Extraction and Physicochemical Characterization of Chitin from Cicada orni Sloughs of the South-Eastern French Mediterranean Basin

Chitin is a structural polysaccharide of the cell walls of fungi and exoskeletons of insects and crustaceans. In this study, chitin was extracted, for the first time in our knowledge, from the Cicada orni sloughs of the south-eastern French Mediterranean basin by treatment with 1 M HCl for demineralization, 1 M NaOH for deproteinization, and 1% NaClO for decolorization. The different steps of extraction were investigated by Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric Analysis (TGA), and Scanning Electron Microscopy (SEM). Results demonstrated that the extraction process was efficiently performed and that Cicada orni sloughs of the south-eastern French Mediterranean basin have a high content of chitin (42.8%) in the α-form with a high degree of acetylation of 96% ± 3.4%. These results make Cicada orni of the south-eastern French Mediterranean basin a new and promising source of chitin. Furthermore, we showed that each step of the extraction present specific characteristics (for example FTIR and XRD spectra and, consequently, distinct absorbance peaks and values of crystallinity as well as defined values of maximum degradation temperatures identifiable by TGA analysis) that could be used to verify the effectiveness of the treatments, and could be favorably compared with other natural chitin sources.


Introduction
Chitin is a copolymer of N-acetyl-d-glucosamine and d-glucosamine units linked with β-(1-4) glycosidic bond, composed predominantly of N-acetyl-d-glucosamine units and represents the second most abundant polysaccharide after cellulose [1]. Due to their excellent properties, including biocompatibility, nontoxicity [2], and biodegradability [3], chitin and its derivatives, such as chitosan (deacetylated form of chitin), have recently become of great interest for medical and pharmaceutical applications [4,5]. Even though chitosan is mostly used for biomedical applications due to its higher solubility in water and other traditional solvents such as acetic acid [6], chitin can be solubilized in some other solvents, such as ionic liquids [7,8]. Due to the versatile properties of the material, it can be used to fabricate scaffolds [3], hydrogels [9], or microspheres [10], showing promising physiochemical, mechanical, and biological properties for different applications, such as in the biomedical field for wound healing [11], vascularization [12], and bone repair [7]. Chitin represents the primary structural Figure 1 shows pictures of the samples corresponding to all the steps of chitin extraction with their relative weights after each step. Raw material corresponds to cicada sloughs washed and dried at 50 • C for 24 h and represents the initial quantity (5.4 g). The decolorized samples correspond to the final product chitin. After each step, the powder was dried and weighed, and the difference of weight before and after the demineralization and deproteinization processes was used to calculate, as described in Equation (2), the percentage content of minerals and proteins present in the raw material. The mineral and protein contents were estimated to be 14.8% and 30.4%, respectively. The chitin content (%) of Cicada orni sloughs of the south-eastern French Mediterranean basin studied, defined in the following as Cicada orni (CO), and calculated using the Equation (1), was 42.6%. The chitin, protein, and mineral content of Cicada orni sloughs are similar to the only reported study on chitin extraction from cicada sloughs. According to Sajomsang and Gonil [35] cicada sloughs consist of approximately 37% chitin, 40% proteins, and 12% minerals. Only one other study reported the chitin content of cicada but, this time, from the whole body of six cicada species collected from the Mediterranean region of Turkey. The chitin content was in the range of 4.97-8.84% [30]. Table 1 summarizes some of the chitin content from different sources. The chitin content obtained in this study is higher compared to other sources, (Table 1), including seafood, which represent the primary sources of chitin. According to these results, extracting chitin from cicada sloughs offers the advantage of the production of higher quantities of final product for a given initial source biomass. However, a disadvantage of this source of chitin is that its availability is limited to a few months of the year. Comparable chitin contents have been reported for squid gladius but, in that organism, the chitin is type β [38]. study is higher compared to other sources, (Table 1), including seafood, which represent the primary sources of chitin. According to these results, extracting chitin from cicada sloughs offers the advantage of the production of higher quantities of final product for a given initial source biomass. However, a disadvantage of this source of chitin is that its availability is limited to a few months of the year. Comparable chitin contents have been reported for squid gladius but, in that organism, the chitin is type β [38].

