [3]. Squid is another important source of chitin in which it exists in the -isomorph which was found to show higher solubility, higher reactivity and higher affinity toward solvents and swelling than ␣-chitin. These characteristics are due to weaker intermolecular hydrogen bonding ascribable to the parallel arrangement of the main chains [4]. ␥-Chitin, a third chitin allomorph, has also been described [5,6], but from a detailed analysis, it seems that it is just a variant of the ␣-family [7]. Several techniques to extract chitin from different sources have been reported. The most common method is referred to the chemical procedure which involves various major steps. Firstly, shells
∗ Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail address: [email protected] (S. Hajji).
are ground and minerals, mainly calcium carbonate, are removed (demineralization) using dilute acidic medium (usually HCl) at ambient temperature. Secondly, the proteins are extracted (deproteinization) from the residual material by treatment with aqueous solutions of NaOH or KOH. These traditional chemical methods create a disposal problem due to the large amounts of toxic waste which would pollute the environment. In addition, this process becomes expensive due to enforced environmental controls and disposal measures [8]. To overcome the disadvantage of chemical treatments, some efforts have been directed toward its substitution by more eco-friendly processes such as bacterial fermentation [9] or treatment by proteolytic enzymes which have been applied for the deproteinization of crustacean wastes [9–12]. Because solid state chitin has a compact structure, it remains insoluble in most solvents. Therefore, usually chemical deacetylation is performed to produce the most common derivative named chitosan [13]. In acidic conditions, chitosan becomes positively charged due to NH2 protonation and soluble in aqueous medium. Under these conditions, this polymer has numerous physiological and biological properties with great potential in a wide range of industries such as cosmetology (lotions, hair additives, body creams) [14,15], food (coating, preservative, antioxidant, antimicrobial) [16], biotechnology (chelator, emulsifier, flocculent) [17],
0141-8130/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
G Model BIOMAC 4125 1–9
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81
pharmacology and medicine (fibers, drugs, membranes, artificial organs) [18] and agriculture (soil modifier, films, fungicide) [19]. Chitosan can be characterized in terms of its quality, intrinsic properties (purity, molar mass, viscosity, and acetylation degree) and physical forms [20]. It was reported that both acetylation degree and molar mass are important chemical characteristics, which could influence the performance of chitosan in many of its applications [21]. The aim of this work was to extract chitin from different Tunisian marine sources using chemical demineralization and enzymatic deproteinization (with Bacillus mojavensis A21 proteases) and then to compare their physicochemical characteristics using XRD, FTIR and 13 C NMR spectroscopy. Following, the chitins obtained were deacetylated to chitosans. Acetylation degree and molar mass of each chitosan were examined. To the best of our knowledge, this present work is the first systematic trial to investigate the extraction of chitin and chitosan from different indigenous sources in Tunisia.
88 89 90
91
100
The moisture and ash contents were determined at 105 ◦ C and 550 ◦ C, respectively, according to the AOAC [22] standard methods 930.15 and 942.05. Total nitrogen content of each raw material was determined by using the Kjeldahl method. Separately, for each raw sample, pure chitin is prepared to determine its nitrogen contribution allowing to estimate the crude protein content by multiplying nitrogen content attributed to protein by the factor of 6.25 [23]. Lipids were determined gravimetrically by soxhlet extraction using hexane.
101
2.3. Chemical demineralization
92 93 94 95 96 97 98 99
102 103 104 105 106 107 108 109 110 111 112 113
114
115 116 117
2.5. Deproteinization of shell waste by proteases
Demineralization was carried out at room temperature using 0.55 M hydrochloric acid baths. Each bath was performed with 100 ml of acid solution and 10 g of raw material. The number of baths and their duration (between 15 and 60 min) were dependent upon the source [24]. Demineralization step was followed by pH evolution toward neutrality due to acid consumption. The end of the repeated series of baths was indicated by stability of medium acidity. Demineralized materials were filtered through four layers of gauze using a vacuum pump, washed to neutrality with deionized water and dried for 1 h at 60 ◦ C. Demineralization degree (DDM) was expressed as a percentage and computed by the following equation [25]: [(AO × O) − (AR × R)] %DDM = × 100 AO × O
(1)
where AO and AR are ash contents (%) before and after demineralization, respectively, while O and R represent the mass (g) of initial and demineralized residue respectively on dry weight basis.
