Comparison of the physicochemical and structural characteristics of enzymatic produced chitin and commercial chitin

BIOMAC-12969; No of Pages 7 International Journal of Biological Macromolecules 139 (2019) xxx

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Comparison of the physicochemical and structural characteristics of enzymatic produced chitin and commercial chitin Moosavi-Nasab Marzieh a,⁎, Forghani Zahra b, Ebrahimi Tahereh b, Khoshnoudi-Nia Sara a,⁎ a b

Seafood Processing Research Group & Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran

a r t i c l e

i n f o

Article history: Received 13 May 2019 Received in revised form 30 July 2019 Accepted 30 July 2019 Available online 31 July 2019 Keywords: White shrimp Mild chemical procedure Enzyme-assisted chitin production

a b s t r a c t White shrimp (Litopenaeus vannamei) waste is a good source of chitin. However, the chemical chitin extraction generates a large amount of environmental-pollution and increases partial deacetylation of chitin. Therefore, the use of biological extraction is an interesting alternative. This study aimed to isolate chitin by Trypsin and Ficin with/without sodium-metabisulfite and investigate their properties as compared to commercial chitin. Shell demineralization and deproteinization were done by lactic-acid and proteolytic-enzyme (0.1–0.05%w/w). The enzymatic deproteinized shells were subjected to mild-alkali treatment (0–2% NaOH; 60 °C and 30 min). After demineralization the ash content was decreased to ~1.1%. The combination of enzymatic and mild chemical treatments exhibited the 92% deproteinization. The enzymatic deproteinization was higher in presence of sodium-metabisulphite (p N 0.05). The CI of enzymatic-chitin was more than commercial-chitin (p b 0.05). Therefore, a smooth with high molecular packing microstructure was observed in enzymatic- chitins. The DA% of enzymatic chitins (81–83%) were higher than chemical (p b 0.05). The chitins with DA between 70 and 90% and low protein content, is considered as good final products. Therefore, the enzyme-assisted chitin extraction generated the high grade chitin under mild and semi-ecofriendly conditions. This method is a promising alternative method for chemical extraction which can retain the native microstructure of chitin. © 2019 Published by Elsevier B.V.

1. Introduction Seafood is considered as one of the most important protein sources in the world. However, seafood (especially crustaceans such as shrimp, prawn, crab and crayfish) processing industry generates a significant volume of the non-edible part (mainly exoskeletons and heads), so that 50–70% of crustacean is non edible and waste [1,2]. The dry basis global annual production of shell waste from crustacean harvest is estimated over 3.14 million metric tons in which large amounts of these shells, as wastes of seafood industry, have been released to environment. However, shrimp bio-waste can be a good source for production chitin [3]. Chitin as an insoluble linear homopolymer of β-(1 → 4)-Nacetyl-D-glucosamine is the second most abundant biopolymer in nature after cellulose. Chitin crystalline structures include three allomorphic forms: α, β and γ. In α-chitin, the adjacent layers are arranged in different directions. This type of chitin is, as the most abundant allomorph, found in hard structures. In β-chitin, these layers have the same direction (parallel-type). In γ–chitin, 3rd layer is an opposing ⁎ Corresponding authors at: School of Agriculture, Shiraz University, PO Box: 7144165186, Shiraz, Iran. E-mail addresses: [email protected] (M.-N. Marzieh), [email protected] (K.-N. Sara).

