Enhancing Edible Bird Nest Cleaning Process by Immobilizing Cross-Linked Enzyme Aggregates (CLEAs) of Keratinase

Abstract

Edible bird's nest (EBN) is a premium functional food prized for its nutritional and medicinal properties but conventionally cleaned through laborious and chemically intensive processes that compromise safety and quality. In this study, we developed a reusable cross-linked enzyme aggregate (CLEA) form of keratinase as an eco-efficient biocatalyst for EBN cleaning. The immobilized keratinase (keratinase-CLEA) exhibited optimal performance at 50°C and pH 6, retaining up to 40% residual activity after five reuse cycles. Structural and morphological analyses (FTIR, FESEM) confirmed successful cross-linking and stable aggregate formation, while kinetic evaluation revealed enhanced catalytic turnover (V_max = 9.77 mmol·L⁻¹·min⁻¹). When applied in EBN cleaning, keratinase-CLEA reduced soaking time to 30 min while maintaining the nest's structural integrity and nutritional composition. Compared with free enzymes or conventional water cleaning, the reusable CLEA system provides a scalable, cost-effective, and environmentally sustainable bioprocess, marking the first report demonstrating a recyclable immobilized enzyme platform for EBN purification. This work introduces a practical biocatalytic processing model applicable to high-value animal-derived food materials, supporting green manufacturing within the functional food industry.

Keywords

biocatalysis / cross-linked enzyme aggregates (CLEA) / edible bird nest / enzyme immobilization / food process engineering / keratinase

References

[1] Abdul Wahab, M. K. H., H. A. El-Enshasy, F. D. A. Bakar, A. M. A. Murad, J. M. Jahim, and R. M. Illias. 2019. “Improvement of Cross-Linking and Stability on Cross-Linked Enzyme Aggregate (CLEA)-Xylanase by Protein Surface Engineering.” Process Biochemistry 86, no. June: 40–49. https://doi.org/10.1016/j.procbio.2019.07.017.

[2] Abdullah, A. H. D., S. Chalimah, I. Primadona, and M. H. G. Hanantyo. 2018. “Physical and Chemical Properties of Corn, Cassava, and Potato Starchs.” IOP Conference Series: Earth and Environmental Science 160, no. 1: 012003. https://doi.org/10.1088/1755-1315/160/1/012003.

[3] Akkas, T., A. Zakharyuta, A. Taralp, and C. W. Ow-Yang. 2020. “Cross-Linked Enzyme Lyophilisates (CLELs) of Urease: A New Method to Immobilize Ureases.” Enzyme and Microbial Technology 132, no. February 2019: 109390. https://doi.org/10.1016/j.enzmictec.2019.109390.

[4] Badrulzaman, S. Z. S., A. W. Aminan, A. N. M. Ramli, R. Che Man, and N. I. Wan Azelee. 2021. “Extraction and Characterization of Keratin From Chicken and Swiftlet Feather.” Materials Science Forum 1025: 157–162. https://doi.org/10.4028/www.scientific.net/MSF.1025.157.

[5] Bai, W., X. Liu, Q. Fan, J. Lian, and B. Guo. 2023. “Study of the Antiaging Effects of Bird's Nest Peptide Based on Biochemical, Cellular, and Animal Models.” Journal of Functional Foods 103, no. January: 105479. https://doi.org/10.1016/j.jff.2023.105479.

[6] Bilal, M., S. Noreen, M. Asgher, and S. Parveen. 2021. “Development and Characterization of Cross-Linked Laccase Aggregates (Lac-CLEAs) From Trametes Versicolor IBL-04 as Ecofriendly Biocatalyst for Degradation of Dye-Based Environmental Pollutants.” Environmental Technology & Innovation 21: 101364. https://doi.org/10.1016/j.eti.2021.101364.

[7] Bouguerra, O. M., R. A. Wahab, F. Huyop, et al. 2024. “An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications.” Applied Biochemistry and Biotechnology 196, no. 9: 5711–5739. https://doi.org/10.1007/s12010-023-04809-y.

[8] Bradford, M. M. 1976. “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.” Analytical Biochemistry 72, no. 1/2: 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.

[9] Charoenwongpaiboon, T., R. Pichyangkura, R. A. Field, and M. H. Prousoontorn. 2019. “Preparation of Cross-Linked Enzyme Aggregates (CLEAs) of an Inulosucrase Mutant for the Enzymatic Synthesis of Inulin-Type Fructooligosaccharides.” Catalysts 9, no. 8: 641. https://doi.org/10.3390/catal9080641.

[10] Dai, Y., J. Cao, Y. Wang, Y. Chen, and L. Jiang. 2021. “A Comprehensive Review of Edible Bird's Nest.” Food Research International 140, no. October 2020: 109875. https://doi.org/10.1016/j.foodres.2020.109875.