(2)
where PO and PR are the protein concentrations (%) before and after hydrolysis, respectively, while O and R represent the mass (g) of original sample and hydrolyzed residue respectively on dry weight basis. 2.6. Deacetylation of chitin
2.7. Physicochemical characterization of chitins and chitosans
121 122 123 124 125 126 127 128 129 130 131
133 134 135 136 137 138 139 140 141 142 143 144 145
146
147 148 149 150
152 153 154 155 156
157
2.7.1. Fourier transform infrared spectroscopy (FTIR) Infrared spectra were obtained using a Perkin Elmer type FTIR 1000 spectrometer at room temperature and KBr pellets. The sample pellets were prepared at a pressure of 5 tons for 2 min. Pellets were scanned at room temperature (25 ◦ C) in the 400–4000 cm−1 spectral range [28]. The incertitude of this measurement was 2 cm−1 . In the case of overlapping peaks, deconvolution was performed to calculate the contribution of the individual peaks using Dmfit program [29] with Gaussian and/or Lorentzian shapes to estimate the area related to the specific vibration of each selected peak. Spectra were corrected for the baseline and the absorbance was normalized between 0 and 1. The acetylation degree (DA) of chitins and chitosans was calculated according to the method proposed by Moore and Roberts [30], as follows:
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
120
151
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 a chitosan perfectly soluble in might acidic conditions. After filtration, the residue was washed with deionised water, and the crude chitosan was recovered by drying in a dry heat incubator at 50 ◦ C during 12 h.
A1650 /A3450 DA (%) = × 100 1.33
119
132
2.2. Chemical analysis of the raw materials
2.1. Preparation of raw material
87
B. mojavensis A21 was isolated from marine water in Sfax by Haddar et al. [26]. The growth medium used for protease production was composed of (g/L): hulled grain of wheat, 30.0; yeast extract, 1.0; CaCl2 , 2.0; MgSO4 , 1.0; K2 HPO4 , 0.3; KH2 PO4 , 1.0; and NaCl, 2.0. The pH medium was adjusted to 9.0. Media were autoclaved at 121 ◦ C for 20 min. Cultivations were conducted in 250 ml Erlenmeyer flasks with a working volume of 25 ml. Incubations were carried out in an orbital shaking incubator at 30 ◦ C and 200 rpm for 24 h. The cultures were centrifuged at 10,000 rpm for 15 min 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. [27] using casein as a substrate.
[(PO × O) − (PR × R)] %DDP = × 100 PO × O
83
86
118
Shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells and cuttlefish (Sepia officinalis) bones were obtained in fresh conditions from a fishery products and processing plant located at Sfax, Tunisia. They were washed thoroughly with tap water, dessicated at room temperature and milled (sieved from 2 mm to 5 mm). After drying, they were kept at room temperature until used.
2. Materials and methods
85
2.4. Crude enzyme preparation
B. mojavensis A21 crude enzyme preparation was selected for its deproteinization efficiency [10]. Deproteinization tests were carried out in a thermostated stirred Pyrex reactor (300 ml). The demineralized shells homogenate (15 g) were mixed with 45 ml distilled water. The pH and temperature of the mixture were adjusted to pH 9.0 (with NaOH 4%, w/v) and 50 ◦ C. The shell waste proteins were digested with crude enzyme using different Enzyme/Substrate ratios (E/S) during 3 h. The reaction was stopped by heating at 90 ◦ C for 20 min to inactivate enzymes. The solid phase was washed, pressed manually through four layers of gauze and then dried at 60 ◦ C during 12 h. Deproteinization degree (DDP) was expressed as percentage and computed by the following equation [25]:
82
84
ARTICLE IN PRESS S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2
(3)
158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
173
ARTICLE IN PRESS
G Model BIOMAC 4125 1–9
S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
174 175 176 177 178
The amide-I band ( = 1650 cm−1 ) was used as the analytical band and the hydroxyl band ( = 3450 cm−1 ) as the internal reference band. The factor ‘1.33’ denoted the value of the ratio of A1655 /A3450 for fully N-acetylated chitin. Errors in %DA were less than 20% of their nominal value.
