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Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species
Journal Pre-proof Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species
Mahmut Kabalak, Doruk Aracagök, Murat Torun PII:
S0141-8130(19)40346-2
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.194
Reference:
BIOMAC 14222
To appear in:
International Journal of Biological Macromolecules
Received date:
16 December 2019
Accepted date:
21 December 2019
Please cite this article as: M. Kabalak, D. Aracagök and M. Torun, Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.194
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© 2018 Published by Elsevier.
Journal Pre-proof EXTRACTION, CHARACTERIZATION AND COMPARISON OF CHITINS FROM LARGE BODIED FOUR COLEOPTERA AND ORTHOPTERA SPECIES Mahmut Kabalak1*, Doruk Aracagök1, Murat Torun2 1 2
Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, Turkey.
Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe, Ankara, Turkey. E-mail adresses: [email protected], [email protected], [email protected] *
Corresponding author: [email protected]
ABSTRACT Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758)
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(Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae))
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for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%,
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10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614°C to 748°C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity
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index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales.
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Elemental analysis have been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD
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and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.
1. Introduction
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KEYWORDS: Coleoptera, Orthoptera, Biopolymer, Chitin, Extraction, Characterization
Chitin, a naturally abundant mucopolysaccharide, and the supporting material of crustaceans, insects, etc., is well known to consist of 2-acetamido-2-deoxy-(3-D-glucose) through a β-(1 4) linkage, which can be degraded by chitinase. Its immunogenicity is exceptionally low, in spite of the presence of nitrogen. It is a highly insoluble material resembling cellulose in its solubility and low chemical reactivity. It may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group. Like cellulose, it functions naturally as a structural polysaccharide [1]. Chitin and its main derivative chitosan have various applications in medicine, pharmacy, biotechnology, environmental, and food engineering because of their nontoxicity, biodegradability, biocompatibility, antimicrobial, and antioxidant properties [2]. There are many studies on extraction and determining physicochemical properties of chitin of different organisms (insects, myriapods, arachnids, molluscs, fungi, bryozoan, fish etc.) which were published by different authors [2 – 32]. According to those literature, it is understood that different organisms and even their different body parts having chitin composition with different physicochemical characteristics that also depends on isolation method and the source of chitin [2, 8, 10, 11, 17].
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Journal Pre-proof Some living organisms are evaluated as potential chitin supply which may be used for further processes, mainly conversion to water soluble chitosan for practical applications. Chitin was extracted from blue crabs, Callinectes sapidus, and converted to chitosan which was used for the synthesis of cryogel scaffolds which are potentials for tissue engineering after characterization of chitosan [33]. Antibacterial properties of chitosan from chitin extracted from the waste of Litopenaeus vannamei shrimps was also evaluated [34]. Physicochemical characterization of chitin from Parapenaeus longirostris shrimp shell waste and its chitosan derivative was examined as well [35]. Insects are alternative chitin sources for technology and further processes. There are researches in literature to produce and characterize chitin from different insect taxonomy. Chitin was extracted from housefly, Musca domestica, pupa shells and manufactured to chitosan. Percent yield of conversion, viscosity and degree of deacetylation of chitosan was discussed as well [36]. In another study, structure of chitin was discussed isolated
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from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia in which the form of the chitin was α-form. Its surface morphologies and cristallinity was dicussed and compared with commercial chitin from shrimp [37].
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The isolation of chitin from insect (Melolontha melolontha) was examined and its physicochemical properties was compared with isolated crustacean species from Oniscus asellus [12]. The antimicrobial and antioxidant
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activities of chitin and chitosan from Cosmopolitan Orthoptera Species (insecta) was examined well on Calliptamus barbarus and Oedaleus decorus species as well as physicochemical behaviors of the species was
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discussed in details [15]. In another study, chitin was extracted from the Beetle Holotrichia parallela motschulsky for analysis of chemical structures and physicochemical properties and authors suggest Holotrichia
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parallela is an alternative source of chitin [30], other studies follow the same for chitin from cicada sloughs [38] and from bumblebee (Bombus terrestris) [34]. The effect of the taxonomic relations on physicochemical
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properties of chitin was discussed for 16 arthropod species as a chitin source [2] and the chitin structures from seven orthoptera species was compared [17]. The change of the physicochemical properties of chitin was examined for Vespa crabro (wasp) at larvae, pupa and adult stages during development [26]. It seems to be
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insects are promising alternative sources for chitin extraction in the use of daily life however the physicochemical properties should be evaluated to use the chitin or its derivatives in a proper section.
In this study, the chitin contents of 4 species belonging to 2 orders were determined for the first time. Bradyporus (Callimenus) sureyai Ünal, 2011 belongs to the genus Bradyporus Charpentier, 1825 (Orthoptera: Tettigonidae) which is known as the Armoured Crickets or Glandular Bush-crickets. They can be easily recognized from a distance by their loud calling songs heard usually in the afternoon and early evening. Their most characteristic behaviour is the ejaculation of haemolymph from just under the posterior margin of the metanotum for defensive purposes [39]. Gryllotalpa gryllotalpa (Linnaeus, 1758) is called as “Mole crickets”, which is a member of the family of Gryllotalpidae are distributed throughout temperate and tropical regions. These insects are best known for their digging forelimbs and singing from specialized burrows in the soil [40-42]. European mole cricket spent nearly all their lives underground. They damage vegetable gardens, seedling beds, eat seed, cereals, potato and almost all vegetables. Shoots and young plants often perish after damage [43-47].
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Journal Pre-proof Polyphylla fullo (Linnaeus, 1758) is a major polyphagous agricultural pest because the larvae feed on the roots of many important cultivated plants. It has a 2- to 3-year life cycle with three larval stages that feed on plant roots causing extensive damage, and, in severe infestations, can cause yield loss and plant death [48]. Lucanus cervus (Linnaeus, 1758) (stag beetle) (Coleoptera: Scarabaeidae) is often cited as an indicator of ancient oak forest with ancient trees and large dimensions of dead wood [49]. The larva lives on underground woody debris, mostly of oak [50].
Main objects of this study are extractions of chitins of four species, characterizations of extracted chitins by several methods and comparisons physicochemical behaviours of chitins of examined species for the first time. Chitins are used in many areas, the crystallinity, surface morphology and thermal behaviours are important parameters that determines the subject chitin to be used. In this study, properties of chitins from large bodied
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four coleoptera and orthoptera species were discussed after several characterization methods and these species
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are suggested as alternative chitin sources for various areas according to the observed results.
2. Materials and methods
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2.1. Specimens and materials
Specimens were taken from dry unlabelled insect collection from Hacettepe University Faculty of Science,
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Department of Biology Applied Entomology Laboratory. Hydrochloric acid, sodium hydroxide, chloroform, ethyl acetate and methyl alcohol were all supplied from Sigma-Aldrich which were used for isolation of chitin
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2.2. Specimen preparation
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from species. All reagents were analytical grade and used without further purification.
Specimens present in collection were killed by using ethyl acetate and dried at room temperature up to a constant
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weight then powdered by using liquid nitrogen in the mortar.
