Green and Facile Production of Chitin from Crustacean Shells Using a

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Agricultural and Environmental Chemistry

Green and Facile Production of Chitin from Crustacean Shells Using a Natural Deep Eutectic Solvent Wen-Can Huang, Dandan Zhao, Na Guo, Changhu Xue, and Xiangzhao Mao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03847 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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Journal of Agricultural and Food Chemistry

Green and Facile Production of Chitin from Crustacean Shells Using a Natural Deep Eutectic Solvent Wen-Can Huanga§, Dandan Zhaoa§, Na Guoa, Changhu Xuea,b, Xiangzhao Maoa,b* a

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

b

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China

* Corresponding author: Xiangzhao Mao Address: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China Tel.: +86-532-82032660 Fax: +86-532-82031789 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Natural deep eutectic solvents (NADESs) are sustainable, nontoxic and biodegradable solvents, which are composed of natural primary metabolites. A green and efficient approach based on choline chloride-malic acid, a NADES, was developed for extracting chitin from crustacean shells, and its effectiveness for demineralization and deproteinization

was

determined.

Fourier

transform

infrared

(FT-IR),

thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were used to investigate changes in the chemical composition of extracted chitin. The results revealed that most of the minerals and proteins were removed from the shrimp shells by using a NADES with the assistance of microwave irradiation. The quality of the obtained chitin was superior, and it displayed a relative crystallinity of 71%. All of these results were achieved without using harsh chemicals, which can raise environmental issues. This study provides a green and facile approach for chitin production from crustacean shells and reveals the potential of NADESs for applications in the extraction of biopolymers from natural sources.

KEYWORDS Natural deep eutectic solvents, Primary metabolites, Crustacean shells, Shrimp shells, Chitin, Biopolymers


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INTRODUCTION Chitin,

a

linear

polysaccharide

composed

of

β-1,4-glycosidic

linked

N-acetyl-D-glucosamine units,1-2 is one of the most abundant natural polymers after cellulose.1-2 This polymer is primarily found in the exoskeletons of crustaceans, insects, and microorganisms.3-4 Chitin has several desirable characteristics, such as biodegradability, biocompatibility, renewability, and sustainability.5-6 Thus, chitin materials have recently received great attention and have been widely used in various fields including the food, pharmaceutical, medical, and agricultural industries.7 The main sources of chitin are shells of two crustaceans, namely, shrimp and crabs.8-9 Crustacean shells are composed of chitin, protein, and mineral salts.10-11 The chitin network in crustacean shells are embedded and stiffened by a mineral-protein matrix.10 Thus, demineralization and deproteinization process are required to obtain chitin from crustacean shells. In industrial processing, chitin is produced from crustacean shells using acid demineralization followed by alkali deproteinization.9 However, the use of acid and alkali in the process of chitin production has caused serious environmental problems.12 Many approaches without using harsh chemicals have been developed for chitin extraction from crustacean shells such as ionic liquid extraction, enzymatic reactions and microbial fermentation.1, 12 Among these approaches, ionic liquid is considered a promising solvent for chitin production due to several advantages, including low vapor pressure, non-flammability, and excellent solubility.1 Nevertheless, many



reports pointed out the hazardous properties of ionic liquids (ILs) such as toxicity and non-biodegradability.13-14 These hazardous properties of ILs significantly limit the various applications of ILs. Deep eutectic solvents (DESs), an alternative for ILs, exhibit similar properties to those of ILs,15 but DESs are more advantageous due to their biodegradability, low cost, and simple synthetic process.16-18 Natural deep eutectic solvents (NADESs) are DESs, which are composed of natural compounds,13 particularly primary metabolites common in living cells.19-20 NADESs possess excellent properties as solvents,21 and apart from exhibiting similar characteristics to ILs and DESs, NADESs present better properties during extraction.20 In this study, a green and facile approach based on choline chloride-malic acid, a NADES, was developed for extracting chitin from shrimp shells. To the best of our knowledge, this is the first report on the use of NADESs for chitin extraction from shrimp shells. The extracted chitin was characterized using scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The losses of the NADES were investigated via nuclear magnetic resonance (NMR).

