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Destabilizations of casein micelles can be observed after modifications of physico-chemical conditions or their proteolysis with as consequence gelation or sedimentation. In this context, the objective of this work were to 1.appreciate the variability of this destabilization of UHT milk during their storage as a function of the strains of Pseudomonas and 2.understand the physico-chemical modifications of casein micelles induced by 9 strains of Pseudomonas fluorescens and also by AprX, an extracellular protease produced by one strain of Pseudomonas fluorescens F. After inoculation of raw milk by these strains or after addition of different concentrations of purified AprX protease in raw milk and UHT treatment, milk destabilization was determined at macroscopic, colloidal and molecular levels. For experiments testing the strain variability, 5 on the 9 tested strains were highly destabilizing. For experiment with purified AprX enzyme, destabilizations were also observed. In all these cases, instabilities were visual (presence of sediment) and increasing as a function of time (after several days or weeks depending on the strains and the concentrations of added enzyme). The analyses of the destabilized UHT milks revealed the presence of aggregates. The zeta potentials and hydrations of casein micelles decreased. At molecular level, we determined a significant proteolysis. The determination by liquid chromatography coupled to mass spectrometry of one part of the released peptides indicated that the α s1-, α s2-, β - and κ-caseins were hydrolyzed with a quantitative preference for β -casein. The nature of the different peptides released was similar for all destabilized UHT milks. 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.

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.

International audience

International audience

International audience

International audience

International audience

International audience