Elucidating mechanisms of thermodynamic detoxification and conformational engineering of Lemna minor RuBisCO: in situ pickering armor cross-linking in cryoresistant hybrid cheese models
DOI:
https://doi.org/10.15587/1729-4061.2026.365023Keywords:
alternative proteins, biomass upcycling, heavy metal removal, multiphase rheology, phytoremediationAbstract
This study focuses on the design of cryoresistant buffalo cheese models, in which the lipid phase is volumetrically replaced by a Pickering emulsion gel based on a RuBisCO isolate derived from Lemna minor biomass. The core problem addressed is the limited upcycling potential of this phytoremediant into sustainable alternative proteins, caused by heavy metal hyperaccumulation during biomass harvesting in contaminated military and industrial biotopes. Results demonstrate that a thermodynamic detoxification protocol (acid shock at pH 3.0 and dialysis) reduced the initial concentrations of Pb (6.9 mg/kg) and Cd (0.6 mg/kg) to < 0.10 mg/kg (efficiencies of ≥ 98.5% and ≥ 83.3%, respectively) and Cu levels by ≥ 84.6%, ensuring the production of a decontaminated isolate. The resulting isolate underwent conformational engineering (a pH-shift from 11.0 to 7.0) to form nanoparticles that stabilized the initial Pickering emulsion. Following ionotropic gelation, the emulsion was utilized for the volumetric replacement of animal fat by up to 75%. Gel integration substantially enhanced the water-holding capacity of the system (up to 87%) and minimized cryo-syneresis (from 14% to 1.5%). During the Schreiber test (85°C), the hybrid model exhibited a transition from a viscoelastic melt to a thermostable gel with suppressed lipid exudation. These macroscopic effects are attributed to enhanced polymer hydration, Donnan osmotic swelling, and the spatial restriction of capillary water mobility. The observed structural transition and thermostability are likely driven by synergistic in situ interfacial cross-linking of the Pickering armor, disulfide bond formation, and thermally induced macromolecular consolidation indicative of late-stage Maillard reactions. The stability of this rheological test system confirms the efficacy of upcycling phytoremediants for non-food applications and the feasibility of extrapolating the protocol to standard food raw materials
References
- Liu, Y., Helikh, A., Filon, A., Duan, Z. (2023). Sausage technology for food sustainability: recipe, color, nutrition, structure. Eastern-European Journal of Enterprise Technologies, 4 (11 (124)), 47–58. https://doi.org/10.15587/1729-4061.2023.286323
- Helikh, A., Filon, A. (2025). Biochemical variability of vegetable juice powders: a key factor in modulating the physicochemical properties and safety profile of vegan fermented sausages. Technology Audit and Production Reserves, 4 (3 (84)), 52–59. https://doi.org/10.15587/2706-5448.2025.334830
- Helikh, A., Filon, A. (2025). Nanocomposite Biopolymer Coating with β-Nanochitosan for Preserving Snail Fillets: A Synergistic Antimicrobial System with Monarda punctata Oil. 2025 IEEE 15th International Conference Nanomaterials: Applications & Properties (NAP), MTFC12–1. https://doi.org/10.1109/nap68437.2025.11216270
- Muller, T., Bazinet, L. (2025). Water lentil (duckweed) protein purification by chemical (HCl) and electrochemical (Electrodialysis with bipolar membranes) acidification: Composition, structure and functional properties vs commercial protein isolates. Food Chemistry, 489, 144901. https://doi.org/10.1016/j.foodchem.2025.144901
