Achatina achatina (AA) is a rich source of collagen due to its large size, but it is underutilized. Type I collagen was extracted from AA to serve as an alternative to existing collagen sources. The collagen was extracted at varying alkaline and temperature conditions to determine the optimal parameters that would give a high yield of acid-soluble collagen. The extracted collagen was characterised using X-ray diffraction, Fourier transform infrared (FTIR) spectrometry, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) to confirm the integrity and purity of the extracted collagen. The type of collagen was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The α-1, α-2, and dimer electrophoresis bands confirmed that the collagen is type I, and the XRD data supported the findings. The highest collagen yield was obtained at 4 ℃ for 48 h, which decreased with increasing temperature due to the instability of the protein in acid at high temperatures. A cytotoxicity test was conducted using an Alamar blue assay. The AA collagen-treated normal prostate cell line (PNT2) showed no significant difference from the untreated control cells. The high-quality type I collagen extracted from AA has the potential for biomedical and other industrial applications.

1. Sorushanova A, Delgado LM, Wu Z, et al. The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development. Advanced Materials. 2018; 31(1). doi: 10.1002/adma.201801651

2. Kular JK, Basu S, Sharma RI. The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering. Journal of Tissue Engineering. 2014; 5: 204173141455711. doi: 10.1177/2041731414557112

3. Miyazaki K, Oyanagi J, Hoshino D, et al. Cancer cell migration on elongate protrusions of fibroblasts in collagen matrix. Scientific Reports. 2019; 9(1). doi: 10.1038/s41598-018-36646-z

4. Smith PC, Martínez C, Martínez J, et al. Role of Fibroblast Populations in Periodontal Wound Healing and Tissue Remodeling. Frontiers in Physiology. 2019; 10. doi: 10.3389/fphys.2019.00270

5. Silvipriya K, Kumar K, Bhat A, et al. Collagen: Animal Sources and Biomedical Application. Journal of Applied Pharmaceutical Science. Published online 2015: 123-127. doi: 10.7324/japs.2015.50322

6. Dellaquila A, Campodoni E, Tampieri A, et al. Overcoming the Design Challenge in 3D Biomimetic Hybrid Scaffolds for Bone and Osteochondral Regeneration by Factorial Design. Frontiers in Bioengineering and Biotechnology. 2020; 8. doi: 10.3389/fbioe.2020.00743

7. Al-Atif H. Collagen Supplements for Aging and Wrinkles: A Paradigm Shift in the Field of Dermatology and Cosmetics. Dermatology Practical & Conceptual. Published online February 2, 2022: e2022018. doi: 10.5826/dpc.1201a18

8. Ramadass SK, Perumal S, Gopinath A, et al. Sol–Gel Assisted Fabrication of Collagen Hydrolysate Composite Scaffold: A Novel Therapeutic Alternative to the Traditional Collagen Scaffold. ACS Applied Materials & Interfaces. 2014; 6(17): 15015-15025. doi: 10.1021/am502948g

9. Indriani S, Benjakul S, Kishimura H, et al. Impact of extraction condition on the yield and molecular characteristics of collagen from Asian bullfrog (Rana tigerina) skin. LWT. 2022; 162: 113439. doi: 10.1016/j.lwt.2022.113439

10. Matinong AME, Chisti Y, Pickering KL, et al. Collagen Extraction from Animal Skin. Biology. 2022; 11(6): 905. doi: 10.3390/biology11060905

11. Monsur HA, Jaswir I, Salleh HM, et al. Effects of Pretreatment on Properties of Gelatin from Perch (Lates Niloticus) Skin. International Journal of Food Properties. 2014; 17(6): 1224-1236. doi: 10.1080/10942912.2012.685676

12. Liu S, Liu W, Li G. Effect of ionic liquids pretreatment on the extraction of collagen from calf skin. Journal of the American Leather Chemists Association. 2019; 114: 391–399.

13. Liu D, Wei G, Li T, et al. Effects of alkaline pretreatments and acid extraction conditions on the acid-soluble collagen from grass carp (Ctenopharyngodon idella) skin. Food Chemistry. 2015; 172: 836-843. doi: 10.1016/j.foodchem.2014.09.147

14. Jafari H, Lista A, Siekapen MM, et al. Fish Collagen: Extraction, Characterization, and Applications for Biomaterials Engineering. Polymers. 2020; 12(10): 2230. doi: 10.3390/polym12102230

15. Blidi O El, Omari N El, Balahbib A, et al. Extraction methods, characterization and biomedical applications of collagen: A review. Biointerface Research in Applied Chemistry. 2021; 11: 13587–13613.

