Effects of different heat treatments on structural and functional properties of acid-precipitated Buffalo Milk protein fractions.

Acid-precipitated buffalo milk protein fractions are casein-rich fractions with potential nutritional and functional value. However, heat treatment of milk before acid precipitation may markedly alter their structural organization and functional behavior. Buffalo milk was subjected to three thermal conditions: 65 °C for 30 min, 80 °C for 15 s, and 135 °C for 4 s. Acid-precipitated buffalo milk protein fractions, potentially containing co-precipitated whey proteins, were recovered at pH 4.6 to evaluate heat-induced structural and functional changes. Structural characteristics were analyzed using circular dichroism, FT-IR spectroscopy, fluorescence spectroscopy, and scanning electron microscopy. We also tested surface hydrophobicity, emulsifying and foaming properties, antioxidant activity, solubility, and storage stability to assess function. The results revealed that mild and moderate heating induced limited structural alterations. The acid-precipitated protein fractions underwent partial conformational rearrangement, and their surface hydrophobicity increased. These changes improved emulsifying and foaming performance.The 135 °C treatment caused pronounced changes in micellar organization and promoted extensive aggregation. The α-helix level decreased, random coil structures increased, and large uneven aggregates appeared. This severe structural disruption reduced surface hydrophobicity, thereby decreasing the functionality and dispersion stability of the acid-precipitated protein fractions. These findings clarify how thermal intensity governs the structure-function relationship of acid-precipitated buffalo milk protein fractions, and they support better choices of heat treatment in buffalo dairy processing to improve product quality and functionality.