direction relative to the two others. These two last chitin forms are found in flexible structures [4]. Chitin is widely distributed as a structural component of crustacean exoskeletons, insects and other arthropods, as well as a component of the cell walls of most fungi and some algae [5,6]. However, the main sources of raw material for the production of commercial chitin are cuticles of various crustaceans. Biodegradability, biocompatibility, and non-toxicity are some of the great functional properties of chitin and its derivatives (such as chitosan and chito-oligosaccharides) that make them applicable in various field such as pharmacy, medicine, agriculture, textiles, cosmetics, waste water treatment and food industry [7–10]. White shrimp (Litopenaeus vannamei) is one of the most commercially exploited shrimp species in Asia and the Americas, which also produces a large amount of shrimp bio-waste. Chitin existing in the animal world is closely associated with proteins, minerals, lipids and pigments. Therefore, the production of chitin from shell waste involved three steps: demineralization, deproteinization and decolorization [11–13]. For extracting chitin from shell wastes chemical [14,15], physical microbiological [10,14] and enzymatic methods [10,16] have been used. However, due to covalent bond of shell compounds, especially protein, with chitin [17,18], deproteinization and demineralization procedures industrially rely on sever and corrosive basic (sodium hydroxide: NaOH) and

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acidic (hydrochloric acid: HCl) treatment, respectively [19,20]. Harsh chemical treatment result in produce large amounts of environmental pollution and damage human health. Furthermore, the use of chemical treatment can lead to hydrolysis of the polysaccharide chains as well as intensify partial deacetylation of chitin and consequently, generate inconsistent and undesirable physicochemical properties in final chitin quality [21,22]. Nowadays, the use of milder and more eco-friendly methods for extraction chitin have attracted widespread attention to overcome the environmental issues associated to harsh acid-base treatments [1]. A good alternative for chemical extraction of chitin can be the use of various proteolytic enzymes [3,23] along with mild acidic and basic treatments. Several authors reported the application of different proteolytic enzymes such as Bacillus mojavensis A21 crude protease [24,25], Streptomyces griseus protease [3], alkaline proteases from P. segnis viscera [26], fish protease [27], Alcalase [28] and trypsin [16] to extract the chitin from crustaceans in more eco-friendly manner. However, the long fermentation period (48–120 h) besides high amount of residual protein and more cost of are some of limitation of biological chitin extraction as compared to chemical methods. Therefore, the improvement of biological chitin extraction methods is necessary [29]. For this purpose, in current study the commercial enzymes were used. These enzymes can be used in a low concentration and consequently it is not very expensive. Moreover, the use of pure enzymes for chitin extraction takes less time as compared to crude enzyme. Also, the combination of enzyme extraction and mild chemical process can be improved the performance of chitin preparation. To the best of our knowledge, no studies have reported the enzymatic preparation of chitin from white shrimp shell waste using different trypsin and ficin concentration with/without sodium metabisulfite together with mild chemical treatments. Therefore, the goals of this study were to 1) investigate the synergic effect of several parameters such as enzyme concentration, incubation time and addition of sodium metabisulfite along with a mild alkaline treatment on efficiency of deproteinization and demineralization level of white shrimp shells; 2) evaluate the physicochemical and structural characteristics of enzymatic produced chitin by scanning electron microscopy (SEM), X-ray diffraction (XRD) and elemental analysis 3) compared to the physicochemical properties of chitin extracting by enzyme and chemical methods (commercial chitin). 2. Materials and methods

2.4. Enzymatic deproteinization Deproteinization of the demineralized shells was done using Trypsin and Ficin. Trypsin and Ficin need pH 7.7 and 7.5 respectively for maximum activity. The phosphate buffers with the above these was prepared and in the treatment with sodium metabisulphite, this material was added to the buffer solutions (2 g/kg). demineralized shells were ground with respective buffer solution in the ratio 1:3 and then enzyme was added in the ratio 1:1000, 1:2000 and 1:3000 w/w (0.1, 0.05, 0.033% w/w) on the basis of protein content in the shell waste. The whole mixture was stirred continuously at ambient temperature. The pH of mixture was controlled by adding phosphoric acid (1%) at a regular interval [34]. The mixture was taken out after 1, 2, 3 and 4 h. The enzymatic reaction was stopped by increasing temperature to 60 °C. The mixture was filtered through filter cloth then it was washed with distilled water to neutrality. Buffer mixture at pH 7.7 and 7.5 without related enzymes were used as control runs [35]. 2.5. Mild alkali treatment The enzymatic deproteinized shell waste was subjected to mild alkali treatment at 60 °C for 30 min, ratio of solids to NaOH solution of 1:10 (w/v). The concentration of NaOH was considered from 0 to 2% [36]. 2.6. Decolorization process The deproteinized and demineralized shells were decolorized with acetone for 30 min and then washed with distillated water and dried for 4 h at room temperature [37]. 2.7. X-ray diffraction X-ray diffraction (XRD) (D500 Siemens Bruker XRD diffractometer, model D8-Advance, Bruker Germany) was used to detect the crystallinity and structural characterization of various extracted chitins. Cu Kαradiation (at 45 kV and 40 Ma) was considered as the X-ray source. The crystalline index (CI) was determined by following formula:

CI 110 % ¼ ½ðI110 −Iam Þ=I110   100:

2.1. Materials Shell waste (5 Kg) from white shrimp (Litopenaeus vannamei) was supplied from the shrimp processing plant located in Bushehr (Iran). Shrimp shell waste was washed with tap water and dried at 60 °C for 2 h. Then, the waste was ground by a kitchen miller (Pars Khazar Co. Rasht, Iran) to powder. Commercial chitin and Trypsin, Ficin enzymes were obtained from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). All other reagents were high analytical grade.

where I110 is the maximum intensity (arbitrary unit) of the diffraction (110) at 2θ = 19° and Iam is the intensity of the amorphous diffraction at 2θ = 12.6° [38]. 2.8. Scanning electron microscopy (SEM) The surface morphology of chitin was observed by scanning electron microscopy (SEM) (model 5526, Cambridge, UK) operated at 20 kV.

2.2. Chemical analysis

2.9. Degree of acetylation (DA)

The ash and moisture content (%) were evaluated according to the AOAC standard and using the methods 942.05 and 930.15, respectively [30,31]. Moreover, Total nitrogen content was determined based on the Kjeldahl method [32].

The average degree of acetylation (DA) of chitin samples was determined from data of elemental analysis which was carried out by using CHN analyzer (model 2400; Perkin-Elmer, Beaconsfield, United Kingdom). These data were recorded on Thermosinnigan Flash 11-12 series EA [39].

2.3. Demineralization

2.10. Statistical analysis

The demineralization of 100 g of the shells was conducted at 24 °C for 6 h using lactic acid (75.6 g/L) at a ratio of shells to acid of 1:10 (w/v). Demineralized shells were washed with distilled water until achieving a neutral pH [33].

All experiments were carried out at least three times and the findings were reported as mean ± with standard deviation (SD) are reported. Statistical analysis was conducted using the SPSS version 24 software (SPSS Statistical Software, Inc., Chicago, IL, USA). One-way