186
2.7.2. 13 C CP/MAS-NMR spectroscopic analysis Solid-state 13 C CP/MAS NMR spectroscopy was carried out using a Bruker W300 spectrometer with a frequency of 75.5 MHz, 50 ms acquisition time, 8 ms contact time and 5 s repetition time. DA of the samples was determined by dividing the area of the resonance of the methyl group carbon by the average area of the resonances of the glycosyl ring carbon atoms. The DA was calculated using the following relationship [31]:
187
DA (%) =
179 180 181 182 183 184 185
ICH3 (I[C1] + I[C2] + I[C3] + I[C4] + I[C5] + I[C6])/6
(4)
188 189 190
× 100
where I is the area of the particular resonance peaks allowing to get DA % with 5% precision.
203
2.7.3. X-ray powder diffraction (XRD) The structural characterization and the crystallinity of chitin and chitosan were studied by using X-ray diffractometer (D8, Advance Bruker XRD diffractometer, Germany). The X ray powder patterns ˚ The were recorded using Ni-filtered Cu K␣ radiation (k = 1.5406 A). relative intensity was recorded in the scattering range 2 of 5–40◦ . The error of this measurement was ±1◦ . After normalization of XRD peak intensities on the basis of constant total-peak area, various parameters were evaluated through a deconvolution procedure using Origin 6.0 (Microcal Software Inc.). The relative crystallinity of the polymers was calculated by dividing the area of the crystalline peaks by the total area under the curve [32] by using the equation:
204
%Xc =
191 192 193 194 195 196 197 198 199 200 201 202
205 206 207
208 209 210 211 212 213 214 215 216
Fc × 100 (Fc + Fa)
2.7.4. Viscosity average molar mass of chitosan The viscosity measurements were done using an Ubbelohde viscometer and recording the efflux time of the solution in a constant temperature bath of 25 ± 0.1 ◦ C. Chitosans samples were dissolved in a solvent system of 0.3 M acetic acid/0.1 M sodium acetate. Intrinsic viscosity ([]) was obtained from linear plots of reduced viscosity (sp /C) against concentration (C, g/mL), extrapolating to zero concentration. The viscosity average molar mass (MW) of chitosans was estimated using the Mark–Houwink relationship [33]:
217 218
[] = K(MW )
(6)
220
where K = 7.95 × 10−2 and a = 0.79 [34]. The means of four replicates was taken for the viscosity measurements.
221
3. Results and discussion
222
3.1. Chemical composition of raw materials
219
223 224 225 226 227 228
Table 1 Characterization of the dried raw materials from Tunisian sources, purified chitins and chitosans. Shrimp
Crab
Cuttlefish
Raw material
Asha Proteina Chitina Fata
48.8 13.1 37.2 0.9
59.8 12.1 27.4 0.7
90.6 3.4 5.8 0.2
Chitin
Yield of extractionb DA Residual proteinsa Residual ashsa
20 88.5 – 0.4
10 78.5 – 0.5
5 70.1 – 0.8
Chitosan
Yield of extractionb DA Residual proteinsa Residual ashsa
14.9 12 – –
5.3 17 – –
1.2 5 – –
–, not detected. a % on dried material basis. b % on dried raw material.
