Effects of Temperature Treatments on Protein Structural Modifications and Aggregation Behavior in Fermented Milks Heated after Culturing

The texture of ambient yogurt, produced by heating fermented milk after culturing (a process known as secondary pasteurization), is often compromised by graininess and roughness resulting from protein aggregation during heat treatment. However, the underlying mechanisms remain unclear, hindering the development of targeted solutions. This study aimed to elucidate the aggregation process and structural characteristics of particle aggregates in fermented milk during secondary pasteurization (55-85°C, 25 s). Circular dichroism (CD) spectroscopy, surface hydrophobicity assays, and protein concentration measurements were used to analyze protein structural changes and the extent of aggregation. Particle aggregates of varying sizes were intercepted and separated using a combination of standard sieves, followed by gel electrophoresis, chemical force analysis, and scanning electron microscopy (SEM) to reveal their composition, intermolecular interactions, and morphology. Results demonstrate that the unfolding of whey proteins and exposure of hydrophobic domains under acidic conditions (pH ~4.50) are the primary drivers of particle aggregation. Increasing the secondary pasteurization temperature promoted the formation of larger aggregates. Notably, the aggregation mechanism shifted with temperature: below 65°C, growth was mainly governed by the formation of disulfide-linked complexes involving κ-casein, LF, BSA, IgG, β-LG, and α-LA. Above 65°C, aggregate enlargement was dominated by inter-aggregate association, likely facilitated by earlier-formed protein complexes acting as molecular bridges. These findings underscore that enhancing the structural integrity of fermented milk proteins during secondary pasteurization and suppressing hydrophobic and interparticle aggregation are essential for improving product quality. Adjusting pasteurization parameters or optimizing conventional stabilizers, such as anionic polysaccharides, to shield proteins may offer promising strategies.