[11] Darwesh, O. M., I. A. Matter, and M. F. Eida. 2019. “Development of Peroxidase Enzyme Immobilized Magnetic Nanoparticles for Bioremediation of Textile Wastewater Dye.” Journal of Environmental Chemical Engineering 7, no. 1: 102805. https://doi.org/10.1016/j.jece.2018.11.049.

[12] Das, S., A. Das, N. Das, et al. 2024. “Harnessing the Potential of Microbial Keratinases for Bioconversion of Keratin Waste.” Environmental Science and Pollution Research 31, no. 46: 57478–57507. https://doi.org/10.1007/s11356-024-34233-6.

[13] Fan, Q., J. Lian, X. Liu, F. Zou, X. Wang, and M. Chen. 2022. “A Study on the Skin Whitening Activity of Digesta From Edible Bird's Nest: A Mucin Glycoprotein.” Gels 8, no. 1: 1–15. https://doi.org/10.3390/gels8010024.

[14] Gonenc, I., and F. Us. 2019. “Effect of Glutaraldehyde Crosslinking on Degree of Substitution, Thermal, Structural, and Physicochemical Properties of Corn Starch.” Starch - Stärke 71, no. 3/4: 1800046. https://doi.org/10.1002/star.201800046.

[15] Hero, J. S., C. M. Romero, J. H. Pisa, N. I. Perotti, C. Olivaro, and M. A. Martinez. 2018. “Designing Cross-Linked Xylanase Aggregates for Bioconversion of Agroindustrial Waste Biomass Towards Potential Production of Nutraceuticals.” International Journal of Biological Macromolecules 111: 229–236. https://doi.org/10.1016/j.ijbiomac.2017.12.166.

[16] Hu, Y., Y. Lv, H. Tang, et al. 2025. “An Overview of Industrial Enzyme Engineering to Achieve a pH Adaptability-Activity Trade-Off: Current Status and Future Perspectives.” Journal of Agricultural and Food Chemistry 73, no. 33: 20547–20562. https://doi.org/10.1021/acs.jafc.5c03673.

[17] Hui Yan, T., A. S. Babji, S. J. Lim, and S. R. Sarbini. 2021. “A Systematic Review of Edible Swiftlet's Nest (ESN): Nutritional Bioactive Compounds, Health Benefits as Functional Food, and Recent Development as Bioactive ESN Glycopeptide Hydrolysate.” Trends in Food Science & Technology 115: 117–132. https://doi.org/10.1016/j.tifs.2021.06.034.

[18] Hwang, E., S. W. Park, and J.-E. Yang. 2020. “Anti-Aging, Anti-Inflammatory, and Wound-Healing Activities of Edible Bird's Nest in Human Skin Keratinocytes and Fibroblasts.” Pharmacognosy Magazine 16, no. 69: 336–342. https://doi.org/10.4103/pm.pm_326_19.

[19] Ifko, D., K. Vasić, Ž. Knez, and M. Leitgeb. 2023. “(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase From T. Reesei: A Stable and Efficient Biocatalyst.” Molecules 28, no. 3: 1305. https://doi.org/10.3390/molecules28031305.

[20] IndoWalet, M. 2021. Penentuan Kualitas Sarang Walet. https://masindowalet.com/2021/01/18/penentuan-kualitas-sarangwalet/.

[21] Jaafar, N. R., R. Md Illias, N. Jailani, and S. A. Mohamed. 2023. “Characterization of Maltogenic Amylase Activity Recovery: A Potential Approach for Improving Immobilization.” Journal of Bioprocessing and Biomass Technology 2, no. 2: 37–43. https://doi.org/10.11113/bioprocessing.v2n2.34.

[22] Khan, M. R. 2021. “Immobilized Enzymes: A Comprehensive Review.” Bulletin of the National Research Centre 45, no. 1: 207. https://doi.org/10.1186/s42269-021-00649-0.

[23] Khanahmadi, S., F. Yusof, H. Chyuan Ong, A. Amid, and H. Shah. 2016. “Cocoa Pod Husk: A New Source of CLEA-Lipase for Preparation of Low-Cost Biodiesel: An Optimized Process.” Journal of Biotechnology 231: 95–105. https://doi.org/10.1016/j.jbiotec.2016.05.015.

[24] Lee, T. H., C. H. Lee, N. A. Azmi, et al. 2020. “Characterization of Polar and Non-Polar Compounds of House Edible Bird's Nest (EBN) From Johor, Malaysia.” Chemistry and Biodiversity 17, no. 1: e1900419. https://doi.org/10.1002/cbdv.201900419.