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analysis of variance was carried out. The difference of means between treatments was estimated by Tukey test at significance level of 0.05. 3. Results and discussions 3.1. Proximate composition Table 1 presented the content of chemical composition (i.e., minerals (ash), protein and moisture) of the dry weight of shrimp shells. The protein and ash content were found 31.13% and 33.61% respectively. In Rodde et al. study, shell of Pandalus borealis shrimps were analyzed and the levels of protein and ash, based on seasonal variation, were found 33%–40% and 32–38% of dry weight [40], which were relatively in the same range as those measured in present research. However, in Hongkulsup et al. study, the protein and ash of shell shrimp reported 28.4 and 47.66%, respectively [3]. Also, Cardenas et al. reported the higher level of ash, 42.5% (w/w) and lower lever of protein 21.9% (w/ w) for red shrimp shells (Pleuroncodes monodon) [38]. Difference in spices, season of harvest and feeding can be some of reasons for such various results. 3.2. Demineralization In the recovery of chitin from shrimp waste, associated minerals should be removed to enhance accessibility of enzymes to protein and increase the quality of chitin [41]. Moreover, the acidic treatment may likely eliminate the acid-soluble proteins. In general, the low mineral content is one of the most important indicating the suitable quality of chitin [42,43]. Therefore, first demineralization of shrimp shell was carried by lactic acid and then enzymatic deproteinization was done. After demineralization the ash content reach from 33.6% to at around 1.1% (Table 2). Paul et al. also reported that the ash contents of chitin produced by protease and demineralized by HCl were between 0.88 and 1.06% that these values are comparable with the contents founded in current work [43]. Some of reasons that explain the difference between various chitin ash content have been laid on the variation in calculation, analytical and demineralization methods. 3.3. Enzymatic deproteinization of shrimp waste After demineralization, the enzymatic deproteinization of shrimp shells by Ficin and Trypsin with/without sodium metabisulphite was performed under conditions of optimal enzyme activity and stability (pH = 7.7 and 7.5 for Trypsin and Ficin, respectively), and several experimental conditions, including various enzyme concentration (0, 0.1, 0.05 and 0.03% w/w) and different treatment time, (1, 2, 3 and 3 h). The results showed that the increasing incubation time to 2 h has significant effect on the deproteinization rate of shrimp waste (p b 0.05). However, increasing incubation time beyond 2 h, did not have any significant effect on protein level of shrimp waste (p N 0.05) and after this time the amount of protein was almost remained constant (p N 0.05; the results are not shown). Therefore, the best time for deproteinization process was considered 2 h. The effect of type and concentration of enzymes and presence of sodium metabisulphite in 2 h incubation time was presented in Fig. 1. Table 1 Chemical composition of chitins obtained deproteinization with Trypsin, Ficin and sodium metabisulphite. Sample Raw shell waste Chitin (T + SM) Chitin (F + SM)

Protein (%)

Humidity (%) a

30.61 ± 0.27 5.31 ± 0.80 c 6.1 ± 0.77 b

a

4.51 ± 0.29 3.35 ± 0.01b 3.34 ± 0.02 b

Ash (%)

Chitin (%) a

33.66 ± 0.46 1.14 ± 0.02 b 1.09 ± 0.03 b

32.12 ± 0.38a 27.06 ± 0.21b 27.35 ± 0.19b

T: trypsin; F: Ficin; SM: sodium metabisulfite. Means in a column with different letters indicate significant difference between groups (p b 0.05).