the shrimp shells (48.8%). Cuttlefish bones were found to have a low level of protein (3.4%). Whereas, the higher protein contents were found in shrimp (13.1%) and crab shells (12.1%). The three raw materials contain low lipid content (0.2–0.9%). Crab and shrimp shells contain 27.4% and 37.2% chitin, respectively, whereas a lower percentage was found in the cuttlefish bones (5.8%). For the three materials, the most common mineral elements were determined as given in Table 2. In shells, clearly calcium is by far the most abundant element, followed by sodium and magnesium. From the comparison of the results of Table 2, it is shown that the source has an influence on the percent of each element. Cuttlefish bones, having the larger amount of ashes, present also the highest percentage of Ca compared to the other chitin sources. 3.2. Chitin extraction
229 230 231 232 233 234 235 236 237 238 239 240 241
242
(5)
where Fc and Fa are the area of the crystalline peaks at 2 = 20◦ and the amorphous diffraction at 2 = 16◦ , respectively. (rad) is half the Bragg angle corresponding to the crystalline peak.
a
3
Chitin was extracted from three marine sources, shrimp waste, crab shells and cuttlefish bones. The chemical compositions of the raw materials are determined following methods describes in Section 2.2; the values are given in Table 1. The highest percentage of inorganic matter (CaCO3 ) was found in cuttlefish bones 90.6% and 59.8% in crab shells, and the lowest percentage was found in
3.2.1. Chemical demineralization To extract chitin from the different raw materials, associated minerals should be removed as a first stage. As a consequence, the raw materials were subjected to mild acid treatment in order to remove minerals. We were inspired from the process described by Tolaimate et al. [24] with slight modifications, using 0.55 M hydrochloric acid baths at a ratio of 1:10 (w/v) at room temperature. The used conditions were very moderate compared to many other processes described in the literature [35,36]. The number of baths and their duration (between 15 and 60 min) were dependent upon the source following the pH evolution. In fact, the pH increases until the end of the demineralization. When the pH remains stable, it means that the necessary acid number of baths for every species is attained. Residual minerals were determined after each bath in order to control which number of baths is required for efficient demineralization (Table 2). Commonly, on the basis of previous studies [24], multi-stage process was much more efficient than only one step process. In fact, during the first bath, the structure of the treated material was modified to improve the accessibility of acid during further baths. Thus, exhaustion of minerals becomes much easier after the first bath and number of baths required for each waste species depends on the structure and the mineral content. In our study, the results showed that the number of baths necessary for a full demineralization is two baths for the shrimp waste, three for crab shells and four for the cuttlefish bones. One of the factors determining the effectiveness of the demineralization step is the low initial mineral content [24]. The three demineralized materials obtained in this work present metal content as low as those reported by Percot et al. at around 1.8% [37].
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
ARTICLE IN PRESS
G Model BIOMAC 4125 1–9
S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
4
Table 2 Demineralization steps during chitin isolation process. Evolution of the mineral contents after successive acidic baths (0.55 M HCl: 15 min for each bath). Chitin source
Cu (%)
Mn (%)
Zn (%)
Fe (%)
Mg (%)
Na (%)
Ca (%)
Total content (%)
Shrimp
Shells First bath Second bath Third bath Fourth bath
0.061 0.006 0.005 0.003 0.001
0.050 0.110 0.100
G Model BIOMAC 4125 1–9
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural differences between chitin and chitosan extracted from three different marine sources
1
2
3 4 5 6 7
Q1
Sawssen Hajji a,∗ , Islem Younes a , Olfa Ghorbel-Bellaaj a , Rachid Hajji b , Marguerite Rinaudo c , Moncef Nasri a , Kemel Jellouli a a
Laboratory of Enzyme Engineering and Microbiology, National School of Engineering of Sfax, University of Sfax, B.P. 1173, 3038 Sfax, Tunisia Laboratory of Solid State, Faculty of Science, University of Sfax, B.P. 802, 3018 Sfax, Tunisia c Biomaterials Applications, 6, rue Lesdiguières, 38000 Grenoble, France b
8
9
a r t i c l e
i n f o
a b s t r a c t
10 11 12 13 14
Article history: Received 18 November 2013 Received in revised form 11 January 2014 Accepted 17 January 2014 Available online xxx
15
20
Keywords: Chitin Penaeus kerathurus Carcinus mediterraneus Sepia officinalis
21
1. Introduction
16 17 18 19
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Three marine sources of chitin from Tunisia were investigated. Structural differences between ␣-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and -chitin from cuttlefish (Sepia officinalis) bones were indicated by the 13 C NMR, FTIR, and XRD diffractograms. The 13 C NMR analysis showed a splitting of the C3 and C5 carbon signals for ␣-chitin, while that of -chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of ␣-chitin in the region of O H, N H and CO stretching regions. IR and 13 C NMR were used to determine the chitin degree of acetylation. XRD analysis indicated that ␣-chitins were more crystalline polymorph than -chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molar masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source. © 2014 Published by Elsevier B.V.
