Hydrolysis of Casein Micelles by AprX Enzym from Pseudomonas fluorescens Induces their Déstabilisation

Casein micelle is a colloidal structure containing different casein molecules and calcium phosphate. Different forces are involved in this micellar organisation and contribute to their stability. However micellar destabilisations can be observed after modifications of physico-chemical conditions like acidification, calcium addition, intensive heat treatments, high pressure or proteolysis with as consequence the formation of a gel or a sediment. The objective of this work was to understand the physico-chemical modifications of casein micelles by AprX, an extracellular protease produced by a psychrotrophic bacteria Pseudomonas fluorescens commonly found in raw milk. For this, we studied the milk destabilisation at macroscopic, colloidal and molecular levels after 1. Inoculation of raw milk by Pseudomonas fluorescens and UHT treatment; 2. Addition of chromatographically purified AprX protease from Pseudomonas fluorescens in milk. For both types of experiment (addition of bacteria or enzyme), destabilisation appeared visually (presence of sediment) and progressively as a function of time. This destabilisation was also determined by measuring the phosphate stability named Ramsdell test. Aggregates were formed with in parallel decreases in the zeta potential and hydration of casein micelles. At molecular level, peptides were released from casein micelles. By reversed phase liquid chromatography coupled to tandem mass spectrometry, one part of the peptides was identified. The as1-, as2-, ab- and k-caseins were hydrolyzed with a quantitative preference for b-casein.The decrease in the stability of casein micelles will be discussed in relation with the modifications of structure and the observed proteolysis. Potential application of this research in term of detection of unstable UHT milks will be proposed knowing that this enzyme is heat-resistant and its implication in the destabilisation of UHT milk during its storage is often evoked.