2.3. Chitin isolation procedure
In order to demineralize chitin, all samples were treated in 1M HCl solution at 95°C by stirring under reflux for 1 hour. Then, biomasses were separated through filtration with filter paper. Filtrates were washed with distilled water for several times after pH testing. After that samples were treated in 1M NaOH solution at 90°C for 14 hours to remove proteins under reflux. Solutions were filtered again and were washed with distilled for several times water after pH testing. The extracts were left in chloroform-methanol-water (1:2:4, v: v) mixture at room temperature for 1 hour to remove lipids and pigments in the third step, after separation by filtration, the extracts were washed with distilled water for several times. Finally chitin extracts were left to dry at room temperature for 5 days up to a constant weight to determine the chitin percentages and they were stored for further processes.
2.4. Infrared (FT-IR) analysis All chitin samples were characterized spectroscopically by Nicolet IS10 FTIR spectrophotometer (Thermo Scientific) in ATR mode. The resolution of spectra was adjusted as 4 cm-1 with a scanning number of 64. All spectra were recorded between 4000-400 cm-1 wavenumber region and OMNIC program was used for detailed analysis.
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2.5. Thermogravimetric analysis (TGA) TG and DTG curves of chitin samples were recorded with EXSTAR S11 7300 model instrument at a heating rate of 10°C min-1 from 25°C to 800°C under an inert atmosphere by N 2.
2.6. Elemental analysis TruSpec Micro CHNS-O analyzer (Leco Instruments, Stockport, UK) was used to determine the percent composition of carbon, hydrogen and nitrogen elements in isolated chitin samples.
2.7. Scanning electron microscope (SEM) analysis The surface morphologies of extracted chitin samples from four specimens were monitored with Zeiss EVO 50
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model scanning electron microscope. All samples were coated with gold by the sputter coater before image
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analysis.
2.8. X-ray diffraction (XRD) analysis
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X-ray diffraction of powder chitin samples were examined by Rigaku D/MAX 2200 PC model system with tube voltage of 40 kV and current of 40 mA with a scanning speed of 2°/min from 2° to 50° 2θ value. The
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crystallinity index of isolated chitin samples (CrI) were calculated from XRD data by following equation [51]: 𝐼0 − 𝐼𝑎𝑚 𝑥100% 𝐼𝑜
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𝐶𝑟𝐼 =
amorphous region.
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3. Results and discussion
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where I0 is the maximum intensity at 2θ=20° and Iam is the maximum intensity at 2θ=13° corresponding
3.1. Chitin content of examined species at dry basis Chitin contents of the species varied from 9.8% to 11.3% that shows a narrow distribution for samples. Lucanus cervus (Lucanidae) and Polyphylla fullo (Scarabaeidae) belong to the order Coleoptera shows a chitin content of 10.9 and 11.3, respectively while it is reported as 18.2% to 25.2% for the same (Anoplotrupes stercorosus, Blaps tibialis, Cetonia aurata, Geotrupes stercorarius) [2], 15% for Agabus bipustulatus and Holotrichia paralella [30], 20% for Hydrophilus piceus and Leptinotarsa [11] 14% for Melolontha melolotha, [12]. Bradyporus (Callimenus) sureyai (Tettigonidae) and Gryllotalpa gryllotalpa (Gryllotalpidae) belongs to the order Orthoptera have a chitin content of 9.8% and 10.1%, respectively while it is 4.71%-11.84 for male and female grasshoppers (Celes variabilis, Decticus verrucivorus, Melanogryllus desertus, Paracyptera labiata) [18] and 5.3% to 8.3% (Ailopus simulatrix, Ailopus strepens, Duroniella fracta, Duroniella laticornis, Oedipoda miniata, Oedipoda caerulescens, Pyrgomorpha cognate) [17]. The results were relatively low when compared with some reported values which may be due to the environment that species grow as well as it may be due to the conditions during isolation of chitin. However, chitin contents of the orders in this study is close to the average value and these species are alternative sources for the chitin extraction for further processes.
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Journal Pre-proof 3.2. FT-IR analysis The isolated chitin samples from four different species were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) over a range of 4000-400 cm-1 (Figure 1). FT-IR bands of these chitin samples are summarized in Table 1. The observed bands in FT-IR spectra are all similar for four specimens with a small deviation that can be neglected. There are different crystalline forms of chitin which are α, β and γ forms in the nature [52]. The divided bands observed at about 1652-1654 cm-1 and 1619-1620 cm-1 for amide groups confirms α-form chitin for all species in this study [13, 23, 25, 53] that is attributed to hydrogen bonding of amide groups oriented in antiparallel confirmation [54]. Chitin from natural sources are mainly in α-form [55]. The α-form chitin for the order Coleoptera was reported in literature rather than other forms [11, 12, 15, 30, 56] as well as α-form chitin for the order Orthoptera was widely discussed in literature [15, 17, 18, 30, 56]. 1153-1154 cm-1 for oxygen connecting
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two N-acetylglucosamine rings and 1064-1066 cm-1 for C-O bonds in N-acetylglucosamine ring are characteristic to chitin species. FT-IR spectra analysis specifies that the method used for separation of chitin
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from other organic species was successful when the characteristic band for chitin at about 895-896 cm-1 which corresponds the stretching of the β-1,4-glycosidic linkage was observed [37]. Similar FTIR spectra were
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recorded by several authors in literature [13, 15, 23, 25, 30, 53]. The absence of the peak at 1540 cm-1 that is characteristic for protein confirms that isolation is protein free for all species [38] as well as confirmation of the
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3.3. Thermal analysis
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type of crystallinity as α-form demonstrates that the isolation of chitin from four specimens is successful.
In addition to spectroscopic characterization, thermal behaviour of four chitin samples were analysed (Figure 2). Four chitin samples follow similar thermal degradation that is composed of three steps which are based on the
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loss of moisture up to 1000C, then decomposition of main chitin structure with the highest rate and decomposition of residual species [57-59]. All chitin samples left ash even if at higher temperatures. The
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moisture content for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus were 5.2%, 6.0%, 5.9% and 6.6%, respectively. These moisture contents are similar to each other and follows the first degradation step up to 100 0C for each sample. Similar results were discussed in literature as varying between 2,7% to 9% [11, 12, 15, 17, 18, 20, 23, 26]. Four chitin samples are thermally stable for about 300 0C and after that temperature, main thermal decomposition of chitin samples just start up to 420 0C for B. (C.) sureyai, 4120C for G. gryllotalpa, 4150C for P. fullo and 4050C for L. cervus [18, 58, 60] similar with chitin extracted from C. barbarus and O. decorus [15]. Percent decomposition of B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus are 72%, 70%, 73% and 70% respectively at the end of the second decomposition stages that corresponds similar crystallinity for all isolated chitin samples. Similar degradation percent results were determined in literature for α-chitins from class Insecta and class Arachnida [2], from M. melolontha and O. asellus [12], from grasshopper species [17, 18]. Differential thermogravimetric data (DTG) for B. (C.) sureyai is the highest with a value of 382.40C following 379.90C for L. cervus, 374.70C for P. fullo and 374.60C for G. gryllotalpa. Similarly, DTG data in literature for alpha chitin are between 355°-395° [58, 52, 61, 62], as well as chitin extracted from class insecta and class arachnida [2], chitins extracted from shrimp, krill, crayfish and crab [57, 63, 64], chitins extracted from Agabus bipustulatus, Anax imperator, Asellusa aquaticus, Hydrophilus piceus, Notonecta glauca, Ranatra linearis [13], from M. melolontha and O. asellus [12], chitin from other sources [58, 62, 64]. The last step for thermal decomposition
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Journal Pre-proof differs chitin samples from each other, chitin from L. cervus decomposed up to 7480C with releasing ash as 0.6% as well as the same is 6140C with releasing ash as 1.7% for P. fullo. Chitin samples isolated from B. (C.) sureyai and G. gryllotalpa follows similar thermal decomposition at higher temperatures which are up to 671 0C with a release of ash for 3.8% and 6850C with releasing ash as 2.1%, respectively. These different behaviours for the third thermal decomposition step for chitin samples are because of the rearrangement of chemical bonds at higher temperatures. No further thermal decomposition were observed up to 800 0C for the third thermal decomposition stage.