Chitin is the second most abundant polysaccharide in biomass after cellulose. Chitin production was about 25,000 tons in 2006 [1]. This polymer is a linear chain consisting of poly (1–4) N-acetyld-glucosamine. Chitin is usually isolated from the exoskeletons of crustaceans and more particularly from shrimps and crabs where ␣-chitin isomorph is produced [2]. ␣-Chitin has a tightly compact structure due to its crystalline structure in which antiparallel chain favor strong hydrogen bonding [3]. Squid is another important source of chitin in which it exists in the -isomorph which was found to show higher solubility, higher reactivity and higher affinity toward solvents and swelling than ␣-chitin. These characteristics are due to weaker intermolecular hydrogen bonding ascribable to the parallel arrangement of the main chains [4]. ␥-Chitin, a third chitin allomorph, has also been described [5,6], but from a detailed analysis, it seems that it is just a variant of the ␣-family [7]. Several techniques to extract chitin from different sources have been reported. The most common method is referred to the chemical procedure which involves various major steps. Firstly, shells
∗ Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail address: [email protected] (S. Hajji).
are ground and minerals, mainly calcium carbonate, are removed (demineralization) using dilute acidic medium (usually HCl) at ambient temperature. Secondly, the proteins are extracted (deproteinization) from the residual material by treatment with aqueous solutions of NaOH or KOH. These traditional chemical methods create a disposal problem due to the large amounts of toxic waste which would pollute the environment. In addition, this process becomes expensive due to enforced environmental controls and disposal measures [8]. To overcome the disadvantage of chemical treatments, some efforts have been directed toward its substitution by more eco-friendly processes such as bacterial fermentation [9] or treatment by proteolytic enzymes which have been applied for the deproteinization of crustacean wastes [9–12]. Because solid state chitin has a compact structure, it remains insoluble in most solvents. Therefore, usually chemical deacetylation is performed to produce the most common derivative named chitosan [13]. In acidic conditions, chitosan becomes positively charged due to NH2 protonation and soluble in aqueous medium. Under these conditions, this polymer has numerous physiological and biological properties with great potential in a wide range of industries such as cosmetology (lotions, hair additives, body creams) [14,15], food (coating, preservative, antioxidant, antimicrobial) [16], biotechnology (chelator, emulsifier, flocculent) [17],
0141-8130/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
G Model BIOMAC 4125 1–9
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81
pharmacology and medicine (fibers, drugs, membranes, artificial organs) [18] and agriculture (soil modifier, films, fungicide) [19]. Chitosan can be characterized in terms of its quality, intrinsic properties (purity, molar mass, viscosity, and acetylation degree) and physical forms [20]. It was reported that both acetylation degree and molar mass are important chemical characteristics, which could influence the performance of chitosan in many of its applications [21]. The aim of this work was to extract chitin from different Tunisian marine sources using chemical demineralization and enzymatic deproteinization (with Bacillus mojavensis A21 proteases) and then to compare their physicochemical characteristics using XRD, FTIR and 13 C NMR spectroscopy. Following, the chitins obtained were deacetylated to chitosans. Acetylation degree and molar mass of each chitosan were examined. To the best of our knowledge, this present work is the first systematic trial to investigate the extraction of chitin and chitosan from different indigenous sources in Tunisia.
