Granulomorphometry: a suitable tool for identifying hydrophobic and disulfide bonds in ß-lactoglobulin aggregates. Application to the study of ß-lactoglobulin aggregation mechanism between 70 and 95°C.

This work deals with the investigation of ß-lactoglobulin (ß-LG) aggregation by granulomorphometry. In the first part of this study, we showed that the binding interactions involved in aggregate structure could be identified by their appearance in granulomorphometric pictures. The reliability of this analytical approach was demonstrated by comparing the appearance of ß-LG aggregates in the presence and absence of a thiol-blocking agent (N-ethylmaleimide). The translucency of the aggregates was associated with hydrophobic interactions and their opacity was associated with disulfide bonds. We state, based on the morphology of the aggregates, along with the color of protein aggregates and insoluble materials, that hydrophobic interactions had a better water-holding capacity than disulfide bonds. Additionally, our results suggest that disulfide and hydrophobic bonds compete for ß-LG aggregate shaping. In the second part of this work, interesting features of granulomorphometry useful for identifying aggregate binding interactions were highlighted to clarify the effect of temperature on the aggregation mechanisms occurring in a ß-LG concentrate with a moderate calcium content (6.6mmol·L(-1)). Heat treatment experiments were performed between 70 and 95°C, and granulomorphometric measurements (aggregate size, aggregate number, and gray level of the picture) were conducted at different sampling times up to 4h. Results, which were interpreted in light of calculated ß-LG denaturation levels, revealed that the aggregation mechanism could be split into 2 steps. Initially, ß-LG denatured quickly, leading to fast ß-LG aggregation by disulfide bonds. The denaturation rate then declined, which drastically slowed the disulfide aggregation mechanism. From that point on, a second aggregation path became preponderant. It consisted of the agglomeration of small aggregates by hydrophobic interactions and resulted in the formation of large aggregates containing both interaction types. This second aggregation mechanism was clearly favored at high temperatures because it was not detected in our experiments at temperatures below 85°C.

Influence of calcium on ß-lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mechanisms.

Much research dealing with the processing of milk by-products in heat exchangers has noted the key role of calcium in ß-lactoglobulin (ß-LG) fouling behavior. Nevertheless, the manner by which Ca affects ß-LG denaturation has rarely been quantified using reliable kinetic and thermodynamic data. To this end, the influence of Ca on ß-LG denaturation mechanisms in simulated lactoserum concentrates was studied on the laboratory-scale under 100°C by HPLC analysis. The heat-treated solutions were composed of 53.3g/L ß-LG and were enriched in Ca at various concentrations (0, 66, 132, and 264 mg/kg). The kinetic parameters (reaction order, activation energy, and frequency factor) associated with ß-LG denaturation, along with the unfolding and aggregation thermodynamic parameters were deduced from these experiments and discussed with respect to Ca content. We found that the multistage process characterizing ß-LG thermal denaturation is not greatly affected by Ca addition. In fact, the general model subdividing ß-LG denaturation mechanisms in 2 steps, namely, unfolding and aggregation, remained valid for all tested Ca concentrations. The change in the predominant mechanism from unfolding to aggregation was observed at 80°C across the entire Ca concentration range. Moreover, the classical 1.5 reaction order value was unaffected by the presence of Ca. Interpretation of the acquired kinetic data showed that Ca addition led to a significant increase in kinetic rate, and more so in the aggregation temperature range. This indicates that Ca principally catalyzes ß-LG aggregation, by lowering the Coulombian repulsion between the negatively charged ß-LG reactive species, bridging ß-LG proteins, or via an ion-specific conformational change. To a lesser extent, Ca favors ß-LG unfolding, probably by disturbing the noncovalent binding network of native ß-LG. Simultaneously, Ca has a slight protective role on the native and unfolded ß-LG species, as shown by the increase in activation energy with Ca concentration. The calculation of thermodynamic parameters related to ß-LG denaturation confirmed this observation. A threshold effect in Ca influence was noted in this study: no further significant kinetic rate change was observed above 132 mg/kg of Ca; at this concentration, the studied solution was an almost equimolar mixture of ß-LG and Ca. Finally, we simulated the temporal evolution of ß-LG species concentrations at diverse Ca contents at 3 holding temperatures. The simulations were based on the acquired kinetic parameters. This permitted us to highlight the greater effect of Ca on ß-LG denaturation at high Ca content or for short-time heat treatments at temperatures near 100°C, as in heat exchangers.