Page Header Logo Applied Science and Engineering Progress

Home > ONLINE FIRST > Nugroho

Extraction of Chitosan from Black Soldier Fly Larvae Exuviae for Film Material and Its In Vivo Wound Healing Potential

Rudy Agung Nugroho, Retno Aryani, Hadi Kuncoro, Aura Nur Kholifah, Raihatul Jannah, Rudianto Rudianto, Muhammad Vieraldi

Abstract


Chitosan, a biocompatible polysaccharide, is typically sourced from crustaceans, but sustainable alternatives, such as the exuviae of black soldier fly (Hermetia illucens) larvae (BSFL), offer environmentally benign possibilities for biomedical applications. This study aimed to extract and analyze chitosan from BSFL exuviae and to assess its efficacy in accelerating wound healing in a rat model, highlighting the potential of utilizing insect-derived chitosan for in vivo wound repair. Chitosan was derived via deproteination, demineralization, and deacetylation, yielding 36.41–47.55% chitin and 33.97–48.66% chitosan, with a deacetylation degree of 70–80%. Scanning Electron Microscope (SEM) revealed the presence of porous nanofibrous structures. Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the presence of functional groups, specifically N-H/O-H stretches, within the range 3245–3253 cm−1. Films composed of pectin, carboxymethyl cellulose, and chitosan were prepared. In an in vivo wound healing assay, Wistar rats (n = 20) with 10 mm excisional wounds were divided into four treatment groups, including a negative control, a positive control using Bioplacenton, and two groups treated with films containing shrimp or BSFL-chitosan. The BSFL-chitosan group exhibited accelerated wound closure (2.62 ± 0.80 mm on day 4 compared to 7.17 ± 0.93 mm in the negative control; p-value < 0.05). In the wound tissue after 4 days, the protein concentration increased (1648 ± 0.48 μg/mg), and the DNA concentration also rose (4.31 ± 0.02 μg/mg), whereas the hydroxyproline content decreased (0.90 ± 0.05 μg/mL). The chitosan film derived from BSFL is a promising sustainable biomaterial for improved wound healing.

Keywords



[1] G. A. Varshan and S. K. R. Namasivayam, “A green chemistry principle for the biotransformation of fungal biomass derived chitosan into versatile nano scale materials with high biocompatibility and potential biological activities—A review,” BioNanoScience, vol. 14, no. 1, pp. 4145–4166, 2024, doi: 10.1007/s12668-024-01564-0.

[2] S. Gul, Y. Karahan, O. B. Kurtur, and Y. Budama-Kilinc, Cellulose, Chitin, and Chitosan Composite-Based Sustainable Biomaterials, in Sustainable Green Biomaterials. Berlin, Germany: Springer, 2025, pp. 317–342, doi: 10.1007/978-3-031-79062-1_13.

[3] A. K. Maurya, “Sustainable food packaging: A brief review of chitosan-based materials,” International Journal of Science and Engineering Science Research, vol. 1, no. 1, pp. 1–9, 2025, doi: 10.5281/zenodo.15731364.

[4] A. Li et al., “Chitosan-based injectable hydrogel with multifunction for wound healing: A critical review,” Carbohydrate Polymers, vol. 333, 2024, Art. no. 121952, doi: 10.1016/j.carbpol.2024.121952.

[5] S. M. Mawazi et al., “Recent applications of chitosan and its derivatives in antibacterial, anticancer, wound healing, and tissue engineering fields,” Polymers, vol. 16, no. 10, pp. 1–34, 2024, doi: 10.3390/polym16101351.

[6] M. Nasaj et al., “Factors influencing the antimicrobial mechanism of chitosan action and its derivatives: A review,” International Journal of Biological Macromolecules, vol. 277, no. 2, pp. 1–13, 2024, doi: 10.1016/j.ijbiomac.2024.134321.

[7] R. Cassano et al., “Chitosan hemostatic dressings: Properties and surgical applications,” Polymers, vol. 16, no. 13, pp. 1–11, 2024, doi: 10.3390/polym16131770.

[8] A. Haider et al., “Chitosan as a tool for tissue engineering and rehabilitation: Recent developments and future perspectives–A review,” International Journal of Biological Macromolecules, vol. 278, no. 1, 2024, Art. no. 134172, doi: 10.1016/j.ijbiomac.2024.134172.

