2026 Covalent protein-phenolic modification – Effect of the phenolic compound structure on protein modification and conformational changes

Authors:
Solange M.L. Ha a b, Kerstin Schild a c, Timon R. Heyn c, Anna-Kristina Marel d, Karin Schwarz c, Wouter J.C. de Bruijn b, Julia K. Keppler a

Journal:
Food Hydrocolloids, Volume 174, May 2026, 112405,doi.org/10.1016/j.foodhyd.2025.112405

Institute:
a Laboratory of Food Process Engineering, Wageningen University & Research, Bornse Weilanden 9, 6708 WG, Wageningen, the Netherlands
b Laboratory of Food Chemistry, Wageningen University & Research, Bornse Weilanden 9, 6708 WG, Wageningen, the Netherlands
c Christian-Albrechts-University of Kiel, Institute of Human Nutrition and Food Science, Division of Food Technology, Heinrich-Hecht Platz 10, 24118, Kiel, Germany
d Department of Food Technology and Bioprocess Engineering, Max Rubner-Institute, Federal Research Institute of Nutrition and Food, 76131, Karlsruhe, Germany


Abstract:
Phenolic compounds can undergo auto-oxidation, especially at alkaline pH, forming reactive o-quinones that bind covalently to proteins and may affect the protein structure, solubility, and functional properties. Yet, it is still unclear how the phenolic compounds' structure and properties affect their reaction with proteins, and how they influence the resulting changes in protein structure and functionality. Therefore, the model protein β-lactoglobulin (BLG) was incubated with ten common phenolic compounds at pH 8.5 for 24 h at a phenolic-to-protein molar ratio of 5:1. RP-HPLC was used to screen for covalent modifications. Protein structural changes were investigated using MALDI-TOF-MS, OPA and Ellman's assays, ATR-FTIR, tryptophan fluorescence quenching, SDS-PAGE, and SEC. Protein functionality changes were determined by oil droplet size measurement after emulsion formation via high pressure homogenization.
Only phenolic compounds with a di or-trihydroxybenzene moiety resulted in noteworthy BLG modification (>40 %), with an average of one phenolic compound bound per protein molecule, primarily on the thiol groups of the cysteine residues. Modification decreased the protein's α-helices and shifted the intramolecular β-sheets to higher wavelengths. Protein modification resulted in the formation of smaller oil droplets (from 1.38 to 2 μm) at low homogenization pressure. A positive relationship was found between the presence of a carboxyl group in the phenolic compounds, the unfolding of the protein's tertiary structure (R2 = 0.95), and smaller oil droplet size. The insights from this study support the selection of phenolic compounds for targeted protein modifications or for avoiding undesired protein modifications.