Heat-induced aggregation of β-lactoglobulin was investigated as a function of pH, heating temperature, and NaCl concentration by measurements of reaction kinetics, differential scanning calorimetry, and light scattering. The aggregation can be well interpreted using a reaction scheme consisting of two steps:  a denaturation equilibrium, with a first-order unfolding reaction, followed by second-order aggregation reactions. Denaturation becomes rate limiting at high heating temperature, pH values close to the isoelectric point of the protein, and high NaCl concentration. At neutral pH a maximum is seen in the overall reaction rate as a function of NaCl concentration, which is explained by a stabilizing, salting-out effect of the salt in combination with an increase in the rate of successive aggregation reactions. At high NaCl concentrations physical bonding becomes increasingly important; large aggregates that continue to grow in time are formed, and two phases are distinguished in the aggregation step. The onset time of the secondary aggregation is related to a critical concentration of primary (denatured or small, aggregated) particles. Keywords: Whey; β-lactoglobulin; aggregation; kinetics; light scattering

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SummaryIn situ light scattering, where light scattered from a sample is measured directly while the sample is heated in the instrument, is presented as a simple and effective technique for studying the heat-induced aggregation of β-lactoglobulin. This technique was shown to be applicable not only to monitoring the initial aggregation steps, but to following the overall aggregation process with time. The experiments gave results similar to measurements carried out after a heat-quench treatment, but were more informative. From experiments on a standard NIZO β-lactoglobulin sample, a strongly desalted standard NIZO sample, different genetic variants of β-lactoglobulin and a mixture of these, we concluded that the standard NIZO sample was suitable for studying heat-induced aggregation. This sample has been investigated more extensively. Results with β-lactoglobulin (10–100 g/1) at 65 °C fitted a kinetic model for the denaturation and aggregation of β-lactoglobulin. This model, which held for β-lactoglobulin dissolved in water at near neutral pH and at 60–75 °C, recognizes an initiation, propagation and termination reaction, by analogy with polymer radical chemistry. It gave a quantitatively correct description of the dependence of the scattering intensity on the initial β-lactoglobulin concentration. Salt composition, pH and temperature strongly influenced the aggregation of β-lactoglobulin. Particle size increased with salt concentration in the range studied (up to 20 mM-NaCl and 1·0 mM-CaCl2). When the pH increased from 6°9 to 8·0 particle size was strongly reduced, whereas it strongly increased when pH was lowered to 6·2. Between 61·5 and 70 °C temperature did not affect particle size, whereas aggregation rate strongly increased. These effects could be incorporated in the kinetic model via the reaction constants of the reaction kinetic pathway.

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Whey proteins are globular, heat-sensitive proteins. The gel structure, the formation of this structure, and the rheological properties of particulate whey protein isolate (WPI) gels have been investigated. On increasing the NaCl concentration, the permeability of the WPI gels increased, indicating a coarsening of the gel structure, confirmed by confocal scanning laser microscopy pictures. Only a part of the total amount of protein present contributed to the gel network at the gel point (the primary spatial structure). Large variations were observed in the amount of aggregated material at the gel point (and thus the primary spatial structure) as a function of NaCl concentration, due to differences in the kinetics of the denaturation/aggregation process. After the gel point more protein is incorporated in the gel network by "thickening" the strands in the gel and "decorating" the pores in the gel, apparently without changing the gross spatial structure. Power law behavior was found for the permeability dependence of aged gels on the amount of aggregated material at the gel point. For various salt concentrations the curves coincided to one master curve. This power law behavior is consistent with a primary spatial structure of fractal flocs with a fractal dimensionality of 2.4. The elastic modulus is remarkably related (via a power law) with the total amount of protein contributing to the gel network, in contrast to permeability.