[9] A. Ewais et al., “Fully deacetylated chitosan from shrimp and crab using minimum heat input,” Egyptian Journal of Chemistry, vol. 66, no. 2, pp. 321–337, 2023, doi: 10.21608/ejchem.2022.136694.6020.

[10] B. T. Iber et al., “A review of various sources of chitin and chitosan in nature,” Journal of Renewable Materials, vol. 10, no. 4, pp. 1–27, 2022, doi: 10.32604/JRM.2022.018142.

[11] B.-Q. Yuan et al., “Physical and chemical characterization of chitin and chitosan extracted under different treatments from black soldier fly,” International Journal of Biological Macromolecules, vol. 279, no. 2, pp. 1–12, 2024, doi: 10.1016/j.ijbiomac.2024.135228.

[12] H. González-Lara et al., “Black soldier fly culture as a source of chitin and chitosan for its potential use in concrete: An overview,” Polymers, vol. 17, no. 6, pp. 1–15, 2025, doi: 10.3390/polym17060717.

[13] R. A. Nugroho et al., “Nutritive value, material reduction, biomass conversion rate, and survival of black solider fly larvae reared on palm kernel meal supplemented with fish pellets and fructose,” International Journal of Tropical Insect Science, vol. 43, no. 4, pp. 1243–1254, 2023, doi: 10.1007/s42690-023-01032-4.

[14] R. A. Nugroho et al., “Bioconversion of biowaste by black soldier fly larvae (Hermetia illucens L.) for dried larvae production: A life cycle assessment and environmental impact analysis,” F1000Research, vol. 12, no. 1, pp. 1–9, 2023, doi: 10.12688/f1000research.132371.1.

[15] A. Santoso et al., “Sustainability index analysis of the black soldier fly (Hermetia illucens) cultivation from food waste substrate,” Global Journal of Environmental Science and Management, vol. 9, no. 4, pp. 851-870, 2023, doi: 10.22035/gjesm.2023.04.

[16] B. Fabian et al., “Transformations of head structures during the larval development of the black soldier fly Hermetia illucens (Stratiomyidae, Diptera),” Journal of Morphology, vol. 286, no. 4, pp. 1–15, 2025.

[17] V. J. Kemboi et al., “Biocontrol potential of chitin and chitosan extracted from black soldier fly pupal exuviae against bacterial wilt of tomato,” Microorganisms, vol. 10, no. 1, pp. 1–13, 2022, doi: 10.3390/microorganisms10010165.

[18] E. Mirwandhono et al., “Isolation and characterization of chitosan from black soldier fly exuviae,” IOP Conference Series: Earth and Environmental Science, vol. 1362, no. 1, pp. 1–6, 2024.

[19] R. D. Sharma and P. Thangadurai, “Chitin from Hermetia illucens exuviae: A sustainable biopolymer yielding composition-defined functional oligomers with antioxidant cytoprotection,” International Journal of Biological Macromolecules, vol. 3, no. 1, pp. 1–13, 2025, doi: 10.1016/j.ijbiomac.2025.149453.

[20] E. Fricke, R. Saborowski, and M. J. Slater, “Utility of by‐products of black soldier fly larvae (Hermetia illucens) production as feed ingredients for Pacific Whiteleg shrimp (Litopenaeus vannamei),” Journal of the World Aquaculture Society, vol. 55, no. 4, pp. 1–16, 2024, doi: 10.1111/jwas.13070.

[21] A. Mannucci et al., “From food waste to functional biopolymers: Characterization of chitin and chitosan produced from prepupae of black soldier fly reared with different food waste-based diets,” Foods, vol. 13, no. 2, pp. 1–14, 2024, doi: 10.3390/foods13020278.

[22] A. Riofrio and H. Baykara., “Environmental impact of chitosan production from black soldier flies using life cycle assessment,” Materials Science Forum, vol. 1108, no. 1, pp. 163–171, 2023, doi: 10.4028/fp-10p7DY.

[23] H. Teo et al., “Antibacterial properties of chitosan isolated from the black soldier fly, Hermetia illucens,” Sains Malaysiana, vol. 5, no. 12, pp. 3923–3935, 2022, doi: 10.17576/jsm-2022-5112-05.