3.4. SEM analysis Scanning Electron Microscope (SEM) analysis were recorded for surface morphology of isolated chitin samples. Chitin isolated from B. (C.) sureyai, G. gryllotalpa and P. fullo have a microfibrillar structure with large porosity
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(Figure 3), large porous surface is homogeneous for B. (C.) sureyai as well as it is regional for P. fullo and in addition to large ones, relatively small porosity was observed for G. gryllotalpa and P. fullo. Similar surface
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morphologies were recorded for α-chitins from C. marginatus, L. equestris, B. lapidaries and C. aenea [2]. The diameters of the large pores are about 10 μm for B. (C.) sureyai although there is a distribution for P. fullo
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between 4-5 μm and between 12-17 μm for G. gryllotalpa. The diameters of small pores for G. gryllotalpa and P. fullo changes from 200 nm to 400 nm. The surface of the G. gryllotalpa and P. fullo have both micropores
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and nanopores. The chitin isolated from L. cervus has different morphology than other isolated chitin samples, it has a complex microfibrillar structure with porosity similar with alpha chitin extracted from Duroniella fracta
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[17] that diameters ranged in a narrow size as 1-2 μm (Figure 3). Chitin isolated from adult Vespa crabro had micropores between diameter of 3-5 μm [26]. The diameter of the pores of the isolated chitin from M. Melontha
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and O. asellus were detected as 185-400 nm and 100-250 nm, respectively [12], 180-260 nm for the chitin extracted from male and female grasshopper species [18], It was reported that chitin extracted from O. caerulescens species only consist of fibrils without pores [17], some includes both fibrils and pores [65] and the
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others do not have fibrils and pores [68]. In this study, the isolated chitin from all specimens were both have fibrils and pores that changes according to the source and there is no any relation between the taxonomy and isolation procedure with the surface of the chitin [2]. The thickness of the fibrils for L. cervus was observed as relatively higher than other species from the images. Variation of wings, legs and antenna in the organisms also changes the surface of the chitin [26]. The surface morphology of chitin such as pore size and fibril characteristics are important and has to be known before application. It is not possible to discuss a standard surface morphology for α-chitin although they are from same genus [17].
3.5. Elemental analysis Elemental analysis results of four chitin samples are shown in table 2. During isolation of chitin from insects, proteins were completely removed and the assay elements are all constituents of the chitin. The carbon element percent in B. (C.) sureyai is relatively higher than the others as well as hydrogen and nitrogen elements percent are close to each other. The carbon element percents were determined as 46.6, 44.2, 45.4 and 45.9 for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus, respectively. The carbon atom percent of extracted α-chitin from species in literature was determined as 39.2-49.0% [18, 20, 62] in which the experimental results of the four species are acceptable for carbon content in this study. The percentage of nitrogen atom in chitin is one of the
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Journal Pre-proof most important parameter that corresponds to the purity of chitin. The nitrogen content for the pure chitin was determined to be 6.89% [30, 38, 62]. If nitrogen content of chitin is higher than 6.89%, protein structures are not removed completely from the structure as well as if it is lower, inorganic structures are still in the structure [30, 35, 62] which may be Ca, Mg, K, Al, P, S, Si and Fe elements as detected in a cicada sloughs specimens [62]. The nitrogen element percents were determined as 5.3, 5.0, 5.1 and 5.3 for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus, respectively in this study. It seems to be the inorganic structures were not completely removed from chitin during isolation process. N atom contents for M. melolontha and O. asellus were determined as 6.72% and 4.7%, respectively [12], chitin from shrimp as 4.85% and bumblebee as 5.92% [38]. The N atom content of the chitin isolated from four specimens in this study is comparable with literature data. The ratio of the carbon element to nitrogen element for each species changes from 8.8 to 8.5. Chitin naturally found with proteins, pigments and minerals. The presence of the impurities in extracted chitin changes the
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percent of atoms [66] that is due to analytical methods and purification processes [62]. The results confirmed
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that the chitin structure retained during isolation.
3.6. XRD analysis
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All species of chitin samples have similar X-ray diffractometry (XRD) which are two strong peaks approximately at 2θ values of 9.5° and 19.5° and five peaks with a low intensity at 2θ values of approximately
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12.5°, 23.8°, 26.2°, 28° and 39°. Different chitin samples from different sources were characterized by XRD [52] in which this form corresponds to α-form of chitin [13, 30, 67]. XRD data are given in figure 4 and Table 3 with
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crystallinity index (CrI) calculated from peak areas from recorded XRD. Crystallinity index of examined chitin species changes from 86.1 (P. fullo) to 80.6 (G. gryllotalpa) [13, 30, 67]. The crystallinity index for the
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orthopteran chitin (B. (C.) sureyai, G. gryllotalpa) is similar with commercial one (78%) [17]. The crystallinity index for the chitin extracted from specimens with hard cuticles are relatively higher [30]. The CI for the chitin extracted from shrimp and a beetle species were at about 89% [30], it is 76.2% for α-chitin from shrimp, 82.7%
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for lobster α-chitin [68]. The different crystallinity index of the chitins extracted from specimens in this study vary with the hardness of the cuticles. In this study, crystallinity index of P. fullo (86.1) and L. cervus (85.2) are relatively higher that is attributed to the hardness of the cuticle. TGA data confirms the crystallinity index of the chitin extracted from species in this study since thermal resistance of the chitin increase with crystallinity. This result is harmonious with results of Kaya et al. [2] in which, two Coleoptera species have higher crystallinity index values (Cetonia aurata with 86.3 and Anoplotrupes stercorosus with 83.5) than all other examined species. The peaks observed at about 2θ values of 26° and 28° are due to the inorganic structures that supports elemental analysis results in which there are low nitrogen atom percents in this study. The crystallinity index of isolated chitins from some organisms were detected as between 47% and 92% and that is attributed to the isolation method and the type of species [30, 37, 67]. The crystallinity index of the isolated chitins from specimens are similar when compared with earlier data discussed above. The low crystallinity of the extracted chitin was reported as a presence of catechol remaining during isolation [69]. The relatively high crystallinity index of the isolated chitins from organisms confirms that isolation of chitin from organic compounds is successful.
