Comparison of the Loading of Lactoferrin Extracted from Camel Milk on Pectin and Chitosan Nanoparticles

Authors

  • Saeid Zibaee *

    Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO),  Mashhad P.O. Box 9183896516, Iran

  • Somayeh Deljoo Aval

    Department of Biology, Payam Noor University of Mashhad, Mashhad 9186143546, Iran

  • Shiva Soleimani

    Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Rasht Branch, Rasht 9151216534, Iran

  • Nilofar Shakibapour

    Faculty of Science, Ferdowsi University of Mashhad, Mashhad 9178175154, Iran

DOI:

https://doi.org/10.55121/fds.v3i1.943

Keywords:

Microencapsulation , Camel Milk Lactoferrin, Pectin, Chitosan

Abstract

Lactoferrin is a bioactive ingredient that is sensitive to environmental stresses and needs the use of microencapsulation, notwithstanding all the biological activities and effects it exerts. Pectin is a polysaccharide that acts as an anion for ion–ion interactions. Chitosan is a biodegradable and nontoxic material. In this study, lactoferrin was extracted and purified, pectin and chitosan nanoparticles were prepared, and finally, the lactoferrin was microencapsulated in the nanoparticles. After determining the final loading efficiencies of the lactoferrin, SEM and subsequent zeta potential and particle size measurements confirmed the lactoferrin loading onto the pectin and chitosan nanoparticles. The findings confirmed the presence of lactoferrin in the 0.5, 0.6, 0.7, and 0.8 M (mol·L−1) NaCl fractions through an SDS-PAGE and tetramethylbenzidine test. The loadings indicated that lactoferrin was indeed loaded, with the final loading efficiencies for pectin and chitosan being 84% and 81.5%, respectively. SEM confirmed the loading and showed that the emulsion structures present at the time of production and attachment were very uniform, with a consistent distribution of spherical particles. The average size of lactoferrin-loaded pectin nanoparticles was 276.3 nm, with a surface charge of –35 mV. Loading of lactoferrin onto the polysaccharides pectin and chitosan resulted in a shift toward more negative zeta potential values.

References

[1] Meleti, E., Koureas, M., Manouras, A., et al., 2025. Bioactive Peptides from Dairy Products: A Systematic Review of Advances, Mechanisms, Benefits, and Functional Potential. Dairy. 6(6), 65. DOI: https://doi.org/10.3390/dairy6060065

[2] Rascón-Cruz, Q., Espinoza-Sánchez, E.A., Siqueiros-Cendón, T.S., et al., 2021. Lactoferrin: A Glycoprotein Involved in Immunomodulation, Anticancer, and Antimicrobial Processes. Molecules. 26(1), 205. DOI: https://doi.org/10.3390/molecules26010205

[3] Chesneau, C., Larue, L., Belbekhouche, S., et al., 2023. Design of Tailor-Made Biopolymer-Based Capsules for Biological Application by Combining Porous Particles and Polysaccharide Assembly. Pharmaceutics. 15(6), 1718. DOI: https://doi.org/10.3390/pharmaceutics15061718

[4] Korkmaz, K., Tunçtürk, Y., 2024. Encapsulation Applications and Current Studies in Food Technology. Journal of the Institute of Science and Technology. 14(4), 1550–1561. DOI: https://doi.org/10.21597/jist.1472879

[5] Eker, F., Duman, H., Ertürk, M., et al., 2024. The Potential of Lactoferrin as Antiviral and Immune-Modulating Agent in Viral Infectious Diseases. Frontiers in Immunology. 15, 1402135. DOI: https://doi.org/10.3389/fimmu.2024.1402135

[6] Jiang, S., Mo, F., Li, W., et al., 2024. Deep Learning-Driven Optimization of Antihypertensive Properties from Whey Protein Hydrolysates: A Multienzyme Approach. Journal of Agricultural and Food Chemistry. 73(2), 1373–1388. DOI: https://doi.org/10.1021/acs.jafc.4c10830

[7] Mohammed, A.S.A., Naveed, M., Jost, N., et al., 2021. Polysaccharides; Classification, Chemical Properties, and Future Perspective Applications in Fields of Pharmacology and Biological Medicine (A Review of Current Applications and Upcoming Potentialities). Journal of Polymers and the Environment. 29(8), 2359–2371. DOI: https://doi.org/10.1007/s10924-021-02052-2

[8] Mardani, M., Siahtiri, S., Besati, M., et al., 2024. Microencapsulation of Natural Products Using Spray Drying: An Overview. Journal of Microencapsulation. 41(7), 649–678. DOI: https://doi.org/10.1080/02652048.2024.2389136

[9] Timilsena, Y.P., Akanbi, T.O., Khalid, N., et al., 2019. Complex Coacervation: Principles, Mechanisms and Applications in Microencapsulation. International Journal of Biological Macromolecules. 121, 1276–1286. DOI: https://doi.org/10.1016/j.ijbiomac.2018.10.144