MATERIALS AND METHODS Materials. Choline chloride was purchased from Yuanye Bio-Technology (China); malic acid, hydrochloric acid, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); and dimethyl sulfoxide was purchased from Cambridge Isotope Laboratories, Inc. (USA). The shrimp shells were


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dried in an oven and then ground in a blender before extraction. Synthesis of a natural deep eutectic solvent (NADES). Choline chloride and malic acids were mixed at a molar ratio 1:1, and then the mixture was heated at 80 °C with constant stirring until a homogeneous liquid was formed. Chitin extraction by the NADES. A schematic depiction of chitin extraction from the shrimp shells using the NADES is shown in Figure 1. To extract chitin, the shrimp shells were added into the NADES with various shrimp shell/NADES ratios of 1:5, 1:10, and 1:20. Next, the mixture was heated under 700 w microwave irradiation (Haier MZC-2070M1) for different durations of time (1 min, 3 min, 7 min, and 9 min). During microwave irradiation, 3 s pulses was taken in each minute to avoid overheating of the NADES. Then, chitin was separated from the NADES by centrifugation. The chitin was collected and thoroughly washed with distilled water, followed by drying. The NADES was recycled three times without purification, and the reusability of the NADES was evaluated. The mineral content of the samples was determined by heating the samples at 525 °C in a muffle furnace for 5 h. The protein content of the samples was measured by Bradford method.22 The deproteinization efficiency was determined by measuring the deduction of proteins in extracted chitin from the proteins contained in the raw shrimp shells and the percentage of the deproteinization efficiency (DP) is expressed as DP (%) = [ (Po - P) / Po ] × 100%

(1)

where Po and P are the protein content in the raw and NADES-treated samples,



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respectively. In comparison with the chitin extracted by the NADES, the acid/alkali extraction of chitin from shrimp shells was carried out.8 To perform demineralization, 30 g samples were treated with a 1 M HCl solution (300 mL) for 1 h at room temperature. Next, the demineralized samples were collected by centrifugation, followed by washing with distilled water. Then, deproteinization was conducted by treating the collected samples with 2.5 M NaOH solution (600 mL) for 3 h at 90 °C. The resulting chitin was washed with distilled water and dried in a vacuum oven. Characterization. The surface morphologies of the shrimp shells before and after the NADES treatment were observed under a scanning electron microscope (SEM; Quanta FEG250). Prior to imaging, all the samples were freeze dried and attached to the metal stubs using conductive carbon tape, and then the samples were sputter-coated with a platinum film. The samples were analyzed at an acceleration voltage of 10 kV. Fourier transform infrared (FT-IR) spectra were recorded in the range of 4000 to 500 cm-1 using a FT-IR spectrometer (Thermo Scientific Nicolet iS10). The XRD patterns were recorded using an X-ray diffractometer (XRD; Bruker D2 Phaser) with Cu Kα radiation (λ = 1.78892 nm) at 40 kV. The diffraction data were obtained with 2θ range from 5° to 60° at a scanning rate of 5°/min. The relative crystallinity index (CrI) was calculated by the Segal method.23 CrI (%) = [ (I110 - Iam) / I110 ] × 100

(2)

where I110 is the peak intensity of the diffraction for the (110) plane at 2θ ≈ 20°


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and Iam is the intensity of the amorphous diffraction at 2θ ≈ 18°. Thermogravimetric analysis (TGA) was carried out using a thermogravimetric analyzer (NETZSCH TG 209 F3) at a heating rate of 10 °C/min under a nitrogen atmosphere. The NADES losses were determined using NMR spectroscopy (Agilent ProPulse 500 MHz). After removing the chitin, the NADES were identified by 1H NMR using dimethyl sulfoxide as an internal standard.

RESULTS AND DISCUSSION Demineralization

and

Deproteinization

of

the

Shrimp

Shells.