88 89 90
91
100
The moisture and ash contents were determined at 105 ◦ C and 550 ◦ C, respectively, according to the AOAC [22] standard methods 930.15 and 942.05. Total nitrogen content of each raw material was determined by using the Kjeldahl method. Separately, for each raw sample, pure chitin is prepared to determine its nitrogen contribution allowing to estimate the crude protein content by multiplying nitrogen content attributed to protein by the factor of 6.25 [23]. Lipids were determined gravimetrically by soxhlet extraction using hexane.
101
2.3. Chemical demineralization
92 93 94 95 96 97 98 99
102 103 104 105 106 107 108 109 110 111 112 113
114
115 116 117
2.5. Deproteinization of shell waste by proteases
Demineralization was carried out at room temperature using 0.55 M hydrochloric acid baths. Each bath was performed with 100 ml of acid solution and 10 g of raw material. The number of baths and their duration (between 15 and 60 min) were dependent upon the source [24]. Demineralization step was followed by pH evolution toward neutrality due to acid consumption. The end of the repeated series of baths was indicated by stability of medium acidity. Demineralized materials were filtered through four layers of gauze using a vacuum pump, washed to neutrality with deionized water and dried for 1 h at 60 ◦ C. Demineralization degree (DDM) was expressed as a percentage and computed by the following equation [25]: [(AO × O) − (AR × R)] %DDM = × 100 AO × O
(1)
where AO and AR are ash contents (%) before and after demineralization, respectively, while O and R represent the mass (g) of initial and demineralized residue respectively on dry weight basis.
(2)
where PO and PR are the protein concentrations (%) before and after hydrolysis, respectively, while O and R represent the mass (g) of original sample and hydrolyzed residue respectively on dry weight basis. 2.6. Deacetylation of chitin
2.7. Physicochemical characterization of chitins and chitosans
121 122 123 124 125 126 127 128 129 130 131
133 134 135 136 137 138 139 140 141 142 143 144 145
146
147 148 149 150
152 153 154 155 156
157
2.7.1. Fourier transform infrared spectroscopy (FTIR) Infrared spectra were obtained using a Perkin Elmer type FTIR 1000 spectrometer at room temperature and KBr pellets. The sample pellets were prepared at a pressure of 5 tons for 2 min. Pellets were scanned at room temperature (25 ◦ C) in the 400–4000 cm−1 spectral range [28]. The incertitude of this measurement was 2 cm−1 . In the case of overlapping peaks, deconvolution was performed to calculate the contribution of the individual peaks using Dmfit program [29] with Gaussian and/or Lorentzian shapes to estimate the area related to the specific vibration of each selected peak. Spectra were corrected for the baseline and the absorbance was normalized between 0 and 1. The acetylation degree (DA) of chitins and chitosans was calculated according to the method proposed by Moore and Roberts [30], as follows:
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
120
151
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 a chitosan perfectly soluble in might acidic conditions. After filtration, the residue was washed with deionised water, and the crude chitosan was recovered by drying in a dry heat incubator at 50 ◦ C during 12 h.
A1650 /A3450 DA (%) = × 100 1.33
119
132
2.2. Chemical analysis of the raw materials
2.1. Preparation of raw material
87
B. mojavensis A21 was isolated from marine water in Sfax by Haddar et al. [26]. The growth medium used for protease production was composed of (g/L): hulled grain of wheat, 30.0; yeast extract, 1.0; CaCl2 , 2.0; MgSO4 , 1.0; K2 HPO4 , 0.3; KH2 PO4 , 1.0; and NaCl, 2.0. The pH medium was adjusted to 9.0. Media were autoclaved at 121 ◦ C for 20 min. Cultivations were conducted in 250 ml Erlenmeyer flasks with a working volume of 25 ml. Incubations were carried out in an orbital shaking incubator at 30 ◦ C and 200 rpm for 24 h. The cultures were centrifuged at 10,000 rpm for 15 min 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. [27] using casein as a substrate.
[(PO × O) − (PR × R)] %DDP = × 100 PO × O
83
86
118
Shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells and cuttlefish (Sepia officinalis) bones were obtained in fresh conditions from a fishery products and processing plant located at Sfax, Tunisia. They were washed thoroughly with tap water, dessicated at room temperature and milled (sieved from 2 mm to 5 mm). After drying, they were kept at room temperature until used.