[25] Loh, S.-P., S.-H. Cheng, and W. Mohamed. 2022. “Edible Bird's Nest as a Potential Cognitive Enhancer.” Frontiers in Neurology 13, no. May: 865671. https://doi.org/10.3389/fneur.2022.865671.

[26] Mahmod, S. S., F. Yusof, M. S. Jami, S. Khanahmadi, and H. Shah. 2015. “Development of an Immobilized Biocatalyst With Lipase and Protease Activities as a Multipurpose Cross-Linked Enzyme Aggregate (Multi-CLEA).” Process Biochemistry 50, no. 12: 2144–2157. https://doi.org/10.1016/j.procbio.2015.10.008.

[27] Mohammad Munawar, T., K. Aruna, and A. V. N. Swamy. 2012. “Studies on Production of Keratinase From Different Keratin Substrates Using Bacillus Licheniformis Strains.” International Journal of Pharmaceutical Research and Development 4, no. 7: 64–68.

[28] Moktip, T., L. Salaipeth, A. E. Cope, M. J. Taherzadeh, T. Watanabe, and P. Phitsuwan. 2025. “Current Understanding of Feather Keratin and Keratinase and Their Applications in Biotechnology.” Biochemistry Research International 2025, no. 1: 6619273. https://doi.org/10.1155/bri/6619273.

[29] Naseer, S., J. Ouyang, X. Chen, et al. 2020. “Immobilization of β-glucosidase by Self-Catalysis and Compared to Crosslinking With Glutaraldehyde.” International Journal of Biological Macromolecules 154: 1490–1495. https://doi.org/10.1016/j.ijbiomac.2019.11.030.

[30] Ng, C. H., P. L. Tang, and Y. Y. Ong. 2023. “Enzymatic Hydrolysis Improves Digestibility of Edible Bird's Nest (EBN): Combined Effect of Pretreatment and Enzyme.” Journal of Food Measurement and Characterization 17, no. 1: 549–563. https://doi.org/10.1007/s11694-022-01648-z.

[31] Parveen, S., M. Asgher, and M. Bilal. 2021. “Lignin Peroxidase-Based Cross-Linked Enzyme Aggregates (LiP-CLEAs) as Robust Biocatalytic Materials for Mitigation of Textile Dyes-Contaminated Aqueous Solution.” Environmental Technology & Innovation 21: 101226. https://doi.org/10.1016/j.eti.2020.101226.

[32] Perwez, M., J. Ahmed Mazumder, and M. Sardar. 2019. “Preparation and Characterization of Reusable Magnetic Combi-CLEA of Cellulase and Hemicellulase.” Enzyme and Microbial Technology 131: 109389. https://doi.org/10.1016/j.enzmictec.2019.109389.

[33] Rahayu, S., M. T. Suhartono, W. Suryapratama, and M. Bata. 2017. “Keratinolytic Enzymes for Cleaning Edible Bird's Nest.” Biosciences Biotechnology Research Asia 14, no. 3: 989–996. https://doi.org/10.13005/bbra/2533.

[34] Rahayu, S., W. Suryapratama, F. M. Suhartati, M. Bata, E. A. Rimbawanto, and B. Hartoyo. 2024. “Quality of Edible Bird's Nest Treated by Keratinolytic Enzymes-Based Cleaning Solution.” Food Research 8, no. 2: 299–307. https://doi.org/10.26656/fr.2017.8(2).287.

[35] Rathankumar, A. K., S. SaiLavanyaa, K. Saikia, et al. 2019. “Systemic Concocting of Cross-Linked Enzyme Aggregates of Candida Antarctica Lipase B (Novozyme 435) for the Biomanufacturing of Rhamnolipids.” Journal of Surfactants and Detergents 22, no. 3: 477–490. https://doi.org/10.1002/jsde.12266.

[36] Razib, M. S. M., R. N. Z. R. A. Rahman, F. M. Shariff, and M. S. M. Ali. 2020. “Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease.” Catalysts 10, no. 1: 55. https://doi.org/10.3390/catal10010055.

[37] Rehman, S., H. N. Bhatti, M. Bilal, and M. Asgher. 2016. “Cross-Linked Enzyme Aggregates (CLEAs) of Pencilluim Notatum Lipase Enzyme With Improved Activity, Stability and Reusability Characteristics.” International Journal of Biological Macromolecules 91: 1161–1169. https://doi.org/10.1016/j.ijbiomac.2016.06.081.

[38] Royvaran, M., A. Taheri-Kafrani, A. Landarani Isfahani, and S. Mohammadi. 2016. “Functionalized Superparamagnetic Graphene Oxide Nanosheet in Enzyme Engineering: A Highly Dispersive, Stable and Robust Biocatalyst.” Chemical Engineering Journal 288: 414–422. https://doi.org/10.1016/j.cej.2015.12.034.