- Müller, M., Holderer, O., Schwärzer, K., Wiese-Klinkenberg, A., Förster, B., Förster, S. et al. (2025). Proteins derived from green biomass: Alfalfa (Medicago sativa L.) and water lentil concentrate (Lemna minor L.) in the focus as stabilizers for emulsions. Food Hydrocolloids for Health, 8, 100233. https://doi.org/10.1016/j.fhfh.2025.100233
- Pérez-Vila, S., Fenelon, M. A., O’Mahony, J. A., Gómez-Mascaraque, L. G. (2022). Extraction of plant protein from green leaves: Biomass composition and processing considerations. Food Hydrocolloids, 133, 107902. https://doi.org/10.1016/j.foodhyd.2022.107902
- Wang, X., Huang, J.-H., Meng, B., Mao, K., Zheng, M., Tan, A. et al. (2024). LmGSTF3 Overexpression Enhances Cadmium Tolerance in Lemna minor. Environmental Science & Technology, 59 (5), 2711–2721. https://doi.org/10.1021/acs.est.4c08749
- Zhytniakivska, O., Tarabara, U., Vus, K., Trusova, V., Gorbenko, G. (2024). Interaction of Heavy Metals with β-Lactoglobulin: Molecular Dynamics Study. East European Journal of Physics, 4, 511–517. https://doi.org/10.26565/2312-4334-2024-4-60
- Takano, C., Shigemasa, Y., Aoki, K., Maulidin, I., Kawasaki, S., Nakashima, K. (2025). Protein- and Polysaccharide-Based Heavy Metal Removal Materials: Batch and Continuous Systems. ACS Sustainable Resource Management, 2 (11), 2095–2102. https://doi.org/10.1021/acssusresmgt.5c00270
- Sternke‐Hoffmann, R., Liu, C., Wang, X., Pupart, H., Sun, X., Dreiser, J. G.-H. et al. (2026). Protein‐Driven Copper Redox Regulation: Uncovering the Role of Disulphide Bonds and Allosteric Modulation. Angewandte Chemie International Edition, 65 (15). https://doi.org/10.1002/anie.202519673
- Helikh, A., Filon, A. (2025). Study of the amino acid profile of alternative proteins (Helix pomatia, Lissachatina fulica, Helix aspersa) and their potential application in a healthy diet: optimization of a modern brandade recipe. Technology Audit and Production Reserves, 2 (3 (82)), 71–79. https://doi.org/10.15587/2706-5448.2025.326896
- Park, J. I., McClements, D. J., Choi, S. J. (2025). Impact of small molecule surfactant type and oil phase composition on Ostwald ripening in model food emulsions. Food Science and Biotechnology, 34 (13), 3067–3076. https://doi.org/10.1007/s10068-025-01954-4
- Geng, Q., Zhou, L., Gao, K., Yi, X., Wu, Z., Chen, H. (2026). pH-Shifting-Mediated Coassembly with Polyphenols Reduces Allergenicity and Maintains Functionality of Peanut Protein. Journal of Agricultural and Food Chemistry, 74 (15), 12448–12460. https://doi.org/10.1021/acs.jafc.6c00749
- Huang, G., Liu, G., Xu, Z., Jiang, L., Zhang, Y., Sui, X. (2023). Stability, rheological behavior and microstructure of Pickering emulsions co-stabilized by soy protein and carboxymethyl chitosan. Food Hydrocolloids, 142, 108773. https://doi.org/10.1016/j.foodhyd.2023.108773
- Dobson, S., Marangoni, A. G. (2024). Fat stabilization techniques for the reduction of oil loss in high protein plant-based cheese. Food Hydrocolloids, 156, 110362. https://doi.org/10.1016/j.foodhyd.2024.110362
- Yang, J., Monta, K., Ishikawa, D., Fujii, T. (2025). Analysis of syneresis phenomenon of surimi gel due to freezing and thawing. Food Science and Technology Research, 31 (3), 205–213. https://doi.org/10.3136/fstr.fstr-d-24-00189
