Aggregation of β-lactoglobulin studied by in situ light scattering

Abstract

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.

Influence of moderate electric fields on gelation of whey protein isolate

“…It is known that denaturation of lg under neutral pH and low ionic strength in the temperature range of 60-70 o C forms transparent dispersions, containing relatively small polymeric protein particles with sizes < 100 nm (Hoffmann, Roefs, Verheul, VanMil, & DeKruif, 1996;Roefs & De Kruif, 1994). Native-PAGE provided evidence for the formation of high molecular mass aggregates of whey proteins after heating treatments of WPI solutions at 85 o C (Fig.…”

“…The aggregation mechanism of WPI at neutral pH is close to that of pure lg (Mahmoudi, Mehalebi, Nicolai, Durand, & Riaublanc, 2007). Therefore, this aggregation behavior is considered to be the result of unfolding, and subsequent aggregation of lg, which occur through: disulphide exchange reactions (propagation) (Kazmierski & Corredig, 2003); reactions between reactive, unfolded intermediates and native lg molecules; and reactions by thiol groupthiol group (termination) of reactive intermediates (Hoffmann et al, 1996;Roefs & De Kruif, 1994). During thermal exposure at pH values above the isoelectric point, the unfolding of the protein's native globular structure exposes the hydrophobic region of lg, containing disulphide bonds and the free SH group (Laligant, Dumay, Valencia, Cuq, & Cheftel, 1991).…”

Association behavior of native ?-lactoglobulin

“…The sample contains the genetic variants A and B in a nearly 1:1 ratio. 36 The pure genetic variants of ␤-lg (sample codes N␤-lg A and N␤-lg B) were prepared from the milk of homozygous cows. The ␤-lg samples were prepared by dissolving the ␤-lg powders in 99% D 2 O containing 0 or 0.1M NaCl; 0.02% NaN 3 was added in order to avoid microbial spoilage.…”

“…Protein concentrations were determined by reversed-phase high performance liquid chromatography as described earlier. 36 The pH values given were direct readings of the pH meter. The samples were mounted in sealed quartz cuvettes with a 2 mm path length.…”

Production of Whey Protein-Based Aggregates Under Ohmic Heating

“…The turbidity of the treated WPI solutions was proven to be highly dependent on both the presence of electric fields (independently of their intensity) and the applied heating CUT. Thus, differences found between OH and conventional heating treatments under identical thermal conditions can be explained by the fact that type and number (or size) of aggregates produced varied under the electrical effects of OH (Hoffmann et al 1996;Prabakaran and Damodaran 1997). When CUT is reduced from 100 to 10 s, differences towards conventional heating (at 0 V cm −1 ) are even more noticeable independently of the electric field applied.…”