Proteolysis of ultra high temperature-treated casein micelles by AprX enzyme from Pseudomonas fluorescens F induces their destabilisation

Destabilisation of ultra high temperature (UHT) treated milk has been linked to residual proteolytic activity after UHT treatment. To understand the physico-chemical modifications of casein micelles by the protease AprX, produced by Pseudomonas fluorescens F, this enzyme was purified and added to raw milk before UHT treatment. Destabilisation of the UHT milk, over three months of storage, was investigated at macroscopic, colloidal and molecular scales. A visual destabilisation appeared progressively over time. At colloidal scale, aggregates were formed and a parallel decrease in zeta potential and hydration of casein micelles was observed. At molecular scale, peptides were released from casein micelles and identified by reversed-phase liquid chromatography coupled with tandem mass spectrometry. The αS1-, αS2-, β- and κ-caseins were hydrolysed, with a preference for β-casein. The results were consistent with the proposition that proteolysis by Ps. fluorescens leading to the destabilisation of milk was due to the activity of AprX.