Multi-Scale Structural Analysis and Hierarchical Discrimination of Natural Chitin from Diverse Biological Sources
DOI:
https://doi.org/10.48048/tis.2026.12776Keywords:
Chitin, Comprehensive structural characterization, Hierarchical organization, SR-FTIR spectroscopy, Multivariate analysis, Principal component analysis, Marine biomaterials, Insect biomaterials, Chitin, Comprehensive structural characterization, Hierarchical organization, SR-FTIR spectroscopy, Multivariate analysis, Principal component analysis, Marine biomaterials, Insect biomaterialsAbstract
Chitin, one of the most abundant sustainable biopolymers in nature, exhibits source-dependent structural variations that significantly influence its functional properties and applications. This study presents a multi-scale morphological analysis combined with hierarchical multivariate discrimination to comprehensively characterize chitin extracted from 5 diverse biological sources: marine mollusks (cockle, mussel), marine crustacean (shrimp), and terrestrial insects (cricket, silkworm pupa). Chitin was extracted through sequential deproteinization, neutralization, and demineralization processes. Scanning electron microscopy at 25,000× magnification revealed distinct morphological characteristics across all sources. Marine mollusk chitin exhibited highly organized fibrous (cockle) and compact plate-like (mussel) structures, while shrimp demonstrated extensive porous networks. Multi-scale analysis (5,000× - 25,000×) of insect chitin revealed a remarkable hierarchical organization with a consistent sheet-like (cricket) and wrinkled (silkworm pupa) morphologies maintained across all magnification levels, distinguishing them from marine sources. Extraction yields varied significantly (4.09% - 15.07%), with marine sources (shrimp, cockle, mussel) achieving the highest yields, correlating with their high-density structures. Degree of acetylation ranged from 68.20 ± 1.00% (cricket) to 83.39 ± 1.26% (shrimp) to 89.15 ± 1.52% (silkworm pupa), while true protein content ranged from 4.50 ± 0.34% (shrimp) to 8.19 ± 0.31% (cockle). Synchrotron Radiation-coupled Fourier-transform infrared (SR-FTIR) spectroscopy combined with hierarchical multivariate analysis (PCA) successfully discriminated all sources. Comprehensive PCA (73% variance) separated marine mollusks from other sources, but shrimp, cricket, and silkworm pupa remained overlapping. Focused PCA performed specifically on these 3 non-marine sources (71% variance) successfully resolved their subtle spectral differences. These findings establish structure-property relationships across multiple scales, providing practical guidance for selecting chitin sources for biomaterial applications. The multi-scale approach reveals that hierarchical organization in insect chitin influences extraction efficiency and molecular properties.
HIGHLIGHTS
- Integrated multi-scale characterization (SEM, synchrotron-FTIR, PCA) provides comprehensive assessment of source-dependent chitin hierarchical organization beyond single-method approaches
- Distinct hierarchical features in insect chitin compared to marine sources, affecting extraction efficiency and molecular composition
- Non-destructive chitin source identification and property prediction using synchrotron-FTIR with PCA discrimination
- Targeted selection of chitin for specific biomaterial and bioengineering applications based on source-dependent structural features
- Comprehensive multi-scale comparison of chitin from diverse biological sources as baseline data for chitin standardization
GRAPHICAL ABSTRACT
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AK Azad, T Bhattacharya, MS Hasnain, G Tripathi and AK Nayak. Chitin- and chitosan-based nanomaterials for therapeutic applications. In: MS Hasnain, AK Nayak and TM Aminabhavi (Eds.). Polymeric nanosystems. Academic Press, Oxford, England, 2023, p. 173-205.
D Elieh-Ali-Komi and MR Hamblin. Chitin and chitosan: Production and application of versatile biomedical nanomaterials. International Journal of Advanced Research 2016; 4, 411-427.
M Kumi, T Wang, O Ejeromedoghene, J Wang, P Li and W Huang. Exploring the potentials of chitin and chitosan-based bioinks for 3D-printing of flexible electronics: The future of sustainable bioelectronics. Small Methods 2024; 8, 2301341.