[24] M. A. Cisternas and K. Varaprasad, Chitosan-based Biomaterials for Hemostasis and Wound Healing. Berlin, Heidelberg: Springer, 2025, pp. 367–401, doi: 10.1007/12_2024_179.

[25] V. Singh et al., “Silk fibroin hydrogel: A novel biopolymer for sustained release of vancomycin drug for diabetic wound healing,” Journal of Molecular Structure, vol. 1286, no. 1, 2023, Art. no. 135548, doi: 10.1016/j.molstruc.2023.135548.

[26] D. A. Sandakila et al., “Ethanolic extract of black soldier fly (Hermetia illucens L.) larvae for wound healing in mice (Mus musculus L.),” Aceh Journal of Animal Science, vol. 9, no. 3, pp. 119-127, 2024, doi: 10.13170/ajas.9.3.42024.

[27] F. Akbari et al., “In vitro and in vivo wound healing activity of Astragalus floccosus Boiss.(Fabaceae),” Advances in Pharmacological and Pharmaceutical Sciences, vol. 2022, no. 1, pp. 1–15, 2022, doi: 10.1155/2022/7865015.

[28] H. Yao et al., “Fabrication and performance evaluation of gelatin/sodium alginate hydrogel-based macrophage and MSC cell-encapsulated paracrine system with potential application in wound healing,” International Journal of Molecular Sciences, vol. 24, no. 2, pp. 1–15, 2023, doi: 10.3390/ijms24021240.

[29] N. I. Ibrahim et al., “The effects of aqueous extract of Labisia pumila (Blume) Fern.-Vill. Var. Alata on wound contraction, hydroxyproline content and histological assessments in superficial partial thickness of second-degree burn model,” Frontiers in Pharmacology, vol. 13, no. 1, pp. 1–15, 2022, doi: 10.3389/fphar.2022.968664.

[30] S. R. Tobat et al., “Wound healing activity of phyllanthin-rich sub-fractions ointment: Isolated from meniran (Phyllanthus niruri L.) Leaf in experimental rats using hydroxyproline as biochemical marker,” Tropical Journal of Natural Product Research, vol. 8, no. 7, pp. 1–15, 2024, doi: 10.26538/tjnpr/v8i7.15.

[31] L. Natrayan et al., “Optimization process of potassium carbonate activated carbon through jute-based core materials by using artificial neural network with response surface methodology,” Adsorption Science & Technology, vol. 2023, no. 1, pp. 1–14, 2023, doi: 10.1155/2023/8674382.

[32] N. Pragadish et al., “A short review on AWJM of natural fibre reinforced composite materials,” Applied Mechanics and Materials, vol. 912, no. 1, pp. 123–139, 2023, doi: 10.4028/p-bya4d7.

[33] P. Chankachang et al., “Preparation and properties of chitosan/gelatin film containing capsaicinoid for hemostasis and antibacterial,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 694, no. 1, pp. 1–11, 2024, doi: 10.1016/j.colsurfa.2024.134078.

[34] C. N. Elangwe et al., “A review on chitosan and cellulose hydrogels for wound dressings,” Polymers, vol. 14, no. 23, pp. 1–17, 2022, doi: 10.3390/polym14235163.

[35] L. Elviri et al., “Controlled local drug delivery strategies from chitosan hydrogels for wound healing,” Expert opinion on drug delivery, vol. 14, no. 7, pp. 897–908, 2017, doi: 10.1080/17425247.2017.1247803.

[36] J. Shah et al., “Recent advancements in chitosan-based biomaterials for wound healing,” Journal of Functional Biomaterials, vol. 16, no. 2, pp. 1–18, 2025, doi: 10.3390/jfb16020045.

[37] M. K. Lagat et al., “Antimicrobial activity of chemically and biologically treated chitosan prepared from black soldier fly (Hermetia illucens) pupal shell waste,” Microorganisms, vol. 9, no. 12, pp. 1–15, 2021, doi: 10.3390/microorganisms9122417.

[38] D. I. Sánchez-Machado et al., “Measurement of the degree of deacetylation in chitosan films by FTIR, 1H NMR and UV spectrophotometry,” MethodsX, vol. 12, no. 1, pp. 1–7, 2024, doi: 10.1016/j.mex.2024.102583.