2. Materials and methods
85
2.4. Crude enzyme preparation
B. mojavensis A21 crude enzyme preparation was selected for its deproteinization efficiency [10]. Deproteinization tests were carried out in a thermostated stirred Pyrex reactor (300 ml). The demineralized shells homogenate (15 g) were mixed with 45 ml distilled water. The pH and temperature of the mixture were adjusted to pH 9.0 (with NaOH 4%, w/v) and 50 ◦ C. The shell waste proteins were digested with crude enzyme using different Enzyme/Substrate ratios (E/S) during 3 h. The reaction was stopped by heating at 90 ◦ C for 20 min to inactivate enzymes. The solid phase was washed, pressed manually through four layers of gauze and then dried at 60 ◦ C during 12 h. Deproteinization degree (DDP) was expressed as percentage and computed by the following equation [25]:
82
84
ARTICLE IN PRESS S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2
(3)
158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
173
ARTICLE IN PRESS
G Model BIOMAC 4125 1–9
S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
174 175 176 177 178
The amide-I band ( = 1650 cm−1 ) was used as the analytical band and the hydroxyl band ( = 3450 cm−1 ) as the internal reference band. The factor ‘1.33’ denoted the value of the ratio of A1655 /A3450 for fully N-acetylated chitin. Errors in %DA were less than 20% of their nominal value.
186
2.7.2. 13 C CP/MAS-NMR spectroscopic analysis Solid-state 13 C CP/MAS NMR spectroscopy was carried out using a Bruker W300 spectrometer with a frequency of 75.5 MHz, 50 ms acquisition time, 8 ms contact time and 5 s repetition time. DA of the samples was determined by dividing the area of the resonance of the methyl group carbon by the average area of the resonances of the glycosyl ring carbon atoms. The DA was calculated using the following relationship [31]:
187
DA (%) =
179 180 181 182 183 184 185
ICH3 (I[C1] + I[C2] + I[C3] + I[C4] + I[C5] + I[C6])/6
(4)
188 189 190
× 100
where I is the area of the particular resonance peaks allowing to get DA % with 5% precision.
203
2.7.3. X-ray powder diffraction (XRD) The structural characterization and the crystallinity of chitin and chitosan were studied by using X-ray diffractometer (D8, Advance Bruker XRD diffractometer, Germany). The X ray powder patterns ˚ The were recorded using Ni-filtered Cu K␣ radiation (k = 1.5406 A). relative intensity was recorded in the scattering range 2 of 5–40◦ . The error of this measurement was ±1◦ . After normalization of XRD peak intensities on the basis of constant total-peak area, various parameters were evaluated through a deconvolution procedure using Origin 6.0 (Microcal Software Inc.). The relative crystallinity of the polymers was calculated by dividing the area of the crystalline peaks by the total area under the curve [32] by using the equation:
204
%Xc =
191 192 193 194 195 196 197 198 199 200 201 202
205 206 207
208 209 210 211 212 213 214 215 216
Fc × 100 (Fc + Fa)
2.7.4. Viscosity average molar mass of chitosan The viscosity measurements were done using an Ubbelohde viscometer and recording the efflux time of the solution in a constant temperature bath of 25 ± 0.1 ◦ C. Chitosans samples were dissolved in a solvent system of 0.3 M acetic acid/0.1 M sodium acetate. Intrinsic viscosity ([]) was obtained from linear plots of reduced viscosity (sp /C) against concentration (C, g/mL), extrapolating to zero concentration. The viscosity average molar mass (MW) of chitosans was estimated using the Mark–Houwink relationship [33]:
217 218
[] = K(MW )
(6)
220
where K = 7.95 × 10−2 and a = 0.79 [34]. The means of four replicates was taken for the viscosity measurements.