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As shown in Fig. 1 the minimum amount of protein was obtained when a concentration of 0.1% w/w Trypsin enzyme incorporation with sodium metabisulfite was used (~82% deproteinization degree). The results showed that the presence of sodium metabisulphite has a significant impact on deproteinization process in control sample. Although, samples were treated with sodium metabisulfite (without enzyme), showed more deproteinization (p b 0.05), there was not any significant synergistic effect between enzyme and sodium metabisulphite. However, enzymatic deproteinization was more in presence of sodium metabisulphite as compared to samples treated with enzyme alone (p N 0.05). The effect of sodium metabisulphite on improving the efficiency of protein extraction can be attributed to its effect on reducing disulfide bonds and increasing protein solubility [44]. Moreover, a comparison between the impact of ficin and trypsin on deproteinization of shell shrimps showed that the performance of trypsin was slightly better than ficin (p b 0.05). Paul et al., the percent of protein removal from black tiger shrimp shell waste using cocktail and commercial protease after 4-days incubation reported 80% and 73.33%, respectively. These authors the possible reason for the low protein removal related to Maillard reaction connected to high-temperature during drying. In Maillard reaction, protein was cross-linked with sugars and other compounds and this complex is resistant to protease treatment [43]. Chakrabarti [34] reported that the amount of deproteinization from brown shrimp shell waste by trypsin was slightly more than those obtaining by papain and pepsin. The best enzymatic deproteinization rate was recorded 82.2% during 2 h treatment with trypsin at ambient temperature. Younes et al. reported that the protein level of shrimp shell in optimal experimental condition (E/S ratio: 7.75 U/mg, temperature: 60 °C and incubation time 6 h) by B. mojavensis A21 crude protease reached from 27% to 12% [22], which this result is a little better than the findings of current study that obtained in only 2 h treatment. Also, Busto and Healy reached to a maximum value of 82% with Pseudomonas maltophilia after six days [45]. Chemical composition of produced chitin after deproteinization by Trypsin, Ficin and sodium metabisulphite was reported in Table 1. 3.4. Mild alkali deproteinization The enzymatic deproteinization rate from shrimp shell cannot reach to 100%, because the enzyme cannot access to some proteins in the inner layer of shrimp shell which are protected against the protease attacks by the minerals and outer layer chitin [22]. Therefore, shrimp waste deproteinized by enzymatic treatment was subjected to different concentrations of NaOH (0–2%) to increasing deproteinization degree. The results of Table 2 show that using 0.5% NaOH can reach protein content of chitin produced with Trypsin and ficin from 5.31 and 6.10 to 2.35 and 2.8% (deproteinization degree reached to 92%) respectively. The result showed that increasing NaOH concentration above 0.5% has not significant effect on the protein content of chitin (p N 0.5). Therefore, the mild alkaline treatment (0.5% NaOH, 60 °C, 0.5 h.) may be sufficient for further protein recovery from the precipitate. Application of lower concentration of alkaline solution in comparison with commercial processes (2–10%) can be considered as an advantage from the economic as well as environmental aspects [37]. The Ficin and Trypsin treatments along with mild alkali deproteinization reached the chitin content of shell waste reached from 32.12 ± 0.36 to 27.35 ± 0.19 and 27.06 ± 0.21%, respectively confirming a good yield for the chitin extraction. Other studies reported comparable yields of chitin. For example, Abdou et al. used chemical treatment for extraction of chitin and reported that the chitin content of Brown shrimp (Penaeus aztecus) and pink shrimp (penaeus durarum) shells reported 21.53 and 23.72%, respectively. These authors used chemical treatment for extraction of chitin [24]. Furthermore, Paul et al. reported that the chitin content of shell waste after deproteinization with crude protease, commercial protease, alkali and acidic treatment reached from 34.23 to 26.43, 21.21, 10.19, 27.28%

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Table 2 Effect of NaOH concentration on the residual protein content of produced chitins. NaOH concentration

0%

0.5%

1%

1.5%

2%

Chitin (T + SM)

5.31 ± 0.80Ba 6.1 ± 0.77Aa

2.35 ± 0.36Bb 2.8 ± 0.39Ab

2.33 ± 0.37Bb 2.76 ± 0.33Ab

2.27 ± 0.21Bb 2.75 ± 0.36Ab

2.2 ± 0.39Bb 2.7 ± 0.24Ab

Chitin (F + SM)

T: trypsin; F: Ficin; SM: sodium metabisulfite. Means in a column with different capital letters indicate significant difference between groups (p b 0.05). Means in a row with different small letters indicate significant difference between various NaOH treatments (p b 0.05).

[43] which the result of enzymatic and alkali deproteinization were comparable with those obtained in current work. 3.5. Crystallinity index The degree of crystallinity index and amorphousness of chitin were studied using XRD patterns. XRD patterns of chitins extracted by trypsin, ficin besides commercial chitin are shown in Fig. 2. Depending on the producing method of chitin, different XRD patterns were observed. The XRD patterns of the extracted chitins by ficin and trypsin show a sharp peak around 19.3–19.6°, and several weak peaks were noticed around 12.6°-13.3, 20.7–21.1°, 23.1–23.8°, and 26.4–26.8. It is reported in the previous studies that these peaks confirm α-form of extracted chitin from crustaceans. Also a sharp peak near 9.6° was reported by several studies. [1,25,46–49]. In current study the XRD patterns of chitins have been obtained in the range of 10°-50°, therefore probably this peak was missed. Kaya et al. [50] reported that the peaks showed α-chitin were not limited to the four at 9.6°, 19.6°, 21.1° and 23.7° but others around 12–13° and 26° also covered. The locations of these peaks were can be varied in regarding the species that used as a chitin source [50]. These authors reported the peak at 38.84°–39.52° for chitins extracted from six different aquatic organisms. In current study, two other peaks were also observed about 30.2° and 34.3° which probably related to species of chitin source. In commercial chitin three sharp peaks were found in the XRD pattern at around 12.7°, 20.55° and 23.5° and five weak peak at about