FTIR Analysis
Since chitin is a copolymer of N-acetyl-d-glucosamine and d-glucosamine, DA is an important factor used to measure the quality of chitin, being obtained from the ratio of acetylated group (N-acetyl-d-glucosamine) to deacetylated (d-glucosamine) amino groups [15]. FTIR spectra were used to (1) to analyze the demineralization, deproteinization, and decolorization processes, (2) to characterize the extracted chitin, and (3) to calculate the degree of acetylation. Figure 2 shows the FTIR spectra of raw material, demineralized, deproteinized, and decolorized samples. The trend of the absorbance of all samples is similar to the typical trend of chitin [52]. There is no remarkable difference between raw material and demineralized sample spectra. According to Gbenebor,[53], virgin crab and shrimp shell spectra present typical peaks for CaCO 3 at 1473 and 874 cm −1 and 1443 and 874 cm −1 , respectively, and these peaks reduced in intensity after demineralization with 1 M HCl. Raw material spectra present only small peaks at these wavelengths. The absence of these peaks might be due to the lower mineral content cicada sloughs (12-14%) [35] compared with shrimp (30-40%) [34,48] and crab shells (30.3-44%) [14,48]. Instead, there are differences between the demineralized and deproteinized samples. According to Hassainia, α-chitin presents two absorption peaks at about 1660 and 1627 cm −1 (due to the C=O stretching vibration secondary amide stretch of the amide I), while β-chitin presents only one band at 1656 cm −1 [15]. After deproteinization, the presence of two distinct peaks at about 1650 and 1621 cm -1 confirms α-chitin structure. These peaks are attributed to vibration modes of amide I, so their resolution in two separate peaks is probably due to the elimination of proteins [54]. In fact, only one peak is present in the raw material and demineralized sample. Deproteinized and decolorized samples present the same peaks, proving that pigments cannot be identified from these spectra. The degree of acetylation (DA) was calculated using the Brugnerotto expression (Equation (4)), which was the most reliable method in previous studies and in agreement with H-NMR and C-NMR data [55]. The peak at 1320 cm −1 is specific of N-acetyl glucosamine and it is used for the determination of the DA with the peak at 1420 cm −1 [52]. DA was calculated using the average of the absorption values at 1420 and 1320 cm −1 (see Figure S1 in Supplementary Materials for details about the baselines used to calculate the DA). The resulting DA was 96.3 ± 3.4. Table 2 summarizes the DA and crystallinity index (CrI) (that will be discussed below) of chitin from different sources. The obtained DA was similar to that obtained by Sajomsang and Gonil from cicada sloughs [35] and comparable to that reported in the literature from different sources ( Table 2). The DA is the parameter that affects the solubility of chitin and its derivatives the most. In fact, when the DA is below 50%, the product is already called chitosan, and its solubility in acidic solutions increases with the reduction of the DA [56].

X-Ray Diffraction
In nature, chitin is arranged in crystalline microfibrils [60]. The crystalline structure can be analyzed by X-ray diffraction (XRD). Figure 3 shows the XRD patterns of the demineralized, deproteinized, and decolorized steps of chitin extraction. All samples present six peaks at about 9.3°, 12.7°, 19.5°, 20.8°, 23.4°, and 26.3° that correspond to the six typical crystalline planes (020), (021), (110), (120), (101), and (130), respectively, except for the peak at 20.8° (120), which was decreased in the deproteinized sample and absent in the decolorized one. The positions and the amplitude of these peaks are in good agreement with those reported for five other sources of chitin, including cicada sloughs [33]. According to Jang [17], α-chitin has four crystalline reflections at 9.6, 19.6, 21.1, and 23.7, while β-chitin has two crystalline reflections at 9.1 and 20.3. Cicada orni results indicate that the