221
3. Results and discussion
222
3.1. Chemical composition of raw materials
219
223 224 225 226 227 228
Table 1 Characterization of the dried raw materials from Tunisian sources, purified chitins and chitosans. Shrimp
Crab
Cuttlefish
Raw material
Asha Proteina Chitina Fata
48.8 13.1 37.2 0.9
59.8 12.1 27.4 0.7
90.6 3.4 5.8 0.2
Chitin
Yield of extractionb DA Residual proteinsa Residual ashsa
20 88.5 – 0.4
10 78.5 – 0.5
5 70.1 – 0.8
Chitosan
Yield of extractionb DA Residual proteinsa Residual ashsa
14.9 12 – –
5.3 17 – –
1.2 5 – –
–, not detected. a % on dried material basis. b % on dried raw material.
the shrimp shells (48.8%). Cuttlefish bones were found to have a low level of protein (3.4%). Whereas, the higher protein contents were found in shrimp (13.1%) and crab shells (12.1%). The three raw materials contain low lipid content (0.2–0.9%). Crab and shrimp shells contain 27.4% and 37.2% chitin, respectively, whereas a lower percentage was found in the cuttlefish bones (5.8%). For the three materials, the most common mineral elements were determined as given in Table 2. In shells, clearly calcium is by far the most abundant element, followed by sodium and magnesium. From the comparison of the results of Table 2, it is shown that the source has an influence on the percent of each element. Cuttlefish bones, having the larger amount of ashes, present also the highest percentage of Ca compared to the other chitin sources. 3.2. Chitin extraction
229 230 231 232 233 234 235 236 237 238 239 240 241
242
(5)
where Fc and Fa are the area of the crystalline peaks at 2 = 20◦ and the amorphous diffraction at 2 = 16◦ , respectively. (rad) is half the Bragg angle corresponding to the crystalline peak.
a
3
Chitin was extracted from three marine sources, shrimp waste, crab shells and cuttlefish bones. The chemical compositions of the raw materials are determined following methods describes in Section 2.2; the values are given in Table 1. The highest percentage of inorganic matter (CaCO3 ) was found in cuttlefish bones 90.6% and 59.8% in crab shells, and the lowest percentage was found in
3.2.1. Chemical demineralization To extract chitin from the different raw materials, associated minerals should be removed as a first stage. As a consequence, the raw materials were subjected to mild acid treatment in order to remove minerals. We were inspired from the process described by Tolaimate et al. [24] with slight modifications, using 0.55 M hydrochloric acid baths at a ratio of 1:10 (w/v) at room temperature. The used conditions were very moderate compared to many other processes described in the literature [35,36]. The number of baths and their duration (between 15 and 60 min) were dependent upon the source following the pH evolution. In fact, the pH increases until the end of the demineralization. When the pH remains stable, it means that the necessary acid number of baths for every species is attained. Residual minerals were determined after each bath in order to control which number of baths is required for efficient demineralization (Table 2). Commonly, on the basis of previous studies [24], multi-stage process was much more efficient than only one step process. In fact, during the first bath, the structure of the treated material was modified to improve the accessibility of acid during further baths. Thus, exhaustion of minerals becomes much easier after the first bath and number of baths required for each waste species depends on the structure and the mineral content. In our study, the results showed that the number of baths necessary for a full demineralization is two baths for the shrimp waste, three for crab shells and four for the cuttlefish bones. One of the factors determining the effectiveness of the demineralization step is the low initial mineral content [24]. The three demineralized materials obtained in this work present metal content as low as those reported by Percot et al. at around 1.8% [37].
Please cite this article in press as: S. Hajji, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.045
243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
ARTICLE IN PRESS
G Model BIOMAC 4125 1–9
S. Hajji et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
4
Table 2 Demineralization steps during chitin isolation process. Evolution of the mineral contents after successive acidic baths (0.55 M HCl: 15 min for each bath). Chitin source
Cu (%)
Mn (%)
Zn (%)
Fe (%)
Mg (%)
Na (%)
Ca (%)
Total content (%)
Shrimp
Shells First bath Second bath Third bath Fourth bath
0.061 0.006 0.005 0.003 0.001
0.050 0.110 0.100
