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Protein nanofibrils (PNFs) have been prepared by whey protein fibrillation at low pH and in the presence of different metal ions. The effect of the metal ions was systematically studied both in terms of PNF suspension gelation behavior and fibrillation kinetics. A high valence state and a small ionic radius ( e.g ., Sn 4+ ) of the metal ion resulted in the formation of hydrogels already at a metal ion concentration of 30 mM, whereas an intermediate valence state and larger ionic radius (Co 2+ , Ni 2+ , Al 3+ ) resulted in the hydrogel formation occurring at 60 mM. A concentration of 120 mM of Na + was needed to form a PNF hydrogel, while lower concentrations showed liquid behaviors similar to the reference PNF solution where no metal ions had been introduced. The hydrogel mechanics were investigated at steady-state conditions after 24 h of incubation/gelation, revealing that more acidic (smaller and more charged) metal ions induced ca . 2 orders of magnitude higher storage modulus as compared to the less acidic metal ions (with smaller charge and larger radius) for the same concentration of metal ions. The viscoelastic nature of the hydrogels was attributed to the ability of the metal ions to coordinate water molecules in the vicinity of the PNFs. The presence of metal ions in the solutions during the growth of the PNFs typically resulted in curved fibrils, whereas an upper limit of the concentration existed when oxides/hydroxides were formed, and the hydrogels lost their gel properties due to phase separation. Thioflavin T (ThT) fluorescence was used to determine the rate of the fibrillation to form 50% of the total PNFs ( t  1/2 ), which decreased from 2.3 to ca . 0.5 h depending on the specific metal ions added.

Protein Nanofibrils and Their Hydrogel Formation with Metal Ions