4. Conclusions
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Journal Pre-proof In this study, the chitin contents and the physicochemical comparison of four species belonging to two insect orders were determined for the first time. Bradyporus (Callimenus) sureyai (Tettigonidae) and Gryllotalpa gryllotalpa (Gryllotalpidae) belong to the order Orthoptera, while Lucanus cervus (Lucanidae) and Polyphylla fullo (Scarabaeidae) belong to the order Coleoptera. FT-IR analysis of these four isolated species confirmed that isolation of chitin from other organic species is successful and FT-IR spectra are same for these four specimens. TGA analysis recorded that the moisture content of four chitin specimens are similar with each other, and all of them have similar TGA degradation data up to medium temperatures. At high temperatures, L. cervus is the most stable specimen that decomposed thermally up to 748°C with 0.6% residue. This is different from the isolated chitin P. fullo in the same order Coleoptera that is 614°C with 1.7% residue. For chitin samples B. (C.) sureyai and G. gryllotalpa belong to the order Orthoptera have similar decomposition temperatures around 675°C. SEM images supports TGA data since B. (C.) sureyai, G. gryllotalpa and P. fullo specimens have microfibrillar
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structure with large numbers of porosity, L. cervus have microfibrillar structure with relatively lower numbers of porosity as well as the thickness of the fibrils for L. cervus was observed as relatively higher than other species
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from the images that increases the thermal stability. Elemental Analysis results concluded that the content of C, N and H atoms are similar for four isolated specimens. When N atom content of all species compared with
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literature, there are inorganic residues in the isolated chitin samples. XRD results showed that the crystallinity index for four specimens are higher than 80% for all compared species. The isolated chitin specimens are
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characterized as α-form from FT-IR spectra and XRD as well as the isolated chitins were from species with a hard cuticle. Examined species are large bodied and it makes them suitable for chitin extraction by using less
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specimens rather than other small bodied insects and arthropods. This situation could make them potential chitin sources without extinctions, if they could be reared in laboratory. After rearing, chitins of these species could be
potential further studies.
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used for producing chitosan, which can be tested for antimicrobial, antioxidant, antitumor researches etc. in
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Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
This study was presented as "Extraction and physicochemical properties of chitins from four different insect species" in 3rd SEAB2017: The International Symposium on Eurasian Biodiversity, Minsk, Belarus in 2017. Language and grammar control and editing of manuscript were made via Hacettepe University Technology Transfer Center.
Authors' Contributions Mahmut Kabalak conceived research, supplied insect samples. Yusuf Doruk Aracagök and Murat Torun conducted experiments. Mahmut Kabalak and Murat Torun evaluated results, wrote the manuscript, read and approved the manuscript.
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64. Abdou, E.S. Nagy, K.S.A. Elsabee, M.Z. Extraction and characterization of chitin and chitosan from local sources. Bioresour. Technol. 99 (2008) 1359-1367. 65. Mushi, N.E., Butchosa, N., Salajkova, M., Zhou, Q., Berglund, L.A. Nanostructured membranes based on native chitin nanofibers prepared by mild process Carbohydrate Polymers 112 (2014) 255-263. 66. Yen, M.T., Mau, J.L. P Physico-chemical characterization of fungal chitosan from shiitake stipes LWT-Food Science and Technology, 40(3) (2007) 472-479. 67. Ifuku, S. Nomura, R. Morimoto, M. Saimoto, H. Preparation of Chitin Nanofibers from Mushrooms. Materials. 4 (2011) 1417-1425. 68. Kumirska, J., , Weinhold, M.X., Thöming J., Stepnowski, P. Biomedical Activity of Chitin/Chitosan Based Materials— Influence of Physicochemical Properties Apart from Molecular Weight and Degree of NAcetylation. Polymers 3 (2011) 1875-1901. 69. Zhang, M., Haga, A., Sekiguchi, H. Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia International Journal of Biological Macromolecules 27 (2000) 99-105.
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Figure 1. Standardized FT-IR spectra of the chitin specimens isolated from Bradyporus (C.) sureyai,
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Gryllotalpa gryllotalpa, Polyphylla fullo, and Lucanus cervus.
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Table 1. Characteristic FT-IR bands for four different chitin samples. BS: Bradyporus (C.) sureyai, GG: Gryllotalpa gryllotalpa, LC: Lucanus cervus, PF: Polyphylla fullo. Wavenumber (cm-1) frequency
N-H stretching
GG
LC
PF
3432
3434
3435
3431
3258
3259
3257
3259
3101
3100
3101
3101
2924
2923
2922
2925
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CH3 and CH2 stretching
BS
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O-H stretching
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Vibration type
CH3 stretching
2877
2878
2875
2877
C=O amide stretching
1652
1651
1654
1654
1620
1620
1619
1620
N-H bend (amide)
1553
1552
1551
1553
CH2 bending
1420
1426
1424
1423
CH bending
1375
1375
1375
1375
CH2 wagging
1307
1307
1307
1307
NH bending
1259
1259
1259
1259
CH stretching
1202
1202
1202
1203
C-O-C stretching for bridge
1154
1154
1153
1154
1112
1112
1113
1113
C-O-C stretching in phase ring
1065
1064
1066
1066
C-O stretching in phase ring
1009
1009
1009
1009
oxygen N-acetylglucosamine in ring stretching mode
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952
952
952
CH ring stretching
895
896
895
895
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CH3 wagging
Figure 2. TGA/DTG curves of chitin specimens isolated from Bradyporus (C.) sureyai, Gryllotalpa gryllotalpa,
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Polyphylla fullo and Lucanus cervus.
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Figure 3. Scanning electron microscopy (SEM) images of chitin specimens isolated from a. Bradyporus (C.)
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sureyai, b. Gryllotalpa gryllotalpa, c. Polyphylla fullo, and d. Lucanus cervus.
Table 2. Elemental analysis results of the isolated chitin specimens.
C
H
N
C/N
46.6±0.1
7.7±0.1
5.3±0.1
8.8
Gryllotalpa gryllotalpa
44.2±0.1
7.6±0.1
5.0±0.1
8.8
Polyphylla fullo
45.4±0.1
7.5±0.1
5.1±0.1
8.9
Lucanus cervus
45.9±0.1
7.6±0.1
5.3±0.1
8.5
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Bradyporus (C.) sureyai
a
Content %
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Species
b
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c
d
Figure 4. X-ray diffractograms (XRD) of chitin isolated from a. Bradyporus (C.) sureyai, b. Gryllotalpa
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gryllotalpa, c. Polyphylla fullo, and d. Lucanus cervus.
