Use of high-performance size exclusion chromatography to characterize protein aggregation in commercial whey protein concentrates

Protein aggregation profiles of seven commercial whey protein concentrates (WPCs) with protein contents ranging from 32% to 81% were investigated by high-performance size-exclusion chromatography (HPSEC). Two protein fractions were identified: the aggregated protein fraction (APF) and the native protein fraction (NPF). Important differences were found in the proportions of APF ranging from 10% to 29% of the total protein. Centrifugation at 48,000×g for 45 min at 25 °C achieved nearly complete separation of the two fractions for all samples based on HPSEC analysis of both the supernatant and pellet. NPF was shown to contain multiple peaks separable by HPSEC. Electrophoresis (native-PAGE, reducing SDS-PAGE and non-reducing SDS-PAGE) demonstrated that aggregates were bound together by covalent and non-covalent bonds. This study shows that HPSEC provides a quantitative measurement of WPC protein aggregation, which is a useful parameter to be determined prior to WPCs application in food formulations.