R Ridwanto, VD Rosa, Z Rani and ZPA Fauzi. Utilization of chitosan from fresh water lobster (Cherax quadricarinatus) shells in anti-acne gel preparations. Trends in Sciences 2024; 21(2), 7243.
RAF Jasim. Medical, pharmaceutical, and biomedical applications of chitosan: A review. Medical Journal of Babylon 2021; 18(4), 291-294.
Chitin derivatives market overview, Available at: https://www.industryresearch.biz/market-reports/chitin-derivatives-market-100764, accessed November 2025.
KY Zhu, H Merzendorfer, W Zhang, J Zhang and S Muthukrishnan. Biosynthesis, turnover, and functions of chitin in insects. Annual Review of Entomology 2016; 61, 177-196.
B Moussian. Chitin: Structure, chemistry and biology. Advances in Experimental Medicine and Biology 2019; 1142, 5-18.
I Younes and M Rinaudo. Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs 2015; 13, 1133-1174.
T Hahn, A Roth, R Ji, E Schmitt and S Zibek. Current state of chitin purification and chitosan production from insects. Journal of Chemical Technology & Biotechnology 2020; 95(11), 2775-2795.
RA Muzzarelli. Chitin nanostructure in living organisms. In: N Gupta (Ed.). Chitin: Formation and diagenesis. Springer, Dordrecht, Netherlands, 2011, p. 1-34.
NAZ Abidin, F Kormin, NAZ Abidin, NAFM Anuar and MFA Bakar. The potential of insects as alternative sources of chitin: An overview on the chemical method of extraction from various sources. International Journal of Molecular Sciences 2020; 21, 4978.
RL Lavall, OBG Assis and SP Campana-Filho. β-chitin from the pens of Loligo sp.: Extraction and characterization. Bioresource Technology 2007; 98(13), 2465-2472.
Lalarukh, SM Hussain, S Ali, AF Zahoor, H Azmat, N Nazish, MA Alshehri, D Riaz, E Naeem and Mahrukh. (2024). Innovation of advanced polymers from seafood waste: Applications of chitin and chitosan. Polymers for Advanced Technologies 2024; 35(6), e6471.
M Triunfo, E Tafi, A Guarnieri, V Longo, R Russo, R Carotenuto, D Montesano, R Di Sanzo, S Moliterni, D Coppola, M Gallo, TS Tiuman, M Francavilla and P Di Pierro. Characterization of chitin and chitosan derived from Hermetia illucens, a further step in a circular economy process. Scientific Reports 2022; 12, 6613.
M Chalghaf, K Charradi, R Ksouri, QA Alsulami, A Jaouani, SMAS Keshk and EA Hayouni. Physicochemical characterization of chitin extracted by different treatment sequences from an edible insect. International Journal of Biological Macromolecules 2023; 253(6), 127156.
AI Zamri, NF Latiff, QH Abdullah and F Ahmad. Extraction and optimization of chitosan from razor clam (Ensis arcuatus) shells by using response surface methodology (RSM). Food Research 2020; 4, 674-678.
R Varma and S Vasudevan. Extraction, characterization, and antimicrobial activity of chitosan from horse mussel Modiolus modiolus. ACS Omega 2020; 5(32), 20224-20230.
BA Alimi, S Pathania, J Wilson, B Duffy and JMC Frias. Extraction, quantification, characterization, and application in food packaging of chitin and chitosan from mushrooms: A review. International Journal of Biological Macromolecules 2023; 237, 124195.
C Yang, X Chen, Z Chen, Y Zhao, R Yang, Y Xia, Q Zeng, Y He and H Lan. Source-dependent variations in chitin: a comparative study on Antarctic krill, white shrimp and crayfish. Frontiers in Marine Science 2025; 12, 1592331.