XRD peaks at 2θ
Bradyporus (C.) sureyai
9.62, 12.5, 19.72, 23.74, 26.22, 27.8, 39.2
83.1
Gryllotalpa gryllotalpa
9.44, 12.3, 19.41, 23.31, 26.2, 27.9, 39.0
80.6
Polyphylla fullo
9.2, 12.4, 19.46, 23.50, 26.21, 28.1, 39.5
86.1
Lucanus cervus
9.67, 12.40, 19.60, 23.41, 26.26, 39.1
85.2
CrI (%)
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Species
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Table 3. X-ray diffraction data of isolated chitin specimens.
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Mahmut Kabalak, Doruk Aracagök, Murat Torun PII:
S0141-8130(19)40346-2
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.194
Reference:
BIOMAC 14222
To appear in:
International Journal of Biological Macromolecules
Received date:
16 December 2019
Accepted date:
21 December 2019
Please cite this article as: M. Kabalak, D. Aracagök and M. Torun, Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.194
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© 2018 Published by Elsevier.
Journal Pre-proof EXTRACTION, CHARACTERIZATION AND COMPARISON OF CHITINS FROM LARGE BODIED FOUR COLEOPTERA AND ORTHOPTERA SPECIES Mahmut Kabalak1*, Doruk Aracagök1, Murat Torun2 1 2
Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, Turkey.
Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe, Ankara, Turkey. E-mail adresses: [email protected], [email protected], [email protected] *
Corresponding author: [email protected]
ABSTRACT Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758)
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(Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae))
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for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%,
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10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614°C to 748°C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity
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index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales.
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Elemental analysis have been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD
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and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.
1. Introduction
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KEYWORDS: Coleoptera, Orthoptera, Biopolymer, Chitin, Extraction, Characterization
Chitin, a naturally abundant mucopolysaccharide, and the supporting material of crustaceans, insects, etc., is well known to consist of 2-acetamido-2-deoxy-(3-D-glucose) through a β-(1 4) linkage, which can be degraded by chitinase. Its immunogenicity is exceptionally low, in spite of the presence of nitrogen. It is a highly insoluble material resembling cellulose in its solubility and low chemical reactivity. It may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group. Like cellulose, it functions naturally as a structural polysaccharide [1]. Chitin and its main derivative chitosan have various applications in medicine, pharmacy, biotechnology, environmental, and food engineering because of their nontoxicity, biodegradability, biocompatibility, antimicrobial, and antioxidant properties [2]. There are many studies on extraction and determining physicochemical properties of chitin of different organisms (insects, myriapods, arachnids, molluscs, fungi, bryozoan, fish etc.) which were published by different authors [2 – 32]. According to those literature, it is understood that different organisms and even their different body parts having chitin composition with different physicochemical characteristics that also depends on isolation method and the source of chitin [2, 8, 10, 11, 17].
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Journal Pre-proof Some living organisms are evaluated as potential chitin supply which may be used for further processes, mainly conversion to water soluble chitosan for practical applications. Chitin was extracted from blue crabs, Callinectes sapidus, and converted to chitosan which was used for the synthesis of cryogel scaffolds which are potentials for tissue engineering after characterization of chitosan [33]. Antibacterial properties of chitosan from chitin extracted from the waste of Litopenaeus vannamei shrimps was also evaluated [34]. Physicochemical characterization of chitin from Parapenaeus longirostris shrimp shell waste and its chitosan derivative was examined as well [35]. Insects are alternative chitin sources for technology and further processes. There are researches in literature to produce and characterize chitin from different insect taxonomy. Chitin was extracted from housefly, Musca domestica, pupa shells and manufactured to chitosan. Percent yield of conversion, viscosity and degree of deacetylation of chitosan was discussed as well [36]. In another study, structure of chitin was discussed isolated
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from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia in which the form of the chitin was α-form. Its surface morphologies and cristallinity was dicussed and compared with commercial chitin from shrimp [37].
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The isolation of chitin from insect (Melolontha melolontha) was examined and its physicochemical properties was compared with isolated crustacean species from Oniscus asellus [12]. The antimicrobial and antioxidant
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activities of chitin and chitosan from Cosmopolitan Orthoptera Species (insecta) was examined well on Calliptamus barbarus and Oedaleus decorus species as well as physicochemical behaviors of the species was
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discussed in details [15]. In another study, chitin was extracted from the Beetle Holotrichia parallela motschulsky for analysis of chemical structures and physicochemical properties and authors suggest Holotrichia
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parallela is an alternative source of chitin [30], other studies follow the same for chitin from cicada sloughs [38] and from bumblebee (Bombus terrestris) [34]. The effect of the taxonomic relations on physicochemical
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properties of chitin was discussed for 16 arthropod species as a chitin source [2] and the chitin structures from seven orthoptera species was compared [17]. The change of the physicochemical properties of chitin was examined for Vespa crabro (wasp) at larvae, pupa and adult stages during development [26]. It seems to be
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insects are promising alternative sources for chitin extraction in the use of daily life however the physicochemical properties should be evaluated to use the chitin or its derivatives in a proper section.
In this study, the chitin contents of 4 species belonging to 2 orders were determined for the first time. Bradyporus (Callimenus) sureyai Ünal, 2011 belongs to the genus Bradyporus Charpentier, 1825 (Orthoptera: Tettigonidae) which is known as the Armoured Crickets or Glandular Bush-crickets. They can be easily recognized from a distance by their loud calling songs heard usually in the afternoon and early evening. Their most characteristic behaviour is the ejaculation of haemolymph from just under the posterior margin of the metanotum for defensive purposes [39]. Gryllotalpa gryllotalpa (Linnaeus, 1758) is called as “Mole crickets”, which is a member of the family of Gryllotalpidae are distributed throughout temperate and tropical regions. These insects are best known for their digging forelimbs and singing from specialized burrows in the soil [40-42]. European mole cricket spent nearly all their lives underground. They damage vegetable gardens, seedling beds, eat seed, cereals, potato and almost all vegetables. Shoots and young plants often perish after damage [43-47].
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Journal Pre-proof Polyphylla fullo (Linnaeus, 1758) is a major polyphagous agricultural pest because the larvae feed on the roots of many important cultivated plants. It has a 2- to 3-year life cycle with three larval stages that feed on plant roots causing extensive damage, and, in severe infestations, can cause yield loss and plant death [48]. Lucanus cervus (Linnaeus, 1758) (stag beetle) (Coleoptera: Scarabaeidae) is often cited as an indicator of ancient oak forest with ancient trees and large dimensions of dead wood [49]. The larva lives on underground woody debris, mostly of oak [50].
Main objects of this study are extractions of chitins of four species, characterizations of extracted chitins by several methods and comparisons physicochemical behaviours of chitins of examined species for the first time. Chitins are used in many areas, the crystallinity, surface morphology and thermal behaviours are important parameters that determines the subject chitin to be used. In this study, properties of chitins from large bodied
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four coleoptera and orthoptera species were discussed after several characterization methods and these species
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are suggested as alternative chitin sources for various areas according to the observed results.