The

demineralization and deproteinization efficiency of the NADES were determined with various shrimp shell/NADES ratios and microwave irradiation times. After the treatment of the NADES, more than 99% of the calcium carbonate was removed from the shrimp shells via the reaction with malic acid after 9 min of microwave irradiation at all the tested shrimp shell/NADES ratios (Figure 2a). As shown in Figure 2b, the deproteinization

efficiency

was

continuously

increased

when

the

shrimp

shell/NADES ratios increased from 1:5 to 1:20. These results indicate that the treatment of the NADES at higher shrimp shell/NADES ratios is beneficial for the deproteinization efficiency. Microwave irradiation time is another factor that influences the deproteinization efficiency. The deproteinization efficiency kept increasing with increasing irradiation time. The maximal deproteinization of the NADES was as high as 93.8% at the shrimp shell/NADES ratio of 1:20 after 9 min of



microwave irradiation. SEM. The morphologies of the shrimp shells, acid/alkali-extracted chitin and NADES-extracted chitin were analyzed using scanning electron microscopy (SEM), as presented in Figure 3. From the SEM observations, there was a significant change in the morphology and surface of the extracted chitin compared with those of the shrimp shells. A rough surface without pores were observed for the shrimp shells due to the presence of minerals and proteins.24 In the case of the NADES-extracted chitin, because of the removal of the minerals and proteins, a smooth surface with pores was clearly evident.24 The morphology of the acid/alkali-extracted chitin was similar to that of the NADES-extracted chitin. FT-IR. The FT-IR spectra of the NADES-extracted chitin, acid/alkali-extracted chitin and shrimp shells are presented in Figure 4a. In the spectra of NADES-extracted chitin and acid/alkali-extracted chitin, absorption peaks at 3449 cm−1 and 3271 cm−1 are attributed to the O-H and N-H stretching vibration, respectively.25 The absorption peaks at 1664 cm−1 and 1625 cm−1 correspond to the amide I band.25 These bands are associated with the typical features of α-chitin. In contrast, the spectrum of shrimp shells is significantly different from the NADES-extracted chitin and acid/alkali-extracted chitin spectra. In the spectrum of shrimp shells, the amide I band was not split due to the amide peaks of protein overlapping with the chitin amide I band, indicating that the NADES is able to remove proteins from the shrimp shells. XRD. XRD analysis was used to determine the crystal structure and relative


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crystallinity of the extracted chitin and shrimp shells. XRD patterns of the NADES-extracted chitin, acid/alkali-extracted chitin, shrimp shells and CaCO3 are presented in Figure 4b. The NADES-extracted samples exhibited the typical diffraction peaks at 2θ = 9.08°, 12.77°, 19.21°, 22.78°, 26.28°, and 27.98°, which indicated the crystal lattice type of α-chitin. The diffraction peaks of CaCO3 at approximately 2θ = 29.55° was not shown in the NADES-extracted chitin, indicating that CaCO3 was removed by the NADES. The XRD patterns of chitin extracted by the NADES were in good accord with those of the chitin obtained by the conventional chemical process and suggests that the chitin extracted by the NADES was free from minerals. Furthermore, the crystallinity index of the samples was calculated according to the Segal method. The CrI of chitin was calculated to be 70.91% using the NADES method and 65.41% using the traditional chemical process, while the CrI of the shrimp shells was calculated to be 48.30%. The increase in CrI after NADES treatment was due to the removal of the minerals and proteins from the shrimp shells. The increase in CrI suggests that the minerals and proteins were removed from the shrimp shells by the NADES treatment. Thermal stability. The thermal stability was assessed and the thermogravimetric (TG) curves are presented in Figure 4c. The initial mass loss of the NADES-extracted chitin and acid/alkali-extracted chitin was observed at approximately 300 °C and was attributed to the decomposition of the chitin.26 The mass loss in the shrimp shells occurred at approximately 200 °C due to the decomposition of proteins. The absence of mass loss in the NADES-extracted chitin observed in the range from 200−250 °C,