X-ray Diffraction
In nature, chitin is arranged in crystalline microfibrils [60]. The crystalline structure can be analyzed by X-ray diffraction (XRD). Figure 3 shows the XRD patterns of the demineralized, deproteinized, and decolorized steps of chitin extraction. All samples present six peaks at about 9.3 • , 12.7 • , 19.5 • , 20.8 • , 23.4 • , and 26.3 • that correspond to the six typical crystalline planes (020), (021), (110), (120), (101), and (130), respectively, except for the peak at 20.8 • (120), which was decreased in the deproteinized sample and absent in the decolorized one. The positions and the amplitude of these peaks are in good agreement with those reported for five other sources of chitin, including cicada sloughs [33]. According to Jang [17], α-chitin has four crystalline reflections at 9.6, 19.6, 21.1, and 23.7, while β-chitin has two crystalline reflections at 9.1 and 20.3. Cicada orni results indicate that the extracted chitin is the α structure. Crystallinity index (CrI) is an important characteristic influencing the physical and biological properties of chitin, including solubility and biodegradability [59]. In particular, the higher the degree of crystallinity, the lower the solubility [60] and, accordingly, the biodegradability [61].
CrI was calculated using Equation (5). Results show that CrI was 68.6% for the raw material, 55.8% for the demineralized sample, 73.4% for the deproteinized sample, and 72.1% for the decolorized sample. Our hypothesis is that the removal of minerals may induce a diminution of CrI because of the absence of calcium carbonate crystals. After deproteinization, the increase of CrI may be due to a rearrangement of polymer chains after the removal of proteins. The CrI of chitin extracted from CO (72.1%) is within this range of chitin extracted in the literature from different sources, between 57.8% and 89.7% (Table 2).
Molecules 2020, 25, x FOR PEER REVIEW 6 of 12 extracted chitin is the α structure. Crystallinity index (CrI) is an important characteristic influencing the physical and biological properties of chitin, including solubility and biodegradability [59]. In particular, the higher the degree of crystallinity, the lower the solubility [60] and, accordingly, the biodegradability [61]. CrI was calculated using Equation (5). Results show that CrI was 68.6% for the raw material, 55.8% for the demineralized sample, 73.4% for the deproteinized sample, and 72.1% for the decolorized sample. Our hypothesis is that the removal of minerals may induce a diminution of CrI because of the absence of calcium carbonate crystals. After deproteinization, the increase of CrI may be due to a rearrangement of polymer chains after the removal of proteins. The CrI of chitin extracted from CO (72.1%) is within this range of chitin extracted in the literature from different sources, between 57.8% and 89.7% (Table 2).

Thermogravimetric Analysis
Thermogravimetric analysis (TGA), a measure of the change in mass of a substance as a function of temperature, is another important factor that defines the possible application of chitin [62] and helps to identify the type of chitin. TGA of raw material, demineralized, deproteinized, and decolorized samples is shown in Figure 4A. The maximum weight loss for all samples was obtained between 200 and 400 °C , and was 63.9% for the raw material, 68.4% for the demineralized sample, 55.8% for the deproteinized sample, and 63.9% for the decolorized sample. The maximum weight loss obtained from chitin extracted from cicada sloughs by Sajomsang and Gonil [37] was 66.4%, while the maximum weight loss obtained from chitin extracted from the whole body of cicadas was in the range of 72.2-88.3% [30]. Figure 4B shows the derivative thermogravimetric analysis (DTG) of the four samples. The thermal degradation of raw material occurs in at least four different degradation steps, that of demineralized and deproteinized samples in three steps, and that of decolorized samples in two steps. The temperature at which the maximum degradation of the samples occurred was 371.4, 357.6, 359.8, and 375.1 °C for raw material, the demineralized, deproteinized, and decolorized specimen steps, respectively. According to the literature, the maximum distortion temperature of α-chitin is usually higher than 350 °C , while the value of for βchitin is usually lower than 350 °C [16]. The maximum degradation temperature of chitin from CO

Thermogravimetric Analysis
Thermogravimetric analysis (TGA), a measure of the change in mass of a substance as a function of temperature, is another important factor that defines the possible application of chitin [62] and helps to identify the type of chitin. TGA of raw material, demineralized, deproteinized, and decolorized samples is shown in Figure 4A. The maximum weight loss for all samples was obtained between 200 and 400 • C, and was 63.9% for the raw material, 68.4% for the demineralized sample, 55.8% for the deproteinized sample, and 63.9% for the decolorized sample. The maximum weight loss obtained from chitin extracted from cicada sloughs by Sajomsang and Gonil [37] was 66.4%, while the maximum weight loss obtained from chitin extracted from the whole body of cicadas was in the range of 72.2-88.3% [30]. Figure 4B shows the derivative thermogravimetric analysis (DTG) of the four samples. The thermal degradation of raw material occurs in at least four different degradation steps, that of demineralized and deproteinized samples in three steps, and that of decolorized samples in two steps. The temperature at which the maximum degradation of the samples occurred was 371.4, 357.6, 359.8, and 375.1 • C for raw material, the demineralized, deproteinized, and decolorized specimen steps, respectively. According to the literature, the maximum distortion temperature of α-chitin is usually higher than 350 • C, while the value of for β-chitin is usually lower than 350 • C [16]. The maximum degradation temperature of chitin from CO was 375.1 • C, confirming that it is α-chitin. This result is similar to the maximum degradation temperature of chitin extracted from cicada sloughs described by Sajomsang and Gonil [35], which was 362 • C, while for chitin extracted from the whole body of cicadas, it was in the range of 339.9-412.7 • C [30]. All samples presented a peak around 100 • C corresponding to water evaporation. The peak around 306 • C in raw material decreased in the demineralized sample and disappeared in deproteinized and decolorized samples. This suggests that it could be related to the demineralization process.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 was 375.1 °C, confirming that it is α-chitin. This result is similar to the maximum degradation temperature of chitin extracted from cicada sloughs described by Sajomsang and Gonil [35], which was 362 °C , while for chitin extracted from the whole body of cicadas, it was in the range of 339.9-412.7 °C [30]. All samples presented a peak around 100 °C corresponding to water evaporation. The peak around 306 °C in raw material decreased in the demineralized sample and disappeared in deproteinized and decolorized samples. This suggests that it could be related to the demineralization process.  Figure 5 shows scanning electron microscopy (SEM) images of the surface of the raw material ( Figure 5A), and demineralized ( Figure 5B) and decolorized ( Figure 5C) specimens. Raw material surface exhibits a rough morphology. We hypothesize that chitin has a lamellar organization intercalated with some granular material (that could correspond to minerals and proteins) that seems to decrease in demineralized and in decolorized samples, where only the flat lamellar structure seems to remain. This decrease could demonstrate the effective removal of minerals and proteins, respectively [35].