[39] H. Akram et al., “Low solvothermal synthesis and characterization of hollow nanospheres molybdenum sulfide,” Journal of Nanoscience and Nanotechnology, vol. 12, no. 8, pp. 6679–6685, 2012, doi: 10.1166/jnn.2012.4561.

[40] N. Akram et al., “Fabrication and in vitro biological assay of thermo-mechanically tuned chitosan reinforced polyurethane composites,” Molecules, vol. 28, no. 20, pp. 1–21, 2023, doi: 10.3390/molecules28207218.

[41] W. M. Abdeltwab et al., “Antimicrobial effect of chitosan and nano-chitosan against some pathogens and spoilage microorganisms,” Journal of Advanced Laboratory Research in Biology, vol. 10, no. 1, pp. 8–15, 2019, doi:

[42] M. Chandrasekaran, K. D. Kim, and S. C. Chun, “Antibacterial activity of chitosan nanoparticles: A review,” Processes, vol. 8, no. 9, pp. 1–21, 2020, doi: 10.3390/pr8091173.

[43] T. Dai et al., “Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects,” Expert review of anti-infective therapy, vol. 9, no. 7, pp. 857–879, 2011, doi: 10.1586/eri.11.59.

[44] R. Jayakumar et al., “Biomaterials based on chitin and chitosan in wound dressing applications,” Biotechnology advances, vol. 29, no. 3, pp. 322–337, 2011, doi: 10.1016/j.biotechadv.2011.01.005.

[45] A. Waśko et al., “The first report of the physicochemical structure of chitin isolated from Hermetia illucens,” International Journal of Biological Macromolecules, vol. 92, no. 1, pp. 316–320, 2016, doi: 10.1016/j.ijbiomac.2016.07.038.

[46] J. Wasko et al., “Conjugates of chitosan and calcium alginate with oligoproline and oligohydroxyproline derivatives for potential use in regenerative medicine,” Materials, vol. 13, no. 14, pp. 1–28, 2020, doi: 10.1016/j.ijbiomac.2016.07.038.

[47] H. El Knidri et al., “Eco-friendly extraction and characterization of chitin and chitosan from the shrimp shell waste via microwave irradiation,” Process Safety and Environmental Protection, vol. 104, no. 1, pp. 395–405, 2016, doi: 10.1016/j.psep.2016.09.020.

[48] N. G. Kandile et al., “Extraction and characterization of chitosan from shrimp shells,” Open Journal of Organic Polymer Materials, vol. 8, no. 3, pp. 33–42, 2018, doi: 10.4236/ojopm.2018.83003.

[49] A. J. da Silva Lucas et al., “Extraction, physicochemical characterization, and morphological properties of chitin and chitosan from cuticles of edible insects,” Food chemistry, vol. 343, no. 1, pp. 1–11, 2021, doi: 10.1016/j.foodchem.2020.128550.

[50] K. Mohan et al., “Recent insights into the extraction, characterization, and bioactivities of chitin and chitosan from insects,” Trends in Food Science and Technology, vol. 105, no. 1, pp. 17–42, 2020, doi: 10.1016/j.tifs.2020.08.016.

[51] Z. Sheng et al., “Preparation, physicochemical properties and antimicrobial activity of chitosan from fly pupae,” Heliyon, vol. 8, no. 10, pp. 1–8, 2022, doi: 10.1016/j.heliyon.2022.e11168 External Link.

[52] M. Aider, “Chitosan application for active bio-based films production and potential in the food industry,” LWT-Food Science and Technology, vol. 43, no. 6, pp. 837–842, 2010, doi: 10.1016/j.lwt.2010.01.021.

[53] K. u. Rehman et al., “Insect-derived chitin and chitosan: A still unexploited resource for the edible insect sector,” Sustainability, vol. 15, no. 6, pp. 1–34, 2023, doi: 10.3390/su15064864.

[54] P. Feng et al., “Chitosan-based functional materials for skin wound repair: Mechanisms and applications,” Frontiers in Bioengineering and Biotechnology, vol. 9, no. 1, pp. 1–15, 2021, doi: 10.3389/fbioe.2021.650598.

