Thermal effects of sonication promote non-covalent amorphous aggregation of in-situ Pinto bean protein

Hydration is a critical pre-processing step in bean processing, as it softens the seeds for consumption and reduces anti-nutritional factors. Conventional thermal hydration is energy-intensive and has a high carbon footprint. Ultrasound-assisted hydration has been proposed as a greener alternative, yet its effects on in-situ proteins remain unclear, particularly given the complex interplay of thermal and physical sonication effects. To address this, we compared four different hydration strategies for pinto beans: isothermal hydration at 25 °C, ultrasound-assisted hydration with and without temperature control, and high-temperature thermal hydration. Isolated proteins were characterised by chromatography, electrophoresis, calorimetry, and infrared spectroscopy, and their functional properties were evaluated using rheology and texture analysis. Isothermal hydration produced the highest protein purity and the strongest gels. Temperature-controlled ultrasound resulted in weaker gels despite similar molecular features, likely due to co-extracted matrix components inhibiting protein–protein crosslinking. Ultrasound without temperature control and high-temperature hydration generated proteins with higher solubility and firmer textures but dominated by large non-covalent aggregates that acted as inert fillers. The contrasting outcomes of the two ultrasound treatments underscore the importance of thermal management and the need for mechanistic understanding under realistic processing conditions to support industrial applications.