POLYMORPHIC CHARACTERIZATION OF CHITIN EXTRACTED FROM SELECTED INSECT SPECIES

ABSTRACT

There are numerous natural sources of chitin worldwide, with crustacean shells—harvested primarily for their meat—being the most common. However, the physicochemical properties of extracted chitin vary significantly depending on the type of raw material used. In this study, chitin substances isolated from insects native to the Fergana Valley—Melolontha melolontha, Leptinotarsa decemlineata, and Eurygaster integriceps—are examined as alternative sources of chitin. The polymorphic forms of the extracted chitin are characterized using infrared (IR) spectroscopy based on the vibrational frequencies of the amide I group, and X-ray diffraction (XRD) analysis. The height and width of the diffraction peaks are utilized to determine the degree of crystallinity and support the identification of specific polymorphic structures.

АННОТАЦИЯ

Во всём мире существует множество природных источников хитина, наиболее распространённым из которых являются панцири ракообразных, собираемые в основном ради мяса. Однако физико-химические свойства извлечённого хитина значительно варьируются в зависимости от используемого сырья. В данном исследовании рассматриваются хитинсодержащие вещества, выделенные из насекомых, обитающих в Ферганской долине — Melolontha melolontha, Leptinotarsa decemlineata и Eurygaster integriceps, — в качестве альтернативных источников хитина. Полиморфные формы извлечённого хитина охарактеризованы с использованием инфракрасной (ИК) спектроскопии на основе колебательных частот амидной группы I, а также методом рентгеновской дифракции (XRD). Высота и ширина дифракционных пиков используются для определения степени кристалличности и подтверждения идентификации конкретных полиморфных структур.

Keywords: insect, raw material, infrared spectrum, X-ray phase diffractogram, chitin, chitosan.

Ключевые слова: насекомое, сырье, инфракрасный спектр, рентгеновская фазовая дифрактограмма, хитин, хитозан.

Introduction

Chitin has three primary natural sources: the exoskeletons of crustaceans, the cuticle of insects, and the cell walls of filamentous fungi. It serves as a structural material in many animal species and primarily performs a supportive function. Chemically, chitin is a β-1,4-linked homopolymer of N-acetylglucosamine [1].

Figure 1. Structural formula of chitin

Three crystalline forms of naturally occurring chitin have been identified: α-, β, and γ-chitin. Among them, α-chitin is the most stable and widely distributed form, characterized by antiparallel alignment of its monomeric chains. This configuration facilitates strong intermolecular hydrogen bonding. In contrast, β-chitin consists of parallel chains, resulting in weaker intermolecular interactions and, consequently, lower stability compared to α-chitin. γ-chitin is relatively rare in nature and is considered a hybrid structure that contains both parallel and antiparallel arrangements of α- and β-chitin [2].

Figure 2. Polymorphic configurations of chitin

According to the literature, X-ray diffraction patterns of α-chitin extracted from various raw sources such as crab, shrimp, gammarus, krill, honeybee, and other insects typically exhibit sharp peaks at 9º and 19º, along with additional, less intense peaks at 12º, 23º, and 26º. For β-chitin, the diffractograms show broader and lower-intensity peaks around 9.1º and 20.3º. In the case of γ-chitin, two distinct and intense peaks are observed at 9.6º and 19.8º in the X-ray diffractogram [3,4].

Materials and methods

To extract chitin, insect species commonly found in the Fergana Valley with high reproductive capacity, relatively large body size, and thick exoskeletons were selected. These included the May beetle (Melolontha melolontha), the Colorado potato beetle (Leptinotarsa decemlineata), and the sunn pest (Eurygaster integriceps).

Initially, the collected raw materials were dried and ground into powder. The following sequential processing steps were carried out:

Defatting using ethyl acetate.

Demineralization with diluted HCl solution.

Deproteinization using a mild NaOH solution.

Depigmentation with 3% hydrogen peroxide (H₂O₂).

To characterize the structure of the obtained chitin samples, various physicochemical analysis methods were employed, including:

Infrared (IR) spectroscopy – conducted in powder form using the INVENIO S (BRUKER, 2021; ATR range: 4000–400 cm⁻¹).

X-ray diffraction (XRD) – structural analysis was performed to assess the crystallinity of chitin, chitosan, and synthesized N-acyl derivatives of chitosan using an XRD-MiniFlex-600 diffractometer (Rigaku, Japan, 2021). Analysis conditions: CuKα₁–α₂ radiation (Kα₁: Kα₂ ratio 50%), wavelength λ = 0.15406 nm, 40 kV voltage, 30 mA current, scan range 0º–70º, scan rate 2°/min, and step size 0.05°.

Results and discussions

The infrared (IR) spectra of chitin samples extracted by the classical method from Melolontha melolontha, Leptinotarsa decemlineata, and Eurygaster integriceps are presented in Figures 3 to 5.

Figure 3. IR spectrum of chitin extracted from Leptinotarsa decemlineata

Figure 4. IR spectrum of chitin extracted from Melolontha melolontha

Figure 5. IR spectrum of chitin extracted from Eurygaster integriceps

In the IR spectrum shown in Figure 3, the presence of a single, undivided absorption band at 1654 cm⁻¹, characteristic of the amide (I) group, indicates that the chitin extracted from Leptinotarsa decemlineata is in a crystalline form, corresponding to the β-chitin polymorphic structure.

In Figure 4, the IR spectrum of chitin from Melolontha melolontha exhibits a partially split amide (I) peak, which suggests that the sample corresponds to the γ-chitin polymorphic form.

Meanwhile, the IR spectrum in Figure 5 of the chitin extracted from Eurygaster integriceps displays two fully resolved absorption bands at 1656 cm⁻¹ and 1623 cm⁻¹, associated with the amide (I) group. This indicates that the sample exhibits a structure typical of α-chitin.

In the diffractogram of chitin extracted from Leptinotarsa decemlineata (Figure 6), two intense peaks are observed at 9.8º and 19.9º, which are characteristic of the β-chitin polymorphic form. The diffractogram of chitin from Melolontha melolontha (Figure 7) displays sharp peaks at 9.4º and 19.34º, along with weaker peaks at 12.2º, 23.43º, and 27.2º, indicating the presence of γ-chitin. Meanwhile, the diffractogram of chitin obtained from Eurygaster integriceps (Figure 8) shows distinct and sharp peaks at 9.1º and 19º, which are typical for α-chitin.

Figure 6. XRD diffractogram of chitin extracted from Leptinotarsa decemlineata

Figure 7. XRD diffractogram of chitin extracted from Melolontha melolontha

Figure 8. XRD diffractogram of chitin extracted from Eurygaster integriceps

The presence of sharp and well-defined peaks in the diffractogram indicates a high degree of crystallinity in the material, as well as the existence of strong intermolecular and intramolecular hydrogen bonding.

Conclusion

In this study, chitin samples extracted using a classical method from several insect species commonly found in the Fergana Valley were analyzed through their IR spectra and X-ray diffraction (XRD) patterns. Based on the presence or absence of splitting in the characteristic vibration frequencies of the amide (I) group in the IR spectra, as well as the height and width of peaks in the diffractograms, it was determined that the chitin from Melolontha melolontha corresponds to the γ-polymorph, that from Leptinotarsa decemlineata to the β-polymorph, and that from Eurygaster integriceps to the α-polymorphic form. Deacetylation of the obtained chitin increases its amorphous nature, resulting in the formation of chitosan, which can be effectively used as a sorbent.

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