2. Materials and methods
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2.1. Specimens and materials
Specimens were taken from dry unlabelled insect collection from Hacettepe University Faculty of Science,
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Department of Biology Applied Entomology Laboratory. Hydrochloric acid, sodium hydroxide, chloroform, ethyl acetate and methyl alcohol were all supplied from Sigma-Aldrich which were used for isolation of chitin
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2.2. Specimen preparation
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from species. All reagents were analytical grade and used without further purification.
Specimens present in collection were killed by using ethyl acetate and dried at room temperature up to a constant
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weight then powdered by using liquid nitrogen in the mortar.
2.3. Chitin isolation procedure
In order to demineralize chitin, all samples were treated in 1M HCl solution at 95°C by stirring under reflux for 1 hour. Then, biomasses were separated through filtration with filter paper. Filtrates were washed with distilled water for several times after pH testing. After that samples were treated in 1M NaOH solution at 90°C for 14 hours to remove proteins under reflux. Solutions were filtered again and were washed with distilled for several times water after pH testing. The extracts were left in chloroform-methanol-water (1:2:4, v: v) mixture at room temperature for 1 hour to remove lipids and pigments in the third step, after separation by filtration, the extracts were washed with distilled water for several times. Finally chitin extracts were left to dry at room temperature for 5 days up to a constant weight to determine the chitin percentages and they were stored for further processes.
2.4. Infrared (FT-IR) analysis All chitin samples were characterized spectroscopically by Nicolet IS10 FTIR spectrophotometer (Thermo Scientific) in ATR mode. The resolution of spectra was adjusted as 4 cm-1 with a scanning number of 64. All spectra were recorded between 4000-400 cm-1 wavenumber region and OMNIC program was used for detailed analysis.
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2.5. Thermogravimetric analysis (TGA) TG and DTG curves of chitin samples were recorded with EXSTAR S11 7300 model instrument at a heating rate of 10°C min-1 from 25°C to 800°C under an inert atmosphere by N 2.
2.6. Elemental analysis TruSpec Micro CHNS-O analyzer (Leco Instruments, Stockport, UK) was used to determine the percent composition of carbon, hydrogen and nitrogen elements in isolated chitin samples.
2.7. Scanning electron microscope (SEM) analysis The surface morphologies of extracted chitin samples from four specimens were monitored with Zeiss EVO 50
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model scanning electron microscope. All samples were coated with gold by the sputter coater before image
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analysis.
2.8. X-ray diffraction (XRD) analysis
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X-ray diffraction of powder chitin samples were examined by Rigaku D/MAX 2200 PC model system with tube voltage of 40 kV and current of 40 mA with a scanning speed of 2°/min from 2° to 50° 2θ value. The
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crystallinity index of isolated chitin samples (CrI) were calculated from XRD data by following equation [51]: 𝐼0 − 𝐼𝑎𝑚 𝑥100% 𝐼𝑜
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𝐶𝑟𝐼 =
amorphous region.
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3. Results and discussion
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where I0 is the maximum intensity at 2θ=20° and Iam is the maximum intensity at 2θ=13° corresponding
3.1. Chitin content of examined species at dry basis Chitin contents of the species varied from 9.8% to 11.3% that shows a narrow distribution for samples. Lucanus cervus (Lucanidae) and Polyphylla fullo (Scarabaeidae) belong to the order Coleoptera shows a chitin content of 10.9 and 11.3, respectively while it is reported as 18.2% to 25.2% for the same (Anoplotrupes stercorosus, Blaps tibialis, Cetonia aurata, Geotrupes stercorarius) [2], 15% for Agabus bipustulatus and Holotrichia paralella [30], 20% for Hydrophilus piceus and Leptinotarsa [11] 14% for Melolontha melolotha, [12]. Bradyporus (Callimenus) sureyai (Tettigonidae) and Gryllotalpa gryllotalpa (Gryllotalpidae) belongs to the order Orthoptera have a chitin content of 9.8% and 10.1%, respectively while it is 4.71%-11.84 for male and female grasshoppers (Celes variabilis, Decticus verrucivorus, Melanogryllus desertus, Paracyptera labiata) [18] and 5.3% to 8.3% (Ailopus simulatrix, Ailopus strepens, Duroniella fracta, Duroniella laticornis, Oedipoda miniata, Oedipoda caerulescens, Pyrgomorpha cognate) [17]. The results were relatively low when compared with some reported values which may be due to the environment that species grow as well as it may be due to the conditions during isolation of chitin. However, chitin contents of the orders in this study is close to the average value and these species are alternative sources for the chitin extraction for further processes.
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Journal Pre-proof 3.2. FT-IR analysis The isolated chitin samples from four different species were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) over a range of 4000-400 cm-1 (Figure 1). FT-IR bands of these chitin samples are summarized in Table 1. The observed bands in FT-IR spectra are all similar for four specimens with a small deviation that can be neglected. There are different crystalline forms of chitin which are α, β and γ forms in the nature [52]. The divided bands observed at about 1652-1654 cm-1 and 1619-1620 cm-1 for amide groups confirms α-form chitin for all species in this study [13, 23, 25, 53] that is attributed to hydrogen bonding of amide groups oriented in antiparallel confirmation [54]. Chitin from natural sources are mainly in α-form [55]. The α-form chitin for the order Coleoptera was reported in literature rather than other forms [11, 12, 15, 30, 56] as well as α-form chitin for the order Orthoptera was widely discussed in literature [15, 17, 18, 30, 56]. 1153-1154 cm-1 for oxygen connecting
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two N-acetylglucosamine rings and 1064-1066 cm-1 for C-O bonds in N-acetylglucosamine ring are characteristic to chitin species. FT-IR spectra analysis specifies that the method used for separation of chitin
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from other organic species was successful when the characteristic band for chitin at about 895-896 cm-1 which corresponds the stretching of the β-1,4-glycosidic linkage was observed [37]. Similar FTIR spectra were
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recorded by several authors in literature [13, 15, 23, 25, 30, 53]. The absence of the peak at 1540 cm-1 that is characteristic for protein confirms that isolation is protein free for all species [38] as well as confirmation of the
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3.3. Thermal analysis
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type of crystallinity as α-form demonstrates that the isolation of chitin from four specimens is successful.