Extraction of Chitin from Cicada orni
Cicada orni sloughs were taken in August 2017 in the south-eastern French Mediterranean basin, especially in La Seyne sur Mer in a place called PinRolland. Chitin was extracted with a modified protocol based on Luo et al.'s work [33]. Cicada orni sloughs were washed with distilled water and dried in an oven at a temperature of 50 °C for 24 h. Then they were mechanically ground in a mortar. Specimen were demineralized with 1 M HCl aqueous solution (1:15 ratio of solid sample to solution) in a 30 °C water bath at 200 rpm for 2 h to remove calcium carbonate and other calcium salts. Then they were filtered out and washed with deionized water until neutral pH was detected. The reaction of demineralization is the following [63]: (1)  Figure 5 shows scanning electron microscopy (SEM) images of the surface of the raw material ( Figure 5A), and demineralized ( Figure 5B) and decolorized ( Figure 5C) specimens. Raw material surface exhibits a rough morphology. We hypothesize that chitin has a lamellar organization intercalated with some granular material (that could correspond to minerals and proteins) that seems to decrease in demineralized and in decolorized samples, where only the flat lamellar structure seems to remain. This decrease could demonstrate the effective removal of minerals and proteins, respectively [35].

Scanning Electron Microscopy
Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 was 375.1 °C, confirming that it is α-chitin. This result is similar to the maximum degradation temperature of chitin extracted from cicada sloughs described by Sajomsang and Gonil [35], which was 362 °C , while for chitin extracted from the whole body of cicadas, it was in the range of 339.9-412.7 °C [30]. All samples presented a peak around 100 °C corresponding to water evaporation. The peak around 306 °C in raw material decreased in the demineralized sample and disappeared in deproteinized and decolorized samples. This suggests that it could be related to the demineralization process.

Extraction of Chitin from Cicada orni
Cicada orni sloughs were taken in August 2017 in the south-eastern French Mediterranean basin, especially in La Seyne sur Mer in a place called PinRolland. Chitin was extracted with a modified protocol based on Luo et al.'s work [33]. Cicada orni sloughs were washed with distilled water and dried in an oven at a temperature of 50 °C for 24 h. Then they were mechanically ground in a mortar. Specimen were demineralized with 1 M HCl aqueous solution (1:15 ratio of solid sample to solution) in a 30 °C water bath at 200 rpm for 2 h to remove calcium carbonate and other calcium salts. Then they were filtered out and washed with deionized water until neutral pH was detected. The reaction of demineralization is the following [63]: (1)