P Stempflé, X Bourrat, O Pantalé, RK Njiwa, J Jehl, A Domatti and E Lopez. Multiscale structure of nacre biomaterial: Thermomechanical behavior and wear processes. Materials Science & Engineering C 2018; 91, 78-93.
W Lin, P Liu, S Li, J Tian, W Cai, X Zhang, J Peng, C Miao, H Zhang, P Gu, Z Wang, Z Zhang and T Luo. Multi-scale design of the chela of the hermit crab Coenobita brevimanus. Acta Biomaterialia 2021; 127, 229-241.
Z Mei, P Kuzhir and G Godeau. Update on chitin and chitosan from insects: Sources, production, characterization, and biomedical applications. Biomimetics 2024, 9, 297.
M Wang, X Lu, X Yin, Y Tong, W Peng, L Wu, H Li, Y Yang, J Gu, T Xiao, M Chen and J Zhang. Synchrotron radiation-based Fourier-transform infrared spectromicroscopy for characterization of the protein/peptide distribution in single microspheres. Acta Pharmaceutica Sinica B 2015; 5(3), 270-276.
Y Guo, T Chen, S Wang, X Zhou, H Zhang, D Li, N Mu, M Tang, M Hu, D Tang, Z Yang, J Zhong, Y Tang, H Feng, X Zhang and H Wang. Synchrotron radiation-based FTIR microspectroscopic imaging of traumatically injured mouse brain tissue slices. ACS Omega 2020; 5, 29698-29705.
MA Islam, MN Hasan, MSH Evan, MJ Uddin, WS Tulin, MS Islam, MU Khandaker, IMM Rahman and FI Chowdhury. Chitin nanofibers: Recent advances in preparation and applications in biomedical and beyond. Royal Society of Chemistry 2025, 15, 14655-14690
M Salavati. Mechanical properties of α-chitin and chitosan biocomposite: A molecular dynamic study. Journal of Composites Science 2023; 7, 464.
P Baharlouei and A Rahman, Chitin and chitosan: Prospective biomedical applications in drug delivery, cancer treatment, and wound healing. Marine Drugs 2022, 20, 460.
Y Kim, Z Zharkinbekov, K Raziyeva, L Tabyldiyeva, K Berikova, D Zhumagul, K Temirkhanova and A Saparov. Chitosan-based biomaterials for tissue regeneration. Pharmaceutics 2023, 15, 807.
ZM Zia. Experiment on the chemical extraction of chitin and chitosan from the exoskeletons of indigenous prawn species and their biomedical application. International Journal of Zoology and Applied Biosciences 2019; 4(3), 106-112.
AOAC International. Protein (crude) in seafood, Kjeldahl method (method 984.13). In: Official methods of analysis of AOAC International. 20th ed. AOAC International, Rockville, United States, 2016.
A Baxter, M Dillon, KDA Taylor and GAF Roberts. Improved method for i.r. determination of the degree of N-acetylation of chitosan. International Journal of Biological Macromolecules 1992; 14(3), 166-169.
S Muthukrishnan, S Mun, MY Noh, ER Geisbrecht and Y Arakane. Insect cuticular chitin contributes to form and function. Current Pharmaceutical Design 2020; 26(29), 3530-3545.
VBS Chan, MB Johnstone, AP Wheeler and AS Mount. Chitin facilitated mineralization in the eastern oyster. Frontiers in Marine Science 2018; 5, 347.
L Addadi, D Joester, F Nudelman and S Weiner. Mollusk shell formation: A source of new concepts for understanding biomineralization processes. Chemistry - A European Journal 2006; 12(4), 980-987.
H Izadi, H Asadi and M Bemani. Chitin: A comparison between its main sources. Frontiers in Materials 2025; 12, 1537067.
LD Tolesa, BS Gupta and MJ Lee. Chitin and chitosan production from shrimp shells using ammonium-based ionic liquids. International Journal of Biological Macromolecules 2019; 130, 818-826.