16. Schmidt MM, Dornelles RCP, Mello RO, et al. Collagen extraction process. International Food Research Journal. 2016; 23: 913–922.

17. Engmann FN, Afoakwah NA, Darko PO, et al. Proximate and Mineral Composition of Snail (Achatina achatina). Any Nutritional Justification for Acclaimed Health Benefits? 2013; 3: 8–15.

18. Roobak Kumar A, Reddy PK, MS U, et al. Occurrence of giant African snail, Achatina fulica Bowditch in coffee growing areas of Karnataka and its management. Journal of Entomology and Zoology Studies. 2018; 6: 134–137.

19. Gołdyn B, Kaczmarek Ł, Roszkowska M, et al. Urban Ecology of Invasive Giant African SnailAchatina fulica(Férussac) (Gastropoda: Achatinidae) on its First Recorded Sites in the Ecuadorian Amazon. American Malacological Bulletin. 2017; 35(1): 59-64. doi: 10.4003/006.035.0108

20. Jimoh AO. Rendimiento reproductivo de caracoles tendidos alimentados con forrajes y diferentes mezclas de concentrados. Archivos de Zootecnia. 2021; 70(272): 338-342. doi: 10.21071/az.v70i272.5572

21. Ngbolua K te N, Djolu RD, Selenga MD, et al. Breeding of the Giant African Edible Snail (Achatina achatina Linnaeus, 1758) in Gbado-Lite city, Democratic Republic of the Congo. Britain International of Exact Sciences (BIoEx) Journal. 2022; 4(2): 92-101. doi: 10.33258/bioex.v4i2.730

22. Xu S, Yang H, Shen L, et al. Purity and yield of collagen extracted from southern catfish (Silurus meridionalis Chen) skin through improved pretreatment methods. International Journal of Food Properties. 2017; 20(sup1): S141-S153. doi: 10.1080/10942912.2017.1291677

23. Song X, Li Z, Li Y, et al. Typical structure, biocompatibility, and cell proliferation bioactivity of collagen from Tilapia and Pacific cod. Colloids and Surfaces B: Biointerfaces. 2022; 210: 112238. doi: 10.1016/j.colsurfb.2021.112238

24. Jeong HS, Venkatesan J, Kim SK. Isolation and characterization of collagen from marine fish (Thunnus obesus). Biotechnology and Bioprocess Engineering. 2013; 18(6): 1185-1191. doi: 10.1007/s12257-013-0316-2

25. Noorzai S, J.R. Verbeek C. Collagen: From Waste to Gold. In: Biotechnological Applications of Biomass. Epub ahead of print; 2021.

26. Cliche S, Amiot J, Avezard C, et al. Extraction and characterization of collagen with or without telopeptides from chicken skin. Poultry Science. 2003; 82(3): 503-509. doi: 10.1093/ps/82.3.503

27. Sotelo CG, Comesaña MB, Ariza PR, et al. Characterization of Collagen from Different Discarded Fish Species of the West Coast of the Iberian Peninsula. Journal of Aquatic Food Product Technology. 2015; 25(3): 388-399. doi: 10.1080/10498850.2013.865283

28. McAlinden A, Hudson DM, Fernandes AA, et al. Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc. Matrix Biology Plus. 2021; 12: 100077.

29. Gallagher SR. SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE). Current Protocols Essential Laboratory Techniques. 2012; 6(1). doi: 10.1002/9780470089941.et0703s06

30. De Sousa Moraes PRF, Saska S, Barud H, et al. Bacterial cellulose/collagen hydrogel for wound healing. Mater Res. 2016; 19: 106–116.

31. Buchanan JK, Zhang Y, Holmes G, et al. Role of X‐ray Scattering Techniques in Understanding the Collagen Structure of Leather. Chemistry Select. 2019; 4(48): 14091-14102. doi: 10.1002/slct.201902908

32. Bak S, Lee S, Choi C, et al. Assessment of the Influence of Acetic Acid Residue on Type I Collagen during Isolation and Characterization. Materials. 2018; 11(12): 2518. doi: 10.3390/ma11122518

33. Sumbono A, Rossarie D. Characterization of Physical and Chemical Properties of Collagen Golden Apple Snail (Pomacea canaliculata). Epub ahead of print; 2020.