Extraction of Chitin from Cicada orni
Cicada orni sloughs were taken in August 2017 in the south-eastern French Mediterranean basin, especially in La Seyne sur Mer in a place called PinRolland. Chitin was extracted with a modified protocol based on Luo et al.'s work [33]. Cicada orni sloughs were washed with distilled water and dried in an oven at a temperature of 50 • C for 24 h. Then they were mechanically ground in a mortar. Specimen were demineralized with 1 M HCl aqueous solution (1:15 ratio of solid sample to solution) in a 30 • C water bath at 200 rpm for 2 h to remove calcium carbonate and other calcium salts. Then they were filtered out and washed with deionized water until neutral pH was detected. The reaction of demineralization is the following [63]: In more detail, HCl reacts with calcium carbonate to produce an aqueous solution of calcium chloride (CaCl 2 ) and CO 2 . Specimens were dried in the oven at 50 • C for 24 h and weighed to calculate the amount on minerals present in Cicada orni sloughs. For deproteinization, a treatment with 1 M NaOH solution (1:15 ratio of solid sample to solution) was done, and then refluxed at 90 • C for 2 h to remove proteins. The specimen was also dried in the oven at 50 • C for 24 h and weighed to calculate the amount of protein present in Cicada orni sloughs. Insoluble material was filtered and washed extensively with deionized water until neutral pH. For the decolorization, the sample was treated with 1% sodium hypochlorite (NaClO) (1:30 ratio of solid sample to solution) for 30 min at 25 • C room temperature, filtered, and washed with distilled water, and treated another two times with 1% NaClO for 10 min, until the water was transparent. After this last step, specimens were washed extensively to remove any residual chemicals.

Chitin Content
Chitin content was calculated by measuring the weight of samples before and after the extraction (always dried in an oven at 60 • C overnight). The chitin content (%) was calculated using the following formula: Chitin content (%) = Dried chitin extracted (g) Raw material (g) × 100 (2) The mineral content (%) of cicada sloughs was calculated using the following formula: where W1 is the raw material and W2 is the weight of the dried sample after demineralization. The protein content was calculated using the same formula, attributing the weight of the dried demineralized sample to W1 and the weight of the dried deproteinized sample to W2. The weight of the remaining material after the decolorization process represents the dried chitin extracted.

FTIR Analysis
Dried powder of raw material, demineralized, deproteinized, and decolorized Cicada orni samples were used for FTIR analysis. Spectra were collected in a range between 4000 to 400 cm −1 with a Nicolet 6700 Spectrophotometer by accumulation of 64 scans with a resolution of 4 cm −1 in KBr pellets (1 mg of the sample in 100 mg of KBr). The degree of acetylation (DA) was calculated from the Absorbance (A) ratios according to the Brugnerotto method [52]: where the values of the absorbance at 1420 cm −1 and 1320 cm −1 were calculated using the baselines shown in Figure S1. The DA was expressed as average of results from eight FTIR spectra.

X-ray Diffraction
Powder X-ray diffraction (XRD) (D8 Advance, Bruker, Billerica, MA, USA) analysis of demineralized, deproteinized, and decolorized steps were record using a D8 Advance X-ray diffractometer. Data were collected at a scan rate of 1 • /min with the scan angle from 5-40 • . The crystallinity indexes (CrI) were calculated using the following equation [57]: CrI (%) = I 110 − I am I 110 × 100 where I 110 is the maximum intensity of the crystalline region at 20 • and I am is the maximum intensity of amorphous diffraction at 16 • .

Thermogravimetric Analysis
The thermal degradation properties of demineralized, deproteinized, and decolorized powdered specimens were analyzed using a thermogravimetric analyzer equipped of a Setaram microbalance ((Setsys Ev. 1750, Setaram, Kep Technologies, Lyon, France). The samples (10 mg) were heated from 20 • C to 1000 • C at a constant heating rate of 10 • C/min under argon atmosphere.

Scanning Electron Microscopy
The surface morphologies of the samples were observed using a Quanta 650-FEG (FEI, Hillsboro, OR, USA) scanning electron microscope (SEM), model, manufacturer, city, country. The samples (raw material, demineralized, deproteinized, and decolorized) were dried, fixed on an adhesive tape, and coated with a gold layer. The images were taken at an acceleration voltage of 5 kV and with a magnification of 10 µm.

Conclusions
In this study, we extracted chitin from Cicada orni sloughs of the south-eastern French Mediterranean basin using a chemical method. We obtained a chitin yield of 42.6%, which is higher than that of other studies. FTIR, DRX, and TGA analysis confirmed that the chitin extracted from Cicada orni is in the α-form, with a DA of 99.6% ± 3.4%, a CrI of 72.1%, and a maximum degradation temperature of 375.1 • C. Cicada orni sloughs of the south-eastern French Mediterranean basin show great potential as an ecological alternative source of chitin. Furthermore, for the first time, we analyzed and described all the steps of the extraction process to compare the effectiveness of the treatments and different extraction conditions. For example, FTIR spectra showed the formation of two distinct peaks at 1650 and 1621 cm −1 after the deproteinization step that seem to be due to the elimination of proteins or, at least, to their decrease.