N Sayari, A Sila, BE Abdelmalek, RB Abdallah, S Ellouz-Chaabouni, A Bougatef and R Balti. Chitin and chitosan from the Norway lobster by-products: Antimicrobial and anti-proliferative activities. International Journal of Biological Macromolecules 2016; 87, 163-171.
M Agarwal, MK Agarwal, N Shrivastav, S Pandey and PA Gaur. A simple and effective method for preparation of chitosan from chitin. International Journal of Life Sciences Research 2018; 4, 1721-1728.
M Jones, M Kujundzic, S John and A Bismarck. Crab vs. Mushroom: A review of crustacean and fungal chitin in wound treatment. Marine Drugs 2020; 18, 64.
MAS Siddiqui, MS Rabbi, RU Ahmed, F Alam, MAM Hossain, S Ahsan and NM Miah. Bioinspired composite structures: A comprehensive review of natural materials, fabrication methods, and engineering applications. Composites Part C: Open Access 2025; 17, 100578.
SO Andersen. Insect cuticular sclerotization: A review. Insect Biochemistry and Molecular Biology 2010; 40(3), 166-178.
MM Abo Elsoud and EM El Kady. Current trends in fungal biosynthesis of chitin and chitosan. Bulletin of the National Research Centre 2019; 43, 59.
X Hu, Z Tian, X Li, S Wang, H Pei, H Sun and Z Zhang. Green, simple, and effective process for the comprehensive utilization of shrimp shell waste. ACS Omega 2020; 5, 19227-19235.
J Kumirska, M Czerwicka, Z Kaczyński, A Bychowska, K Brzozowski, J Thöming and P Stepnowski. Application of spectroscopic methods for structural analysis of chitin and chitosan. Marine Drugs 2010; 8, 1567-1636.
S Hasan, VM Boddu, DS Viswanath and TK Ghosh. Chitin and Chitosan. Springer, Cham, Switzerland, 2022, p. 79-102.
Y Ogawa, CM Lee, Y Nishiyama and SH Kim. Absence of sum frequency generation in support of orthorhombic symmetry of α chitin. Macromolecules 2016; 49, 7025-7031.
J Nie, X Wu, J Wei, Y Li, H Xie, W Wang, H Wang, H Ping, B Li and Z Fu. Regulating growth of strontium carbonate in self-assembled chiral chitin matrices with robust mechanical properties. ACS Applied Materials & Interfaces 2025; 17, 25849-25860.
R Seoudi and AMA Nada. Molecular structure and dielectric properties studies of chitin and its treated by acid, base and hypochlorite. Carbohydrate Polymers 2007; 68, 728-733.
L Ogresta, F Nekvapil, T Tamas, L Barbu-Tudoran, M Suciu, R Hirian, M Aluas, G Lazar, E Levei, B Glamuzina and SC Pinzaru. Rapid and application-tailored assessment tool for biogenic powders from crustacean shell waste: Fourier transform-infrared spectroscopy complemented with x ray diffraction, scanning electron microscopy, and nuclear magnetic resonance spectroscopy. Omega 2021; 6, 27773-27780.
KM Kocot, F Aguilera, C McDougall, DJ Jackson and BM Degnan. Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization. Frontiers in Zoology 2016; 13, 23.
X Zhang, J Yuan, F Li and J Xiang. Chitin Synthesis and degradation in crustaceans: A genomic view and application. Marine Drugs 2021; 19, 153.
MH Younus, GG Ali and HA Salih. The reinforced optical fiber sensing with bilayer AuNPs/SiC for pressure measurement: Characterization and optimization. Journal of Physics: Conference Series 2021; 1795, 012002.
GG Ali, AZ Khalil and OA Alhamd. Growth and characterization of cadmium and nickel nanocomposites on porous silicon for some antibacterial properties. Trends in Sciences 2025; 22(8), 10054.
OA Alhamd, GG Ali and MSH Aljuboori. Study and characterization of copper and titanium oxides nanostructures for some molecular and biological applications. Trends in Sciences 2024; 21(4), 7402.
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