[10] Alrosan, M., Al-Rabadi, N., Alu’datt, M.H., et al., 2025. Complex Coacervation of Plant-Based Proteins and Polysaccharides: Sustainable Encapsulation Techniques for Bioactive Compounds. Food Engineering Reviews. 17, 1059–1082. DOI: https://doi.org/10.1007/s12393-025-09408-7

[11] McClements, D.J., 2015. Encapsulation, Protection, and Release of Hydrophilic Active Components: Potential and Limitations of Colloidal Delivery Systems. Advances in Colloid and Interface Science. 219, 27–53. DOI: https://doi.org/10.1016/j.cis.2015.02.002

[12] Burapapadh, K., Takeuchi, H., Sriamornsak, P., 2016. Development of Pectin Nanoparticles through Mechanical Homogenization for Dissolution Enhancement of Itraconazole. Asian Journal of Pharmaceutical Sciences. 11(3), 365–375. DOI: https://doi.org/10.1016/j.ajps.2015.07.003

[13] Vosoughi, P., Naghib, S.M., Jafari, T., et al., 2025. Chitosan-Encapsulated Lipid-Based Nanovesicles for Therapeutic Applications and Tissue Engineering: A Review. Carbohydrate Polymer Technologies and Applications. 10, 100805. DOI: https://doi.org/10.1016/j.carpta.2025.100805

[14] Ahmed, T.A., Aljaeid, B.M., 2016. Preparation, Characterization, and Potential Application of Chitosan, Chitosan Derivatives, and Chitosan Metal Nanoparticles in Pharmaceutical Drug Delivery. Drug Design, Development and Therapy. 10, 483–507. DOI: https://doi.org/10.2147/DDDT.S99651

[15] Raei, M., Rajabzadeh, G., Zibaei, S., et al., 2015. Nano-Encapsulation of Isolated Lactoferrin from Camel Milk by Calcium Alginate and Evaluation of Its Release. International Journal of Biological Macromolecules. 79, 669–673. DOI: https://doi.org/10.1016/j.ijbiomac.2015.05.048

[16] Raei, M., Shahidi, F., Farhoodi, M., et al., 2017. Application of Whey Protein-Pectin Nano-Complex Carriers for Loading of Lactoferrin. International Journal of Biological Macromolecules. 105, 281–291. DOI: https://doi.org/10.1016/j.ijbiomac.2017.07.037

[17] Shahidi, F., Athiyappan, K.D., 2025. Polyphenol–Polysaccharide Interactions: Molecular Mechanisms and Potential Applications in Food Systems – A Comprehensive Review. Food Production, Processing and Nutrition. 7(1), 42. DOI: https://doi.org/10.1186/s43014-025-00322-3

[18] Aval, S.D., Zibaei, S., Ramshini, H., et al., 2021. Loading Lactoferrin Derived from Camel Milk on Pectin Nanoparticles and Studying Its Effect on the MCF-7 Cancer Cell Line. Journal of Research in Agriculture and Animal Science. 8(5), 36–43. Available from: https://www.questjournals.org/jraas/papers/v8-i5/F08053643.pdf

[19] Isa, M.T., Abdulkarim, A.Y., Bello, A., et al., 2024. Synthesis and Characterization of Chitosan for Medical Applications: A Review. Journal of Biomaterials Applications. 38(10), 1036–1057. DOI: https://doi.org/10.1177/08853282241243010

[20] Giles, H., Gallagher, J., Warren-Walker, D., et al., 2025. An Investigative Study into the Suitability of the Bradford Assay for Rapid Protein Determination in Whey. Food Chemistry. 499, 147375. DOI: https://doi.org/10.1016/j.foodchem.2025.147375

[21] McClements, D.J., 2014. Nanoparticle- and Microparticle-Based Delivery Systems: Encapsulation, Protection and Release of Active Compounds. CRC Press: Boca Raton, FL, USA.

[22] Alagarsamy, S., Kandasamy, S., Kandasamy, R., et al., 2024. Polymeric Nanoparticles for Oral Delivery of Biopharmaceuticals: An Overview. Journal of Innovations in Applied Pharmaceutical Science. 9(1), 1–8. DOI: https://doi.org/10.37022/jiaps.v9i1.565

[23] Yang, Z., Jiang, C., Xiang, Q., et al., 2025. Probing the Stability of Emulsified Asphalts: A Dual Analysis of Zeta Potential and Particle Size. Fuel. 396, 135266. DOI: https://doi.org/10.1016/j.fuel.2025.135266

[24] Berne, B.J., Pecora, R., 2000. Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics. Courier Corporation: Mineola, NY, USA.

