Elettra | IUVS, SAXS Proteins are the architects of both biological structure and food texture. Whether inside a living cell or in a spoonful of yogurt, they rarely act in isolation. In crowded environments, like the interior of a cell or dense food matrix, proteins interact, fold, and sometimes misfold, leading to aggregation. This process is central not only to many cellular mechanisms but also to food processing. While protein aggregation has long intrigued scientists, the intricate relation between molecular interactions, supramolecular structure, and the resulting macroscopic properties of these aggregates such as viscosity and texture are still not fully understood, especially in systems where different proteins coexist. This study investigates how two well-known milk-derived proteins, β-lactoglobulin (BLG) and bovine serum albumin (BSA), interact and aggregate when mixed under thermal and acidic conditions, mimicking scenarios commonly encountered in food production. The focus is not only understanding how these proteins aggregate, but more importantly how the nature and morphology of the resulting aggregates govern the rheological behavior of the system, in particular its ability to form hydrogels. These hydrogels are crucial to food structure, providing desirable textures and mechanical stability. To trace the full evolution of BLG and BSA from monomers in dilute solution to highly crowded gel networks, we have employed a comprehensive suite of state-of the-art techniques, including UV Resonance Raman Spectroscopy (UVRR) and SmallAngle X-ray Scattering (SAXS) at Elettra Sincrotrone Trieste, Fourier-Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD), Scanning Electron Microscopy (SEM), and Heterodyne Transient Grating Spectroscopy (HD-TG). Some of the main results are presented in Fig. 1. Overall, the joint combination of these techniques allowed for a detailed mapping of structural transitions across molecular to macroscopic scales. The findings reveal that BLG and BSA follow distinctly different aggregation pathways. When subjected to heat and low pH, BLG predominantly forms long amyloid fibrils characterized by highly ordered cross-β architecture. While amyloids are often associated with neurodegenerative diseases, they are increasingly recognized in soft matter field for their mechanical strength and functional properties. In contrast, BSA aggregates into amorphous and disordered structures that lack the spectroscopic hallmarks of amyloids. Interestingly, when BLG and BSA are mixed, they do not co-aggregate into hybrid fibrils. Instead, the presence of BSA influences the aggregation kinetics of BLG, notably accelerating its amyloid formation without incorporating into the fibrils themselves. Rather, BSA loosely associates with the growing fibrillar network, often appearing to wrap around or bridge BLG amyloids, resulting in complex, mixed morphologies that depend on the relative concentrations of each protein. As protein concentration increases, approaching levels typical in food systems, the heat-induced aggregation leads to the formation of hydrogel networks. These proteinbased hydrogels are soft materials and their mechanical behavior is intimately related to the composition and structure of the underlying protein aggregates. Notably, the study finds that the amount of amyloid fibrils within the gel strongly determines its structure and viscosity. High BLG content produces an extended fibrillar network with high characteristic correlation length of the matrix and a higher viscosity, while BSA-rich formulations yield shorter characteristic correlation length of the matrix and less viscous systems. This ability to modulate viscosity by adjusting protein composition and processing conditions presents a valuable tool for food technologists. It enables the tuning of consistency and mouthfeel in both dairy-based and plant-based products. Importantly, it has been highlights that food texture is not merely a macroscopic attribute but it emerges from the molecularscale organization and structure of its components. The multi-level process, from protein unfolding to aggregation and system percolation, ultimately defines the hydrogels structure and performance. By finely controlling the ratio of amyloid and amorphous aggregates, and the extent of molecular crowding and thermal treatment, it becomes possible to design composite gels with tailored structural and mechanical profiles. This bottomup approach to food design could open new possibilities for creating functional materials that are not only appealing in texture but also nutritionally optimized and environmentally sustainable. This work provides a detailed and nuanced picture of how two structurally different proteins behave individually and in tandem under realistic food processing conditions. It maps their behavior across molecular, supramolecular, and macroscopic dimensions, offering a robust framework for the rational design of composite hydrogel matrices. How whey proteins aggregates: Uncovering amyloid formation in mixed systems Elettra Highlights 2024–2025 54
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