34. Riaz T, Zeeshan R, Zarif F, et al. FTIR analysis of natural and synthetic collagen. Applied Spectroscopy Reviews. 2018; 53(9): 703-746. doi: 10.1080/05704928.2018.1426595

35. Sousa RO, Martins E, Carvalho DN, et al. Collagen from Atlantic cod (Gadus morhua) skins extracted using CO2 acidified water with potential application in healthcare. Journal of Polymer Research. 2020; 27(3). doi: 10.1007/s10965-020-02048-x

36. Zarai Z, Balti R, Mejdoub H, et al. Process for extracting gelatin from marine snail (Hexaplex trunculus): Chemical composition and functional properties. Process Biochemistry. 2012; 47: 1779–1784.

37. Kaewdang O, Benjakul S, Kaewmanee T, et al. Characteristics of collagens from the swim bladders of yellowfin tuna (Thunnus albacares). Food Chem. 2014; 155: 264–270.

38. Raut SK, Barker GM. Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture. Molluscs as crop pests. Published online January 2002: 55-114. doi: 10.1079/9780851993201.0055

39. Eze CN, Ohanele OO. Heavy Metals and Parasites in African Giant Snail (Achatina achatina) from Three Communities in Ogoni, Rivers State, Nigeria. IOSR Journal of Agriculture and Veterinary Science. 2021; 14: 27–35.

40. Mbah JO, Osondu-Anyanwu C, Imalele Ee. Parasitic Profile of Five Species of Terrestrial Achatina Snail in Cross River State, Nigeria: Public Health Implications. Uttar Pradesh J Zool 2022; 43: 13–21.

41. Zaelani B, Safithri M, Tarman K, et al. Collagen isolation with acid soluble method from the skin of Red Snapper (lutjanus sp.). IOP Conference Series: Earth and Environmental Science. 2019; 241: 012033. doi: 10.1088/1755-1315/241/1/012033

42. Blanco M, Vázquez JA, Pérez-Martín RI, et al. Collagen Extraction Optimization from the Skin of the Small-Spotted Catshark (S. canicula) by Response Surface Methodology. Marine Drugs. 2019; 17(1): 40. doi: 10.3390/md17010040

43. Jatto OE, Asia IO, Medjor WE. Proximate and Mineral Composition of Different Species of Snail Shell. Pacific Journal of Science and Technology. 2010; 11: 416–419.

44. Jadamba B, Urnukhsaikhan E, Gantulga A, et al. The Characterization of Acid Soluble Collagen from Sheep. Tail Tendon. 2021; 2: 37–42.

45. Cheng X, Shao Z, Li C, et al. Isolation, Characterization and Evaluation of Collagen from Jellyfish Rhopilema esculentum Kishinouye for Use in Hemostatic Applications. PLOS ONE. 2017; 12(1): e0169731. doi: 10.1371/journal.pone.0169731

46. Wahyuningsih R, Rusman, Nurliyani, et al. Optimization of acid soluble collagen extraction from Indonesian local “Kacang” goat skin and physico-chemical properties characterization. Chem Eng Trans. 2018; 63: 703–708.

47. Matmaroh K, Benjakul S, Prodpran T, et al. Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus). Food Chemistry. 2011; 129(3): 1179-1186. doi: 10.1016/j.foodchem.2011.05.099

48. Senaratne LS, Park PJ, Kim SK. Isolation and characterization of collagen from brown backed toadfish (Lagocephalus gloveri) skin. Bioresource Technology. 2006; 97(2): 191-197. doi: 10.1016/j.biortech.2005.02.024

49. Sibilla S, Godfrey M, Brewer S, et al. An Overview of the Beneficial Effects of Hydrolysed Collagen as a Nutraceutical on Skin Properties: Scientific Background and Clinical Studies. The Open Nutraceuticals Journal. 2015; 8(1): 29-42. doi: 10.2174/1876396001508010029

50. Bolke L, Schlippe G, Gerß J, et al. A Collagen Supplement Improves Skin Hydration, Elasticity, Roughness, and Density: Results of a Randomized, Placebo-Controlled, Blind Study. Nutrients. 2019; 11(10): 2494. doi: 10.3390/nu11102494

51. Wiatrak B, Kubis-Kubiak A, Piwowar A, Barg E. PC12 cell line: cell types, coating of culture vessels, differentiation and other culture conditions. Cells. MDPI; 2020; 9(4): 958.

52. Li J, Li Y, Li Y, Yang Z, Jin H. Physicochemical Properties of Collagen from Acaudina Molpadioidesand its Protective Effects against H2O2-Induced Injury in RAW264. 7 Cells. Marine Drugs. 2020; 18(7).