[25] Xiang, Q., Li, J., Wu, J., et al., 2025. Investigation of Dispersion Stability of Regenerated Asphalt Emulsions through Electric Potential-Particle-Surface Triad. Construction and Building Materials. 493, 143212. DOI: https://doi.org/10.1016/j.conbuildmat.2025.143212

[26] Yuan, Y., Ma, M., Xu, Y., et al., 2023. Construction of Biopolymer-Based Nanoencapsulation of Functional Food Ingredients Using the pH-Driven Method: A Review. Critical Reviews in Food Science and Nutrition. 63(22), 5724–5738. DOI: https://doi.org/10.1080/10408398.2021.2023858

[27] Maheshwari, S., Singh, A., Wasim, R., et al., 2025. Revealing the Potential of Mucoadhesive Ocular Nanoparticles for Enhanced Drug Delivery. Current Nanomedicine. 15(2), 129–141. DOI: https://doi.org/10.2174/0124681873284203240328102643

[28] Zafar, A., Arshad, R., UrRehman, A., et al., 2023. Recent Developments in Oral Delivery of Vaccines Using Nanocarriers. Vaccines. 11(2), 490. DOI: https://doi.org/10.3390/vaccines11020490

[29] Vikal, A., Maurya, R., Dubey, A., et al., 2024. Miscellaneous Targeting Approaches: Fundamentals of Gene Delivery and Overview of Colon, Liver, Macrophage, Mitochondrial, and M-Cell Targeting. In: Yadav, A.K., Jain, K. (Eds.). Novel Carrier Systems for Targeted and Controlled Drug Delivery. Springer Nature: Singapore. pp. 501–531. DOI: https://doi.org/10.1007/978-981-97-4970-6_19

[30] McClements, D.J., 2015. Nanoscale Nutrient Delivery Systems for Food Applications: Improving Bioactive Dispersibility, Stability, and Bioavailability. Journal of Food Science. 80(7), N1602–N1611. DOI: https://doi.org/10.1111/1750-3841.12919

[31] Muniyandy, S., 2025. Pectin Beads in Drug Delivery: Extraction, Formulation, and Pharmaceutical Applications. International Journal of Applied Pharmaceutics. 17(5), 64–73. DOI: https://doi.org/10.22159/ijap.2025v17i5.54903

[32] Jan, M., Shalla, A.H., Majid, K., 2025. Development of a Chitosan-Based Smart Hydrogel Composite as a Controlled Release Drug Delivery Agent. Langmuir. 41(44), 29668–29677. DOI: https://doi.org/10.1021/acs.langmuir.5c03866

[33] Duarte, L.G.R., Ferreira, N.C.A., Fiocco, A.C.T.R., et al., 2023. Lactoferrin-Chitosan-TPP Nanoparticles: Antibacterial Action and Extension of Strawberry Shelf-Life. Food and Bioprocess Technology. 16(1), 135–148. DOI: https://doi.org/10.1007/s11947-022-02927-9

[34] Zibaee, S., Soleimani, S., Khalilollahi, F., et al., 2024. Effect of Lactoferrin Loaded on Chitosan against Staphylococcus Aureus and Pseudomonas Aeruginosa. Applied Microbiology: Theory & Technology. 5(2), 110–121. DOI: https://doi.org/10.37256/amtt.5220244614

[35] Valenti, P., Antonini, G., 2005. Lactoferrin: An Important Host Defence against Microbial and Viral Attack. Cellular and Molecular Life Sciences. 62(22), 2576. DOI: https://doi.org/10.1007/s00018-005-5372-0

[36] Másson, M., 2021. Antimicrobial Properties of Chitosan and Its Derivatives. In: Jayakumar, R., Prabaharan, M. (Eds.). Chitosan for Biomaterials III: Structure-Property Relationships. Springer International Publishing: Cham, Switzerland. pp. 131–168.

[37] Rubio-Martin del Campo, K.N., Rivas-Gastelum, M.F., Garcia-Amezquita, L.E., et al., 2025. From Nature to Science: A Review of the Applications of Pectin-Based Hydrogels. Macromol. 5(4), 58. DOI: https://doi.org/10.3390/macromol5040058

[38] Jafernik, K., Ładniak, A., Blicharska, E., et al., 2023. Chitosan-Based Nanoparticles as Effective Drug Delivery Systems—A Review. Molecules. 28(4), 1963. DOI: https://doi.org/10.3390/molecules28041963

[39] Schrope, J.H., Robertson, T.F., Sarris, M., et al., 2025. Chemical and Mechanical Regulation of Leukocyte Migration. Cold Spring Harbor Perspectives in Biology. a041752. DOI: https://doi.org/10.1101/cshperspect.a041752

[40] Hua, S., Marks, E., Schneider, J.J., et al., 2015. Advances in Oral Nano-Delivery Systems for Colon Targeted Drug Delivery in Inflammatory Bowel Disease: Selective Targeting to Diseased versus Healthy Tissue. Nanomedicine: Nanotechnology, Biology and Medicine. 11(5), 1117–1132. DOI: https://doi.org/10.1016/j.nano.2015.02.018

[41] Lu, K., Zhu, X.-Y., Li, Y., et al., 2023. Progress in the Preparation of Prussian Blue-Based Nanomaterials for Biomedical Applications. Journal of Materials Chemistry B. 11(24), 5272–5300. DOI: https://doi.org/10.1039/d2tb02617a

[42] Hu, K., McClements, D.J., 2015. Fabrication of Biopolymer Nanoparticles by Antisolvent Precipitation and Electrostatic Deposition: Zein-Alginate Core/Shell Nanoparticles. Food Hydrocolloids. 44, 101–108. DOI: https://doi.org/10.1016/j.foodhyd.2014.09.015

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