In addition to spectroscopic characterization, thermal behaviour of four chitin samples were analysed (Figure 2). Four chitin samples follow similar thermal degradation that is composed of three steps which are based on the
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loss of moisture up to 1000C, then decomposition of main chitin structure with the highest rate and decomposition of residual species [57-59]. All chitin samples left ash even if at higher temperatures. The
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moisture content for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus were 5.2%, 6.0%, 5.9% and 6.6%, respectively. These moisture contents are similar to each other and follows the first degradation step up to 100 0C for each sample. Similar results were discussed in literature as varying between 2,7% to 9% [11, 12, 15, 17, 18, 20, 23, 26]. Four chitin samples are thermally stable for about 300 0C and after that temperature, main thermal decomposition of chitin samples just start up to 420 0C for B. (C.) sureyai, 4120C for G. gryllotalpa, 4150C for P. fullo and 4050C for L. cervus [18, 58, 60] similar with chitin extracted from C. barbarus and O. decorus [15]. Percent decomposition of B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus are 72%, 70%, 73% and 70% respectively at the end of the second decomposition stages that corresponds similar crystallinity for all isolated chitin samples. Similar degradation percent results were determined in literature for α-chitins from class Insecta and class Arachnida [2], from M. melolontha and O. asellus [12], from grasshopper species [17, 18]. Differential thermogravimetric data (DTG) for B. (C.) sureyai is the highest with a value of 382.40C following 379.90C for L. cervus, 374.70C for P. fullo and 374.60C for G. gryllotalpa. Similarly, DTG data in literature for alpha chitin are between 355°-395° [58, 52, 61, 62], as well as chitin extracted from class insecta and class arachnida [2], chitins extracted from shrimp, krill, crayfish and crab [57, 63, 64], chitins extracted from Agabus bipustulatus, Anax imperator, Asellusa aquaticus, Hydrophilus piceus, Notonecta glauca, Ranatra linearis [13], from M. melolontha and O. asellus [12], chitin from other sources [58, 62, 64]. The last step for thermal decomposition
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Journal Pre-proof differs chitin samples from each other, chitin from L. cervus decomposed up to 7480C with releasing ash as 0.6% as well as the same is 6140C with releasing ash as 1.7% for P. fullo. Chitin samples isolated from B. (C.) sureyai and G. gryllotalpa follows similar thermal decomposition at higher temperatures which are up to 671 0C with a release of ash for 3.8% and 6850C with releasing ash as 2.1%, respectively. These different behaviours for the third thermal decomposition step for chitin samples are because of the rearrangement of chemical bonds at higher temperatures. No further thermal decomposition were observed up to 800 0C for the third thermal decomposition stage.
3.4. SEM analysis Scanning Electron Microscope (SEM) analysis were recorded for surface morphology of isolated chitin samples. Chitin isolated from B. (C.) sureyai, G. gryllotalpa and P. fullo have a microfibrillar structure with large porosity
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(Figure 3), large porous surface is homogeneous for B. (C.) sureyai as well as it is regional for P. fullo and in addition to large ones, relatively small porosity was observed for G. gryllotalpa and P. fullo. Similar surface
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morphologies were recorded for α-chitins from C. marginatus, L. equestris, B. lapidaries and C. aenea [2]. The diameters of the large pores are about 10 μm for B. (C.) sureyai although there is a distribution for P. fullo
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between 4-5 μm and between 12-17 μm for G. gryllotalpa. The diameters of small pores for G. gryllotalpa and P. fullo changes from 200 nm to 400 nm. The surface of the G. gryllotalpa and P. fullo have both micropores
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and nanopores. The chitin isolated from L. cervus has different morphology than other isolated chitin samples, it has a complex microfibrillar structure with porosity similar with alpha chitin extracted from Duroniella fracta
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[17] that diameters ranged in a narrow size as 1-2 μm (Figure 3). Chitin isolated from adult Vespa crabro had micropores between diameter of 3-5 μm [26]. The diameter of the pores of the isolated chitin from M. Melontha
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and O. asellus were detected as 185-400 nm and 100-250 nm, respectively [12], 180-260 nm for the chitin extracted from male and female grasshopper species [18], It was reported that chitin extracted from O. caerulescens species only consist of fibrils without pores [17], some includes both fibrils and pores [65] and the
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others do not have fibrils and pores [68]. In this study, the isolated chitin from all specimens were both have fibrils and pores that changes according to the source and there is no any relation between the taxonomy and isolation procedure with the surface of the chitin [2]. The thickness of the fibrils for L. cervus was observed as relatively higher than other species from the images. Variation of wings, legs and antenna in the organisms also changes the surface of the chitin [26]. The surface morphology of chitin such as pore size and fibril characteristics are important and has to be known before application. It is not possible to discuss a standard surface morphology for α-chitin although they are from same genus [17].
3.5. Elemental analysis Elemental analysis results of four chitin samples are shown in table 2. During isolation of chitin from insects, proteins were completely removed and the assay elements are all constituents of the chitin. The carbon element percent in B. (C.) sureyai is relatively higher than the others as well as hydrogen and nitrogen elements percent are close to each other. The carbon element percents were determined as 46.6, 44.2, 45.4 and 45.9 for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus, respectively. The carbon atom percent of extracted α-chitin from species in literature was determined as 39.2-49.0% [18, 20, 62] in which the experimental results of the four species are acceptable for carbon content in this study. The percentage of nitrogen atom in chitin is one of the
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Journal Pre-proof most important parameter that corresponds to the purity of chitin. The nitrogen content for the pure chitin was determined to be 6.89% [30, 38, 62]. If nitrogen content of chitin is higher than 6.89%, protein structures are not removed completely from the structure as well as if it is lower, inorganic structures are still in the structure [30, 35, 62] which may be Ca, Mg, K, Al, P, S, Si and Fe elements as detected in a cicada sloughs specimens [62]. The nitrogen element percents were determined as 5.3, 5.0, 5.1 and 5.3 for B. (C.) sureyai, G. gryllotalpa, P. fullo and L. cervus, respectively in this study. It seems to be the inorganic structures were not completely removed from chitin during isolation process. N atom contents for M. melolontha and O. asellus were determined as 6.72% and 4.7%, respectively [12], chitin from shrimp as 4.85% and bumblebee as 5.92% [38]. The N atom content of the chitin isolated from four specimens in this study is comparable with literature data. The ratio of the carbon element to nitrogen element for each species changes from 8.8 to 8.5. Chitin naturally found with proteins, pigments and minerals. The presence of the impurities in extracted chitin changes the
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percent of atoms [66] that is due to analytical methods and purification processes [62]. The results confirmed
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that the chitin structure retained during isolation.
3.6. XRD analysis
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All species of chitin samples have similar X-ray diffractometry (XRD) which are two strong peaks approximately at 2θ values of 9.5° and 19.5° and five peaks with a low intensity at 2θ values of approximately
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12.5°, 23.8°, 26.2°, 28° and 39°. Different chitin samples from different sources were characterized by XRD [52] in which this form corresponds to α-form of chitin [13, 30, 67]. XRD data are given in figure 4 and Table 3 with
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crystallinity index (CrI) calculated from peak areas from recorded XRD. Crystallinity index of examined chitin species changes from 86.1 (P. fullo) to 80.6 (G. gryllotalpa) [13, 30, 67]. The crystallinity index for the
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orthopteran chitin (B. (C.) sureyai, G. gryllotalpa) is similar with commercial one (78%) [17]. The crystallinity index for the chitin extracted from specimens with hard cuticles are relatively higher [30]. The CI for the chitin extracted from shrimp and a beetle species were at about 89% [30], it is 76.2% for α-chitin from shrimp, 82.7%
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for lobster α-chitin [68]. The different crystallinity index of the chitins extracted from specimens in this study vary with the hardness of the cuticles. In this study, crystallinity index of P. fullo (86.1) and L. cervus (85.2) are relatively higher that is attributed to the hardness of the cuticle. TGA data confirms the crystallinity index of the chitin extracted from species in this study since thermal resistance of the chitin increase with crystallinity. This result is harmonious with results of Kaya et al. [2] in which, two Coleoptera species have higher crystallinity index values (Cetonia aurata with 86.3 and Anoplotrupes stercorosus with 83.5) than all other examined species. The peaks observed at about 2θ values of 26° and 28° are due to the inorganic structures that supports elemental analysis results in which there are low nitrogen atom percents in this study. The crystallinity index of isolated chitins from some organisms were detected as between 47% and 92% and that is attributed to the isolation method and the type of species [30, 37, 67]. The crystallinity index of the isolated chitins from specimens are similar when compared with earlier data discussed above. The low crystallinity of the extracted chitin was reported as a presence of catechol remaining during isolation [69]. The relatively high crystallinity index of the isolated chitins from organisms confirms that isolation of chitin from organic compounds is successful.