- Zimmerman, B. K., Datta, B., Shi, R., Schulman, R., Nguyen, T. D. (2024). A reactive electrochemomechanical theory for growth and remodeling of polyelectrolyte hydrogels and application to dynamic polymerization of DNA hydrogels. Journal of the Mechanics and Physics of Solids, 186, 105568. https://doi.org/10.1016/j.jmps.2024.105568
- Cao, L., Lu, W., Mata, A., Nishinari, K., Fang, Y. (2020). Egg-box model-based gelation of alginate and pectin: A review. Carbohydrate Polymers, 242, 116389. https://doi.org/10.1016/j.carbpol.2020.116389
- Fomich, M., Yuan, Y., Smith, M. D., Krishnan, H. B., Dia, V., Wang, T. (2025). Glycosylated peptides isolated from cheese whey have antifreezing activity. Food Chemistry, 469, 142530. https://doi.org/10.1016/j.foodchem.2024.142530
- Lin, X., Zhang, C., Hu, S., Chen, R. (2024). Heterogeneous ice nucleation of salt solution in porous media. The Journal of Chemical Physics, 160 (9). https://doi.org/10.1063/5.0190862
- Zhao, Q., Hong, X., Fan, L., Liu, Y., Li, J. (2023). Freeze-thaw stability and rheological properties of high internal phase emulsions stabilized by phosphorylated perilla protein isolate: Effect of tea saponin concentration. Food Hydrocolloids, 134, 108001. https://doi.org/10.1016/j.foodhyd.2022.108001
- Anuar, M. N. B., Zuo, J. (2025). Enhancing Yellow Pea Protein Extraction and Purification Through Ultrafiltration. Membranes, 15 (11), 326. https://doi.org/10.3390/membranes15110326
- Tan, Y., Zhang, Z., McClements, D. J. (2023). Preparation of plant-based meat analogs using emulsion gels: Lipid-filled RuBisCo protein hydrogels. Food Research International, 167, 112708. https://doi.org/10.1016/j.foodres.2023.112708
- Du, L., Ru, Y., Weng, H., Zhang, Y., Chen, J., Xiao, A., Xiao, Q. (2024). Agar-gelatin Maillard conjugates used for Pickering emulsion stabilization. Carbohydrate Polymers, 340, 122293. https://doi.org/10.1016/j.carbpol.2024.122293
- El Hosry, L., Elias, V., Chamoun, V., Halawi, M., Cayot, P., Nehme, A., Bou-Maroun, E. (2025). Maillard Reaction: Mechanism, Influencing Parameters, Advantages, Disadvantages, and Food Industrial Applications: A Review. Foods, 14 (11), 1881. https://doi.org/10.3390/foods14111881
- Khan, M. A., Wani, G. A., Majid, H., Farooq, F. U., Reshi, Z. A., Husaini, A. M., Shah, M. A. (2020). Differential Bioaccumulation of Select Heavy Metals from Wastewater by Lemna minor. Bulletin of Environmental Contamination and Toxicology, 105 (5), 777–783. https://doi.org/10.1007/s00128-020-03016-3
- Syrpas, M., Valanciene, E., Augustiniene, E., Malys, N. (2021). Valorization of Bilberry (Vaccinium myrtillus L.) Pomace by Enzyme-Assisted Extraction: Process Optimization and Comparison with Conventional Solid-Liquid Extraction. Antioxidants, 10 (5), 773. https://doi.org/10.3390/antiox10050773
- Kratochvil, D., Volesky, B. (1998). Advances in the biosorption of heavy metals. Trends in Biotechnology, 16 (7), 291–300. https://doi.org/10.1016/s0167-7799(98)01218-9
- Mao, Y., Huang, W., Jia, R., Bian, Y., Pan, M.-H., Ye, X. (2023). Correlation between Protein Features and the Properties of pH-Driven-Assembled Nanoparticles: Control of Particle Size. Journal of Agricultural and Food Chemistry, 71 (14), 5686–5699. https://doi.org/10.1021/acs.jafc.3c00147
- Kazemipour, N., Salehi Inchebron, M., Valizadeh, J., Sepehrimanesh, M. (2016). Clotting characteristics of milk by Withania coagulans: Proteomic and biochemical study. International Journal of Food Properties, 20 (6), 1290–1301. https://doi.org/10.1080/10942912.2016.1207664
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