[55] D. S. R. Rajkumar, K. Keerthika, and V. Vijayaragavan, “Chitosan-based biomaterial in wound healing: A review,” Cureus, vol. 16, no. 2, pp. 1–18, 2024, doi: 10.7759/cureus.55193.

[56] A. Ehterami et al., “In vitro and in vivo study of PCL/COLL wound dressing loaded with insulin-chitosan nanoparticles on cutaneous wound healing in rats model,” International Journal of Biological Macromolecules, vol. 117, no. 1, pp. 601–609, 2018, doi: 10.1016/j.ijbiomac.2018.05.184.

[57] J. Natarajan et al., “Nanostructured lipid carriers of pioglitazone loaded collagen/chitosan composite scaffold for diabetic wound healing,” Advances in Wound Care, vol. 8, no. 10, pp. 499–513, 2019, doi: 10.1089/wound.2018.083.

[58] S. Dhivya, V. V. Padma, and E. Santhini, “Wound dressings–a review,” BioMedicine, vol. 5, no. 4, pp. 24–28, 2015, doi: 10.7603/s40681-015-0022-9.

[59] J. Boateng and O. Catanzano, “Advanced therapeutic dressings for effective wound healing—A review,” Journal of Pharmaceutical Sciences, vol. 104, no. 11, pp. 3653–3680, 2015, doi: 10.1002/jps.24610.

[60] G. Rivero, D. M. D. P. Pereira, C. Pablo, and G. A. Abraham, Nanofibrous scaffolds for skin tissue engineering and wound healing applications, in Tissue engineering using ceramics and polymers. Amsterdam, Netherlands: Elsevier, 2022, pp. 645–681, doi: 10.1016/B978-0-12-820508-2.00020-9

[61] M. A. Matica et al., “Chitosan as a wound dressing starting material: Antimicrobial properties and mode of action,” International journal of molecular sciences, vol. 20, no. 23, pp. 1–33, 2019, doi: 10.3390/ijms20235889.

[62] J. M. Budinčić et al., “Exuviae of Tenebrio molitor larvae as a source of chitosan: Characterisation and possible applications,” Applied Sciences, vol. 15, no. 17, pp. 1–25, 2025, doi: 10.3390/app15179285.

[63] J. R. B. Witono et al., “Strategic advances in efficient chitin extraction from black soldier fly puparia: Uncovering the potential for direct chitosan production,” Polysaccharides, vol. 6, no. 2, pp. 1–17, 2025, doi: 10.3390/polysaccharides6020026.

[64] M. Giani et al., “Hermetia illucens-derived chitosan as a promising sustainable biomaterial for wound healing applications: development of sponge-like scaffolds,” International Journal of Biological Macromolecules, vol. 304, no. 2, pp. 1–14, 2025, doi: 10.1016/j.ijbiomac.2025.140903.

[65] M. Triunfo et al., “A comprehensive characterization of Hermetia illucens derived chitosan produced through homogeneous deacetylation,” International Journal of Biological Macromolecules, vol. 271, no. 1, pp. 1–13, 2024, doi: 10.1016/j.ijbiomac.2024.132669.

[66] E. Mirwandhono et al., “Isolation and characterization of chitosan from black soldier fly exuviae,” IOP Conference Series: Earth and Environmental Science, vol. 1362, no. 1, pp. 1–6, 2024, doi: 10.1088/1755-1315/1362/1/012013.

[67] L. Queiroz et al., “Physico-chemical and colloidal properties of protein extracted from black soldier fly (Hermetia illucens) larvae,” International Journal of Biological Macromolecules, vol. 186, no. 1, pp. 714–723, 2021, doi: 10.1016/j.ijbiomac.2021.07.081.

[68] S. A. Siddiqui et al., “The potential of chitin and chitosan from dead black soldier fly (BSF) (Hermetia illucens) for biodegradable packaging material – A critical review,” Process Safety and Environmental Protection, vol. 189, no. 1, pp. 1342–1367, 2024, doi: 10.1016/j.psep.2024.06.108.

[69] A. Marangon et al., “Chitin extracted from black soldier fly larvae at different growth stages,” Polymers (Basel), vol. 16, no. 20, pp. 1–12, 2024, doi: 10.3390/polym16202861.