26.5°, 30.4°, 34.1°, 39.6° and 47.2° (Fig. 2). In previous study, the relatively similar additional sharp peaks were recognized in the XRD pattern of crab shell [25,51]. Therefore, in this research the commercial chitin was properly prepared from crab shells. Furthermore, the XRD analysis can be applied to detect the crystallinity index of the isolated chitin. All chitin samples showed strong reflections at 2θ around 19–20° and minor reflections at higher 2θ values. The values of crystalline index (CI) of the chitins isolated from shrimp shells by Trypsin and Ficin as well as commercial chitin were listed in Table 3. This data showed that the crystalline index of chitin producing by enzymatic treatment is more than the crystalline index of commercial chitin (Trypsin: 67.5%, Ficin: 67% and commercial chitin: 58.4%). Previous studies showed that CI values of different organisms are varied between 43 and 91.7% [52]. In previous studies, the CI values were recorded as 67.8% for crab, 62% for cuttlefish [25] and between 63 and 67% for seven grasshopper species [52]. Moreover, Hajji et al. obtained a shrimp α-chitin without residual protein and salts that showed 64.1% crystallinity [25]. These results are relatively similar to current study findings. However, several studies reported the higher CI values for some of species such as Norway lobster (79%) and shrimp (89.17%), noted that the CI value of shrimp were around 89.17% and [49,53]. A lower crystallinity index the of polysaccharide shows disruption of intra/inter-molecular hydrogen bonds which may probably facilitate the chemical modifications in next processing steps [54]. Overall, the results emphasizes that crystalline index value is very depending on species [25,48]. 3.6. Degrees of acetylation (DA) DA is the most important factor which effect on the chitin and chitosan applications (such as, biodegradability, chemical modifications procedure and solubility). N-acetyl-D-glucosamine is the basic repeating unit of chitin. Most of the C-2 amino groups of chitin are acetylated and some of them are free [25,55]. Therefore, depending on chitin source and morphology as well as extraction method, chitin samples can have different degrees of acetylation [46]. The average degree of acetylation of chitin samples were presented in Table 3. The results show that the DA% of enzymatic produced chitins were significantly higher than that obtained in commercial chitin (p b 0.05). Although,

Fig. 1. The effect of type and concentration of enzymes with or without sodium metabisulphite on protein content of white shrimp shell during 2 h incubation time. Means with different letters are significantly different (p b 0.05).

