The aim of this study was to gain a better understanding of the effect of buttermilk constituents on the heat-induced aggregation of whey proteins. Spray-dried buttermilk protein concentrates were prepared and powders were hydrated in liquid whey protein concentrate. Samples were heated up to 90 degrees C (pH 4.6) in the presence or absence of a thiol-blocking agent. SDS-PAGE gels under reducing conditions showed that considerable amounts of protein had already lost the native form prior to heating. Confocal images confirmed the presence of protein aggregates prior to heating. The results suggest overall that reactive particles from buttermilk were formed during the processing steps, leading to interactions between protein and components from milk fat globule membrane through both thiol/disulfide exchanges and hydrophobic interactions. It appeared that these "pre-formed" particles could act as aggregation nuclei during heat-induced denaturation of whey proteins. (C) 2014 Elsevier Ltd. All rights reserved.