[70] D. Purkayastha and S. Sarkar, “Physicochemical structure analysis of chitin extracted from pupa exuviae and dead imago of wild black soldier fly (Hermetia illucens),” Journal of Polymers and the Environment, vol. 28, no. 2, pp. 445–457, 2020, doi: 10.1007/s10924-019-01620-x.

[71] E. Mirwandhono et al., “Isolation and characterization of chitosan from black soldier fly exuviae,” IOP Conference Series: Earth and Environmental Science, vol. 1362, no. 1, pp. 1–6, 2024, doi: 10.1088/1755-1315/1362/1/012013.

[72] M. Triunfo et al., “Characterization of chitin and chitosan derived from Hermetia illucens, a further step in a circular economy process,” Scientific Reports, vol. 12, no. 1, pp. 1–17, 2022, doi: 10.1038/s41598-022-10423-5.

[73] D. Archana, J. Dutta, and P. Dutta, “Evaluation of chitosan nano dressing for wound healing: Characterization, in vitro and in vivo studies,” International Journal of Biological Macromolecules, vol. 57, no. 1, pp. 193–203, 2013, doi: 10.1016/j.ijbiomac.2013.03.002.

[74] H. Bai et al., “Regulation of inflammatory microenvironment using a self-healing hydrogel loaded with BM-MSCs for advanced wound healing in rat diabetic foot ulcers,” Journal of Tissue Engineering, vol. 11, no. 1, pp. 1–13, 2020, doi: 10.1177/2041731420947242.

[75] C. Viezzer et al., “A new waterborne chitosan-based polyurethane hydrogel as a vehicle to transplant bone marrow mesenchymal cells improved wound healing of ulcers in a diabetic rat model,” Carbohydrate Polymers, vol. 231, no. 1, pp. 1–10, 2020, doi: 10.1016/j.carbpol.2019.115734.

[76] M. S. Al-saggaf, “Formulation of insect chitosan stabilized silver nanoparticles with propolis extract as potent antimicrobial and wound healing composites,” International Journal of Polymer Science, vol. 2021, no. 1, pp. 1–9, 2021, doi: 10.1155/2021/5578032.

[77] C. S. Liew et al., “Chitin and chitosan production from black soldier fly larvae (Hermertia illucens) as bioresource: Current progress, applications, challenges and way forwards,” Waste and Biomass Valorization, vol. 17, no. 1, pp. 1–12, 2026, doi: 10.1007/s12649-025-03175-6.

[78] X. Che et al., “Application of chitosan-based hydrogel in promoting wound healing: A review,” Polymers, vol. 16, no. 3, pp. 1–20, 2024, doi: 10.3390/polym16030344.

[79] A. Haider et al., “Chitosan as a tool for tissue engineering and rehabilitation: Recent developments and future perspectives – A review,” International Journal of Biological Macromolecules, vol. 278, no. 1, pp. 1–13, 2024, doi: 10.1016/j.ijbiomac.2024.134172.

[80] S. Pramanik et al., “Chitosan alchemy: transforming tissue engineering and wound healing,” RSC advances, vol. 14, no. 27, pp. 19219–19256, 2024, doi: 10.1039/d4ra01594k.

[81] M. Mahmoodi et al., “Evaluation of gene expression levels in diabetic rat skin wound healing treated with chitosan/curcumin nanoparticles-loaded sodium alginate/chitosan hydrogels,” International Journal of Biological Macromolecules, vol. 324, no. 1, pp. 1–14, 2025, doi: 10.1016/j.ijbiomac.2025.147220.

[82] D. Kathyayani et al., “Insights into the physicochemical characteristics and miscibility of chitosan/polypeptide blends: Promising material for wound healing in sprague–dawley rats,” ACS Biomaterials Science & Engineering, vol. 10, no. 9, pp. 5807–5821, 2024, doi: 10.1021/acsbiomaterials.4c01123.

[83] Y. Li et al., “Therapeutic effects of EGF-modified curcumin/chitosan nano-spray on wound healing,” Regenerative Biomaterials, vol. 8, no. 2, pp. 1–9, 2021, doi: 10.1093/rb/rbab009.

Full Text: PDF

DOI: 10.14416/j.asep.2026.06.008

Refbacks

  • There are currently no refbacks.


Copyright © 2025 by King Mongkut’s University of Technology North Bangkok. All rights reserved.