[39] Subramaniam, Y., Y. C. Faib, and E. S. L. Ming. 2015. “Edible Bird Nest Processing Using Machine Vision and Robotic Arm.” Journal Teknologi 72, no. 2: 85–88. https://doi.org/10.11113/jt.v72.3889.

[40] Tai, S. K., Z. Hamzah, Q. H. Ng, and C. S. Tan. 2020. “Surface Morphology Study on Unclean, Commercial, and Bromelain Treated Edible Bird Nest (EBN) Using Scanning Electron Microscope.” IOP Conference Series: Materials Science and Engineering 932, no. 1: 012013. https://doi.org/10.1088/1757-899X/932/1/012013.

[41] Torabizadeh, H., and M. Mikani. 2018. “Kinetic and Thermodynamic Features of Nanomagnetic Cross-Linked Enzyme Aggregates of Naringinase Nanobiocatalyst in Naringin Hydrolysis.” International Journal of Biological Macromolecules 119: 717–725. https://doi.org/10.1016/j.ijbiomac.2018.08.005.

[42] Tork, S. E., Y. E. Shahein, A. E. El-Hakim, A. M. Abdel-Aty, and M. M. Aly. 2013. “Production and Characterization of Thermostable Metallo-Keratinase From Newly Isolated Bacillus Subtilis NRC 3.” International Journal of Biological Macromolecules 55: 169–175. https://doi.org/10.1016/j.ijbiomac.2013.01.002.

[43] Verma, R., A. Kumar, and S. Kumar. 2019. “Synthesis and Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Thermostable Xylanase From Geobacillus Thermodenitrificans X1.” Process Biochemistry 80: 72–79. https://doi.org/10.1016/j.procbio.2019.01.019.

[44] Wahab, W. A. A. 2025. “Review of Research Progress in Immobilization and Chemical Modification of Microbial Enzymes and Their Application.” Microbial Cell Factories 24, no. 1: 167. https://doi.org/10.1186/s12934-025-02791-0.

[45] Xu, M., D. Ji, Y. Deng, and D. Agyei. 2020. “Preparation and Assessment of Cross-Linked Enzyme Aggregates (CLEAs) of β-Galactosidase From Lactobacillus Leichmannii 313.” Food and Bioproducts Processing 124: 82–96. https://doi.org/10.1016/j.fbp.2020.08.004.

[46] Xu, M.-Q., S.-S. Wang, L.-N. Li, J. Gao, and Y.-W. Zhang. 2018. “Combined Cross-Linked Enzyme Aggregates as Biocatalysts.” Catalysts 8, no. 10: 460. https://doi.org/10.3390/catal8100460.

[47] Yamaguchi, H., Y. Kiyota, and M. Miyazaki. 2018. “Techniques for Preparation of Cross-Linked Enzyme Aggregates and Their Applications in Bioconversions.” Catalysts 8, no. 5: 174. https://doi.org/10.3390/catal8050174.

[48] Yeo, B.-H., Y. Rukayadi, S.-F. Wong, C.-P. Tan, and O.-M. Lai. 2023. “Physicochemical Properties of Raw Cleaned Edible Bird's Nest After Different Primary Processing Including a New Cleaning Method.” Sains Malaysiana 52, no. 12: 3421–3435. https://doi.org/10.17576/jsm-2023-5212-06.

[49] Yeo, B.-H., T.-K. Tang, S.-F. Wong, et al. 2021. “Potential Residual Contaminants in Edible Bird's Nest.” Frontiers in Pharmacology 12: 1–15. https://doi.org/10.3389/fphar.2021.631136.

[50] Zeng, W.-W., and L.-S. Lai. 2019. “Anti-Melanization Effects and Inhibitory Kinetics of Tyrosinase of Bird's Nest Fern (Asplenium Australasicum) Frond Extracts on Melanoma and Human Skin.” Journal of Bioscience and Bioengineering 127, no. 6: 738–743. https://doi.org/10.1016/j.jbiosc.2018.11.005.

[51] Zhang, R., J. Sun, Y. Sun, et al. 2025. “Bacillus Subtilis SLXX08 Thermostable Keratinase for Decomposition and Bioconversion of Feather Waste and Dehairing.” Journal of Biotech Research 20: 291–304.

[52] Zulkifli, A. S., A. S. Babji, S. J. Lim, A. H. Teh, N. M. Daud, and H. A. Rahman. 2019. “Effect of Different Hydrolysis Time and Enzymes on Chemical Properties, Antioxidant and Antihyperglycemic Activities of Edible Bird Nest Hydrolysate.” Malaysian Applied Biology 48, no. 2: 149–156.

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2025 The Author(s). Food Bioengineering published by John Wiley & Sons Australia, Ltd. on behalf of State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.