Methods to determine denaturation and aggregation of proteins in low-, medium- and high-heat skim milk powders

Abstract

Skim milk powders (SMPs) of different heat classifications are used in recombined milks and milk products. These SMPs are broadly classified as low-, medium- and high-heat powders, based on their whey protein nitrogen index (WPNI). The WPNI is a measure of undenatured whey protein nitrogen (WPN) content (expressed as milligrams of WPN per gram of powder). This heat classification, based on the WPNI, gives an indirect indication of the denaturation and aggregation of whey proteins and thus the severity of the heat treatments that were used during the manufacture of milk powders. The severity of heat treatment has an impact on the functional properties of the resultant powders or their suitability for different applications. In the present study, we measured the WPNIs of a range of SMPs using a dye binding method (reference method) and attempted to corre- late these results with the WPNI predicted using Fourier transform near infra-red (FT-NIR) spectra, and with denaturation and aggregation of proteins as analysed using various polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE) methods. In most cases, FT-NIR spec- troscopy provided a rapid method for predicting the WPNI of milk powders, with good correlation (R 2 = 0.985). The correlation was used to successfully predict the WPNI of a new set of powders, and the method could potentially be used to determine the WPNI routinely using appropriate con- trols. The denaturation and aggregation of native monomeric whey proteins as analysed by PAGE and CE correlated well with the WPNI of the respective SMP samples. Modified one-dimensional sodium dodecyl sulfate (SDS)-PAGE and two-dimensional SDS- and then reduced SDS-PAGE gave an indication of the type and composition of disulfide-linked protein aggregates and showed some interesting differences between possible protein-protein interactions involved in the manufacture of low-, medium- and high-heat SMPs. The low-heat powder (WPNI 6.76 mg WPN·g −1 powder) retained most of the whey proteins in the native state. In contrast, the high-heat powder (WPNI 0.33 mg WPN·g −1 powder) contained a comparatively small proportion of native whey proteins, although some α-lactalbumin was present. The degree of denaturation of β-lactoglobulin appeared to be crucial and could be related to the WPNI. skim milk powders / WPNI / protein denaturation and aggregation / 1D and 2D gel elec- trophoresis / FT-NIR