β-Lactoglobulin nanofibrils: Effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions

Almost all published studies of heat-induced β-lactoglobulin self-assembly into amyloid-like fibrils at low pH and low ionic strength have involved heating at 80 °C, and the effect of heating temperature on self-assembly has received little attention. Here we heated β-lactoglobulin at pH 2 and 75 °C, 80 °C, 90 °C, 100 °C, 110 °C or 120 °C and investigated the kinetics of self-assembly (using Thioflavin T fluorescence), the morphology of fibrils, and the rheological properties of fibril dispersions. Self-assembly occurred at all temperatures tested. Thioflavin T fluorescence increased sigmoidally at all temperatures, however it decreased sharply with >3.3 h heating at 110 °C and with >5 h heating at 120 °C. The sharp decreases were attributed partly to local gelation, but destruction of fibrils may have occurred at 120 °C. Thioflavin T fluorescence results indicated that maximal rates of fibril formation increased with increasing temperature, especially above 100 °C, but fibril yield (maximum Thioflavin T fluorescence) was not affected by temperature. At 100 °C and 110 °C, fibrils were slightly shorter than at 80 °C, but otherwise they looked very similar. Fibrils made by heating at 120 °C for 1 h were also similar, but heating at 120 °C for 8 h gave predominantly short fibrils, apparently the products of larger fibrils fragmenting. Heating at 100 °C gave consistently higher viscosity than at 80 °C, and heating for >2 h at 120 °C decreased viscosity, which may have been linked with fibril fragmentation seen in micrographs.