4. Conclusions
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Journal Pre-proof In this study, the chitin contents and the physicochemical comparison of four species belonging to two insect orders were determined for the first time. Bradyporus (Callimenus) sureyai (Tettigonidae) and Gryllotalpa gryllotalpa (Gryllotalpidae) belong to the order Orthoptera, while Lucanus cervus (Lucanidae) and Polyphylla fullo (Scarabaeidae) belong to the order Coleoptera. FT-IR analysis of these four isolated species confirmed that isolation of chitin from other organic species is successful and FT-IR spectra are same for these four specimens. TGA analysis recorded that the moisture content of four chitin specimens are similar with each other, and all of them have similar TGA degradation data up to medium temperatures. At high temperatures, L. cervus is the most stable specimen that decomposed thermally up to 748°C with 0.6% residue. This is different from the isolated chitin P. fullo in the same order Coleoptera that is 614°C with 1.7% residue. For chitin samples B. (C.) sureyai and G. gryllotalpa belong to the order Orthoptera have similar decomposition temperatures around 675°C. SEM images supports TGA data since B. (C.) sureyai, G. gryllotalpa and P. fullo specimens have microfibrillar
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structure with large numbers of porosity, L. cervus have microfibrillar structure with relatively lower numbers of porosity as well as the thickness of the fibrils for L. cervus was observed as relatively higher than other species
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from the images that increases the thermal stability. Elemental Analysis results concluded that the content of C, N and H atoms are similar for four isolated specimens. When N atom content of all species compared with
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literature, there are inorganic residues in the isolated chitin samples. XRD results showed that the crystallinity index for four specimens are higher than 80% for all compared species. The isolated chitin specimens are
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characterized as α-form from FT-IR spectra and XRD as well as the isolated chitins were from species with a hard cuticle. Examined species are large bodied and it makes them suitable for chitin extraction by using less
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specimens rather than other small bodied insects and arthropods. This situation could make them potential chitin sources without extinctions, if they could be reared in laboratory. After rearing, chitins of these species could be
potential further studies.
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used for producing chitosan, which can be tested for antimicrobial, antioxidant, antitumor researches etc. in
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Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
This study was presented as "Extraction and physicochemical properties of chitins from four different insect species" in 3rd SEAB2017: The International Symposium on Eurasian Biodiversity, Minsk, Belarus in 2017. Language and grammar control and editing of manuscript were made via Hacettepe University Technology Transfer Center.
Authors' Contributions Mahmut Kabalak conceived research, supplied insect samples. Yusuf Doruk Aracagök and Murat Torun conducted experiments. Mahmut Kabalak and Murat Torun evaluated results, wrote the manuscript, read and approved the manuscript.
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Figure 1. Standardized FT-IR spectra of the chitin specimens isolated from Bradyporus (C.) sureyai,
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Gryllotalpa gryllotalpa, Polyphylla fullo, and Lucanus cervus.
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Table 1. Characteristic FT-IR bands for four different chitin samples. BS: Bradyporus (C.) sureyai, GG: Gryllotalpa gryllotalpa, LC: Lucanus cervus, PF: Polyphylla fullo. Wavenumber (cm-1) frequency
N-H stretching
GG
LC
PF
3432
3434
3435
3431
3258
3259
3257
3259
3101
3100
3101
3101
2924
2923
2922
2925
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CH3 and CH2 stretching
BS
na
O-H stretching
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Vibration type
CH3 stretching
2877
2878
2875
2877
C=O amide stretching
1652
1651
1654
1654
1620
1620
1619
1620
N-H bend (amide)
1553
1552
1551
1553
CH2 bending
1420
1426
1424
1423
CH bending
1375
1375
1375
1375
CH2 wagging
1307
1307
1307
1307
NH bending
1259
1259
1259
1259
CH stretching
1202
1202
1202
1203
C-O-C stretching for bridge
1154
1154
1153
1154
1112
1112
1113
1113
C-O-C stretching in phase ring
1065
1064
1066
1066
C-O stretching in phase ring
1009
1009
1009
1009
oxygen N-acetylglucosamine in ring stretching mode
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952
952
952
CH ring stretching
895
896
895
895
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CH3 wagging
Figure 2. TGA/DTG curves of chitin specimens isolated from Bradyporus (C.) sureyai, Gryllotalpa gryllotalpa,
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Polyphylla fullo and Lucanus cervus.
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Figure 3. Scanning electron microscopy (SEM) images of chitin specimens isolated from a. Bradyporus (C.)
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sureyai, b. Gryllotalpa gryllotalpa, c. Polyphylla fullo, and d. Lucanus cervus.
Table 2. Elemental analysis results of the isolated chitin specimens.
C
H
N
C/N
46.6±0.1
7.7±0.1
5.3±0.1
8.8
Gryllotalpa gryllotalpa
44.2±0.1
7.6±0.1
5.0±0.1
8.8
Polyphylla fullo
45.4±0.1
7.5±0.1
5.1±0.1
8.9
Lucanus cervus
45.9±0.1
7.6±0.1
5.3±0.1
8.5
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Bradyporus (C.) sureyai
a
Content %
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Species
b
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c
d
Figure 4. X-ray diffractograms (XRD) of chitin isolated from a. Bradyporus (C.) sureyai, b. Gryllotalpa
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gryllotalpa, c. Polyphylla fullo, and d. Lucanus cervus.
XRD peaks at 2θ
Bradyporus (C.) sureyai
9.62, 12.5, 19.72, 23.74, 26.22, 27.8, 39.2
83.1
Gryllotalpa gryllotalpa
9.44, 12.3, 19.41, 23.31, 26.2, 27.9, 39.0
80.6
Polyphylla fullo
9.2, 12.4, 19.46, 23.50, 26.21, 28.1, 39.5
86.1
Lucanus cervus
9.67, 12.40, 19.60, 23.41, 26.26, 39.1
85.2
CrI (%)
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Species
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Table 3. X-ray diffraction data of isolated chitin specimens.
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