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Fig. 2. X-ray diffraction pattern of produced chitin with trypsin (green line), produced chitin with ficin (blue line) and commercial chitin (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the DA% of chitin produced by trypsin (DA% = 83.3 ± 0.98) was more than that generated by ficin (DA% = 81.01 ± 1.07), but the difference between two chitins were not significant (p N 0.05). The results were comparable with that obtained in previous studies. For example, Manni et al. calculated the DA values of 89.5 and 77% for shrimp chitins prepared by protease from Bacillus cereus SV1 and by alkali [56]. Also, Hajji et al. reported the DA values of 88.5% and 78.6% for shrimp and crab α-chitins deproteinized by proteases shells, respectively by B. mojavensis A21 crude enzyme [25]. Paul et al. [43] also reported that the DA of chitin obtained by chemical procedure was lower (81.47%) than that isolated by protease-lactic (90.83%) and protease-acetic (92.67%). However, the DAs reported by these authors, were more than those obtained in our study, in some case. DA value depends on the raw material and the deproteinization processes [56]. Observed differences in the degree of acetylation values can be caused by deacetylation of polysaccharide occurring in the mild alkali treatment. Harsh alkaline and acidic treatments in deproteinization and demineralization procedures of shell used in commercial chitin production can cause undesirable deacetylation of chitin [57]. The previous study reported that the use of inorganic acids such as HCl for demineralization of chitin have negative effects on the molecular mass, the DA and subsequently intrinsic characteristics of chitin [58,59]. Therefore, the use of enzyme besides mild alkali treatment in deproteinization step of chitin production and also mild acidic demineralization by lactic acid result in lower deacetylation in enzymatic produced chitin. It confirmed that αchitin shows a higher resistance against deacetylation [60] so it was expected that the DA index is high in α-chitin. A good chitin has a DA range of 70–90% and low protein content [25]. Thus, the chitins produced by ficin and trypsin are better final products than commercial one.

Table 3 Crystallinity index (CI) and degree of acetylation (DA) of produced chitins and commercial chitin. Sample Chitin (T + SM) Chitin (F + SM) Commercial chitin

CI (%)

DA (%) a

67.5 ± 0.19 67 ± 0.22a 58.4 ± 0.28b

83.3 ± 0.98a 81.01 ± 1.07a 61.8 ± 0.91c

T: trypsin; F: ficin; SM: sodium metabisulfite. Means in a column with different letters indicate significant difference between groups (p b 0.05).

3.7. SEM To better understand chitin morphology, various chitin samples were observed by SEM. Fig. 3 shows SEM photographs of produced chitin with enzymes and commercial chitin. Chitin and chitosan can be classified into three groups: (1) without porosity and micro/ nanofibrillar structure, (2) with porosity or micro/nanofibrillar structure, and (3) with microfibrillar and without porosity structure [50]. A smooth and microfibrillar structure with high molecular packing was observed in enzymatic produced chitins. Previous studies showed that the chitin with a fibrillar surface morphology has a great potential for use in textiles [24]. Al Sagheer et al. exhibited a very uniform with a lamellar organization and dense structure for α-chitin extracted from marine sources in Arabian Gulf such as Penaeus semisulcatus [51]. One reason for microfibrillar and packing structure of enzymatic chitins is a higher crystallinity index (Table 3) of them and subsequently higher inter- or intra-molecular hydrophobic interactions and hydrogen bonding [38,61]. 4. Conclusions In this study, chitins were extracted from white shrimp shell, by treatment with lactic acid for demineralization. Afterwards, the combination of enzymatic and mild alkali deproteinization carried out using trypsin and ficin with/without sodium metabisulfite. High deproteinization and demineralization rates were obtained by these treatments. Moreover, the enzymatic chitins (especially chitin produced by trypsin) exhibited higher degree of acetylation and crystalline index as well as a smooth microfibrillar crystalline structure as compared to commercial one (the chitins with DA values between 70 and 90% and low protein content, can be considered as good final products). In current study we used mild chemical treatment to improve the deproteinization and deproteinization performance. Moreover, the extraction process was carried out based on commercial enzyme which was chemically isolated. Therefore, the introduced method cannot be considered as a quite eco-friendly procedure. However, the chitin extraction in a completely green way, is time-consuming and expensive. Further research is needed to introduce a more industrial, cheaper and eco-friendly method. Therefore, the treatments were used to chitin extraction had a high potential to generate the high grade chitin from white shrimp shell waste under milder and eco-friendly conditions as compared to chemical treatment.

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Declaration of competing interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with animals performed by any of the authors. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent Not applicable. References

Fig. 3. SEM micrograph of produced chitin with a) trypsin, b) ficin and c) commercial chitin.

Acknowledgment The authors gratefully acknowledge the financial support from Shiraz University of Iran (Grant Number: GR-AGR-56).

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