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The amount of heat-denatured whey protein is typically determined by pH 4.6 precipitation. Using this method, a significant amount of nondenatured protein was reported even after long heating times. Apparently, a fraction of the unfolded protein refolds into the "native" state rather than form aggregates. This fact is known and has been explained using kinetic models. How the conditions affect the refolding and aggregation is, however, not fully understood. Therefore, this study investigates the unfolding, refolding, and aggregation process of β-lactoglobulin using circular dichroism and size-exclusion chromatography to characterize different folding variants and to quantify their content. The proteins remaining in solution at pH 4.6 were confirmed to be native-like. The nonaggregated fraction contains proteins with a native-like and two types of non-native-like conformations. The nonaggregated fraction increased with decreasing temperature (60-90 °C) and concentration (1-50 g/L) and increasing electrostatic repulsion (pH 7-8; 0-50 mM). The native-like fraction in the nonaggregated fraction was independent of pH, ionic strength, and concentration but increased with decreasing temperature.

Processing beverages containing high concentrations of globular proteins represents a technological challenge due to their instability during heating caused by protein aggregation and gelation. Aggregation of whey protein mixtures was investigated in aqueous model systems at pH 3.5, 4.0, and 4.5 at heating conditions resembling conventional industrial treatment (90 °C for 30 s). The extent of aggregation progressively decreased moving away from the pI. Protein aggregates became smaller and had a more open structure compared to higher pH values. Significant loss of protein dispersibility occurred at pH 4.0 and 4.5 above the denaturation T of whey protein (∼70 °C), at which aggregation was caused by intermolecular hydrophobic interactions. Accessible thiol groups were detected in the heat-treated systems, with a higher intensity at higher pH and increasing extent of aggregation. Intermolecular -S-S- bonding played only a minor role in the aggregation at all conditions studied.

Inclusion of liposoluble bioactive compounds in fortified foods represents a complex challenge due to the labile nature of such compounds and the instability of oil-in-water emulsion-based delivery systems. In the present study, dispersions prepared with 10% (w/w) sunflower oil (SO) or hydrogenated palm kernel oil (HPKO) containing 0.05% (w/w) β-carotene were stabilized by various concentrations of whey protein isolate (WPI) or sodium caseinate (NaCas) (0.1 to 2.0% w/w) in 30% (w/w) sucrose aqueous solutions. Physicochemical characterization of emulsions was done considering the particle size, the particle surface protein coverage, and the physical state of continuous and dispersed phases. Physical stability of the systems and their protection properties on β-carotene were compared. The lipid carrier type and interfacial structure were investigated as the two key factors which regulate the stability of labile lipophilic bioactive molecules in food model systems. Our results showed high β-carotene stability when O/W systems were stable (protein concentration ≥0.8% w/w.) A (partially) solid lipid carrier (HPKO) enhanced protection compared to the liquid carrier (SO) as the bioactive molecules were entrapped in isolated domains within the solid lattice and kept apart from reactive species in the surroundings. NaCas provided a better barrier than WPI due to the different amino acid composition and interface structure which significantly reduced β-carotene degradation rate.