which is typically observed for proteins in shrimp shells, revealed that proteins are removed by the NADES. The weight loss in the shrimp shells appeared at approximately 700 °C and was attributed to the calcium carbonate decomposition into calcium oxide and carbon dioxide.26 The absence of mass loss at 700 °C indicates that the NADES-based method is able to remove calcium carbonate in addition to the proteins. Overall, the morphology, crystallinity, and thermal stability of the NADES-extracted chitin were comparable to those of the chitin extracted by the conventional chemical method, whereas the processing was greener and more efficient. Reuse of the NADES. When choline chloride-malic acid was used to extract chitin from the shrimp shells, a certain amount of malic acid was consumed during the demineralization process. Thus, after each cycle, same amount of malic acid, which was used in the demineralization process, should be added to maintain the composition of the NADES (Figure 1). As shown in Figure 4d, the NADES could be reused 3 times without a significant decrease in the chitin extraction yield. After 3 cycles, the NADES became too viscous to be used in the extraction, which may because proteins existing in the recycled NADES may influence the viscosity of the NADES during reuse. The NADES losses were determined using 1H NMR. The spectrum of the recycled NADES was similar to that of the original NADES, except that the intensity of the malic acid peak decreased. This decrease is because of the consumption of malic acid during the demineralization process. Mechanism of the NADES-based chitin extraction. Demineralization was carried out by the malic acids. When the choline chloride-malic acid was applied to


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the shrimp shells, minerals, which are mostly in the form of crystalline CaCO3,27 were removed by malic acid, leaving the proteins and chitin. The spacing between the chitin-protein fibers was filled with proteins and minerals27; thus, the removal of minerals resulted in weakening of the linkages within the inner structural organization of the shrimp shells. Since the minerals are removed by the malic acids, in order to conduct demineralization, one component of the NADES used in the chitin extraction should be an acid. Due to its high molecular weight and extensive intermolecular and intramolecular hydrogen bond formation, chitin is insoluble in water and most organic solvents.9, 28 The high extractability of chitin with the NADES can be attributed to the hydrogen bond interactions between the NADES and shrimp shell components. Competing hydrogen bond formation between the NADES and carbohydrates cause the breaking of the intramolecular hydrogen bond network, consequently weakening the hydrogen bond interactions in the shrimp shells. As a result, chitin is dissolved in the NADES and separated from the proteins. In this study, a NADES-based extraction method was demonstrated to be a fast, efficient and green approach for extracting chitin from shrimp shells. By using this method, most of the minerals and proteins were removed from the shrimp shells. The characterization results indicated that the quality of extracted chitin was superior. Moreover, the major advantages of NADESs over the other solvents is that NADESs are composed of natural primary metabolites; thus, NADES-based extraction avoids the use hazardous chemicals, which can cause environmental problems. Overall, the NADES-based extraction method is a suitable approach to extract chitin from shrimp



shells and reveals the potential for applications in the extraction of biopolymers from natural sources.

AUTHOR INFORMATION Corresponding Authors Xiangzhao Mao, E-mail: [email protected]

Author Contributions § These authors contributed equally.

ACKNOWLEDGMENTS This work was supported by China Agriculture Research System-48, Major Special Science and Technology Projects in Shandong Province (2016YYSP016), and Ningbo Science and Technology Projects (2017C110006).

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Figure 1. Schematic depiction of chitin production from shrimp shells using choline chloride -malic acid. 1261x480mm (72 x 72 DPI)



Figure 2. (a) Demineralization and (b) deproteinization efficiency of the NADES at shrimp shell/NADES ratios of 1:5, 1:10, and 1:20. 682x277mm (72 x 72 DPI)


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Figure 3. SEM images of the (a) shrimp shells, (b) chitin extracted by the conventional chemical process, and (c) chitin extracted by the NADES-based method. 1190x240mm (72 x 72 DPI)



Figure 4. (a) FT-IR spectra of the shrimp shells, acid/alkali-extracted chitin, and NADES-extracted chitin; (b) XRD curves of the shrimp shells, CaCO3, acid/alkali-extracted chitin, and NADES-extracted chitin; (c) TG curves of the shrimp shells, acid/alkali-extracted chitin, and NADES-extracted chitin; and (d) Reusability of the NADES. 696x542mm (72 x 72 DPI)


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TOC Graphic. A green and efficient approach based on a natural deep eutectic solvent (NADES) was developed for extracting chitin from shrimp shells. 866x482mm (72 x 72 DPI)


Green and Facile Production of Chitin from Crustacean Shells Using a

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